Prebiotics for treating and / or preventing vitamin k2 deficiency

Abstract

The present invention relates to a prebiotic agent for use in the treatment and / or prevention of vitamin K2 deficiency in a subject. The present invention relates to the use of a prebiotic agent in promoting vitamin K2 production in the gut of a subject.

Description

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 28 November 2024 (28.11.2024) WIPO I PCT lllllllllllllllllllllllllllllll^ (10) International Publication Number WO 2024 / 240923 Al International Patent Classification:(51) A61K 31 / 702 (2006.01) A61K31 / 122 (2006.01) A61K35 / 745 (2015.01) A61K35 / 747 (2015.01) A23L 33 / 21 (2016.01) A23L 33 / 125 (2016.01) A23L 33 / 135 (2016.01) A61P19 / 08 (2006.01) (21) International Application Number: PCT / EP2024 / 064337 w o 20 24 / 2 40 92 3 A l IIII III III III III III III III III III IN (22) International Filing Date: 24 May 2024 (24.05.2024) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 23175289.0 25 May 2023 (25.05.2023) EP (71) Applicant: SOCIETE DES PRODUITS NESTLE S.A. [CH / CH]; Avenue Nestle 55, 1800 VEVEY (CH). (72) Inventors: BOULANGE, Claire Laurence Lucie Marie; Chemindes Chenes3, 1610 Oronla Ville (CH). DUBOUX, Stephane; 90, Chemin de la Damage, 1162 St-Prex (CH). CAMPOS GIMENEZ, Esther; Chemin de la Bioleyre 29, 1681 Billens (CH). HORCAJADA, Marie Noelle; 168, Rue de la Pierre, 01170 Echenevex (FR). BONNET, Nico las; 385 rue Beaupre, 74160 Beaumont (FR). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CO, C, DK, DK, DK, DK, DCU DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, LA, LC, LK, LK LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, ST, SL, SL, TSV, TH, SY, SY TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW,CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, ME, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: — as to applicant's entitlement to apply for and be granted a patent (Rule 4.17(H)) — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(iii)) Published: — with international search report (Art. 21(3)) — in black and white; the international application as fded contained color or greyscale and is available for download from PATENTSCOPE (54) Title: PREBIOTICS FOR TREATING AND / OR PREVENTING VITAMIN K2 DEFICIENCY (57) Abstract: The present invention relates to a prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in a subject. The presentinvention relates to the use of a prebiotic agent to promote vitamin K2 production in the gut of a subject. WO 2024 / 240923 PCT / EP2024 / 064337 PREBIOTICS FOR TREATING AND / OR PREVENTING VITAMIN K2 DEFICIENCY FIELD OF THE INVENTION The present invention relates to agents, compositions, and methods for treating and / or preventing vitamin K2 deficiency in a subject. The present invention also relates to agents, compositions, and methods for promoting vitamin K2 production in the gut of a subject. BACKGROUND TO THE INVENTION Vitamin K2 activates vitamin K-dependent proteins that support many biological functions, such as bone mineralization, the inhibition of vascular stiffness, the improvement of endothelial function, the maintenance of strong teeth, brain development, joint health, and optimal body weight (Koziol-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p.78). However, over the last few decades, vitamin K2 intakes among parents and their offspring have decreased significantly,resulting in serious health implications. Moreover, secondary vitamin K2 deficiency can occur in people who consume adequate amounts, but have malabsorption conditions or liver disease, when vitamin K antagonist drugs are used, or following long-term use of antibiotics of glucocorticoids. Thus, there is a demand for new nutritional interventions to treat and / or prevent vitamin K2 deficiency. SUMMARY OF THE INVENTION The present inventors have surprisingly found that vitamin K2 production in the gastrointestinal tract (e.g. via conversion of vitamin K1) is promoted by a prebiotic agent, for example comprising bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), and / or cello-oligosaccharides (COS). In one aspect, the present invention provides a prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a prebiotic agent in the manufacture of a medicament for treating and / orpreventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a prebiotic agent. 1 WO 2024 / 240923 PCT / EP2024 / 064337 The prebiotic agent may treat or prevent vitamin K2 deficiency by promoting vitamin K2 production in the subject’s gut. For example, the prebiotic agent may treat or prevent vitamin K2 deficiency by promoting de novo menaquinone-7 production and / or bioconversion from phylloquinone to menaquinone-4 in the subject’s gut. In another aspect, the present invention provides use of a prebiotic agent to promote vitamin K2 production in the gut of a subject. In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a prebiotic agent. The prebioticagent may be any suitable prebiotic agent that promotes vitamin K2 production in the subject’s gut. Suitably, the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), cellobiose, cello- oligosaccharides (COS), inulin, lactose, fructo-oligosaccharides (FOS), galacto­ oligosaccharides (GOS), and β-glucan. In some embodiments, the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), a human milk oligosaccharide (HMO) mixture, and cello-oligosaccharides (COS). In some embodiments, the prebiotic agent comprises BMOs in an amount of from about 80 wt% to about 100 wt%, with respect to the total weight of the prebiotic agent. In some embodiments, the prebiotic agent comprises or consists of one or more HMO. In some embodiments, the prebiotic agent comprises or consists of at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and / or at least one N-acetylatedoligosaccharide. Suitably, the at least one sialylated oligosaccharide is selected from the group consisting of 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), syalyllacto-N-tetraose b (LSTb), syalyllacto-N-tetraose c (LSTc), disyallacto-N-tetraose, and combinations thereof. In some embodiments, the at least one sialylated oligosaccharide is selected from 3’- sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and combinations thereof. Suitably, the at least one fucosylated oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lacto-N-fucopentaose-l (LNFP-I), lacto- N-fucopentaose-ll (LNFP-II), lacto-N-fucopentaose-lll (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose V(LNnFP-V), lacto-N-difucosylhexaose-l (LNDFH-1), lacto- N-neodifucosylhexaose (LNnDFH), monofucosyllacto-n-hexaose-lll (MFNLH-III) difucosyllacto-N-hexaose-a (DFLNHa) and combinations thereof. In some embodiments, the at leastone fucosylated oligosaccharide is 2’-fucosyllactose (2’FL) and / or difucosyllactose (diFL). Suitably, the at least one N-acetylated oligosaccharide is selected from the group 2 WO 2024 / 240923 PCT / EP2024 / 064337 consisting of N-acetyl-glucosamine, N-acetyl-galactosamines, lacto-N-tetraose (LNT), lacto- N-neotetraose (LNnT), and combinations thereof. In some embodiments, the at least one N- acetylated oligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and combinations thereof. In some embodiments, the prebiotic agent comprises or consists of cello-oligosaccharides (COS). The prebiotic agent may be administered to the subject in any suitable amounts. Suitably, the prebiotic agent is administered to the subject in a total amount of from about 0.5 g / day to about 10 g / day. Suitably, BMOs are administered to the subject in a total amount of from about 0.5 g / day to about 10 g / day. Suitably, HMOs are administered to the subject in a total amount of from about 0.5g / day to about 10 g / day. Suitably, cello-oligosaccharides (COS) are administered to the subject in a total amount of from about 0.5 g / day to about 10 g / day. The prebiotic agent may be administered in combination with vitamin K1. Vitamin K2 may be formed via metabolic conversion of vitamin K1 during its absorption in the intestinal mucosa and in other organs. The prebiotic agent and vitamin K1 may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and vitamin K1 are administered simultaneously. Suitably, vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day. The prebiotic agent may be administered in combination with a probiotic agent. The present inventors have surprisingly found that the production of vitamin K2 in the gastrointestinal tract (e.g. via conversion of vitamin K1) can be further promoted by administration of a probiotic agent. The prebiotic agent and the probiotic agent maybe administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and the probiotic agent are administered simultaneously. The probiotic agent may comprise any suitable probiotic. Suitably, the probiotic agent comprises Lactobacillus rhamnosus, Bifidobacterium infantis and / or Bifidobacterium lactis. Suitably, a probiotic agent is administered to the subject in a total amount of from about 106 cfu / day to about 1012 cfu / day. The prebiotic agent (or combination therewith) may be provided in any suitable form, for example in the form of a composition. The prebiotic agent (or combination therewith) may be provided in the form of a nutritional composition. The prebiotic agent (or combination therewith) may be provided in the form of a medical food product for clinical nutrition. Suitably, the composition comprises the prebiotic agent in a total amount of from about 0.5 g / 100g to about 10 g / 100g, on a dry weight basis. Suitably, the compositioncomprises vitamin 3 WO 2024 / 240923 PCT / EP2024 / 064337 K1 in an amount of about 5 pg / 100g to about 200 pg / 100g, on a dry weight basis. Suitably, the composition comprises a probiotic agent in an amount of about 106cfu / 100g to about 1012 cfu / 100g, on a dry weight basis. The subject may be any suitable subject. Suitably, the subject is a human or an animal. In preferred embodiments, the subject is a human. In some embodiments, the subject is a juvenile, an adolescent, a child, a toddler, or an infant. In some embodiments, the subject is a child, a toddler, or an infant. In other embodiments, the subject is an adult. The subject may have or may be at risk of vitamin K2 deficiency. In some embodiments, the subject has or is at risk of dyslipidemia, diabetes, thalassemia major (TM), cystic fibrosis (CF), inflammatory bowel diseases (IBD), or chronic liver diseases. In some embodiments, the subject has or is at risk of reduced bone growth and / or bone strength. DESCRIPTION OF DRAWINGS Figure 1- Effect of human milk oligosaccharides (HMOs) on vitamin K2 production in a gut model The following groups were evaluated: Blank; Single HMO (1.3g / L 2FL); HMO mix (2.5g / L 2FL, DiFL, LNnT, LNT, and 6SL); B. infantis (107 cfu / ml); Single HMO + B. infantis·, HMO mix + B. infantis. Figure 2 - Effect of oligosaccharides and probiotic agents on vitamin K2 production in a gut model (A) The following groups were evaluated (in the absence of milk matrix): Blank; and HMOs + BMOs (7.2 g / L total). (B) The following groups were evaluated: Blank; MM (a milk matrix comprising a mix of 6 HMOs); MM + B. infantis (1011 cfu / g). Figure 3 - Effect of human milk oligosaccharides (HMOs) on vitamin K2 production in a gut model (A) The following groups were evaluated (in a milk matrix in the absence of L. rhamnosus LPR): Blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 χ 106 cfu / ml). (B) The following groups were evaluated (in a milkmatrix in the presence of 4.5 χ 107 cfu / ml L. rhamnosus LPR): Blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 χ 106 cfu / ml). (C) The following groups were evaluated (in a milk matrix): w / o LPR (no L. rhamnosus LPR added); and w / LPR (4.5 χ 107 cfu / ml L. rhamnosus LPR). 4 WO 2024 / 240923 PCT / EP2024 / 064337 Figure 4 - Preclinical experimental design Faltering growth group (with vit.KI): the number of pups per BALB / c mother has been increased by 50% from D8 to D18 to induce a food restriction. In both groups (normal and faltering growth), weaning happened at D18. Male and female mice then received food ad libitum and a daily nutritional supplementation through pipet feeding for 30 days. Figure 5 A&B - Impact of Vit.K2AD on trabecular BV / TV and cortical BMD assessed through micro-computed tomography on femurs. Micro-computed tomography (pCT UCT35, Scanco Medical AG, Basserdorf, Switzerland) was used to assesstrabecular and cortical microstructure, respectively, at distal metaphysis and midshaft diaphysis of femurs, as previously describedl. Briefly, trabecular and cortical bone regions were evaluated using isotropic 6 pm voxels. For the femoral trabecular region, to eliminate the primary spongiosa, 30 slices of bone under the distal growth plate were not considered. The 80 slices of secondary spongiosa directly below were analyzed. Femoral cortical structure was assessed using 60 continuous CT slides located at the femur midshaft. Morphometric variables were computed from binarized images using direct, three-dimensional techniques that do not rely on prior assumptions about the underlying structure2. For the trabecular bone regions, BV / TV fraction (%) was assessed. For cortical bone at the femoral midshaft, cortical bone mineral density (Ct.BMD) was assessed. Figure 6 A&B - Impact of Vit.K2AD on femur strength assessed through a 3-point bending test A 3-point bending test was performed totest the biomechanical properties of the femur as previously described (C.H. Turner, D.B. Burr, Basic biomechanical measurements of bone: a tutorial, Bone, 1993, 14(4):595-608). The load was applied in a compression mode at a nominal deformation rate of 0.08mm / s until fracture. Load-displacement curves were recorded to determine the force at rupture normalized by the antero-posterior diameter of the femur (Fmax (N) / AP(mm)) and the energy at rupture normalized by the antero-posterior diameter of the femur (Wmax (N) / AP(mm)). DETAILED DESCRIPTION Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will understand that they can combine all 5 WO 2024 / 240923 PCT / EP2024 / 064337 features of the invention disclosed herein without departing from the scope of the invention as disclosed. Any reference to prior art documents in this specification is not to be considered an admission that such prior art iswidely known or forms part of the common general knowledge in the field. All publications mentioned in the specification are herein incorporated by reference. As used in this specification, the words “comprises”, “comprising”, and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean “including, but not limited to“. The terms “comprises”, “comprising”, and similar words also include the term “consisting of“. The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Numeric ranges are inclusive of the numbers defining the range and all percentages disclosed herein are on a w / w basis, unless stated otherwise.As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%. Prebiotics The present invention provides a prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in a subject. As used herein, the term “prebiotic” may refer to a non-digestible component that benefits the subject by selectively stimulating the favourable growth and / or activity of one or more microbial taxa. The prebiotic agent may be any suitable prebiotic agent that promotes vitamin K2 production. Exemplary prebiotics include bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), cellobiose, cello-oligosaccharides (COS), inulin,lactose, fructo­ oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan. In some embodiments, the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), cellobiose, cello- 6 WO 2024 / 240923 PCT / EP2024 / 064337 oligosaccharides (COS), inulin, lactose, fructo-oligosaccharides (FOS), galacto­ oligosaccharides (GOS), and β-glucan. In some embodiments, the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), a human milk oligosaccharide (HMO) mixture, and cello- oligosaccharides (COS). In some embodiments, the prebiotic agent comprises BMOs and HMOs. In some embodiments, the prebiotic agent comprises HMOs. In some embodiments, the prebiotic agent comprises cello-oligosaccharides (COS). Bovine milk oligosaccharides The prebiotic agent used in the present invention may comprise or consist of bovine milk oligosaccharides (BMOs). Oligosaccharides in bovine milk are assembled in themammary gland by combining the monosaccharides glucose (Glc), galactose (Gal), N-acetylglucosamine (GIcNAc), N- acetylgalactosamine, fucose, and the sialic acids N-acetylneuraminic acid and N- glycolylneuraminic acid. The collection of BMOs found in milk and colostrum has been extensively profiled, with 30-50 structures typically being identified in comprehensive studies (see e.g. Robinson, R.C., 2019. Frontiers in nutrition, 6, p.50). Although bovine milk typically contains fewer oligosaccharide structures than human milk, the two share at least ten common structures, including 3'-sialyllactose and 6'-sialyllactose, which comprise a large percentage of the BMO pool (see e.g. Robinson, R.C., 2019. Frontiers in nutrition, 6, p.50). The prebiotic agent may comprise BMOs in an amount of about 50 wt% or more, about 60 wt% or more, about 70 wt% or more, about 80 wt% or more, about 85 wt% or more, about 90 wt% or more, about 95 wt% or more, or about 100 wt%, with respect to the total weightof the prebiotic agent. Human milk oligosaccharides (HMOs) The prebiotic agent used in the present invention may comprise or consist of one or more human milk oligosaccharide (HMO). Many different kinds of HMOs are found in the human milk and are typically based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N- acetylglucosamine with many and varied linkages between them. Almost all HMOs have a lactose moiety at their reducing end while sialic acid and / or fucose (when present) occupy 7 WO 2024 / 240923 PCT / EP2024 / 064337 terminal positions at the non-reducing ends. HMOs can be acidic (e.g. charged sialic acid containing oligosaccharides) or neutral (e.g. fucosylated oligosaccharides). Suitably, the one or more HMO may comprise at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and / or at least one N-acetylated oligosaccharide. In some embodiments, the one or more HMO comprises or consists of at least onefucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylated oligosaccharide. In some embodiments, the one or more HMO comprises or consists of 2’- fucosyllactose (2’FL), difucosyllactose (diFL), 6’-sialyllactose (6’-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). In some embodiments, the one or more HMO comprises or consists of 2’-fucosyllactose (2’FL), difucosyllactose (diFL), 6’-sialyllactose (6’-SL), 3’- sialyllactose (3’-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). In some embodiments (e.g. when the prebiotic agent comprises BMOs), the prebiotic agent comprises about 0.5 wt% to about 2 wt%, with respect to the total weight of the prebiotic agent, of at least one fucosylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent comprises BMOs), the prebiotic agent comprises about 2 wt% to about 6 wt%, with respect to the total weight of the prebiotic agent, of at least one sialylated oligosaccharide.In some embodiments (e.g. when the prebiotic agent comprises BMOs), the prebiotic agent comprises about 1 wt% to about 4 wt%, with respect to the total weight of the prebiotic agent, of at least one N-acetylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent comprises BMOs), the prebiotic agent comprises about 2 wt% to about 6 wt%, with respect to the total weight of the prebiotic agent, of at least one fucosylated oligosaccharide; and about 1 wt% to about 4 wt%, with respect to the total weight of the prebiotic agent, of at least one N-acetylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent comprises BMOs), the prebiotic agent comprises: about 2 wt% to about 6 wt%, with respect to the total weight of the prebiotic agent, of at least one fucosylated oligosaccharide; about 0.5 wt% to about 2 wt%, with respect to the total weight of the prebiotic agent, of at least one sialylated oligosaccharide; and about 1 wt% to about 4 wt%, with respect tothe total weight of the prebiotic agent, of at least one N- acetylated oligosaccharide. In other embodiments (e.g. when the prebiotic agent does not comprise BMOs), the prebiotic agent comprises about 30 to about 80 wt%, about 40 to about 80 wt%, or about 50 to about 70 wt%, with respect to the total weight of the prebiotic agent, of at least one fucosylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent does not comprise BMOs), the prebiotic agent comprises about 10 to about 35 wt%, about 10 to about 30 wt%, 8 WO 2024 / 240923 PCT / EP2024 / 064337 or about 10 to about 25 wt%, with respect to the total weight of the prebiotic agent, of at least one sialylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent does not comprise BMOs), the prebiotic agent comprises about 10 to about 35 wt%, about 15 to about 30 wt%, or about 15 to about 20 wt%, with respect to the total weight of the prebiotic agent, of at least one N-acetylated oligosaccharide. In someembodiments (e.g. when the prebiotic agent does not comprise BMOs), the prebiotic agent comprises or consists of about 30 to about 80 wt%, about 40 to about 80 wt%, or about 50 to about 70 wt%, with respect to the total weight of prebiotic agent, of at least one fucosylated oligosaccharide; and about 10 to about 35 wt%, about 15 to about 30 wt%, or about 15 to about 20 wt%, with respect to the total weight of the prebiotic agent, of at least one N-acetylated oligosaccharide. In some embodiments (e.g. when the prebiotic agent does not comprise BMOs), prebiotic agent comprises or consists of: about 30 to about 80 wt%, about 40 to about 80 wt%, or about 50 to about 70 wt%, with respect to the total weight of the prebiotic agent, of at least one fucosylated oligosaccharide; about 10 to about 35 wt%, about 10 to about 30 wt%, or about 10 to about 25 wt%, with respect to the total weight of the prebiotic agent, of at least one sialylated oligosaccharide; and about 10 to about 35 wt%, about15 to about 30 wt%, or about 15 to about 20 wt%, with respect to the total weight of the prebiotic agent, of at least one N- acetylated oligosaccharide. The HMOs may be obtained by any suitable method. Suitable methods for synthesising oligosaccharides will be well known to those of skill in the art. For example, processes have been developed for producing oligosaccharides by microbial fermentations, enzymatic processes, chemical syntheses, or combinations of these technologies (see e.g. Zeuner et al., 2019. Molecules, 24(11), p.2033). Fucosylated oligosaccharides In some embodiments, the prebiotic agent comprises at least one fucosylated oligosaccharide. Non-limiting example(s) of fucosylated oligosaccharide(s) include: 2’-fucosyllactose (2’FL), 3- fucosyllactose (3FL), difucosyllactose (diFL), lacto-N-fucopentaose, such as lacto-N- fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP- III) or lacto-N-fucopentaose V (LNFP-V), lacto-N-fucohexaose,lacto-N-difucohexaose I, lacto- neofucopentaose V (LNnFP-V), lacto-N-difucosylhexaose-l (LNDFH-1), lacto-N- neodifucosylhexaose (LNnDFH), fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose (such as fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II), monofucosyllacto-n-hexaose- 9 WO 2024 / 240923 PCT / EP2024 / 064337 III (MFNLH-III), difucosyllacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyllacto-N- neohexaose I, difucosyllacto-N-neohexaose II, difucosyllacto-N-hexaose-a (DFLNHa), fucosyl-para-Lacto-N-hexaose, tri-fuco-para-Lacto-N-hexaose I, and combinations thereof. In preferred embodiments, the at least one fucosylated oligosaccharide comprises 2’- fucosyllactose (2’FL), which is typically the most prevalent HMO naturally present in human breast milk. In some embodiments, the at least one fucosylated oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’FL), difucosyllactose (diFL) and combinations thereof. In some embodiments, the at least onefucosylated oligosaccharide comprises or consists of 2’- fucosyllactose (2’FL) and difucosyllactose (diFL). The fucosylated oligosaccharides may be obtained by any suitable method. For example, 2’FL may be produced by biotechnological means using specific fucosyltransferases and / or fucosidases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Alternatively, 2’FL may be produced by chemical synthesis from lactose and free fucose. diFL may be synthesized by enzymatic, biotechnological and / or chemical processes. Sialylated oligosaccharides In some embodiments, the prebiotic agent comprises at least one sialylated oligosaccharide. Non-limiting examples of sialylated oligosaccharides include: 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL),syalyllacto-N-tetraose b (LSTb), syalyllacto-N-tetraose c (LSTc), disyallacto-N-tetraose, and combinations thereof. In some embodiments, the at least one sialylated oligosaccharide is selected from the group consisting of 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and combinations thereof. In some embodiments, the at least one sialylated oligosaccharide comprises or consists of 6’- sialyllactose (6’-SL). In some embodiments, the at least one sialylated oligosaccharide comprises or consists of 6’-sialyllactose (6’-SL) and 3’-sialyllactose (3’-SL). The sialylated oligosaccharides may be obtained by any suitable method. For example, 3’- sialyllactose (3’-SL), and / or 6’-sialyllactose (6’-SL) may be isolated by chromatographic or filtration technology from a natural source such as animal milks. Alternatively, they may be produced by biotechnological means using specific sialyltransferases or sialidases, 10 WO 2024 / 240923 PCT / EP2024 / 064337 neuraminidases, either by an enzyme basedfermentation technology (recombinant or natural enzymes), by chemical synthesis or by a microbial fermentation technology. In the latter case microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures or mixed cultures may be used. Sialyl-oligosaccharide formation can be initiated by acceptor substrates starting from any degree of polymerisation (DP), from DP=1 onwards. Alternatively, sialyllactoses may be produced by chemical synthesis from lactose and free N’-acetylneuraminic acid (sialic acid). Sialyllactoses are also commercially available for example from Kyowa Hakko Kogyo, Japan, or from GeneChem, Republic of Korea. If the prebiotic agent comprises 3’-Sialyllactose (3’-SL) and 6’-Sialyllactose (6’-SL), it may be particularly beneficial if said 3’-Sialyllactose (3’-SL) and 6’-Sialyllactose (6’-SL) are comprised in said nutritional composition in a weight ratio between about 10:1 andabout 1:10, such as between about 10:1 and about 2:1, between about 8:1 and about 3:1, between about 6:1 and about 3:1, between about 5:1 and about 3:1, between about 5:1 and about 4:1, or between about 1:2 and about 1.5:1. N-acetylated oligosaccharides In some embodiments, the prebiotic agent comprises at least one N-acetylated oligosaccharide. Suitably, the at least one N-acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamines and combinations thereof. Non-limiting examples of N-acetylated oligosaccharide(s) include: LNT (lacto-N-tetraose), para-lacto-N- neohexaose (para-LNnH), LNnT (lacto-N-neotetraose) and any combinations thereof. Other examples are lacto-N-hexaose, lacto-N-neohexaose, para- lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-octaose, lacto-N- neooctaose, iso- lacto-N-octaose, para- lacto-N- octaose and lacto-N-decaose. In some embodiments, the at least one N-acetylated oligosaccharide is selected from thegroup consisting of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and combinations thereof. In some embodiments, the at least one N-acetylated oligosaccharide comprises or consists of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). The N-acetylated oligosaccharides may be obtained by any suitable method. For example, LNnT may be synthesised chemically by enzymatic transfer of saccharide units from donor moieties to acceptor moieties using glycosyltransferases. Alternatively, LNnT may be prepared by chemical conversion of Keto-hexoses (e.g. fructose) either free or bound to an 11 WO 2024 / 240923 PCT / EP2024 / 064337 oligosaccharide (e.g. lactulose) into N-acetylhexosamine or an N-acetylhexosamine- containing oligosaccharide. LNT may be synthesized by enzymatic, biotechnological and / or chemical processes. Cellobiose and cello-oligosaccharides (COS) The prebiotic agent used in the present invention may comprise or consist of cellobiose and / or cello-oligosaccharides (COS).Cellobiose is a disaccharide with the formula (C6H7(OH)4O)2O that is derived from the condensation of a pair of β-glucose molecules forming a β(1^4) bond. Cellobiose can be obtained by enzymatic or acidic hydrolysis of cellulose and cellulose-rich materials. Suitably, the cellobiose is in the form of free cellobiose or cello-oligosaccharides. In some embodiments, the cellobiose is in the form of cello-oligosaccharides (COS). Cello- oligosaccharides may refer to oligomers of β-glucose molecules (e.g. from about 2 to about 6 β-glucose molecules) with p-1,4-linkages, and may contain mainly cellobiose. The prebiotic agent used in the present invention may comprise or consist of cello-oligosaccharides (COS). Studies have shown that COS has prebiotic potential (see e.g. Zhong, C., et al., 2020. Journal of agricultural and food chemistry, 68(32), pp.8557-8567). Administration of prebiotics The subject may be administered any suitable amounts of prebiotic, in any suitable form and via anysuitable route of administration (e.g. in any form and via any route described herein). For example, suitable doses of human oligosaccharides are described in e.g. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2015. EFSA Journal, 13(11), p.4299; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(6), p.e05717; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2020. EFSA Journal, 18(5), p.e06097; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2022. EFSA Journal, 20(5), p.e07331; and EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(12), p.e05907. Suitably, the prebiotic agent is administered to the subject in an amount of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, the prebiotic agent is administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitably, the prebioticagent is administered to the subject in an amount of from about 0.5 g / day to about 10 g / day, from about 1 g / day to about 8 g / day or from about 2 g / day to about 5 g / day. 12 WO 2024 / 240923 PCT / EP2024 / 064337 Suitably, BMOs are administered to the subject in an amount of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, BMOs are administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitably, BMOs are administered to the subject in an amount of from about 0.5 g / day to about 10 g / day, from about 1 g / day to about 8 g / day or from about 2 g / day to about 5 g / day. Suitably, HMOs are administered to the subject in an amount of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, HMOs are administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitably, HMOs are administered to the subject in an amount offrom about 0.5 g / day to about 10 g / day, from about 1 g / day to about 8 g / day or from about 2 g / day to about 5 g / day. Suitably, COS is administered to the subject in an amount of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, COS is administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitably, COS is administered to the subject in an amount of from about 0.5 g / day to about 10 g / day, from about 1 g / day to about 8 g / day or from about 2 g / day to about 5 g / day. Combination therapies The prebiotic agent may be administered in combination with one or more further agents, mixtures, or compositions. As used herein, a “combination therapy” may refer to a therapy comprising the administration of two or more agents (e.g. a prebiotic agent, one or more vitamins, and / or a probiotic agent), mixtures, or compositions. The combination may be administered by any suitable route and in any suitableform. Suitably, the combination is administered by oral and / or enteral administration. In preferred embodiments, the combination is administered by oral administration. The combination may be administered separately, simultaneously or sequentially. In preferred embodiments, the combination is administered simultaneously. In one aspect, the present invention provides a combination of a prebiotic agent, one or more vitamin (e.g. vitamin K1) and one or more probiotic for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a combination of a prebiotic agent, one or more vitamin (e.g. vitamin K1) and one or more probiotic in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. 13 WO 2024 / 240923 PCT / EP2024 / 064337 In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the methodcomprising administering to the subject an effective amount of a combination of a prebiotic agent, one or more vitamin (e.g. vitamin K1) and one or more probiotic. In another aspect, the present invention provides use of a combination of a prebiotic agent, one or more vitamin (e.g. vitamin K1) and one or more probiotic to promote vitamin K2 production in the gut of a subject. In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent, one or more vitamin (e.g. vitamin K1) and one or more probiotic. Vitamins The prebiotic agent may be administered in combination with one or more vitamin. The prebiotic agent and one or more vitamin may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and one or more vitamin are administered simultaneously. Vitamins are organicmicronutrients required by the body to carry out a range of normal functions and include vitamin K1, vitamin A, vitamin D, vitamin C, folate, vitamin B3, vitamin B6, vitamin B12, and vitamin E. Suitably, the one or more vitamins may comprise or consist of vitamin K1, vitamin K2, vitamin A, and / or vitamin D. In preferred embodiments, the one or more vitamins comprises or consists of vitamin K1. In one aspect, the present invention provides a combination of a prebiotic agent and one or more vitamins for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a combination of a prebiotic agent and one or more vitamins in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject aneffective amount of a combination of a prebiotic agent and one or more vitamins. In another aspect, the present invention provides use of a combination of a prebiotic agent and one or more vitamins to promote vitamin K2 production in the gut of a subject. 14 WO 2024 / 240923 PCT / EP2024 / 064337 In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and one or more vitamins. Vitamin K1 In preferred embodiments, the prebiotic agent may be administered in combination with vitamin K1. The prebiotic agent and vitamin K1 may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and vitamin K1 are administered simultaneously. In one aspect, the present invention provides a combination of a prebiotic agent and vitamin K1 for use in treating and / or preventing vitamin K2deficiency in a subject. In another aspect, the present invention provides use of a combination of a prebiotic agent and vitamin K1 in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and vitamin K1. In another aspect, the present invention provides use of a combination of a prebiotic agent and vitamin K1 to promote vitamin K2 production in the gut of a subject. In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and vitamin K1. Vitamin K represents a family of fat-soluble compounds with the common chemical structureof 3-substituted 2-methyl-1,4-napthoquinone. It naturally occurs in food as phylloquinone (vitamin K1) and menaquinones (vitamin K2). Vitamin K1 (phylloquinone) may have the general formula below: 15 WO 2024 / 240923 PCT / EP2024 / 064337 Vitamin K1 has a phytyl side chain and is typically the primary dietary form of vitamin K, and is found in dark green leafy vegetables (e.g. spinach, lettuce and other salad plants) and Brassica. The subject may be administered any suitable amounts of vitamin K1, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein). Suitable doses of vitamin K1 are described in e.g. Koziol-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p.78 and EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2017. EFSA Journal, 15(5), p.e04780. Suitably, vitamin K1 is administered to the subject in an amount of at least about 0.2 pg / kg / day, at least about 0.5 pg / kg / day, or at least about 0.8 pg / kg / day.Suitably, vitamin K1 is administered to the subject in an amount of about 2 pg / kg / day or less, about 1.5 pg / kg / day or less, or about 1.2 pg / kg / day or less. Suitably, vitamin K1 is administered to the subject in an amount of from about 0.2 pg / kg / day to about 2 pg / kg / day, from about 0.5 pg / kg / day to about 1.5 pg / kg / day or from about 0.8 pg / kg / day to about 1.2 pg / kg / day. In some embodiments, vitamin K1 is administered to the subject in an amount of about 1 pg / kg / day Suitably, vitamin K1 is administered to the subject in an amount of at least about 5 pg / day, at least about 10 pg / day, at least about 15 pg / day, at least about 20 pg / day, at least about 25 pg / day, or at least about 30 pg / day. Suitably, vitamin K1 is administered to the subject in an amount of about 200 pg / day or less, about 100 pg / day or less, about 90 pg / day or less, about 80 pg / day or less, about 70 pg / day or less, or about 60 pg / day or less. Suitably, vitamin K1 is administered to the subject in an amount of from about 5pg / day to about 200 pg / day, from about 10 pg / day to about 100 pg / day, from about 15 pg / day to about 90 pg / day, from about 20 pg / day to about 80 pg / day, from about 25 pg / day to about 70 pg / day, or from about 30 pg / day to about 60 pg / day. Suitably, the prebiotic agent is administered to the subject in an amount of from about 0.5 g / day to about 10 g / day and vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day. 16 WO 2024 / 240923 PCT / EP2024 / 064337 Vitamin K2 Whereas phylloquinone is a dietary vitamin K found in plants, menaquinones are vitamin K quinones that are both consumed in the diet and produced by the gut microbiota (see e.g. Walther, B., et al., 2013. Advances in nutrition, 4(4), pp.463-473). In some embodiments, the prebiotic agent promotes de novo menaquinone-7 production in the subject’s gut (e.g. by the gut microbiota). In some embodiments, the prebiotic agent promotes bioconversion from phylloquinone to menaquinone-4 in the subject’sgut (e.g. by the gut microbiota). Vitamin K2 (menaquinone) may have the general formula below: Vitamin K2 consists of various forms, differing in the number (n) of isoprenyl units, wherein n may range from 4 to 13. The various forms are indicated by a suffix (-n), for example, menaquinone-4 (abbreviated MK-4) has four isoprene residues (n=4). MK-4 may be formed via metabolic conversion of phylloquinone during its absorption in the intestinal mucosa and in other organs. Other menaquinones may be produced in the gastrointestinal tract by the gut microbiota. The prebiotic agent may be administered in combination with vitamin K2. The prebiotic agent and vitamin K2 may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and vitamin K2 are administered simultaneously. In one aspect, the present invention provides a combination of a prebiotic agent and vitamin K2 for use in treating and / or preventing vitamin K2 deficiency in a subject. Inanother aspect, the present invention provides use of a combination of a prebiotic agent and vitamin K2 in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and vitamin K2. 17 WO 2024 / 240923 PCT / EP2024 / 064337 The subject may be administered any suitable amounts of vitamin K2, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein). Suitable doses of vitamin K2 are described in e.g. Koziol-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p.78 and European Food Safety Authority (EFSA), 2008. EFSA Journal, 6(11), p.822. Suitably, vitamin K2 is administered to the subject in an amount of at least about 0.5 pg / kg / day, at leastabout 1 pg / kg / day, or at least about 2 pg / kg / day. Suitably, vitamin K2 is administered to the subject in an amount of about 10 pg / kg / day or less, about 7.5 pg / kg / day or less, or about 5 pg / kg / day or less. Suitably, vitamin K2 is administered to the subject in an amount of from about 0.5 pg / kg / day to about 10 pg / kg / day, from about 1 pg / kg / day to about 7.5 pg / kg / day or from about 2 pg / kg / day to about 5 pg / kg / day. Suitably, vitamin K2 is administered to the subject in an amount of at least about 5 pg / day, at least about 10 pg / day, at least about 15 pg / day, at least about 20 pg / day, at least about 25 pg / day, or at least about 30 pg / day. Suitably, vitamin K2 is administered to the subject in an amount of about 200 pg / day or less, about 100 pg / day or less, about 90 pg / day or less, about 80 pg / day or less, about 70 pg / day or less, or about 60 pg / day or less. Suitably, vitamin K2 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day, from about 10 pg / day toabout 100 pg / day, from about 15 pg / day to about 90 pg / day, from about 20 pg / day to about 80 pg / day, from about 25 pg / day to about 70 pg / day, or from about 30 pg / day to about 60 pg / day. Vitamin A Vitamin A comprises a family of molecules containing a 20 carbon structure with a methyl substituted cyclohexenyl ring (beta-ionone ring) and a tetraene side chain with a hydroxyl group (retinol), aldehyde group (retinal), carboxylic acid group (retinoic acid), or ester group (retinyl ester) at carbon-15. The term vitamin A may also include provitamin A carotenoids that are dietary precursors of retinol. Of the many carotenoids in nature, several have provitamin A nutritional activity, including a-carotene, β-carotene, and β-cryptoxanthin. The amount of vitamin A may be referred to in retinol equivalents (RE) or retinol activity equivalents (RAE). For dietary provitamin A carotenoids β-carotene, α-carotene, and β- cryptoxanthin, REs have been set at 6, 12, and 12 pg, respectively. Using pg RAE,the vitamin A activity of provitamin A carotenoids is half the vitamin A activity assumed when using pg RE. For dietary provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin, RAEs have been set at 12, 24, and 24 pg, respectively. (See e.g. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, 18 WO 2024 / 240923 PCT / EP2024 / 064337 Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington (DC): National Academies Press (US); 2001.4, Vitamin A). The subject may be administered any suitable amounts of vitamin A, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein). Suitable doses of vitamin A are described in e.g. Ross, A.C. and Moran, N.E., 2020. Current Developments in Nutrition, 4(10), p.nzaa096 and EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA), 2015. EFSA Journal, 13(3), p.4028. Suitably, vitamin A is administered to the subject inan amount of at least about 100 pg RE / day, at least about 200 pg RE / day, or at least about 300 pg RE / day. Suitably, vitamin A is administered to the subject in an amount of about 1000 pg RE / day or less, about 800 pg RE / day or less, about 600 pg RE / day or less, or about 400 pg RE / day or less. Suitably, vitamin A is administered to the subject in an amount of from about 100 pg RE / day to about 1000 pg RE / day, from about 200 pg RE / day to about 800 pg RE / day, from about 300 pg RE / day to about 600 pg RE / day, or from about 300 pg RE / day to about 400 pg RE / day. Suitably, vitamin A is administered to the subject in an amount of at least about 100 pg RE / day, at least about 200 pg RAE / day, or at least about 300 pg RAE / day. Suitably, vitamin A is administered to the subject in an amount of about 1000 pg RAE / day or less, about 800 pg RAE / day or less, about 600 pg RAE / day or less, or about 400 pg RAE / day or less. Suitably, vitamin A is administered to the subject in an amount of from about 100 pgRAE / day to about 1000 pg RAE / day, from about 200 pg RAE / day to about 800 pg RAE / day, from about 300 pg RAE / day to about 600 pg RAE / day, or from about 300 pg RAE / day to about 400 pg RAE / day. Vitamin D Vitamin D is a group of fat-soluble secosteroids including vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Calcitriol (also known as 1,25-dihydroxyvitamin D) is the active form of vitamin D. Suitably, the vitamin D comprises or consists of vitamin D2 and vitamin D3. Suitably, the vitamin D comprises or consists of calcitriol. The subject may be administered any suitable amounts of vitamin D, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein). Suitable doses of vitamin D are described in e.g. Greer, F.R., 2004. The American journal of clinical nutrition, 80(6), pp.1759S-1762S and EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2016. EFSA Journal, 14(10), p.e04547. Suitably, vitamin D isadministered to the subject in an amount of at least about 2.5 pg / day, at least about 5 pg / day, at least about 10 pg / day, or at least about 15 pg / day. Suitably, vitamin 19 WO 2024 / 240923 PCT / EP2024 / 064337 D is administered to the subject in an amount of about 100 pg / day or less, about 75 pg / day or less, or about 50 pg / day or less. Suitably, vitamin D is administered to the subject in an amount of from about 2.5 pg / day to about 100 pg / day, from about 5 pg / day to about 100 pg / day, from about 10 pg / day to about 75 pg / day or from about 15 pg / day to about 50 pg / day. Suitably, vitamin D is administered to the subject in an amount of about 15 pg / day. Vitamin mixture The prebiotic agent may be administered in combination with a vitamin mixture. The prebiotic agent and vitamin mixture may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and vitamin mixture are administered simultaneously. In one aspect, the present invention provides acombination of a prebiotic agent and a vitamin mixture for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a combination of a prebiotic agent and a vitamin mixture in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and a vitamin mixture. In another aspect, the present invention provides use of a combination of a prebiotic agent and a vitamin mixture to promote vitamin K2 production in the gut of a subject. As used herein, a “vitamin mixture” may refer to a mixture of two or more vitamins. Any suitable vitamin mixture may be used. In some embodiments, the vitamin mixture comprises or consists of vitamin K1,vitamin A, and vitamin D. Suitably, vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day, vitamin A is administered to the subject in an amount of from about 100 pg RE / day to about 1000 pg RE / day, and vitamin D is administered to the subject in an amount of from about 2.5 pg / day to about 100 pg / day. In other embodiments, the vitamin mixture comprises or consists of vitamin K2, vitamin A, and vitamin D. Suitably, vitamin K2 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day, vitamin A is administered to the subject in an amount of from 20 WO 2024 / 240923 PCT / EP2024 / 064337 about 100 pg RE / day to about 1000 pg RE / day, and vitamin D is administered to the subject in an amount of from about 2.5 pg / day to about 100 pg / day. In further embodiments, the present invention provides use of a combination of a prebiotic agent and vitamin K2 for treating and / or preventing vitamin K2 deficiency in a subject. In furtherembodiments, the present invention provides use of a combination of a prebiotic agent and vitamin A for treating and / or preventing vitamin K2 deficiency in a subject. In further embodiments, the present invention provides use of a combination of a prebiotic agent and vitamin D for treating and / or preventing vitamin K2 deficiency in a subject. In further embodiments, the present invention provides use of a combination of a prebiotic agent and vitamin K2, vitamin A, vitamin D, or any combination thereof for treating and / or preventing vitamin K2 deficiency in a subject. Probiotics The prebiotic agent may be administered in combination with one or more probiotic. The prebiotic agent and one or more probiotic may be administered separately, simultaneously or sequentially. In preferred embodiments, the prebiotic agent and one or more probiotic are administered simultaneously. As used herein, the term “probiotic” may refer to a component that contains a sufficient number of viablemicroorganisms to alter the gut microbiota of the subject (see e.g. Hill, C., et al., 2014. Nature reviews Gastroenterology & hepatology, 11(8), p.506). Suitably, the probiotic comprises a commercially available probiotic strain and / or a strain which has been shown to have health benefits (See e.g. Fijan, S., 2014. International journal of environmental research and public health, 11(5), pp.4745-4767). Exemplary probiotic microorganisms may include Bifidobacterium, Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and Escherichia coli. In one aspect, the present invention provides a combination of a prebiotic agent and one or more probiotic for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a combination of a prebiotic agent and one or more probiotic in the manufacture of a medicament for treating and / or preventing vitaminK2 deficiency in a subject. 21 WO 2024 / 240923 PCT / EP2024 / 064337 In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and one or more probiotic. In another aspect, the present invention provides use of a combination of a prebiotic agent and one or more probiotic to promote vitamin K2 production in the gut of a subject. In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a combination of a prebiotic agent and one or more probiotic. The one or more probiotic may increase vitamin K2 production. The present inventors have surprisingly found that probiotics may boost endogenous production of vitamin K2, through the production of precursors. In someembodiments, the one or more probiotic comprises or consists of Lacticaseibacillus, Bifidobacterium, Lactobacillus, and / or Limosilactobacillus. In some embodiments, the one or more probiotic comprises or consists of Lactobacillus rhamnosus, Bifidobacterium longum and / or Bifidobacterium animalis. Lactobacillus rhamnosus In some embodiments, the one or more probiotic comprises or consists of Lactobacillus rhamnosus. Lacticaseibacillus rhamnosus (also known as Lactobacillus rhamnosus) is a short Gram­ positive homofermentative facultative anaerobic non-spore-forming rod that often appears in chains. Lactobacillus rhamnosus GG (LGG) is one of the most widely used probiotic strains. Various health effects are well documented (see e.g. Segers, M.E. and Lebeer, S., 2014. Microbial cell factories, 13(1), pp.1-16). L rhamnosus may boost endogenous production of vitamin K2, through the production of precursors. In some embodiments, the one or more probiotic comprises or consists of Lactobacillusrhamnosus LPR. Bifidobacterium longum In some embodiments, the one or more probiotic comprises or consists of Bifidobacterium longum. 22 WO 2024 / 240923 PCT / EP2024 / 064337 Bifidobacterium longum is a bacterium present in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium, B. infantis, B. longum, and B. suis, were unified into a single species named B. longum with the biotypes infantis, longum, and suis, respectively (Sakata, S., et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp.1945-1951). In some embodiments, the one or more probiotic comprises or consists of Bifidobacterium longum ssp. infantis (also known as Bifidobacterium infantis), Bifidobacterium longum ssp. suis (also known as Bifidobacterium suis), and / or Bifidobacterium longum ssp. longum (also known as Bifidobacterium longum). In some embodiments, the one or more probiotic comprises or consists of Bifidobacterium infantis. In someembodiments, the one or more probiotic comprises or consists of Bifidobacterium infantis LMG 11588 or a derivative thereof (e.g. R0033 which has been communicated to the US Food and Drug Administration as generally recognized as safe (GRAS), see e.g. Duboux, S., et al., 2022. Microorganisms, 10(2), p.203). B. infantis may boost endogenous production of vitamin K2, through the production of precursors. Bifidobacterium animalis In some embodiments, the one or more probiotic comprises or consists of Bifidobacterium animalis. Bifidobacterium animalis is a bacterium of the Bifidobacterium genus which can be found in the large intestines of most mammals, including humans. Bifidobacterium animalis and Bifidobacterium lactis were previously described as two distinct species. Presently, both are considered B. animalis with the subspecies Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis (see Masco, L., et al., 2004. International Journal of Systematic andEvolutionary Microbiology, 54(4), pp.1137- 1143). In some embodiments, the one or more probiotic comprises or consists of Bifidobacterium animalis ssp. lactis (also known as Bifidobacterium lactis). For example, the bacterium, Bifidobacterium lactis HN019, has been studied for a variety of traits important to its ability to function as a probiotic (see e.g. Sanders, M.E., 2006. Journal of clinical gastroenterology, 40(9), pp.776-783). Administration of probiotics 23 WO 2024 / 240923 PCT / EP2024 / 064337 The subject may be administered any suitable amounts of probiotics, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein). Suitably, the one or more probiotic is administered to the subject in a total amount of at least about 105 cfu / day, at least about 106 cfu / day, at least about 107 cfu / day, at least about 108 cfu / day, at least about 109 cfu / day, or at least about 1010 cfu / day. Suitably, the one or more probiotic isadministered to the subject in a total amount of about 1012 cfu / day or less, about 1011 cfu / day or less, or about 1010 cfu / day or less. Suitably, the one or more probiotic is administered to the subject in a total amount of from about 106 cfu / day to about 1012 cfu / day, from about 107 cfu / day to about 1011 cfu / day or from about 108 cfu / day to about 1010 cfu / day. Suitably, Lactobacillus rhamnosus is administered to the subject in an amount of at least about 105 cfu / day, at least about 106 cfu / day, at least about 107 cfu / day, at least about 108 cfu / day, at least about 109 cfu / day, or at least about 1010 cfu / day. Suitably, Lactobacillus rhamnosus is administered to the subject in an amount of about 1012 cfu / day or less, about 1011 cfu / day or less, or about 1010 cfu / day or less. Suitably, Lactobacillus rhamnosus is administered to the subject in an amount of from about 106 cfu / day to about 1012 cfu / day, from about 107 cfu / day to about 1011 cfu / day or from about 108 cfu / day to about 1010cfu / day. Suitably, Bifidobacterium longum (e.g. B. inf antis) is administered to the subject in an amount of at least about 105 cfu / day, at least about 106 cfu / day, at least about 107 cfu / day, at least about 108 cfu / day, at least about 109 cfu / day, or at least about 1010 cfu / day. Suitably, Bifidobacterium longum (e.g. B. infantis) is administered to the subject in an amount of about 1012 cfu / day or less, about 1011 cfu / day or less, or about 1010 cfu / day or less. Suitably, Bifidobacterium longum (e.g. B. infantis) is administered to the subject in an amount of from about 106 cfu / day to about 1012 cfu / day, from about 107 cfu / day to about 1011 cfu / day or from about 108 cfu / day to about 1010 cfu / day. Suitably, Bifidobacterium animalis (e.g. B. lactis) is administered to the subject in an amount of at least about 105 cfu / day, at least about 106 cfu / day, at least about 107 cfu / day, at least about 108 cfu / day, at least about 109 cfu / day, or at least about 1010 cfu / day. Suitably,Bifidobacterium animalis (e.g. B. lactis) is administered to the subject in an amount of about 1012 cfu / day or less, about 1011 cfu / day or less, or about 1010 cfu / day or less. Suitably, Bifidobacterium animalis (e.g. B. lactis) is administered to the subject in an amount of from about 106 cfu / day to about 1012 cfu / day, from about 107 cfu / day to about 1011 cfu / day or from about 108 cfu / day to about 1010 cfu / day. 24 WO 2024 / 240923 PCT / EP2024 / 064337 Suitably, the prebiotic agent is administered to the subject in an amount of from about 0.5 g / day to about 10 g / day and one or more probiotic is administered to the subject in a total amount of from about 106 cfu / day to about 1012 cfu / day. Suitably, the prebiotic agent is administered to the subject in an amount of from about 0.5 g / day to about 10 g / day, vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 200 pg / day, and one or more probiotic is administered to the subject in a total amount of from about 106cfu / day to about 1012 cfu / day. Compositions Suitably, the prebiotic agent (or combination therewith) is in the form of a composition. The composition may comprise the combination in any therapeutically effective amount. The composition can be any type of composition in which the combination can be incorporated, such as a composition in the form of a food or beverage product, an animal feed product, a nutritional supplement for human or animal, or a pharmaceutical composition. The composition may be in solid (e.g. powder), liquid or semi-liquid form. The combination may be in the form of a food composition, a pet food composition, a beverage, a nutritional formula, a nutritional supplement, or a nutraceutical. Food and beverage products include all products intended to be consumed orally by human beings, for the purpose of providing nutrition and / or pleasure. It can for example be a nutritional composition, such as for young children. Examples of food and beverage products include dairyproducts such as milk products or yogurts, soups, sauces, sweet and savoury snacks, powdered drinks and cereal products. In some embodiments, the prebiotic agent (or combination therewith) is in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement. In some embodiments, the prebiotic agent (or combination therewith) is in the form of a nutritional composition. As used herein, a “nutritional composition” may mean a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously, and typically includes a lipid or fat source and a protein source. In some embodiments, the prebiotic agent (or combination therewith) is in the form of a medical food product for clinical nutrition. As used herein, a “medical food product for clinical nutrition” may also be known as a “Food for Special Medical Purposes (FSMP)” and refer to specialised foods designed to help meet the nutritional or dietary needs ofsubjects living with 25 WO 2024 / 240923 PCT / EP2024 / 064337 a disease, disorder or medical condition who are temporarily or permanently unable to achieve an adequate nutritional intake from normal foods or through modification of the normal diet. In some embodiments, the prebiotic agent (or combination therewith) is in the form of an infant formula. In this case, said infant formula can be a preterm infant formula, a human milk fortifier, a starter infant formula, a follow-on formula, a baby-food formula, an infant cereal formula, or a growing-up milk. In some embodiments, the prebiotic agent (or combination therewith) is in the form of a growing-up milk. The term “growing-up milk” (or GUM) as used herein refers to a milk formula product given from one year onwards. It is generally a diary-based beverage adapted for the specific nutritional needs of young children (e.g. children aged from about 1 to about 3). Growing-up milk may also be known as “young-child formula” or “toddlers’ milk”.In some embodiments, the prebiotic agent (or combination therewith) is in the form of a milk formula. The term "milk formula" as used herein may refer to a foodstuff intended for e.g. childhood nutrition, which may provide the sole source or supplemental nutrition for children e.g. aged about 3 years or older. In some embodiments, the milk formula is a grow milk. In some embodiments, the composition is in a powder form and reconstituted in an aqueous medium (e.g. water) prior to administration. In other embodiments, the composition is in a liquid form ready for administration (e.g. a ready-to-feed formula). In another embodiment, the prebiotic agent (or combination therewith) is in the form of a supplement. As used herein, a "supplement" or “dietary supplement” may be used to complement the nutrition of a subject (it is typically used as such but it might also be added to any kind of compositions intended to be ingested by the subject). When the composition is a supplement, it can beprovided in the form of unit doses. Supplements are typically present in the form of a liquid, a gel, a powder or a tablet or capsule. Powder supplements typically encompass supplements to be dissolved in water or milk, or to be sprinkled on food or in a beverage. Such supplements are intended to provide additional nutrients and / or a health benefit to the subject consuming it. A supplement can be used for providing nutrients and / or a health benefit to human beings, as well as to animals. In another embodiment, the prebiotic agent (or combination therewith) is in the form of a fortifier. The fortifier can be an infant formula fortifier or growing-up milk fortifier. In another embodiment, the prebiotic agent (or combination therewith) is in the form of a pharmaceutical product. Pharmaceutical products include for example drops, syrups, powder, 26 WO 2024 / 240923 PCT / EP2024 / 064337 tablet or capsule products intended to treat of prevent an adverse medical condition in a subject in needthereof. The prebiotic agent (or combination therewith) can also be in the form of an animal food product or a nutritional supplement for animals. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. Prebiotics A nutritional composition according to the invention may contain any suitable amounts of the prebiotic agent. Suitably, the nutritional composition comprises the prebiotic agent in a total amount of at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4 wt%, or at least about 5 wt% on a dry weight basis. Suitably, the nutritional composition comprises the prebiotic agent in a total amount of about 10 wt% or less, about 8 wt% or less or about 5 wt% or less, on a dry weight basis. Suitably, the nutritional composition comprises the prebiotic agent in a total amount of from about 0.5 wt% to about 10 wt%, about1 wt% to about 8 wt%, or about 2 wt% to about 5 wt%, on a dry weight basis. Suitably, the prebiotic agent comprises BMOs in an amount of from about 80 wt% to about 100 wt%, with respect to the total weight of the prebiotic agent. Suitably, the nutritional composition comprises BMOs in a total amount of at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4 wt%, or at least about 5 wt% on a dry weight basis. Suitably, the nutritional composition comprises BMOs in a total amount of about 10 wt% or less, about 8 wt% or less or about 5 wt% or less, on a dry weight basis. Suitably, the nutritional composition comprises BMOs in a total amount of from about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt%, on a dry weight basis. Suitably, the nutritional composition comprises HMOs in a total amount of at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4wt%, or at least about 5 wt% on a dry weight basis. Suitably, the nutritional composition comprises HMOs in a total amount of about 10 wt% or less, about 8 wt% or less or about 5 wt% or less, on a dry weight basis. Suitably, the nutritional composition comprises HMOs in a total amount of from about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt%, on a dry weight basis. 27 WO 2024 / 240923 PCT / EP2024 / 064337 Suitably, the nutritional composition comprises at least one fucosylated oligosaccharide in an amount of about 0.05 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, or about 0.2 wt% to about 1.5 wt%, on a dry weight basis. Suitably, the nutritional composition comprises at least one sialylated oligosaccharide in an amount of about 0.01 wt% to about 2 wt%, about 0.05 wt% to about 1.5 wt%, or about 0.07 wt% to about 1 wt%, on a dry weight basis. Suitably, the nutritional composition comprises at least one N-acetylated oligosaccharide in an amount ofabout 0.01 wt% to about 1 wt%, about 0.03 wt% to about 0.6 wt%, or about 0.05 wt% to about 0.5 wt%, on a dry weight basis. Suitably, the nutritional composition comprises 2’FL in a total amount of from about 0.05 g / L to about 2 g / L, from about 0.1 g / L to about 1 g / L, from about 0.15 g / L to about 0.8 g / L, from about 0.2 g / L to about 0.7 g / L, or from about 0.25 g / L to about 0.6 g / L (the concentration may refer to the concentration after the composition has been reconstituted e.g. with water). Suitably, the nutritional composition comprises fucosylated oligosaccharide(s) (e.g. 2’FL and / or diFL) in a total amount of from about 0.1 g / L to about 4 g / L, from about 0.1 g / L to about 3.5 g / L, from about 0.15 g / L to about 3 g / L, from about 0.2 g / L to about 2.5 g / L, from about 0.3 g / L to about 2 g / L, from about 0.4 g / L to about 2 g / L, or from about 0.5 g / L to about 2 g / L (the concentration may refer to the concentration after the composition has been reconstituted e.g. with water). In a particularembodiment, the nutritional composition comprises from about 0.2 g / L to about 1.8 g / L of total fucosylated oligosaccharide(s). Suitably, the nutritional composition comprises sialylated oligosaccharide(s) (e.g. 3’- sialyllactose (3’-SL) and / or 6’-sialyllactose (6’-SL) in a total amount of from about 0.05 g / L to about 0.75 g / L, from about 0.05 g / L to about 0.5 g / L, from about 0.1 g / L to about 0.3 g / L, or from about 0.1 g / L to about 0.4 g / L (the concentration may refer to the concentration after the composition has been reconstituted e.g. with water). In a particular embodiment, the nutritional composition comprises from about 0.12 g / L to about 0.4 g / L of total sialylated oligosaccharide(s). Suitably, the nutritional composition comprises N-acetylated oligosaccharide(s) (e.g. LNT and / or LNnT) in a total amount of from about 0.05 g / L to about 0.5 g / L, from about 0.1 g / L to about 0.5 g / L, from about 0.2 g / L to about 0.4 g / L, or about 0.3 g / L (the concentration may refer to theconcentration after the composition has been reconstituted e.g. with water). Suitably, the nutritional composition comprises: 28 WO 2024 / 240923 PCT / EP2024 / 064337 - at least one sialylated oligosaccharide in in a total amount of from about 0.05 g / L to about 0.75 g / L, from about 0.05 g / L to about 0.5 g / L, from about 0.1 g / L to about 0.3 g / L, or from about 0.1 g / L to about 0.4 mg / L; - at least one fucosylated oligosaccharide in a total amount of from about 0.1 g / L to about 4 g / L, from about 0.1 g / L to about 3.5 g / L, from about 0.15 to about 3 g / L, from about 0.2 g / L to about 2.5 g / L, from about 0.3 g / L to about 2 g / L, from about 0.4 g / L to about 2 g / L, or from about 0.5 g / L to about 2 g / L; and / or - at least one N-acetylated oligosaccharide in a total amount of from about 0.05 g / L to about 0.5 g / L, from about 0.1 g / L to about 0.5 g / L, or from about 0.2 g / L to about 0.4 g / L. Suitably, the nutritional composition comprises COS in an amount of at least about 0.5 wt%, at least about 1 wt%, atleast about 2 wt%, at least about 3 wt%, at least about 4 wt%, or at least about 5 wt% on a dry weight basis. Suitably, the nutritional composition comprises COS in an amount of about 10 wt% or less, about 8 wt% or less or about 5 wt% or less, on a dry weight basis. Suitably, the nutritional composition comprises COS in an amount of from about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt%, on a dry weight basis. Vitamins A nutritional composition according to the invention may contain any suitable amounts of vitamins. Suitably, the nutritional composition comprises vitamin K1 in an amount of at least about 5 pg / 100g, at least about 10 pg / 100g, at least about 15 pg / 100g, at least about 20 pg / 100g, at least about 25 pg / 100g, or at least about 30 pg / 100g on a dry weight basis. Suitably, the nutritional composition comprises vitamin K1 in an amount of about 2100 pg / 100g or less, about 100 pg / 100g or less, about 90 pg / 100g or less, about 80 pg / 100g orless, about 70 pg / 100g or less, or about 60 pg / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises vitamin K1 in an amount of from about 5 pg / 100g to about 200 pg / 100g, from about 10 pg / 100g to about 100 pg / 100g, from about 15 pg / 100g to about 90 pg / 100g, from about 20 pg / 100g to about 80 pg / 100g, from about 25 pg / 100g to about 70 pg / 100g, or from about 30 pg / 100g to about 60 pg / 100g. Suitably, the nutritional composition comprises vitamin K2 in an amount of at least about 5 pg / 100g, at least about 10 pg / 100g, at least about 15 pg / 100g, at least about 20 pg / 100g, at least about 25 pg / 100g, or at least about 30 pg / 100g on a dry weight basis. Suitably, the 29 WO 2024 / 240923 PCT / EP2024 / 064337 nutritional composition comprises vitamin K2 in an amount of about 2100 pg / 100g or less, about 100 pg / 100g or less, about 90 pg / 100g or less, about 80 pg / 100g or less, about 70 pg / 100g or less, or about 60 pg / 100g or less, on a dry weight basis. Suitably, thenutritional composition comprises vitamin K2 in an amount of from about 5 pg / 100g to about 200 pg / 100g, from about 10 pg / 100g to about 100 pg / 100g, from about 15 pg / 100g to about 90 pg / 100g, from about 20 pg / 100g to about 80 pg / 100g, from about 25 pg / 100g to about 70 pg / 100g, or from about 30 pg / 100g to about 60 pg / 100g. Suitably, the nutritional composition comprises vitamin A in an amount of at least about 100 pg RE / 100g, at least about 200 pg RE / 100g, or at least about 300 pg RE / 100g, on a dry weight basis. Suitably, the nutritional composition comprises vitamin A in an amount of about 1000 pg RE / 100g or less, about 800 pg RE / 100g or less, about 600 pg RE / 100g or less, or about 400 pg RE / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises vitamin A in an amount of from about 100 pg RE / 100g to about 1000 pg RE / 100g, from about 200 pg RE / 100g to about 800 pg RE / 100g, from about 300 pg RE / 100g to about 600 pg RE / 100g, or from about 300 pg RE / 100g toabout 400 pg RE / 100g, on a dry weight basis. Suitably, the nutritional composition comprises vitamin A in an amount of at least about 100 pg RAE / 100g, at least about 200 pg RAE / 100g, or at least about 300 pg RAE / 100g, on a dry weight basis. Suitably, the nutritional composition comprises vitamin A in an amount of about 1000 pg RAE / 100g or less, about 800 pg RAE / 100g or less, about 600 pg RAE / 100g or less, or about 400 pg RAE / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises vitamin A in an amount of from about 100 pg RAE / 100g to about 1000 pg RAE / 100g, from about 200 pg RAE / 100g to about 800 pg RAE / 100g, from about 300 pg RAE / 100g to about 600 pg RAE / 100g, or from about 300 pg RAE / 100g to about 400 pg RAE / 100g, on a dry weight basis. Suitably, the nutritional composition comprises vitamin D in an amount of at least about 5 pg / 100g, at least about 10 pg / 100g, or at least about 15 pg / 100g, on a dry weight basis. Suitably, the nutritional compositioncomprises vitamin D in an amount of about 100 pg / 100g or less, about 75 pg / 100g or less, or about 50 pg / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises vitamin D in an amount of from about 5 pg / 100g to about 100 pg / 100g, from about 10 pg / 100g to about 75 pg / 100g or from about 15 pg / 100g to about 50 pg / 100g, on a dry weight basis. Probiotics A nutritional composition according to the invention may contain any suitable amounts of probiotic. 30 WO 2024 / 240923 PCT / EP2024 / 064337 Suitably, the nutritional composition comprises the one or more probiotic in a total amount of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at least about 107 cfu / 100g, or at least about 108cfu / 100g, at least about 109cfu / 100g, or at least about 101°cfu / 100g on a dry weight basis. Suitably, the nutritional composition comprises the one or more probiotic in a total amount of about 1012 cfu / 100g or less, about 1011 cfu / 100g or less, about 1010 cfu / 100g or less, on adry weight basis. Suitably, the nutritional composition comprises the one or more probiotic in a total amount of about 106cfu / 100g to about 1012 cfu / 100g, about 107cfu / 100g to about 1011 cfu / 100g, or about 108cfu / 100g to about 1010 cfu / 100g, on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium longum (e.g. B. infantis) in an amount of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at least about 107 cfu / 100g, or at least about 108 cfu / 100g, at least about 109 cfu / 100g, or at least about 1010 cfu / 100g on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium longum (e.g. B. infantis) in an amount of about 1012 cfu / 100g or less, about 1011 cfu / 100g or less, about 1010 cfu / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium longum (e.g. B. infantis) in an amount of about 106 cfu / 100g to about 1012 cfu / 100g, about 107cfu / 100g to about 1011 cfu / 100g, or about108cfu / 100g to about 1010 cfu / 100g, on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium animalis (e.g. B. lactis) in an amount of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at least about 107 cfu / 100g, or at least about 108 cfu / 100g, at least about 109 cfu / 100g, or at least about 1010 cfu / 100g on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium animalis (e.g. B. lactis) in an amount of about 1012 cfu / 100g or less, about 1011 cfu / 100g or less, about 1010 cfu / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises Bifidobacterium animalis (e.g. B. lactis) in an amount of about 106 cfu / 100g to about 1012 cfu / 100g, about 107cfu / 100g to about 1011 cfu / 100g, or about 108cfu / 100g to about 1010 cfu / 100g, on a dry weight basis. Suitably, the nutritional composition comprises Lactobacillus rhamnosus in an amount of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at leastabout 107 cfu / 100g, or at least about 108 cfu / 100g, at least about 109 cfu / 100g, or at least about 1010 cfu / 100g on a dry weight basis. Suitably, the nutritional composition comprises Lactobacillus rhamnosus in an amount of about 1012 cfu / 100g or less, about 1011 cfu / 100g or less, about 1010 cfu / 100g or less, on a dry weight basis. Suitably, the nutritional composition comprises Lactobacillus rhamnosus in an amount of about 106cfu / 100g to about 1012 cfu / 100g, about 107cfu / 100g to about 1011 cfu / 100g, or about 108 cfu / 100g to about 1010 cfu / 100g, on a dry weight basis. 31 WO 2024 / 240923 PCT / EP2024 / 064337 Other components A nutritional composition of the invention may contain a protein source, a carbohydrate source and a lipid source. In some embodiments however, especially if a nutritional composition of the invention is a supplement or a fortifier, there may be only lipids (or a lipid source). The nutritional composition of the invention may comprise about 100 kcal / 100g to about 1000kcal / 100g, about 200 kcal / 100g to about 800 kcal / 100g, or about 400 kcal / 100g to about 600 kcal / 100g, on a dry weight basis. Protein A nutritional composition according to the invention may contain a protein source. The protein may be in an amount of from about 1g to about 4g per 100 kcal, or about 1.5g to about 3g per 100 kcal. Protein sources based on, for example, whey, casein and mixtures thereof may be used as well as plant based protein sources, for example, based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions. In some embodiments the protein source is whey predominant (i.e. more than 50% of proteins are coming from whey proteins, such as 60%> or 70%>). The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. By the term "intact" is meant that the main part of the proteins are intact,i.e. the molecular structure is not altered, for example at least 80% of the proteins are not altered, such as at least 85% of the proteins are not altered, preferably at least 90% of the proteins are not altered, even more preferably at least 95% of the proteins are not altered, such as at least 98% of the proteins are not altered. In a particular embodiment, 100% of the proteins are not altered. The term "hydrolysed" means in the context of the present invention a protein which has been hydrolysed or broken down into its component amino acids. The proteins may be either fully or partially hydrolysed. If hydrolysed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, whey protein hydrolysates may be prepared by enzymatically hydrolysing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockageduring the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10%> by weight of lysine; for example about 7% by weight of lysine which greatly improves the nutritional quality of the protein source. 32 WO 2024 / 240923 PCT / EP2024 / 064337 In one particular embodiment, the proteins of the composition are hydrolysed, fully hydrolysed or partially hydrolysed. The degree of hydrolysis (DH) of the protein can be between 2 and 20, or between 8 and 40, or between 20 and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90. At least 70%, 80%, 85%, 90%, 95% or 97% of the proteins may be hydrolysed. In a particular embodiment, 100% of the proteins are hydrolysed. In one particular embodiment, the proteins of the composition are plant based protein. Carbohydrate A nutritional composition according to the present invention may contain a carbohydrate source. The carbohydrate may be in an amount of from about5g to about 20g per 100 kcal, or about 10g to about 15g per 100 kcal. Any carbohydrate source conventionally found in nutritional compositions such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof may be used although one of the preferred sources of carbohydrates is lactose. Lipid A nutritional composition according to the present invention may contain lipids and essential fatty acids. The lipids may be in an amount of from about 1g to about 10g per 100 kcal, or about 2g to about 6g per 100 kcal. Non-limiting examples of lipids include: palm olein, high oleic sunflower oil, high oleic safflower oil, canola oil, fish oil, coconut oil, bovine milk fat, and combinations thereof. It may be particularly beneficial if the composition comprises fat in an amount of about 25 to about 30g / 100g dry weight of the composition. Non-limiting examples of essential fatty acids include: linoleic acid (LA), α-linolenic acid (ALA). Compositions of the invention may furthercontain gangliosides monosialoganglioside-3 (GM3) and disialogangliosides 3 (GD3), and combinations thereof. Other components A nutritional composition of the invention may also contain all vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins and other nutrients optionally present in the composition of the invention include vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin E, vitamin C, folic acid, 33 WO 2024 / 240923 PCT / EP2024 / 064337 inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population. Ifnecessary, a nutritional composition of the invention may contain emulsifiers and stabilisers such as soy, lecithin, citric acid esters of mono- and diglycerides, and the like. A nutritional composition of the invention may also contain other substances which may have a beneficial effect, especially on bone health or bone development, such as lactoferrin, osteopontin, TGFbeta, slgA, glutamine, nucleotides, nucleosides, and the like. Preparation of compositions The compositions according to the present invention may be prepared by any known or otherwise suitable manner. For example, a nutritional composition may be proposed by blending together a source of protein with a carbohydrate source and a lipid source in appropriate proportions. If used, emulsifiers may be included at this stage. Vitamins and minerals may be added at this stage, but may also be added later to avoid thermal degradation. Water, preferably water which has been subjected to reverse osmosis or deionized water, maythen be added and mixed in to form a liquid mixture. The temperature of mixing is preferably room temperature, but may also be higher. The liquid mixture may then be thermally treated to reduce bacterial loads. The mixture may then be homogenized. If it is desired to produce a powdered composition, the homogenized mixture is dried in a suitable drying apparatus, such as a spray drier or freeze drier and converted into powder. Processes used in the manufacture of formula are based on the concept that the products must be nutritionally adequate and microbiologically safe to consume. Thus, steps that eliminate or restrict microbiological growth are central to production processes. The processing technology for each specific formula is proprietary to the manufacturer but, in general, it involves the preservation of an oil-in-water (o / w) emulsion by dehydration in the case of powder products or, sterilization in the case of ready-to-feed or concentrated liquid products. Powdered formula maybe produced using various processes, such as dry blending dehydrated ingredients to constitute a uniform formula or hydrating and wet-mixing a mixture of macro-ingredients, such as fat, protein and carbohydrate ingredients and then evaporating and spray drying the resultant mixture. A combination of the two processes described above may be used where a base powder is first produced by wet-mixing and spray drying all or some of the macro-ingredients and then dry blending the remaining ingredients, including carbohydrate, minerals and vitamins and other micronutrients, to create a final formula. Liquid 34 WO 2024 / 240923 PCT / EP2024 / 064337 formulae are available in a ready-to-feed format or as a concentrated liquid, which requires dilution, normally 1:1, with water. The manufacturing processes used for these products are similar to those used in the manufacture of recombined milk. If it is desired to produce a liquid formula, the homogenized mixture may be filled into suitable containers,preferably aseptically. However, the liquid composition may also be retorted in the container, suitable apparatus for carrying out the filling and retorting of this nature is commercially available. Subject The subject may be any suitable subject. Suitably, the subject may be a mammal. In preferred embodiments, the subject is a human. In other embodiments, the subject is an animal, preferably wherein the animal is a pet. A pet may be an animal selected from dogs, cats, birds, fish, rodents such as mice, rats, and guinea pigs, rabbits, etc. In some embodiments, the pet is a dog. In some embodiments, the pet is a small dog breed. In some embodiments, the subject is a juvenile. The term “juvenile” may refer to an individual that has not yet reached adulthood. In some embodiments, the subject is an adolescent or a child. The term “adolescent” may refer to an individual during the period from the onset of puberty to adulthood. The term “child” may refer an individual between the stages ofbirth and puberty. In some embodiments, the subject is an infant, a toddler, or a young child. The term “infant” may refer to a subject aged from about 0 years to about 1 year. The term “toddler” may refer to a subject aged from about 1 year to about 3 years. The term “young child” may refer to a subject aged from about 3 years to about 10 years, from about 3 years to about 9 years, from about 3 years to about 8 years, from about 3 years to about 7 years, from about 3 years to about 6 years, or from about 3 years to about 5 years. In some embodiments, the subject is a newborn infant. Newborn infants typically have low vitamin K and may be at risk of vitamin K deficiency bleeding (see e.g. Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p.780). In some embodiments, the subject is a neonate. In some embodiments, the subject is aged about 5 years or less, about 4 years or less, about 3 years or less, about 2 years or less, or about 1 year or less. In some embodiments, the subject isaged about 12 months or less, about 11 months or less, about 10 months or less, about 9 months or less, about 8 months or less, about 7 months or less, or about 6 months or less. In some embodiments, the subject is aged about 0.5 months or older. In some embodiments, the subject is aged from about 0.5 months to about 6 months. 35 WO 2024 / 240923 PCT / EP2024 / 064337 In some embodiments, the subject is an adolescent or an adult. In some embodiments, the subject is an adult. In some embodiments, the subject is elderly. In some embodiments, the subject is at least 60 years of age, at least 65 years of age, at least 70 years of age, at least 75 years of age, or at least 80 years of age. The subject may have or may be at risk of vitamin K2 deficiency. The subject may have or may be at risk of primary vitamin K2 deficiency, i.e. insufficient consumption of vitamin K2. The subject may have or may be at risk of secondary vitamin K2 deficiency. Secondary deficiencies can occur in people who consumeadequate amounts, but have malabsorption conditions, such as cystic fibrosis or chronic pancreatitis, and in people who have liver damage or disease. Secondary vitamin K deficiency can also occur in people who have a prescription for a vitamin K antagonist drug. Long-term antibiotic use and long-term glucocorticoid use can also affect vitamin K levels, particularly in children (see e.g. Koziol-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p.78). In some embodiments, the subject has or is at risk of dyslipidemia, diabetes, thalassemia major (TM), cystic fibrosis (CF), inflammatory bowel diseases (IBD), or liver diseases. In some embodiments, the subject has cystic fibrosis, inflammatory bowel disease (e.g. Crohn’s Disease or ulcerative colitis), liver disease (e.g. chronic liver disease), long-term antibiotic use, or long-term glucocorticoid use. The subject may have or may be at risk of reduced bone growth and / or bone strength. Vitamin K is required for the gamma-carboxylationof osteocalcin in bone and may be required to form strong bones (see e.g. Hamidi, M.S., et al., Journal of clinical densitometry, 16(4), pp.409- 413). The present invention is suitable for children who were born preterm or with low-birth weight or experienced intra-uterine growth retardation or who suffered from growth stunting because of malnutrition or experienced disease such as Crohn’s disease and / or celiac disease and / or cancer or who were treated with drugs leading to malabsorption, anorexia and / or metabolic bone disease, such as chemotherapy drugs and / or corticosteroids. In some embodiments, the subject was born preterm or with low-birth weight or experienced intra-uterine growth retardation, or with intra-uterine malnutrition or suffered growth delay. The present invention is also suitable for subjects at risk of bone disease, having a family history of bone disease, or having already experienced at least one, preferably several, episode(s) of fracture. In some embodiments, thesubject suffered from and / or is suffering from stunted growth. The definition of stunting may refer to the "height for age" value to be less than two standard 36 WO 2024 / 240923 PCT / EP2024 / 064337 deviations of the WHO Child Growth Standards median (see e.g. De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pp.12-26). In some embodiments, the subject suffered from and / or is suffering from faltering growth. The term “faltering growth” may describe a pattern of slower weight gain than expected for age and sex in children and other adolescents (see e.g. King, C. and Davis, T., 2010. European journal of clinical nutrition, 64(1), pp.S11-S13). In some embodiments, the subject suffered from and / or is suffering from growth stunting and / or faltering growth because of malnutrition or experienced disease such as anorexia, Crohn’s disease and / or celiac disease. In some embodiments, the subject suffered from and / or is suffering from growth stunting and / or faltering growth because oftreatment with drugs leading to malabsorption, anorexia and / or metabolic bone disease, such as chemotherapy drugs and / or corticosteroids. In some embodiments, the subject was born preterm or with low-birth weight or experienced intra-uterine growth retardation. The term “preterm infant” may refer to an infant born at least than 37 weeks gestational age. The term “low birth weight infant” may refer to an infant having a live-born weight less than 2,500 g. Possible factors inducing vitamin K deficiency in newborns include poor placental transfer of vitamin, immature gut flora, low vitamin K content in breast milk and substantial differences among individuals, poor intestinal absorption of vitamin K, and low activity level of vitamin K epoxide reductase (see e.g. Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p.780). Methods for treating and / or preventing vitamin K2 deficiency The prebiotic agent (or combination therewith) may be used to treat and / or prevent vitamin K2 deficiency.In one aspect, the present invention provides a prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides use of a prebiotic agent in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a prebiotic agent. The present inventors have surprisingly found that vitamin K2 production in the gastrointestinal tract (e.g. via conversion of vitamin K1) is promoted by a prebiotic agent, for example 37 WO 2024 / 240923 PCT / EP2024 / 064337 comprising bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), and / or cello-oligosaccharides (COS). The prebiotic agent (or a combination therewith) may treat and / or prevent vitamin K2 deficiency by promotingvitamin K2 production in the subject’s gut. For example, the prebiotic agent may treat and / or prevent vitamin K2 deficiency by promoting de novo menaquinone-7 production and / or bioconversion from phylloquinone to menaquinone-4 in the subject’s gut. A vitamin K2 deficiency may be diagnosed by any suitable method known in the art. For example, a vitamin K deficiency may lead to reduced blood clotting, and in severe cases, can result in reduced clotting, increased bleeding, and increased prothrombin time. The diagnosis of vitamin K deficiency bleeding is commonly indicated by a prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT), e.g. a PT international normalized ratio (INR) > 4 or a value > 4 times the normal values in the presence of normal platelet count and fibrinogen level (see e.g. Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p.780). Methods for enhancing bone growth and / or bone strength or preventing bone loss and decreased bone strength.Treating and / or preventing vitamin K2 deficiency in a subject, may thereby enhance bone growth and / or bone strength or prevent bone loss and decreased bone strength in the subject (see e.g. Hamidi, M.S., et al., Journal of clinical densitometry, 16(4), pp.409-413). Within the context of the present invention, the term “enhancing bone growth and / or bone strength, or limiting / preventing bone loss” may refer to, in particular, one or more of the following physiological processes: bone catch-up growth, bone mass acquisition, optimization of peak bone mass, promotion of bone formation, promotion of bone anabolism, promotion of bone mineralization, increase of bone mineral density and micro-architecture, modulation of bone biomechanical properties, and modulation the ratio of bone formation and / or bone resorption, bone mass maintenance, reduction of bone resorption As used herein “bone quality” may refer to aspects of bone composition and structure that contribute to bone strengthindependently of bone mineral density. These include bone turnover, microarchitecture, mineralisation, microdamage and the composition of bone matrix and mineral. Methods to measure bone quality are known in the art. As used herein, “promoting bone growth and / or strength” may refer to the support of normal bone growth and / or strength, for example during childhood and adolescence. Supporting normal bone growth and / or strength may result in normal bone anatomy and physiology. Suitable methods and parameters to determine bone growth and bone strength will be known to the skilled person (see e.g. Donnelly, E., 2011. Clinical Orthopaedics and Related 38 WO 2024 / 240923 PCT / EP2024 / 064337 Research, 469(8), pp.2128-2138). Suitably, normal bone growth and / or strength may be determined using one or more bone parameter selected from: trabecular bone volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medio-lateral diameter,antero-posterior diameter, bone ultimate force (FMax), and bone stiffness. In some embodiments, normal bone growth and / or strength is determined using one or more bone parameter selected from: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax). Suitable methods to determine these parameters will be available to the skilled person. A regular nutritional supply of the composition according to the present invention is also useful for preventing the bone loss that occurs with ageing and / or to protect bone cells during bone aging. In an embodiment, the said composition is for use i) improving bone quality; ii) preventing or treating disorders linked to an imbalance in the relationship between bone formation and bone resorption. By "reduction / inhibition of bone resorption" is meant according to the invention inhibition of the destructive activity of bone tissue by the osteoclast cells. In order to verify that thesupply of the composition in man or animals inhibits bone resorption, the specialist skilled in the art can measure the urinary excretion of desoxypyridinoline as described in the examples, a diminution of the expression of desoxypyridinoline being the reflection of inhibition of bone resorption. The supply of the composition according to the invention to an animal organism induces simultaneously a stimulation of bone formation and inhibition of bone resorption, the overall increase of bone mineralisation, and hence of the bone density, being the result of the induction of these two mechanisms. In order to determine whether a subject presents a state of reduced bone mass and as a consequence requires a supply of the composition according to the present invention, the specialist skilled in the art will be able to refer in particular to the report of the World Health Organisation (WHO) of 1994 entitled "Assessment of fracture risk and its application to screening for post-menopausalosteoporosis" (WHO Technical Series-843). The composition according to the present invention is also designed for Individuals presenting symptoms of bone deficit or likely to suffer from bone deficit, i.e. from an imbalance in the relationship between bone formation and bone resorption which, if it continues, induces a diminution of the bone mass. A composition according to the invention is also designed for 39 WO 2024 / 240923 PCT / EP2024 / 064337 individuals presenting symptoms of bone deficit resulting from a fracture, an operation or also a dental disease. In particular, the composition is designed to prevent or treat diseases selected from osteoporosis, Paget's disease, bone loss or osteolysis observed close to a prosthesis, metastatic bone diseases, hypercalcemia due to a cancer, multiple myelomas, periodontal diseases or osteoarthritis. As has already been mentioned above, many disorders linked to an imbalance of bone metabolism, such as osteoporosis, develop gradually over a longperiod of time and require chronic treatments. Their prevention or their treatment can hence be carried out by means of a regular supply of the composition according to the present invention, preferably in the form of a nutritional composition. Similarly, a regular nutritional supply ofthe composition according to the present invention to individuals, humans or animals, is such as to make possible the production of a high bone density and an elevated peak bone mass by stimulation of bone formation when these individuals attain adult age. A regular nutritional supply ofthe composition according to the present invention is also useful for preventing the bone loss that occurs with ageing (that may lead to osteoporosis) and / or to protect bone cells during bone aging. Thus, in man and other mammals a great variety of disorders are related to abnormal metabolism of bone resorption and bone formation, leading to an imbalance in metabolism or bone remodelling. Ofthe pathological disordersrelated to an imbalance in bone metabolism, particular mention may be made of the disorders or diseases such as osteoporosis, Paget's disease, bone loss or osteolysis observed close to a prosthesis, metastatic bone diseases, hypercalcemia due to a cancer, multiple myelomas and periodontal diseases. Some ofthe disorders or diseases of bone metabolism may be caused by long-term immobilisation, for example long-term hospitalisation or even after a period of weightlessness. Of the disorders linked to abnormal bone resorption, the most common is osteoporosis, the most frequent manifestation of which is observed in women after the start of menopause. Osteoporosis is a systemic skeletal disease characterised by a reduction of the bone mass and a deterioration of the microarchitecture of bone tissue, associated with an increase of the fragility of the bone and its susceptibility to fracture. 40 WO 2024 / 240923 PCT / EP2024 / 064337 Since osteoporosis, like other disorders associated with bone loss,constitutes a chronic disorder, its prevention and its treatment must be planned in the long term. It is currently accepted that early treatment must be preferred because the two critical phases for bone capital are: the period of growth during which the maximal bone mass (peak bone mass) is acquired; ageing which conditions the rate of loss of bone mass. The prevention of osteoporosis must hence no longer be restricted to the elderly individual. Moreover, in man and animals, there are many conditions characterised by the need to increase bone formation. For example, in the case of bone fractures, it is necessary to stimulate bone growth in order to accelerate complete repair of the bone. This need is also present in the periodontal diseases, the metastatic diseases of bone, the osteolytic diseases and the conditions under which repair of the connective tissue is required, for example for the cicatrisation or regeneration of defects or traumatisms of cartilage. The stimulation of bonegrowth is also required in the case of primary and secondary hyperparathyroidism, as well as in osteoporosis associated with diabetes and in osteoporosis associated with glucocorticoids. Methods for promoting catch-up growth Treating and / or preventing vitamin K2 deficiency in a subject, may thereby promote catch-up growth. As used herein, “catch-up growth” may refer to height velocity above the limits of normal for age for at least 1 year after a transient period of growth inhibition and may be complete or incomplete (see e.g. Wit, J.M. and Boersma, B., 2002. Journal of Pediatric Endocrinology and Metabolism, 15, pp. 1229-1242. Suitable method and parameters to determine catch-up growth will be known to the skilled person. Suitably, catch-up growth may be determined using height velocity or height standard deviation score (see e.g. Frongillo, E.A., Leroy, J.L. and Lapping, K., 2019. Advances in Nutrition, 10(3), pp.372-379 and Desmond, C. and Casale, D., 2017. PloS one, 12(12),p.e0189135). In some embodiments, the catch-up growth is determined in absolute terms of linear growth (i.e. the height deficit from the healthy reference population mean is reduced). In some embodiments, the catch-up growth is determined in relative terms of linear growth (i.e. the height-for-age z-score is improved and / or passes the -2SD or -1SD cut-off points). EXAMPLES 41 WO 2024 / 240923 PCT / EP2024 / 064337 The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples. Example 1 - promoting vitamin K2 production in the gastrointestinal tract To evaluate intestinal production of vitamin K2, a Simulator of the Human Microbial Ecosystem (SHIME®) was used (see Van de Wiele, T., et al., 2015. The Impact of Food Bioactives on Health: in vitro and ex vivo models, pp.305-317). SHIME assays typically consist of a colonic fermentation of a selected dose of the testcompound(s) under simulated conditions representative for the gastrointestinal tract of a subject. Production of menaquinone-7 was determined by supercritical fluid chromatography tandem mass spectrometry (SFC-MS / MS). For the current set of experiments a two-stage batch system mimicking the upper gastro­ intestinal tract (Upper GIT, stomach and small intestine) and colonic conditions were used as simplified SHIME® system. A cow milk-based infant beverage, also called infant formula-milk, containing minerals adapted for the age group was used in these studies. In order to simulate the absorptive processes occurring in the small intestine of infants, a dialysis approach was applied by using a cellulose membrane with a cut-off of 14 kDa. By introducing the small intestinal suspension within a dialysis membrane, molecules such as digested amino acids, sugars, micronutrients and minerals were gradually removed from the upper gastro-intestinal matrices. Furthermore, a gradual pH decreaseduring the stomach incubation going from 5.5 to 3.0 during 1 hour of incubation was implemented to simulate the gastric pH of infants. Also, during the first 30 minutes of small intestinal incubation (duodenum), a fixed pH of 4.5 was implemented to allow the available minerals to optimally absorb. In the following 145 minutes of the small intestinal phase (jejunum + ileum), a pH of 7 was introduced. The milk matrix after exposure to gastric and small intestinal conditions was transferred to the colonic compartment containing the fecal sample of an infant. Fresh fecal material was collected from a 12-month-old infant donor. Fecal suspension was prepared and mixed with a protectant. At the start of the short-term colonic incubation, the test ingredients (see below) were added to sugar-depleted nutritional medium containing basal nutrients present in the colon (e.g., host-derived glycans such as mucin). In a first set of experiments, the following groups (test ingredients) were evaluated:Blank; Single HMO (1.3g / L 2FL); HMO mix (2.5g / L 2FL, DiFL, LNnT, LNT, and 6SL); B. infantis (107 42 WO 2024 / 240923 PCT / EP2024 / 064337 cfu / ml); Single HMO + B. infantis·, HMO mix + B. infantis. The results are shown in Figure 1. The HMO mix increased menaquinone-7 production by 65% compared to the negative control. The addition of B. infantis further increased menaquinone-7 production by 163% compared to the negative control. In a second set of experiments, the following groups (test ingredients) were evaluated (in the absence of milk matrix): Blank; and HMOs + BMOs (7.2 g / L total). The composition of HMOs and BMOs is shown in the table below: g / L % of total 2FL 0.252 3.50 DiFL 0.036 0.50 LNnT 0.126 1.75 LNT 0.087 1.21 6SL 0.049 0.68 3SL 0.046 0.64 BMOs 6.602 91.71 Total 7.199 100 The results are shown in Figure 2A. The mixture of BMOs and HMOs increase production of menaquinone-7 by 29% compared to the negative control. It was further shown that a milk matrix comprising a mixture of sixHMOs significantly increases production of menaquinone- 7 and that the addition of B. infantis further increased production (see Figure 2B). In a final set of experiments, the following groups were evaluated (in a milk matrix in the absence or presence of L. rhamnosus LPR): Blank; COS (2700 mg / L); COS (2025 mg / L) + β- glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 χ 106 cfu / ml). The results, in the absence or presence of L rhamnosus LPR, are shown in Figures 3A and 3B, respectively. In the absence of LPR, COS increased the production of MK-7 by 178%. In the presence of LPR, the addition of B. lactis further increase the production of MK-7 by 22%. Further, when all groups were combined, it was shown that the addition of Lactobacillus rhamnosus LPR (4.5 χ 107 cfu / ml) can further increase vitamin K2 production in the gastrointestinal tract by about 40% (see Figure 3C). Example 2 - Preclinical Experimental Results A preclinical experimental setup was followedas shown in Figure 4. The number of pups per BALB / c mother was increased by 50% from D8 to D18 to induce a food restriction. In both groups (normal and faltering growth), weaning happened at D18. Male and female mice then 43 WO 2024 / 240923 PCT / EP2024 / 064337 received food ad libitum and a daily nutritional supplementation through pipet feeding for 30 days. As can be seen from Figure 5 A&B, vitamin K2AD promoted bone quality by improving trabecular bone volume (BV / TV ratio) (trend increase) and cortical bone mineral density (Ct.BMD) (significant increase). Additionally, Figure 6 A&B demonstrates that vitamin K2AD enhanced bone strength by increasing both the force (Fmax) and the energy (Wmax) necessary to reach the point of failure of femurs. EMBODIMENTS Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras). 1. A prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in asubject. 2. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is administered in combination with vitamin K1. 3. The prebiotic agent for use according to para 2, wherein the prebiotic agent and vitamin K1 are administered separately, simultaneously or sequentially, preferably wherein the prebiotic agent and vitamin K1 are administered simultaneously. 4. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is administered in combination with a probiotic agent. 5. The prebiotic agent for use according to para 4, wherein the prebiotic agent and the probiotic agent are administered separately, simultaneously or sequentially, preferably wherein the prebiotic agent and the probiotic agent are administered simultaneously. 6. The prebiotic agent for use according to para 4 or 5, wherein the probiotic agent comprises Lactobacillus rhamnosus, Bifidobacterium infantis and / or Bifidobacterium lactisi. 7. The prebiotic agentfor use according to any preceding para, wherein the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructo­ oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan. 44 WO 2024 / 240923 PCT / EP2024 / 064337 8. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is selected from one or more of: bovine milk oligosaccharides (BMOs), a human milk oligosaccharide (HMO) mixture, and cello-oligosaccharides (COS). 9. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent comprises BMOs in an amount of from about 80 wt% to about 100 wt%, with respect to the total weight of the prebiotic agent. 10. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent comprises or consists of at least one sialylated oligosaccharide, at least one fucosylatedoligosaccharide, and / or at least one N-acetylated oligosaccharide. 11. The prebiotic agent for use according to para 10, wherein the at least one sialylated oligosaccharide is selected from the group consisting of 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), syalyllacto-N-tetraose b (LSTb), syalyllacto-N-tetraose c (LSTc), disyallacto-N- tetraose, and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and combinations thereof. 12. The prebiotic agent for use according to para 10 or 11, wherein the at least one fucosylated oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’FL), 3- fucosyllactose (3FL), difucosyllactose (diFL), lacto-N-fucopentaose-l (LNFP-I), lacto-N- fucopentaose-ll (LNFP-II), lacto-N-fucopentaose-lll (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose V(LNnFP-V), lacto-N-difucosylhexaose-l (LNDFH-1), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyllacto-n-hexaose-lll (MFNLH-III), difucosyllacto-N-hexaose-a (DFLNHa) and combinations thereof, preferably wherein the at least one fucosylated oligosaccharide is 2’-fucosyllactose (2’FL) and / or difucosyllactose (diFL). 13. The prebiotic agent for use according to any of paras 10 to 12, wherein the at least one N- acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N- acetyl-galactosamines, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof, preferably wherein the at least one N-acetylated oligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and combinations thereof. 14. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent comprises: (a) from about 0.5 wt% to about 2 wt% with respect to the total weight of the oligosaccharide mixture, of at least one sialylated oligosaccharide; 45 WO 2024 / 240923 PCT / EP2024 / 064337(b) from about 2 wt% to about 6 wt%, with respect to the total weight of the oligosaccharide mixture, of at least one fucosylated oligosaccharide; and / or (c) from about 1 wt% to about 4 wt%, with respect to the total weight of the oligosaccharide mixture, of at least one N-acetylated oligosaccharide. 15. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent comprises or consists of cello-oligosaccharides (COS). 16. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is administered to the subject in a total amount of from about 0.5 g / day to about 10 g / day. 17. The prebiotic agent for use according to any preceding para, wherein vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 100 pg / day. 18. The prebiotic agent for use according to any preceding para, wherein a probiotic agent is administered to the subject in a total amount of from about 106 cfu / day to about 1012 cfu / day. 19.The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is provided in the form of a nutritional composition. 20. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent is provided in the form of a medical food product for clinical nutrition. 21. The prebiotic agent for use according to para 19 or 20, wherein the composition comprises the prebiotic agent in a total amount of from about 0.5 g / 100g to about 10 g / 100g, on a dry weight basis. 22. The prebiotic agent for use according to any of paras 19 to 21, wherein the composition comprises vitamin K1 in an amount of about 5 pg / 100g to about 100 pg / 100g, on a dry weight basis. 23. The prebiotic agent for use according to any of paras 19 to 22, wherein the composition comprises a probiotic agent in an amount of about 106cfu / 100g to about 1012 cfu / 100g, on a dry weight basis. 24. The prebiotic agent for use according to any preceding para, wherein the subject is an infant,a toddler, or a child. 25. The prebiotic agent for use according to any preceding para, wherein the subject has or is at risk of vitamin K2 deficiency. 46 WO 2024 / 240923 PCT / EP2024 / 064337 26. The prebiotic agent for use according to any preceding para, wherein the subject has or is at risk of dyslipidemia, diabetes, thalassemia major (TM), cystic fibrosis (CF), inflammatory bowel diseases (IBD), or chronic liver diseases. 27. The prebiotic agent for use according to any preceding para, wherein the subject has or is at risk of reduced bone growth and / or bone strength. 28. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting vitamin K2 production in the subject’s gut. 29. The prebiotic agent for use according to any preceding para, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting de novo menaquinone-7 production and / or bioconversion from phylloquinone tomenaquinone-4 in the subject’s gut. 30. Use of a prebiotic agent in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject. 31. A method for treating and / or preventing vitamin K2 deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of a prebiotic agent. 32. Use of a prebiotic agent to promote vitamin K2 production in the gut of a subject. 33. The use according to para 32, wherein the prebiotic agent promotes de novo menaquinone-7 production and / or bioconversion from phylloquinone to menaquinone-4 in the subject’s gut. Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification. 47 WO2024 / 240923 PCT / EP2024 / 064337 CLAIMS 1. A prebiotic agent for use in treating and / or preventing vitamin K2 deficiency in a subject. 2. A prebiotic agent for use according to claim 1, further comprising a vitamin mixture to promote vitamin K2 production in the gut of a subject. 3. A prebiotic agent for use according to claim 1 or 2, wherein the vitamin mixture comprises vitamin K2, A and / or D. 4. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent is administered in combination with vitamin K1. 5. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent is administered in combination with a probiotic agent. 6. The prebiotic agent for use according to claim 5, wherein the probiotic agent comprises Lactobacillus rhamnosus, Bifidobacterium infantis and / or Bifidobacterium lactis. 7. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent is selected from one or more of: bovine milkoligosaccharides (BMOs), human milk oligosaccharides (HMOs), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructo­ oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan, preferably wherein the prebiotic agent is selected from one or more of bovine milk oligosaccharides (BMOs), a human milk oligosaccharide (HMO) mixture, and cello-oligosaccharides (COS). 8. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent comprises BMOs in an amount of from about 80 wt% to about 100 wt%, with respect to the total weight of the prebiotic agent. 9. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent comprises or consists of HMOs, preferably wherein the prebiotic agent comprises or consists of at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and / or at least one N-acetylated oligosaccharide, more preferably wherein: (a) the at least one sialylated oligosaccharide isselected from the group consisting of 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), syalyllacto-N-tetraose b (LSTb), syalyllacto-N-tetraose c (LSTc), disyallacto-N-tetraose, and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from 3’- sialyllactose (3’-SL), 6’-sialyllactose (6’-SL) and combinations thereof; 48 WO 2024 / 240923 PCT / EP2024 / 064337 (b) the at least one fucosylated oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lacto-N- fucopentaose-l (LNFP-I), lacto-N-fucopentaose-ll (LNFP-II), lacto-N-fucopentaose-lll (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose V (LNnFP-V), lacto-N-difucosylhexaose-l (LNDFH-1), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyllacto-n-hexaose-lll (MFNLH-III), difucosyllacto-N-hexaose-a (DFLNHa) and combinations thereof, preferably wherein the at least one fucosylated oligosaccharide is2’-fucosyllactose (2’FL) and / or difucosyllactose (diFL); and / or (c) the at least one N-acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamines, lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), and combinations thereof, preferably wherein the at least one N- acetylated oligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT) and combinations thereof. 10. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent comprises or consists of COS. 11. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent is administered to the subject in a total amount of from about 0.5 g / day to about 10 g / day. 12. The prebiotic agent for use according to any preceding claim, wherein vitamin K1 is administered to the subject in an amount of from about 5 pg / day to about 100 pg / day, and / or wherein a probiotic agent is administered to the subject in a totalamount of from about 106 cfu / day to about 1012 cfu / day. 13. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent is provided in the form of a nutritional composition. 14. The prebiotic agent for use according to any preceding claim, wherein the subject is an infant, a toddler, or a child. 15. The prebiotic agent for use according to any preceding claim, wherein the subject has or is at risk of dyslipidemia, diabetes, thalassemia major (TM), cystic fibrosis (CF), inflammatory bowel diseases (IBD), or chronic liver diseases, and / or wherein the subject has or is at risk of reduced bone growth and / or bone strength. 16. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agent enhances bone growth, bone mineralisation and / or bone strength or limits / prevents bone loss and strength in the subject. 49 WO 2024 / 240923 PCT / EP2024 / 064337 17. The prebiotic agent for use according to any preceding claim, wherein the prebiotic agenttreats and / or prevents vitamin K2 deficiency by promoting vitamin K2 production in the subject’s gut. 18. Use of a prebiotic agent to promote vitamin K2 production in the gut of a subject. 50 WO 2024 / 240923 PCT / EP2024 / 064337 1 / 6 SUBSTITUTE SHEET (RULE 26) WO 2024 / 240923 PCT / EP2024 / 064337 FS8 S JoH “S S Μ & / ΟΟΛ SUBSTITUTE SHEET (RULE 26) WO 2024 / 240923 PCT / EP2024 / 064337 3 / 6 Fi / °°°^ oS a oom "CI W . O SUBSTITUTE SHEET (RULE 26) WO 2024 / 240923 PCT / EP2024 / 064337 4 / 6 F| Λ IU. O(cont’d) Weaning and Randomization DayO Day 8 Day 18 Day 48-51 SUBSTITUTE SHEET (RULE 26) WO 2024 / 240923 PCT / EP2024 / 064337 5 / 6 * p<0.05 FS ®888Sj * S c Noolv SUBSTITUTE SHEET (RULE 26) WO 2024 / 240923 PCT / EP2024 / 064337 6 / 6 * p<0.05 ; ** p<0.01 SUBSTITUTE SHEET (RULE 26) INTERNATIONAL SEARCH REPORT International application No PCT / EP2024 / 064337 A. CLASSIFICATION OF SUBJECT MATTER INV. A61K31 / 702 A61K31 / 122 A61K35 / 745 A61K35 / 747 A23L33 / 21 A23L33 / 125 A23L33 / 135 A61P19 / 08 ADD. According to International PatentClassification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K A61P A23L Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPO-Internal, WPI Data Form PCT / ISA / 210 (second sheet) (April 2005) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X Y X Y WO 2009 / 095240 Al (FRIESLAND BRANDS BV [NL]; SCHAAFSMA ANNE [NL] ET AL.) 6 August 2009 (2009-08-06) page 5 - page 6; examples 2,3,4,9-13 LAWENIUS LINA ET AL: "Development of a synbiotic that protects against ovariectomy-induced trabecular bone loss", AMERICAN JOURNAL OF PHYSIOLOGY: ENDOCRINOLOGY AND METABOLISM., vol. 322, no. 4,1 April 2022 (2022-04-01) , pages E344-E354, XP093097524, US ISSN: 0193-1849, DOI: 10.1152 / ajpendo.00366.2021 page E349, right-hand column - left-hand column page E351, right-hand column; figure 6B - / -- 1,5, 13-18 1-18 1,5, 15-18 1-18 _ χ Further documents are listed in the continuation of Box C. χ See patent family annex. * Special categories of cited documents : ”T” later document published after the international filing date or priority . . . .. . ,. , . . ... . .. . . . ,, date and not in conflict with the application but cited to understand A document defining the general state of the art which is not considered the inci |e or th underlying the invention to be of particular relevance E earlier application or patent but published on or after the international "χ" document of particular relevance;; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive ”L” document which may throw doubts on priority claim(s) or which is step whenthe document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance;; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "0" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art ”P” document published prior to the international filing date but later than the priority date claimed document member of the same patent family Date of the actual completion of the international search 22 July 2024 Date of mailing of the international search report 06 / 08 / 2024 Name and mailing address of the ISA / European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70)340-3016 Authorized officer Pacreu Largo, Marta1 page 1 of 2 INTERNATIONAL SEARCH REPORT International application NoPCT / EP2024 / 064337 C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A KOZIOL-KOZAKOWSKA AGNIESZKA ET AL: "The Impact of Vitamin K2 (Menaquionones) in Children's Health and Diseases: A Review of the Literature", CHILDREN, vol. 9, no. 1, 5 January 2022 (2022-01-05) , page 78, XP093097487, DOI: 10.3390 / children9010078 abstract page 5 - page 10 1-18 A MARESZ KATARZYNA: "Growing Evidence of a Proven Mechanism Shows Vitamin K2 Can Impact Health Conditions Beyond Bone and Cardiovascular", INTEGRATIVE MEDICINE, vol. 20, no. 4, 1 August 2021 (2021-08-01) , pages 34-38, XP093097684, ISSN: 1546-993X page 37 - page 38 1-18 Y WO 2010 / 139690 Al (CHR HANSEN AS [DK]; PEDERSEN MARTIN [DK]) 9 December 2010 (2010-12-09) claims 1-18 A WO 2009 / 146490 Al (BIFFIN JOHN RAY [AU]; REGTOP HUBEERTUS LEONARDUS [AU]) 10 December 2009 (2009-12-10) claims 1-3,8 1-4,15, 17 Y KUANG XIAOTONG ET AL: "Thecombination effect of vitamin K and vitamin D on human bone quality: a meta-analysis of randomized controlled trials", FOOD & FUNCTION, vol. 11, no. 4, 1 January 2020 (2020-01-01), pages 3280-3297, XP093094719, GB ISSN: 2042-6496, DOI: 10.1039 / C9FO03063H abstract 2,3,16 Y WO 2021 / 233960 Al (NESTLE SA [CH]) 25 November 2021 (2021-11-25) claims 1,7,9, 13-16 1 Form PCT / ISA / 210 (continuation of second sheet) (April 2005) page 2 of 2 INTERNATIONAL SEARCH REPORT Information on patent family members International application No PCT / EP2024 / 064337 Patent document Publication Patent family Publication cited in search report date member(s) date WO 2009095240 Al 06-08-2009 CN 101969792 A 09-02-2011 EA 201070893 Al 28-02-2011 EP 2247199 Al 10-11-2010 NL 1034964 C2 30-07-2009 WO 2009095240 Al 06-08-2009 WO 2010139690 Al 09-12-2010 CN 102802647 A 28-11-2012 DK 2437758 T3 21-07-2014 EP 2437758 Al 11-04-2012 HK 1173947 Al 31-05-2013 JP 2012528826 A 15-11-2012 KR 20120016642 A 24-02-2012 US 2012082651Al 05-04-2012 US 2013243742 Al 19-09-2013 WO 2010139690 Al 09-12-2010 wo 2009146490 Al 10-12-2009 AU 2009253839 Al 10-12-2009 CA 2726662 Al 10-12-2009 DE 202009018772 Ul 15-03-2013 EP 2299991 Al 30-03-2011 MY 149641 A 13-09-2013 NZ 589810 A 30-11-2012 US 2011124610 Al 26-05-2011 US 2015164825 Al 18-06-2015 WO 2009146490 Al 10-12-2009 wo 2021233960 Al 25-11-2021 AU 2021275459 Al 24-11-2022 BR 112022022781 A2 13-12-2022 CL 2022003181 Al 10-04-2023 CN 115666585 A 31-01-2023 EP 4153185 Al 29-03-2023 US 2023218648 Al 13-07-2023 WO 2021233960 Al 25-11-2021 Form PCT / ISA / 210 (patent family annex) (April 2005) (19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480032993.2 (22) Application Date 2024.05.24 (30) Priority Data 23175289.0 2023.05.25 EP (85) PCT International Application Entering National Phase Date 2025.11.17 (86) PCT International Application Application Data PCT / EP2024 / 064337 2024.05.24 (87) PCT International Application Publication Data WO2024 / 240923 EN 2024.11.28 (71) Applicant: Nestlé Products Ltd. Address: Switzerland (72) Inventors: C.L.L.M. Blanger, S. Dubois, E. Campos, Jiménez, M.N. Okahada, N. Bonnet (74) Patent Agency: Beijing Zhongzi Law Firm, 11247 Patent Attorneys: Shi Wenjing, Huang Gesheng (51) Int.Cl. A61K 31 / 702 (2006.01)A61K 31 / 122 (2006.01) A61K 35 / 745 (2006.01) A61K 35 / 747 (2006.01) A23L 33 / 21 (2006.01) A23L 33 / 125 (2006.01) A23L 33 / 135 (2006.01) A61P 19 / 08 (2006.01) (54) Invention Title: Prebiotics for the Treatment and / or Prevention of Vitamin K2 Deficiency (57) Abstract: This invention relates to prebiotic preparations for the treatment and / or prevention of vitamin K2 deficiency in subjects. The invention relates to the use of prebiotic preparations in promoting the production of vitamin K2 in the intestines of subjects. Claims (2 pages), Description (29 pages), Drawings (6 pages), CN 121175055 A, 2025.12.19, CN 1 21 17 50 55 A 1. A prebiotic for treating and / or preventing vitamin K2 deficiency in a subject. 2. The prebiotic for the purpose according to claim 1, wherein the prebiotic further comprises a vitamin mixture to promote vitamin K2 production in the intestine of the subject. 3. The prebiotic for the purpose according to claim 1 or 2, wherein the vitamin mixture comprises vitamin K2, A, and / or D. 4. The prebiotic for the purpose according to any preceding claim, wherein the prebiotic is administered in combination with vitamin K1. 5. The prebiotic for the purpose according to any preceding claim, wherein the prebiotic is administered in combination with a probiotic agent. 6. The prebiotic for the purpose according to claim 5, wherein the probiotic agent comprises Lactobacillus rhamnosus, Bifidobacterium infantis, and / or Bifidobacterium lactis. 7. A prebiotic agent for the purpose according to any preceding claim, wherein the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan, preferably wherein the prebiotic agent is selected from one or more of the following: a mixture of bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), and cello-oligosaccharides (COS). 8. A prebiotic agent for the purpose according to any preceding claim, wherein the prebiotic agent comprises about 80% by weight to about 100% by weight of BMO relative to the total weight of the prebiotic agent. 9. A prebiotic agent for the stated purpose according to any preceding claim, wherein the prebiotic agent comprises or is composed of HMO, preferably wherein the prebiotic agent comprises or is composed of at least one sialylated oligosaccharide, at least one fucoidylated oligosaccharide and / or at least one N-acetylated oligosaccharide, more preferably wherein:(a) The at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide, and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof; (b) The at least one fucoidylated oligosaccharide is selected from the group consisting of: 2'-fucosylated lactose (2'FL). 3-Fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), lactose-neofucopentose-V (LNnFP-V), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neodifucosylhexose (LNnDFH), monofucosyllactose-N-hexose-III (MFNLH-III), difucosyllactose-N-hexose-α (DFLNHa) and combinations thereof, preferably wherein the at least one fucoidylated oligosaccharide is 2'-fucosylated lactose (2'FL) and / or difucosylated lactose (diFL); and / or (c) the at least one N-acetylated oligosaccharide is selected from the group consisting of: N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof, preferably wherein the at least one N-acetylated oligosaccharide is selected from lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof. 10. A prebiotic for the purpose according to any preceding claim, wherein the prebiotic comprises or is composed of COS. 11. A prebiotic for the purpose according to any preceding claim, wherein the prebiotic is administered to the subject in a total dose of about 0.5 g / day to about 10 g / day. 12. A prebiotic for the purpose according to any preceding claim, wherein vitamin K1 is administered to the subject at an amount of about 5 µg / day to about 100 µg / day, and / or wherein a probiotic agent is administered to the subject at a total amount of about 10⁶ cfu / day to about 10¹² cfu / day. 13. A prebiotic for the purpose according to any preceding claim, wherein the prebiotic is provided in the form of a nutritional composition. 14. A prebiotic for the purpose according to any preceding claim, wherein the subject is an infant, school-aged child, or young child.15. A prebiotic for the purpose according to any preceding claim, wherein the subject has dyslipidemia, diabetes, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease or is at risk of having said diseases, and / or wherein the subject has reduced bone growth and / or bone strength or is at risk of having said diseases. 16. A prebiotic for the purpose according to any preceding claim, wherein the prebiotic enhances bone growth, bone mineralization, and / or bone strength of the subject, or limits / prevents bone loss and bone strength in the subject. 17. A prebiotic for the purpose according to any preceding claim, wherein the prebiotic treats and / or prevents vitamin K2 deficiency by promoting vitamin K2 production in the gut of the subject. 18. Use of a prebiotic in promoting vitamin K2 production in the gut of a subject. Claims 2 / 2 Page 3 CN 121175055 A Prebiotics for the Treatment and / or Prevention of Vitamin K2 Deficiency Technical Field

[0001] This invention relates to agents, compositions, and methods for the treatment and / or prevention of vitamin K2 deficiency in subjects. The invention also relates to agents, compositions, and methods for promoting the production of vitamin K2 in the gut of subjects. Background Art

[0002] Vitamin K2 activates vitamin K-dependent proteins that support many biological functions, such as bone mineralization, inhibition of vascular stiffness, improvement of endothelial function, maintenance of strong teeth, brain development, joint health, and optimal body weight (Kozio ł-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p. 78).

[0003] However, in recent decades, vitamin K2 intake in parents and their offspring has declined dramatically, leading to serious health problems. Furthermore, secondary vitamin K2 deficiency may occur in individuals with malabsorption syndrome or liver disease who ingest sufficient amounts of vitamin K antagonist drugs, or after prolonged use of antibiotics or glucocorticoids.

[0004] Therefore, new nutritional interventions are needed to treat and / or prevent vitamin K2 deficiency. Summary of the Invention

[0005] The inventors have surprisingly discovered that vitamin K2 production in the gastrointestinal tract (e.g., through the conversion of vitamin K1) is promoted by prebiotics, which include, for example, bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), and / or fiber oligosaccharides (COS).

[0006] In one aspect, the present invention provides prebiotics for treating and / or preventing vitamin K2 deficiency in subjects.

[0007] In another aspect, the present invention provides the use of prebiotics in the manufacture of medicaments for treating and / or preventing vitamin K2 deficiency in subjects.

[0008] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering an effective amount of a prebiotic to the subject.

[0009] The prebiotic may treat or prevent vitamin K2 deficiency by promoting the production of vitamin K2 in the gut of the subject. For example, the prebiotic may treat or prevent vitamin K2 deficiency by promoting the de novo production of methylnaphthoquinone-7 and / or the biotransformation from phylloquinone to methylnaphthoquinone-4 in the gut of the subject.

[0010] In another aspect, the present invention provides the use of a prebiotic in promoting the production of vitamin K2 in the gut of a subject.

[0011] In another aspect, the present invention provides a method for promoting the production of vitamin K2 in the gut of a subject, the method comprising administering an effective amount of a prebiotic to the subject.

[0012] The prebiotic may be any suitable prebiotic that promotes the production of vitamin K2 in the gut of a subject. Suitablely, the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructose oligosaccharides (FOS), galactosidase oligosaccharides (GOS), and β-glucan. In some embodiments, the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), a mixture of human milk oligosaccharides (HMO), and cello-oligosaccharides (COS).

[0013] In some embodiments, the prebiotic agent comprises BMO in an amount of about 80% by weight to about 100% by weight relative to the total weight of the prebiotic agent.

[0014] In some embodiments, the prebiotic agent comprises or consists of one or more HMOs. In some embodiments, the prebiotic agent comprises or consists of at least one sialylated oligosaccharide, at least one fucoidylated oligosaccharide, and / or at least one N-acetylated oligosaccharide. Suitably, at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetrasaccharide b (LSTb), sialyllactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide, and combinations thereof. In some embodiments, at least one sialylated oligosaccharide is selected from 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), and combinations thereof. Suitably, at least one fucoidylated oligosaccharide is selected from the group consisting of: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-I), and so on.II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), lactose-neofofucopentose-V (LNnFP-V), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neofodifucosylhexose (LNnDFH), monofucosyllactose-N-hexose-III (MFNLH-III), difucosyllactose-N-hexose-a (DFLNHa), and combinations thereof. In some embodiments, at least one fucosylated oligosaccharide is 2'-fucosyllactose (2'FL) and / or difucosyllactose (diFL). Suitably, at least one N-acetylated oligosaccharide is selected from the group consisting of: N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof. In some embodiments, at least one N-acetylated oligosaccharide is selected from lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof.

[0015] In some embodiments, the prebiotic contains or is composed of fiber oligosaccharide (COS).

[0016] The prebiotic may be administered to the subject in any suitable amount. Suitably, the prebiotic is administered to the subject in a total amount of about 0.5 g / day to about 10 g / day. Suitably, BMO is administered to the subject in a total amount of about 0.5 g / day to about 10 g / day. Suitably, HMO is administered to the subject in a total amount of about 0.5 g / day to about 10 g / day. Suitablely, fiber oligosaccharides (COS) are administered to the subject in a total amount of about 0.5 g / day to about 10 g / day.

[0017] Prebiotics may be administered in combination with vitamin K1. Vitamin K2 can be formed through the metabolic conversion of vitamin K1 during its absorption in the intestinal mucosa and other organs. Prebiotics and vitamin K1 may be administered alone, simultaneously, or sequentially. In a preferred embodiment, prebiotics and vitamin K1 are administered simultaneously. Suitablely, vitamin K1 is administered to the subject in an amount of about 5 µg / day to about 200 µg / day.

[0018] Prebiotics may be administered in combination with probiotics. The inventors have surprisingly discovered that the production of vitamin K2 in the gastrointestinal tract (e.g., via the conversion of vitamin K1) can be further promoted by the administration of probiotics. Prebiotics and probiotics may be administered alone, simultaneously, or sequentially. In a preferred embodiment, prebiotics and probiotics are administered simultaneously. The probiotic may contain any suitable probiotic. Appropriately, probiotic agents include Lactobacillus rhamnosus, Bifidobacterium infantis, and / or Bifidobacterium lactis.(lactis). Suitablely, the probiotic agent is administered to the subject in a total amount of about 10⁶ cfu / day to about 10¹² cfu / day.

[0019] The prebiotic agent (or combination thereof) may be provided in any suitable form (e.g., in the form of a composition). The prebiotic agent (or combination thereof) may be provided in the form of a nutritional composition. The prebiotic agent (or combination thereof) may be provided in the form of a medical food product for clinical nutrition.

[0020] Suitablely, the composition comprises a prebiotic agent in a total amount based on about 0.5 g / 100 g to about 10 g / 100 g of dry weight. Suitablely, the composition comprises vitamin K1 in an amount based on about 5 µg / 100 g to about 200 µg / 100 g of dry weight. Suitablely, the composition comprises a probiotic agent in an amount based on about 10⁶ cfu / 100 g to about 10¹² cfu / 100 g of dry weight. Instructions 2 / 29, page 5, CN 121175055 A

[0021] The subject can be any suitable subject. Appropriately, the subject is a human or an animal. In a preferred embodiment, the subject is a human. In some embodiments, the subject is a teenager, adolescent, child, toddler, or infant. In some embodiments, the subject is a child, toddler, or infant. In other embodiments, the subject is an adult. The subject may have or be at risk of vitamin K2 deficiency. In some embodiments, the subject has dyslipidemia, diabetes, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease or is at risk of having these diseases. In some embodiments, the subject has decreased bone growth and / or bone strength or is at risk of having these diseases. Figure Descriptions

[0022] Figure 1 - Effect of Human Milk Oligosaccharides (HMOs) on Vitamin K2 Production in an Intestinal Model

[0023] The following groups were evaluated: blank; single HMO (1.3 g / L 2FL); HMO mixture (2.5 g / L 2FL, DiFL, LNnT, LNT, and 6SL); Bifidobacterium infantis (107 cfu / ml); single HMO + Bifidobacterium infantis; HMO mixture + Bifidobacterium infantis.

[0024] Figure 2 - Effect of Oligosaccharides and Probiotics on Vitamin K2 Production in an Intestinal Model

[0025] (A) The following groups were evaluated (in the absence of milk matrix): blank; and HMO + BMO (total 7.2 g / L). (B) The following groups were evaluated: blank; MM (milk matrix containing a mixture of 6 HMOs); MM + Bifidobacterium infantis (1011 cfu / g).

[0026] Figure 3 – Effect of human milk oligosaccharides (HMOs) on vitamin K2 production in an intestinal model

[0027] (A) Evaluation of the following groups (in the milk matrix, in the absence of Lactobacillus rhamnosus LPR): blank; COS(2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + Bifidobacterium lactis (4.5 × 10⁶ cfu / ml). (B) Assess the following groups (in the milk matrix, in the presence of 4.5 × 10⁷ cfu / ml Lactobacillus rhamnosus LPR): blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + Bifidobacterium lactis (4.5 × 10⁶ cfu / ml). (C) The following groups (in the milk matrix) were evaluated: without LPR (without Lactobacillus rhamnosus LPR); and with LPR (4.5 × 10⁷ cfu / ml Lactobacillus rhamnosus LPR).

[0028] Figure 4 – Preclinical experimental design. Growth slowing group (using vit.K1): From D8 to D18, the number of pups per BALB / c mother was increased by 50% to induce food restriction. In both groups (normal and growth slowing), weaning occurred at D18. Male and female mice were then allowed to eat freely and were given daily nutritional supplementation via pipette feeding for 30 days.

[0029] Figures 5A and 5B – Effects of Vit.K2AD on trabecular BV / TV and cortical BMD as assessed by microcomputed tomography on the femur.

[0030] Trabecular and cortical microstructures in the distal metaphysis and axial shaft of the femur were evaluated using microcomputed tomography (µCT UCT35, Scanco Medical AG, Basserdorf, Switzerland), as previously described 1. Briefly, trabecular and cortical bone regions were evaluated using isotropic 6µm voxels. For the femoral trabecular region, 30 pieces of bone beneath the distal growth plate were disregarded to eliminate the predominant cancellous tissue. The 80 pieces of secondary cancellous tissue directly beneath were analyzed. The femoral cortical structure was evaluated using 60 consecutive CT tomographic images located along the femoral axial direction. Morphological variables were calculated from the binarized images using a direct three-dimensional technique independent of prior assumptions about the underlying structure 2. For the trabecular bone region, the BV / TV score (%) was evaluated. For the cortical bone in the midshaft of the femur, cortical bone mineral density (Ct.BMD) was evaluated.

[0031] Figures 6A and 6B – Effect of Vit. K2AD on femoral strength as assessed by the 3-point bending test

[0032] The 3-point bending test was performed to assess the biomechanical properties of the femur, as previously described (C.H. Turner, DB Burr, Basic biomechanical measurements of bone: a tutorial, Bone, 1993, 14).Page 3 / 29, CN 121175055 A (4):595-608). A load was applied in compression mode at a nominal deformation rate of 0.08 mm / s until fracture. Load-displacement curves were recorded to determine the fracture force (Fmax(N) / AP(mm)) normalized to the anterior and posterior diameters of the femur and the fracture energy (Wmax(N) / AP(mm)) normalized to the anterior and posterior diameters of the femur. Detailed Description

[0033] Preferred features and embodiments of the invention will now be described by way of non-limiting examples. Those skilled in the art will understand that they may combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0034] Any references to prior art literature in this specification should not be construed as an admission that such prior art is well-known or part of common knowledge in the art. All publications mentioned in this specification are incorporated herein by reference.

[0035] As used in this specification, the words “comprising,” “including,” and similar terms should not be construed as having an exclusive or exhaustive meaning. In other words, they are intended to mean “including, but not limited to”. The terms “including / comprise” and similar terms also include the term “consisting of”.

[0036] Unless otherwise specified, the practice of the invention will employ conventional techniques within the capabilities of those skilled in the art. Such techniques are described in the literature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0037] Numerical ranges include numerical values ​​that define the range, and unless otherwise stated, all percentages disclosed herein are based on w / w. As used herein, the term “about” means approximately, nearby, roughly, or about. When the term “about” is used in conjunction with a numerical value or range, it modifies the value or range by extending the boundaries to be above and below the stated value. Generally, the terms “about” and “approximately” are used herein to modify numerical values ​​above and below the stated value by 10%.

[0038] Prebiotics

[0039] The present invention provides prebiotic agents for treating and / or preventing vitamin K2 deficiency in subjects.

[0040] As used herein, the term "prebiotic" can refer to an indigestible component that benefits a subject by selectively stimulating the beneficial growth and / or activity of one or more microbial groups. A prebiotic agent can be any suitable prebiotic agent that promotes vitamin K2 production. Exemplary prebiotics include bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cellooligosaccharides (COS), inulin, lactose, fructosaccharides (FOS), galactosaccharides (GOS), and β-glucan.

[0041] In some embodiments, the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cellooligosaccharides (COS), inulin, lactose, fructosaccharides (FOS), galactosaccharides (GOS), and β-glucan.Oligosaccharides (HMOs), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructose oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan.

[0042] In some embodiments, the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), and cello-oligosaccharides (COS). In some embodiments, the prebiotic agent comprises BMOs and HMOs. In some embodiments, the prebiotic agent comprises HMOs. In some embodiments, the prebiotic agent comprises cello-oligosaccharides (COS).

[0043] Bovine Milk Oligosaccharides

[0044] The prebiotic agent used in this invention may comprise or consist of bovine milk oligosaccharides (BMOs).

[0045] Oligosaccharides in bovine milk are assembled in the mammary glands by combining monosaccharides glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine, fucose, and sialic acid N-acetylneuraminic acid and N-hydroxyacetylneuraminic acid. The collection of BMOs found in milk and colostrum has been extensively described, with 30 to 50 structures typically identified in comprehensive studies (see, for example, Robinson, RC, 2019. Frontiers in nutrition, 6, p. 50).

[0046] Although bovine milk generally contains fewer oligosaccharide structures than human milk, both share at least ten common structures, including 3'-sialic acid lactose and 6'-sialic acid lactose, which constitute a large percentage of the BMO library (see, for example, Robinson, RC, 2019. Frontiers in nutrition, 6, p. 50).

[0047] The prebiotic may contain BMO in an amount of about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% by weight relative to the total weight of the prebiotic.

[0048] Human milk oligosaccharides (HMOs)

[0049] The prebiotic used in this invention may contain or be composed of one or more human milk oligosaccharides (HMOs).

[0050] Many different kinds of HMOs are found in human milk and are generally based on combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine with many different bonds between them. Almost all HMOs have lactose molecules at their reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). HMOs can be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucoidylated oligosaccharides).

[0051] Suitablely, one or more HMOs may comprise at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and / or at least one N-acetylated oligosaccharide. In some embodiments, one or more HMOs comprise at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylated oligosaccharide, or are composed of the like. In some embodiments, one or more HMOs comprise 2'-fucosylated lactose (2'FL), difucosylated lactose (diFL), 6'-sialylated lactose (6'-SL), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetrasaccharide (LNnT), or are composed of the like. In some embodiments, one or more HMOs comprise or consist of 2'-fucosylated lactose (2'FL), difucosylated lactose (diFL), 6'-sialylated lactose (6'-SL), 3'-sialylated lactose (3'-SL), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetrasaccharide (LNnT).

[0052] In some embodiments (e.g., when the prebiotic contains BMO), the prebiotic contains at least one fucoidylated oligosaccharide in an amount of about 0.5% to about 2% by weight relative to the total weight of the prebiotic. In some embodiments (e.g., when the prebiotic contains BMO), the prebiotic contains at least one sialylated oligosaccharide in an amount of about 2% to about 6% by weight relative to the total weight of the prebiotic. In some embodiments (e.g., when the prebiotic contains BMO), the prebiotic contains at least one N-acetylated oligosaccharide in an amount of about 1% to about 4% by weight relative to the total weight of the prebiotic.

[0053] In some embodiments (e.g., when the prebiotic contains BMO), the prebiotic contains at least one fucoidylated oligosaccharide at about 2% to about 6% of the total weight of the prebiotic; and at least one N-acetylated oligosaccharide at about 1% to about 4% of the total weight of the prebiotic.

[0054] In some embodiments (e.g., when the prebiotic contains BMO), the prebiotic contains: at least one fucoidylated oligosaccharide at about 2% to about 6% of the total weight of the prebiotic; at least one sialylated oligosaccharide at about 0.5% to about 2% of the total weight of the prebiotic; and at least one N-acetylated oligosaccharide at about 1% to about 4% of the total weight of the prebiotic.

[0055] In other embodiments (e.g., when the prebiotic does not contain BMO), the prebiotic contains at least one fucoidylated oligosaccharide at about 30% to about 80% by weight, about 40% to about 80% by weight, or about 50% to about 70% by weight relative to the total weight of the prebiotic. In some embodiments (e.g., when the prebiotic does not contain BMO), the prebiotic...The prebiotic contains at least one sialylated oligosaccharide at about 10% to about 35% by weight, about 10% to about 30% by weight, or about 10% to about 25% by weight relative to the total weight of the prebiotic. In some embodiments (e.g., when the prebiotic does not contain BMO), the prebiotic contains at least one N-acetylated oligosaccharide at about 10% to about 35% by weight, about 15% to about 30% by weight, or about 15% to about 20% by weight relative to the total weight of the prebiotic.

[0056] In some embodiments (e.g., when the prebiotic does not contain BMO), the prebiotic comprises or is composed of the following, or is described in the following specification (page 5 / 29, CN 121175055 A): at least one fucoidylated oligosaccharide at about 30% to about 80%, about 40% to about 80% or about 50% to about 70% of the total weight of the prebiotic; and at least one N-acetylated oligosaccharide at about 10% to about 35%, about 15% to about 30% or about 15% to about 20% of the total weight of the prebiotic.

[0057] In some embodiments (e.g., when the prebiotic does not contain BMO), the prebiotic comprises or consists of: at least one fucoidylated oligosaccharide at about 30% to about 80%, about 40% to about 80% or about 50% to about 70% of the total weight of the prebiotic; at least one sialylated oligosaccharide at about 10% to about 35%, about 10% to about 30% or about 10% to about 25% of the total weight of the prebiotic; and at least one N-acetylated oligosaccharide at about 10% to about 35%, about 15% to about 30% or about 15% to about 20% of the total weight of the prebiotic.

[0058] HMO can be obtained by any suitable method. Suitable methods for synthesizing oligosaccharides are well known to those skilled in the art. For example, methods have been developed for preparing oligosaccharides by microbial fermentation, enzymatic methods, chemical synthesis, or a combination of these techniques (see, for example, Zeuner et al., 2019. Molecules, 24(11), p. 2033).

[0059] Fucosylated oligosaccharides

[0060] In one embodiment, the prebiotic contains at least one fucosylated oligosaccharide.

[0061] Non-limiting examples of fucosylated oligosaccharides include: 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3FL), difucosylated lactose (diFL), lactose-N-fucopentose (such as, lactose-N-fucopentose I (LNFP-I), lactose-N-fucopentose II (LNFP-II), lactose-N-fucopentose III (LNFP-III), or lactose-N-fucopentose V).(LNFP-V), lactose-N-fucohexose, lactose-N-difucohexose I, lactose-neofucopentose V (LNnFP-V), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neodifucosylhexose-I (LNnDFH), fucose-lactose-N- Hexoses, fucoidyllactose-N-neohexoses (such as fucoidyllactose-N-neohexose I, fucoidyllactose-N-neohexose II), monofucosyllactose-N-hexose III (MFNLH-III), difucosyllactose-N-hexose I, difucosyllactose-N-neohexose, difucosyllactose-N-neohexose I, difucosyllactose-N-neohexose II, difucosyllactose-N-hexose-a (DFLNHa), fucoidyl-para-lactose-N-hexose, trifucosyl-para-lactose-N-hexose I, and combinations thereof.

[0062] In a preferred embodiment, at least one fucoidylated oligosaccharide comprises 2'-fucosyllactose (2'FL), which is generally the most common HMO naturally found in human breast milk.

[0063] In some embodiments, at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosylated lactose (2'FL), difucosylated lactose (diFL), and combinations thereof. In some embodiments, at least one fucosylated oligosaccharide comprises or is composed of 2'-fucosylated lactose (2'FL) and difucosylated lactose (diFL).

[0064] Fucosylated oligosaccharides can be obtained by any suitable method. For example, 2'FL can be prepared by biotechnological means using specific fucosyltransferases and / or fucosidases, through enzyme-based fermentation techniques or microbial fermentation techniques using enzymes (recombinant enzymes or natural enzymes). In the latter case, the microorganisms may express their natural enzymes and substrates, or may be engineered to produce the corresponding substrates and enzymes. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose. diFL can be synthesized by enzymatic, biotechnological, and / or chemical methods.

[0065] Sialinated oligosaccharides

[0066] In some embodiments, the prebiotic agent comprises at least one sialylated oligosaccharide.

[0067] Non-limiting examples of sialylated oligosaccharides include: 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide, and combinations thereof. Specification 6 / 29 pages 9 CN 121175055 A

[0068] In some embodiments, at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyl lactoseSugars (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof. In some embodiments, at least one sialylated oligosaccharide comprises or is composed of 6'-sialyl lactose (6'-SL). In some embodiments, at least one sialylated oligosaccharide comprises or is composed of 6'-sialyl lactose (6'-SL) and 3'-sialyl lactose (3'-SL).

[0069] Sialidized oligosaccharides can be obtained by any suitable method. For example, 3'-sialyl lactose (3'-SL) and / or 6'-sialyl lactose (6'-SL) can be isolated from natural sources (such as animal milk) using chromatographic or filtration techniques. Alternatively, sialylated oligosaccharides can also be prepared by biotechnological means, by enzyme-based (recombinant or natural enzyme) fermentation techniques, by chemical synthesis, or by microbial fermentation techniques, using specific sialyl transferases or sialic acidases, neuraminidases. In the latter case, the microorganism can express its natural enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Single microbial cultures or mixed cultures can be used. The formation of sialic acid oligosaccharides can begin with acceptor substrates initially having any degree of polymerization (DP), starting from DP = 1. Alternatively, sialyl lactose can be produced through the chemical synthesis of lactose and free N'-acetylneuraminic acid (sialic acid). Sialyl lactose is also commercially available from, for example, Kyowa Hakko Kogyo, Japan, or GeneChem, Republic of Korea.

[0070] If the prebiotic contains 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), it may be particularly advantageous that the 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL) are contained in the nutritional composition in a weight ratio between about 10:1 and about 1:10 (such as between about 10:1 and about 2:1, between about 8:1 and about 3:1, between about 6:1 and about 3:1, between about 5:1 and about 3:1, between about 5:1 and about 4:1, or between about 1:2 and about 1.5:1).

[0071] N-acetylated oligosaccharides

[0072] In some embodiments, the prebiotic contains at least one N-acetylated oligosaccharide.

[0073] Suitably, at least one N-acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, and combinations thereof. Non-limiting examples of N-acetylated oligosaccharides include: LNT (lactose-N-tetrasaccharide), p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neohexose), and any combination thereof. Other examples are lactose-N-hexose, lactose-N-neohexose, p-lactose-N-hexose, p-lactose-N-neohexose, lactose-N-octasaccharide, lactose-N-neoctasaccharides, isol-lactose-N-octasaccharides, para-lactose-N-octasaccharides, and lactose-N-decasaccharides.

[0074] In some embodiments, at least one N-acetylated oligosaccharide is selected from the group consisting of lactose-N-tetrasaccharides (LNT), lactose-N-neoctasaccharides (LNnT), and combinations thereof. In some embodiments, at least one N-acetylated oligosaccharide comprises or is composed of lactose-N-tetrasaccharides (LNT) and lactose-N-neoctasaccharides (LNnT).

[0075] N-acetylated oligosaccharides can be obtained by any suitable method. For example, LNnT can be chemically synthesized by enzymatically transferring sugar units from the donor portion to the acceptor portion using a glycosyltransferase. Alternatively, LNnT can be prepared by chemically converting free or oligosaccharide-bound ketohexoses (e.g., fructose) into N-acetylated hexosamine or oligosaccharides containing N-acetylated hexosamine. LNTs can be synthesized by enzymatic, biotechnological, and / or chemical methods.

[0076] Cellobiose and Cellulose Oligosaccharides (COS)

[0077] The prebiotics used in this invention may comprise or consist of cellobiose and / or cellulose oligosaccharides (COS).

[0078] Cellobiose is a disaccharide having the formula (C6H7(OH)4O)2O, which is derived from the condensation of a pair of β-glucose molecules forming a β(1→4) bond. Cellobiose can be obtained by enzymatic or acidic hydrolysis of cellulose and cellulose-rich materials. Suitablely, cellobiose is in the form of free cellobiose or cellulose oligosaccharides.

[0079] In some embodiments, cellobiose is in the form of cellulose oligosaccharides (COS). Cellulose oligosaccharides can refer to oligomers of β-glucose molecules having β-1,4-bonds (e.g., about 2 to about 6 β-glucose molecules) and can primarily contain cellobiose. Prebiotic agents used in this invention may contain or be composed of cellulose oligosaccharides. Studies have shown that COS has prebiotic potential (see, for example, Zhong, C. et al., 2020. Journal of agricultural and food chemistry, 68(32), pp. 8557-8567).

[0080] Administration of prebiotics

[0081] Any suitable amount of prebiotics may be administered to a subject in any suitable form and by any suitable route of administration (e.g., in any form and by any route described herein).

[0082] For example, suitable doses of human oligosaccharides are described in, for example, the following: EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2015. EFSA Journal, 13(11), p. 4299;EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(6), p. e05717; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2020. EFSA Journal, 18(5), p. e06097; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2022. EFSA Journal, 20(5), p. e07331; and EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(12), p. e05907.

[0083] Appropriately, the prebiotic was administered to the subject at a dose of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitablely, the prebiotic is administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitablely, the prebiotic is administered to the subject in an amount of about 0.5 g / day to about 10 g / day, about 1 g / day to about 8 g / day, or about 2 g / day to about 5 g / day.

[0084] Suitablely, BMO is administered to the subject in an amount of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitablely, BMO is administered to the subject in an amount of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitablely, BMO is administered to the subject in an amount of about 0.5 g / day to about 10 g / day, about 1 g / day to about 8 g / day, or about 2 g / day to about 5 g / day.

[0085] Suitably, HMO is administered to the subject at a dose of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, HMO is administered to the subject at a dose of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Suitably, HMO is administered to the subject at a dose of about 0.5 g / day to about 10 g / day, about 1 g / day to about 8 g / day, or about 2 g / day to about 5 g / day.

[0086] Suitably, COS is administered to the subject at a dose of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Suitably, COS is administered to the subject at a dose of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Appropriately, COS was administered to the subjects at doses of about 0.5 g / day to about 10 g / day, about 1 g / day to about 8 g / day, or about 2 g / day to about 5 g / day.

[0087] Combination Therapy

[0088] The prebiotic agent may be administered in combination with one or more other agents, mixtures, or compositions.

[0089] As used herein, "combination therapy" may refer to a therapy comprising the administration of two or more agents (e.g., prebiotic agents, one or more vitamins and / or probiotic agents), mixtures, or compositions.

[0090] The combination may be administered by any suitable route and in any suitable form. Properly, the combination is administered orally and / or enterically. In a preferred embodiment, the combination is administered orally. The combination may be administered alone, simultaneously, or sequentially. In a preferred embodiment, the combination is administered simultaneously.

[0091] In one aspect, the present invention provides a combination of a prebiotic agent, one or more vitamins (e.g., vitamin K1), and one or more probiotics for the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0092] In another aspect, the present invention provides the use of a combination of a prebiotic agent, one or more vitamins (e.g., vitamin K1), and one or more probiotics in the manufacture of a medicament for the treatment and / or prevention of vitamin K2 deficiency in a subject. Specification 8 / 29 pages 11 CN 121175055 A

[0093] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of a combination of a prebiotic, one or more vitamins (e.g., vitamin K1), and one or more probiotics.

[0094] In another aspect, the present invention provides the use of a combination of a prebiotic, one or more vitamins (e.g., vitamin K1), and one or more probiotics in promoting vitamin K2 production in the gut of a subject.

[0095] In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a combination of a prebiotic, one or more vitamins (e.g., vitamin K1), and one or more probiotics.

[0096] Vitamins

[0097] Prebiotics may be administered in combination with one or more vitamins. Prebiotics and one or more vitamins may be administered alone, simultaneously, or sequentially. In a preferred embodiment, prebiotics and one or more vitamins are administered simultaneously.

[0098] Vitamins are organic micronutrients required for a range of normal bodily functions and include vitamin K1, vitamin A, vitamin D, vitamin C, folic acid, vitamin B3, vitamin B6, vitamin B12, and vitamin E. Suitably, one or more vitamins may comprise or consist of vitamin K1, vitamin K2, vitamin A, and / or vitamin D. In a preferred embodiment, one or more vitamins comprise or consist of vitamin K1.

[0099] In one aspect, the present invention provides a combination of a prebiotic and one or more vitamins for treating and / or preventing vitamin K2 deficiency in a subject.

[0100] In another aspect, the present invention provides the use of a combination of a prebiotic and one or more vitamins in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0101] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of a prebiotic and one or more vitamins.

[0102] In another aspect, the present invention provides the use of a combination of a prebiotic and one or more vitamins in promoting vitamin K2 production in the gut of a subject.

[0103] In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a prebiotic and one or more vitamins.

[0104] Vitamin K1

[0105] In a preferred embodiment, the prebiotic may be administered in combination with vitamin K1. The prebiotic and vitamin K1 may be administered alone, simultaneously, or sequentially. In a preferred embodiment, the prebiotic and vitamin K1 are administered simultaneously.

[0106] In one aspect, the present invention provides a combination of a prebiotic and vitamin K1 for treating and / or preventing vitamin K2 deficiency in a subject.

[0107] In another aspect, the present invention provides the use of the combination of a prebiotic and vitamin K1 in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0108] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of the combination of a prebiotic and vitamin K1.

[0109] In another aspect, the present invention provides the use of the combination of a prebiotic and vitamin K1 in promoting vitamin K2 production in the gut of a subject.

[0110] In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of the combination of a prebiotic and vitamin K1.

[0111] Vitamin K represents a fat-soluble compound having a common chemical structure of 3-substituted 2-methyl-1,4-naphthoquinone, family A, page 9 / 29 of the specification, CN 121175055. It is naturally found in food as phylloquinone (vitamin K1) and methylnaphthoquinone (vitamin K2). Vitamin K1 (phylloquinone) can have the following general formula:

[0112]

[0113] Vitamin K1 has a chlorophyll side chain and is generally the main dietary form of vitamin K, and is found in dark green leafy vegetables (e.g., spinach, lettuce, and other salad greens) and Brassica plants.

[0114] Vitamin K1 may be administered to the subject in any suitable form and via any suitable route of administration (e.g., in any form and via any route described herein). Suitable doses of vitamin K1 are described, for example, in: Kozio ł-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p. 78 and EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2017. EFSA Journal, 15(5), p. e04780.

[0115] Suitablely, vitamin K1 is administered to the subject at a dose of at least about 0.2 µg / kg / day, at least about 0.5 µg / kg / day, or at least about 0.8 µg / kg / day. Suitablely, vitamin K1 is administered to the subject at a dose of about 2 µg / kg / day or less, about 1.5 µg / kg / day or less, or about 1.2 µg / kg / day or less. Suitablely, vitamin K1 is administered to the subject at an amount of about 0.2 µg / kg / day to about 2 µg / kg / day, about 0.5 µg / kg / day to about 1.5 µg / kg / day, or about 0.8 µg / kg / day to about 1.2 µg / kg / day. In some embodiments, vitamin K1 is administered to the subject at an amount of about 1 µg / kg / day.

[0116] Suitablely, vitamin K1 is administered to the subject at an amount of at least about 5 µg / day, at least about 10 µg / day, at least about 15 µg / day, at least about 20 µg / day, at least about 25 µg / day, or at least about 30 µg / day. Suitablely, vitamin K1 is administered to the subject at an amount of about 200 µg / day or less, about 100 µg / day or less, about 90 µg / day or less, about 80 µg / day or less, about 70 µg / day or less, or about 60 µg / day or less. Suitablely, vitamin K1 was administered to the subject at an amount of about 5 µg / day to about 200 µg / day, about 10 µg / day to about 100 µg / day, about 15 µg / day to about 90 µg / day, about 20 µg / day to about 80 µg / day, about 25 µg / day to about 70 µg / day, or about 30 µg / day to about 60 µg / day.

[0117] Suitablely, a prebiotic was administered to the subject at an amount of about 0.5 µg / day to about 10 µg / day, and vitamin K1 was administered to the subject at an amount of about 5 µg / day to about 200 µg / day.

[0118] Vitamin K2

[0119] Phloroquinone is a dietary vitamin K found in plants, while methylnaphthoquinone is a vitamin K quinone consumed in the diet and produced by the gut microbiota (see, for example, Walther, B. et al., 2013. Advances in nutrition, 4).(4), pp. 463-473). In some embodiments, the prebiotic promotes the de novo production of methylnaphthoquinone-7 in the gut of the subject (e.g., via the gut microbiota). In some embodiments, the prebiotic promotes the biotransformation from chloroquinone to methylnaphthoquinone-4 in the gut of the subject (e.g., via the gut microbiota).

[0120] Vitamin K2 (methylnaphthoquinone) may have the following general formula: Specification 10 / 29 pages 13 CN 121175055 A

[0121]

[0122] Vitamin K2 is composed of different forms with different numbers (n) of isoprene units, where n may range from 4 to 13. Various forms are indicated by a suffix (-n), for example, methylnaphthoquinone-4 (abbreviated as MK-4) has four isoprene residues (n=4). MK-4 may be formed by metabolic transformation during the absorption of chloroquinone in the intestinal mucosa and other organs. Other methylnaphthoquinones may be produced in the gastrointestinal tract by the gut microbiota.

[0123] Prebiotics may be administered in combination with vitamin K2. Prebiotics and vitamin K2 may be administered alone, simultaneously, or sequentially. In a preferred embodiment, prebiotics and vitamin K2 are administered simultaneously.

[0124] In one aspect, the present invention provides a combination of prebiotics and vitamin K2 for treating and / or preventing vitamin K2 deficiency in a subject.

[0125] In another aspect, the present invention provides the use of the combination of prebiotics and vitamin K2 in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0126] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of the combination of prebiotics and vitamin K2.

[0127] Any suitable amount of vitamin K2 may be administered to the subject in any suitable form and via any suitable route of administration (e.g., in any form and via any route described herein). Suitable doses of vitamin K2 are described, for example, in: Kozio ł-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p. 78 and European Food Safety Authority (EFSA), 2008. EFSA Journal, 6(11), p. 822.

[0128] Suitablely, vitamin K2 is administered to the subject at a dose of at least about 0.5 µg / kg / day, at least about 1 µg / kg / day, or at least about 2 µg / kg / day. Suitablely, vitamin K2 is administered to the subject at a dose of about 10 µg / kg / day or less, about 7.5 µg / kg / day or less, or about 5 µg / kg / day or less. Suitablely, vitamin K2 is administered to the subject at a dose of about 0.5 µg / kg / day to about 10 µg / kg / day, aboutThe subject was administered vitamin K2 at an amount of 1 µg / kg / day to about 7.5 µg / kg / day or about 2 µg / kg / day to about 5 µg / kg / day.

[0129] Suitablely, vitamin K2 was administered to the subject at an amount of at least about 5 µg / day, at least about 10 µg / day, at least about 15 µg / day, at least about 20 µg / day, at least about 25 µg / day, or at least about 30 µg / day. Suitablely, vitamin K2 was administered to the subject at an amount of about 200 µg / day or less, about 100 µg / day or less, about 90 µg / day or less, about 80 µg / day or less, about 70 µg / day or less, or about 60 µg / day or less. Appropriately, vitamin K2 is administered to the subject in amounts of about 5 µg / day to about 200 µg / day, about 10 µg / day to about 100 µg / day, about 15 µg / day to about 90 µg / day, about 20 µg / day to about 80 µg / day, about 25 µg / day to about 70 µg / day, or about 30 µg / day to about 60 µg / day.

[0130] Vitamin A

[0131] Vitamin A comprises a family of molecules containing a 20-carbon structure having a methyl-substituted cyclohexenyl ring (β-ionone ring) and a tetraene side chain having a hydroxyl group (retinol), an aldehyde group (retinaldehyde), a carboxylic acid group (retinoic acid), or an ester group (retinyl ester) at carbon-15. The term vitamin A may also include provitamin A carotenoids as dietary precursors to retinol. Among the many carotenoids in nature, some possess provitamin A nutritional activity, including α-carotene, β-carotene, and β-cryptoxanthin.

[0132] The amount of vitamin A can refer to retinol equivalent (RE) or retinol activity equivalent (RAE). For the dietary provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin, RE is set to 6 μg, 12 μg, and 12 μg, respectively. When using μg RAE, the vitamin A activity of the provitamin A carotenoids is half of the assumed vitamin A activity when using μg RE. For the dietary provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin, RAE is set to 12 μg, 24 μg, and 24 μg, respectively. (See, for example, Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington (DC): National AcademiesPress (US); 2001.4, Vitamin A).

[0133] Any suitable amount of vitamin A may be administered to the subject in any suitable form and by any suitable route of administration (e.g., in any form and by any route described herein). Suitable doses of vitamin A are described, for example, in the following: Ross, AC and Moran, NE, 2020. Current Developments in Nutrition, 4(10), p. nzaa096 and EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA), 2015. EFSA Journal, 13(3), p. 4028.

[0134] Properly, vitamin A is administered to the subject at an amount of at least about 100 µg RE / day, at least about 200 µg RE / day, or at least about 300 µg RE / day. Suitablely, vitamin A is administered to the subject in amounts of about 1000 µg RE / day or less, about 800 µg RE / day or less, about 600 µg RE / day or less, or about 400 µg RE / day or less. Suitablely, vitamin A is administered to the subject in amounts of about 100 µg RE / day to about 1000 µg RE / day, about 200 µg RE / day to about 800 µg RE / day, about 300 µg RE / day to about 600 µg RE / day, or about 300 µg RE / day to about 400 µg RE / day.

[0135] Suitablely, vitamin A is administered to the subject in amounts of at least about 100 µg RE / day, at least about 200 µg RAE / day, or at least about 300 µg RAE / day. Appropriately, vitamin A is administered to the subject in amounts of about 1000 µg RAE / day or less, about 800 µg RAE / day or less, about 600 µg RAE / day or less, or about 400 µg RAE / day or less. Appropriately, vitamin A is administered to the subject in amounts of about 100 µg RAE / day to about 1000 µg RAE / day, about 200 µg RAE / day to about 800 µg RAE / day, about 300 µg RAE / day to about 600 µg RAE / day, or about 300 µg RAE / day to about 400 µg RAE / day.

[0136] Vitamin D

[0137] Vitamin D is a group of fat-soluble open-ring steroids, including vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Calcitriol (also known as 1,25-dihydroxy)Vitamin D is the active form of vitamin D. Suitablely, vitamin D comprises or consists of vitamin D2 and vitamin D3. Suitablely, vitamin D comprises or consists of calcitriol.

[0138] Any suitable amount of vitamin D may be administered to the subject in any suitable form and by any suitable route of administration (e.g., in any form and by any route described herein). Suitable doses of vitamin D are described, for example, in the following: Greer, F.R., 2004. The American journal of clinical nutrition, 80(6), pp. 1759S-1762S and EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2016. EFSA Journal, 14(10), p. e04547.

[0139] Suitablely, vitamin D is administered to the subject in an amount of at least about 2.5 µg / day, at least about 5 µg / day, at least about 10 µg / day, or at least about 15 µg / day. Suitablely, vitamin D is administered to the subject in an amount of about 100 µg / day or less, about 75 µg / day or less, or about 50 µg / day or less. Suitablely, vitamin D is administered to the subject in an amount of about 2.5 µg / day to about 100 µg / day, about 5 µg / day to about 100 µg / day, about 10 µg / day to about 75 µg / day, or about 15 µg / day to about 50 µg / day. Suitablely, vitamin D is administered to the subject in an amount of about 15 µg / day.

[0140] Vitamin Mixture Instructions for Use 12 / 29 pages 15 CN 121175055 A

[0141] Prebiotics may be administered in combination with vitamin mixtures. Prebiotics and vitamin mixtures may be administered alone, simultaneously, or sequentially. In a preferred embodiment, prebiotics and vitamin mixtures are administered simultaneously.

[0142] In one aspect, the present invention provides a combination of a prebiotic and a vitamin mixture for treating and / or preventing vitamin K2 deficiency in a subject.

[0143] In another aspect, the present invention provides the use of the combination of a prebiotic and a vitamin mixture in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0144] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of the combination of a prebiotic and a vitamin mixture.

[0145] In another aspect, the present invention provides the use of the combination of a prebiotic and a vitamin mixture in promoting vitamin K2 production in the gut of a subject.

[0146] As used herein, a “vitamin mixture” may refer to a mixture of two or more vitamins. Any combination may be used.A suitable vitamin mixture.

[0147] In some embodiments, the vitamin mixture comprises or consists of vitamin K1, vitamin A, and vitamin D. Suitablely, vitamin K1 is administered to the subject at an amount of about 5 µg / day to about 200 µg / day, vitamin A is administered to the subject at an amount of about 100 µg RE / day to about 1000 µg RE / day, and vitamin D is administered to the subject at an amount of about 2.5 µg / day to about 100 µg / day.

[0148] In other embodiments, the vitamin mixture comprises or consists of vitamin K2, vitamin A, and vitamin D. Suitablely, vitamin K2 is administered to the subject at an amount of about 5 µg / day to about 200 µg / day, vitamin A is administered to the subject at an amount of about 100 µg RE / day to about 1000 µg RE / day, and vitamin D is administered to the subject at an amount of about 2.5 µg / day to about 100 µg / day.

[0149] In another embodiment, the present invention provides the use of a combination of a prebiotic and vitamin K2 for treating and / or preventing vitamin K2 deficiency in a subject.

[0150] In another embodiment, the present invention provides the use of a combination of a prebiotic and vitamin A for treating and / or preventing vitamin K2 deficiency in a subject.

[0151] In another embodiment, the present invention provides the use of a combination of a prebiotic and vitamin D for treating and / or preventing vitamin K2 deficiency in a subject.

[0152] In another embodiment, the present invention provides the use of a combination of a prebiotic and vitamin K2, vitamin A, vitamin D, or any combination thereof for treating and / or preventing vitamin K2 deficiency in a subject.

[0153] Probiotics

[0154] The prebiotic may be administered in combination with one or more probiotics. The prebiotic and one or more probiotics may be administered alone, simultaneously, or sequentially. In a preferred embodiment, the prebiotic and one or more probiotics are administered simultaneously.

[0155] As used herein, the term “probiotic” may refer to a substance containing a sufficient number of live microorganisms to alter the composition of a subject’s gut microbiota (see, for example, Hill, C. et al., 2014. Nature reviews Gastroenterology & hepatology, 11(8), p. 506). Profitably, probiotics include commercially available probiotic strains and / or strains that have been shown to have health benefits (see, for example, Fijan, S., 2014. International journal of environmental research and public health, 11(5), pp. 4745–4767). Exemplary probiotic microorganisms may include Bifidobacterium, lactobacillus, etc.Lactobacillus, Limosilactobacillus, Lactobacillus paracasei, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc (Leuconostoc), Bacillus, and Escherichia coli.

[0156] In one aspect, the present invention provides a combination of a prebiotic and one or more probiotics for treating and / or preventing vitamin K2 deficiency in a subject.

[0157] In another aspect, the present invention provides the use of a combination of a prebiotic and one or more probiotics in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0158] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering to the subject an effective amount of a prebiotic and a combination of one or more probiotics.

[0159] In another aspect, the present invention provides the use of a combination of a prebiotic and one or more probiotics in promoting vitamin K2 production in the gut of a subject.

[0160] In another aspect, the present invention provides a method for promoting vitamin K2 production in the gut of a subject, the method comprising administering to the subject an effective amount of a prebiotic and a combination of one or more probiotics.

[0161] One or more probiotics can increase vitamin K2 production. The inventors have surprisingly discovered that probiotics can promote endogenous production of vitamin K2 by producing precursors.

[0162] In some embodiments, one or more probiotics comprise or consist of Lactobacillus paracasei, Bifidobacterium, Lactobacillus and / or Lactobacillus reuteri. In some embodiments, one or more probiotics comprise or consist of Lactobacillus rhamnosus, Bifidobacterium longum, and / or Bifidobacterium animalis.

[0163] Lactobacillus rhamnosus

[0164] In some embodiments, one or more probiotics comprise or consist of Lactobacillus rhamnosus.

[0165] Lactobacillus rhamnosus (also known as Lactobacillus rhamnosus) is a short, Gram-positive, homofermentative, facultative anaerobic bacterium that typically occurs in chains.Spore-free. Lactobacillus rhamnosus GG (LGG) is one of the most widely used probiotic strains. Various health effects have been documented (see, for example, Segers, ME and Lebeer, S., 2014. Microbial cell factories, 13(1), pp. 1–16). Lactobacillus rhamnosus can promote endogenous production of vitamin K2 by the production of precursors.

[0166] In some embodiments, one or more probiotics comprise or consist of Lactobacillus rhamnosus LPR.

[0167] Bifidobacterium longum

[0168] In some embodiments, one or more probiotics comprise or consist of Bifidobacterium longum.

[0169] Bifidobacterium longum is a bacterium present in the human digestive tract. In 2002, three previously distinct species of Bifidobacterium (Bifidobacterium infantis, B. longum, and B. suis) were merged with biotypes of Bifidobacterium infantis, B. longum, and B. suis into a single species named Bifidobacterium longum (Sakata, S. et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp. 1945–1951).

[0170] In some embodiments, one or more probiotics comprise or consist of Bifidobacterium longum ssp. infantis (also known as Bifidobacterium infantis), Bifidobacterium longum ssp. suis (also known as Bifidobacterium suis), and / or Bifidobacterium longum ssp. longum (also known as Bifidobacterium longum).

[0171] In some embodiments, one or more probiotics comprise or consist of Bifidobacterium infantis. In some embodiments, one or more probiotics comprise or consist of Bifidobacterium infantis LMG 11588 or a derivative thereof (e.g., R0033, which has been deemed safe by the US Food and Drug Administration, 14 / 29 pages, 17 CN 121175055 A (GRAS), see, for example, Duboux, S. et al., 2022. Microorganisms, 10(2), p. 203). Bifidobacterium infantis can promote endogenous production of vitamin K2 by the production of precursors.

[0172] Bifidobacterium animalis

[0173] In some embodiments, one or more probiotics comprise or consist of Bifidobacterium animalis.

[0174] Bifidobacterium animalis is a bacterium of the genus Bifidobacterium, which can be found in the large intestine of most mammals, including humans. Bifidobacterium animalis and Bifidobacterium lactis were previously described as two distinct species. Currently, both are considered, namely Bifidobacterium animalis, which has both subspecies Bifidobacterium animalis and subspecies Bifidobacterium lactis (see Masco, L. et al., 2004. International Journal of Systematic and Evolutionary Microbiology, 54(4), pp. 1137-1143).

[0175] In some embodiments, one or more probiotics comprise or consist of Bifidobacterium animalis (also known as Bifidobacterium lactis). For example, various traits of the bacterium Bifidobacterium lactis HN019 that are important to its ability to function as a probiotic have been studied (see, for example, Sanders, M.E., 2006. Journal of clinical gastroenterology, 40(9), pp. 776-783).

[0176] Administration of Probiotics

[0177] Any suitable amount of probiotics may be administered to the subject in any suitable form and via any suitable route of administration (e.g., in any form and via any route described herein).

[0178] Suitably, one or more probiotics are administered to the subject in a total amount of at least about 10⁵ cfu / day, at least about 10⁶ cfu / day, at least about 10⁷ cfu / day, at least about 10⁸ cfu / day, at least about 10⁹ cfu / day, or at least about 10¹⁰ cfu / day. Suitably, one or more probiotics are administered to the subject in a total amount of about 10¹² cfu / day or less, about 10¹¹ cfu / day or less, or about 10¹⁰ cfu / day or less. Suitable, one or more probiotics are administered to the subject in a total amount of about 10⁶ cfu / day to about 10¹² cfu / day, about 10⁷ cfu / day to about 10¹¹ cfu / day, or about 10⁸ cfu / day to about 10¹⁰ cfu / day.

[0179] Suitable, *Lactobacillus rhamnosus* is administered to the subject in an amount of at least about 10⁵ cfu / day, at least about 10⁶ cfu / day, at least about 10⁷ cfu / day, at least about 10⁸ cfu / day, at least about 10⁹ cfu / day, or at least about 10¹⁰ cfu / day. Suitable, *Lactobacillus rhamnosus* is administered to the subject in an amount of about 10¹² cfu / day or less, about 10¹¹ cfu / day or less, or about 10¹⁰ cfu / day or less. Appropriately, *Lactobacillus rhamnosus* was administered to the subject at amounts of approximately 10⁶ cfu / day to approximately 10¹² cfu / day, approximately 10⁷ cfu / day to approximately 10¹¹ cfu / day, or approximately 10⁸ cfu / day to approximately 10¹⁰ cfu / day.

[0180] Suitable, *Bifidobacterium longum* (e.g., *Bifidobacterium infantis*) is administered to the subject at an amount of at least about 10⁵ CFU / day, at least about 10⁶ CFU / day, at least about 10⁷ CFU / day, at least about 10⁸ CFU / day, at least about 10⁹ CFU / day, or at least about 10¹⁰ CFU / day. Suitable, *Bifidobacterium longum* (e.g., *Bifidobacterium infantis*) is administered to the subject at an amount of about 10¹² CFU / day or less, about 10¹¹ CFU / day or less, or about 10¹⁰ CFU / day or less. Suitable, *Bifidobacterium longum* (e.g., *Bifidobacterium infantis*) is administered to the subject at an amount of about 10⁶ CFU / day to about 10¹² CFU / day, about 10⁷ CFU / day to about 10¹¹ CFU / day, or about 10⁸ CFU / day to about 10¹⁰ CFU / day.

[0181] Suitably, Bifidobacterium animalis (e.g., Bifidobacterium lactis) is administered to the subject at an amount of at least about 10⁵ CFU / day, at least about 10⁶ CFU / day, at least about 10⁷ CFU / day, at least about 10⁸ CFU / day, at least about 10⁹ CFU / day, or at least about 10¹⁰ CFU / day. Suitably, Bifidobacterium animalis (e.g., Bifidobacterium lactis) is administered to the subject at an amount of about 10¹² CFU / day or less, about 10¹¹ CFU / day or less, or about 10¹⁰ CFU / day or less. Suitably, Bifidobacterium animalis (e.g., Bifidobacterium lactis) is administered to the subject at an amount of about 10⁶ CFU / day to about 10¹² CFU / day, about 10⁷ CFU / day to about 10¹¹ CFU / day, or about 10⁸ CFU / day to about 10¹⁰ CFU / day.

[0182] Suitably, the prebiotic is administered to the subject at an amount of about 0.5 g / day to about 10 g / day, and one or more probiotics are administered to the subject in a total amount of about 10⁶ cfu / day to about 10¹² cfu / day.

[0183] Suitably, the prebiotic is administered to the subject at an amount of about 0.5 g / day to about 10 g / day, vitamin K1 is administered to the subject at an amount of about 5 µg / day to about 200 µg / day, and one or more probiotics are administered to the subject in a total amount of about 10⁶ cfu / day to about 10¹² cfu / day.

[0184] Composition

[0185] Suitably, the prebiotic (or a combination thereof) is in the form of a composition. The composition may contain any therapeutically effective amount of the combination.

[0186] The composition can be any type of composition that can be incorporated into the composition, such as a food or beverage product, an animal feed product, a nutritional supplement for human or animal, or a pharmaceutical composition. The composition can be in solid (e.g., powder), liquid, or semi-liquid form. The composition can be in the form of a food composition, a pet food composition, a beverage, a nutritional formula, a nutritional supplement, or a nutritional product.

[0187] Food and beverage products, intended for the purpose of providing nutrition and / or pleasure, include all products intended for oral consumption by humans. For example, it can be a nutritional composition, such as for young children. Examples of food and beverage products include dairy products, such as dairy products or yogurt, soups, sauces, sweet and savory snacks, powdered beverages, and cereal products.

[0188] In some embodiments, prebiotics (or combinations thereof) are in the form of nutritional compositions, medical food products for clinical nutrition, or supplements.

[0189] In some embodiments, prebiotics (or combinations thereof) are in the form of nutritional compositions. As used herein, “nutritional composition” may mean a composition that provides nutrients to a subject. Such nutritional compositions are typically administered orally or intravenously and typically include a lipid or fat source and a protein source.

[0190] In some embodiments, prebiotics (or combinations thereof) are in the form of medical food products for clinical nutrition. As used herein, “medical food products for clinical nutrition” may also be referred to as “foods for specific medical purposes (FSMP)”, and refers to specialized foods designed to help meet the nutritional or dietary needs of subjects suffering from diseases, conditions, or medical circumstances who are temporarily or permanently unable to obtain adequate nutritional intake from normal foods or by modifying normal diets.

[0191] In some embodiments, the prebiotic (or combination thereof) is in the form of infant formula. In this case, the infant formula may be preterm infant formula, human milk fortifier, stage 1 infant formula, follow-up formula, infant food formula, infant cereal formula, or growing milk.

[0192] In some embodiments, the prebiotic (or combination thereof) is in the form of growing milk. As used herein, the term “growing milk” (or GUM) refers to a dairy formula product provided after one year of age. It is typically a dairy-based beverage suitable for the specific nutritional needs of young children (e.g., children from about 1 year to about 3 years of age). Growing milk may also be referred to as “toddler formula” or “toddler milk”.

[0193] In some embodiments, the prebiotic (or combination thereof) is in the form of a dairy formula. As used herein, the term "dairy formula" may refer to a food intended for, for example, childhood nutrition, which may provide, for example, a sole source or supplemental source of nutrition for children aged about 3 years or older. In some embodiments, the dairy formula is growing milk.

[0194] In some embodiments, the composition is in powder form and is reconstituted in an aqueous medium (e.g., water) prior to application. In other embodiments, the composition is in a ready-to-apply liquid form (e.g., a ready-to-eat formula).

[0195] In another embodiment, the prebiotic (or combination thereof) is in the form of a supplement. As used herein, "supplement" or "dietary supplement" can be used to supplement the nutrition of a subject (which is generally used as such, but it may also be added to foods intended for use by children aged about 3 years or older).(In any kind of composition ingested by the subject). When the composition is a supplement, it may be provided in unit dosage form. Supplementary instructions 16 / 29 pages 19 CN 121175055 A The supplement is generally in the form of liquid, gel, powder or tablet or capsule. Powder supplements generally cover the supplement to be dissolved in water or milk or sprayed in food or beverage. Such supplements are intended to provide additional nutritional and / or health benefits to the subject consuming them. Supplements may be used to provide nutrients and / or health benefits to humans and animals.

[0196] In another embodiment, the prebiotic (or combination thereof) is in the form of a fortifier. The fortifier may be an infant formula fortifier or a growing milk fortifier.

[0197] In another embodiment, the prebiotic (or combination thereof) is in the form of a pharmaceutical product. Pharmaceutical products include, for example, drops, syrups, powders, tablets or capsules designed to treat or prevent adverse medical conditions in subjects in need of them.

[0198] Prebiotics (or combinations thereof) may also be in the form of animal food products or nutritional supplements for animals. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc.

[0199] Prebiotics

[0200] The nutritional composition according to the invention may contain any suitable amount of prebiotics.

[0201] Suitably, the nutritional composition contains a total amount of prebiotics based on a dry weight of at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, or at least about 5% by weight. Suitably, the nutritional composition contains a total amount of prebiotics based on a dry weight of about 10% by weight or less, about 8% by weight or less, or about 5% by weight or less. Suitably, the nutritional composition comprises a prebiotic agent in a total amount based on about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt% of dry weight.

[0202] Suitably, the prebiotic agent comprises BMO in an amount of about 80 wt% to about 100 wt% relative to the total weight of the prebiotic agent. Suitably, the nutritional composition comprises a total amount based on at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4 wt%, or at least about 5 wt% of dry weight. Suitably, the nutritional composition comprises a total amount based on about 10 wt% or less, about 8 wt% or less, or about 5 wt% or less of dry weight. Suitably, the nutritional composition comprises a total amount based on about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt% of dry weight.

[0203] Suitably, the nutritional composition comprises at least about 0.5% by weight, at least about 1% by weight, and at least about 2% by weight based on dry weight.The nutritional composition contains HMO in an amount of about 10% by weight, about 8% by weight, or about 5% by weight, based on a dry weight. The nutritional composition contains HMO in an amount of about 0.5% by weight to about 10% by weight, about 1% by weight to about 8% by weight, or about 2% by weight to about 5% by weight, based on a dry weight.

[0204] The nutritional composition contains at least one fucoidan in an amount of about 0.05% by weight to about 3% by weight, about 0.1% by weight to about 2% by weight, or about 0.2% by weight to about 1.5% by weight, based on a dry weight. The nutritional composition contains at least one sialylated oligosaccharide in an amount of about 0.01% by weight to about 2% by weight, about 0.05% by weight to about 1.5% by weight, or about 0.07% by weight to about 1% by weight, based on a dry weight. Suitably, the nutritional composition comprises at least one N-acetylated oligosaccharide in an amount of about 0.01% to about 1% by weight, about 0.03% to about 0.6% by weight, or about 0.05% to about 0.5% by weight based on dry weight.

[0205] Suitably, the nutritional composition comprises 2'FL (concentration may refer to the concentration of the composition, for example, after reconstruction with water) in a total amount of about 0.05 g / L to about 2 g / L, about 0.1 g / L to about 1 g / L, about 0.15 g / L to about 0.8 g / L, about 0.2 g / L to about 0.7 g / L, or about 0.25 g / L to about 0.6 g / L.

[0206] Suitably, the nutritional composition comprises a total amount of fucoidylated oligosaccharides (e.g., 2′FL and / or diFL) of about 0.1 g / L to about 4 g / L, about 0.1 g / L to about 3.5 g / L, about 0.15 g / L to about 3 g / L, about 0.2 g / L to about 2.5 g / L, about 0.3 g / L to about 2 g / L, about 0.4 g / L to about 2 g / L, or about 0.5 g / L to about 2 g / L (concentration may refer to the concentration of the composition after reconstruction with water, for example). In one specific embodiment, the nutritional composition comprises about 0.2 g / L to about 1.8 g / L of total fucoidylated oligosaccharides.

[0207] Suitably, the nutritional composition comprises a total amount of sialylated oligosaccharides (e.g., 3'-sialyl lactose (3'-SL) and / or 6'-sialyl lactose (6'-SL)) in amounts of about 0.05 g / L to about 0.75 g / L, about 0.05 g / L to about 0.5 g / L, about 0.1 g / L to about 0.3 g / L, or about 0.1 g / L to about 0.4 g / L (concentration may refer to the concentration after the composition has been reconstituted, for example, with water). In one specific embodiment, the nutritional composition comprises about 0.12 g / L to about 0.4 g / L of total sialylated oligosaccharides.

[0208] Suitable, the nutritional composition comprises a total amount of N-acetylated oligosaccharides (e.g., LNT and / or LNnT) of about 0.05 g / L to about 0.5 g / L, about 0.1 g / L to about 0.5 g / L, about 0.2 g / L to about 0.4 g / L or about 0.3 g / L (concentration may refer to the concentration of the composition after reconstruction with water, for example).

[0209] Suitably, the nutritional composition comprises:

[0210] - at least one sialylated oligosaccharide in a total amount of about 0.05 g / L to about 0.75 g / L, about 0.05 g / L to about 0.5 g / L, about 0.1 g / L to about 0.3 g / L, or about 0.1 g / L to about 0.4 mg / L;

[0211] - at least one fucoidylated oligosaccharide in a total amount of about 0.1 g / L to about 4 g / L, about 0.1 g / L to about 3.5 g / L, about 0.15 g / L to about 3 g / L, about 0.2 g / L to about 2.5 g / L, about 0.3 g / L to about 2 g / L, about 0.4 g / L to about 2 g / L, or about 0.5 g / L to about 2 g / L; and / or

[0212] - At least one N-acetylated oligosaccharide in a total amount of about 0.05 g / L to about 0.5 g / L, about 0.1 g / L to about 0.5 g / L, or about 0.2 g / L to about 0.4 g / L.

[0213] Suitably, the nutritional composition comprises COS in an amount of at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, at least about 3 wt%, at least about 4 wt%, or at least about 5 wt% based on dry weight. Suitably, the nutritional composition comprises COS in an amount of about 10 wt% or less, about 8 wt% or less, or about 5 wt% or less based on dry weight. Suitably, the nutritional composition comprises COS in an amount of about 0.5 wt% to about 10 wt%, about 1 wt% to about 8 wt%, or about 2 wt% to about 5 wt% based on dry weight.

[0214] Vitamins

[0215] The nutritional composition according to the invention may contain any suitable amount of vitamins.

[0216] Suitably, the nutritional composition comprises vitamin K1 in an amount of at least about 5 µg / 100g, at least about 10 µg / 100g, at least about 15 µg / 100g, at least about 20 µg / 100g, at least about 25 µg / 100g, or at least about 30 µg / 100g based on dry weight. Suitably, the nutritional composition comprises vitamin K1 in an amount of at least about 2100 µg / 100g or less, about 100 µg / 100g or less, about 90 µg / 100g or less, about 80 µg / 100g or less, about 70 µg / 100g or less, or about 60 µg / 100g or less based on dry weight. Suitably, the nutritional composition comprises vitamin K1 in an amount of at least about 5 µg / 100g to about 200 µg / 100g, about 10 µg / 100g to about 100 µg / 100g, about 15 µg / 100g, or about 15 µg / 100g based on dry weight.Vitamin K1 in amounts from 100g to about 90µg / 100g, about 20µg / 100g to about 80µg / 100g, about 25µg / 100g to about 70µg / 100g, or about 30µg / 100g to about 60µg / 100g.

[0217] Suitably, the nutritional composition comprises vitamin K2 in amounts based on a dry weight of at least about 5µg / 100g, at least about 10µg / 100g, at least about 15µg / 100g, at least about 20µg / 100g, at least about 25µg / 100g, or at least about 30µg / 100g. Appropriately, the nutritional composition contains vitamin K2 in amounts of about 2100 µg / 100g or less, about 100 µg / 100g or less, about 90 µg / 100g or less, about 80 µg / 100g or less, about 70 µg / 100g or less, or about 60 µg / 100g or less based on dry weight. Suitablely, the nutritional composition comprises vitamin K2 in amounts of about 5 µg / 100g to about 200 µg / 100g, about 10 µg / 100g to about 100 µg / 100g, about 15 µg / 100g to about 90 µg / 100g, about 20 µg / 100g to about 80 µg / 100g, about 25 µg / 100g to about 70 µg / 100g, or about 30 µg / 100g to about 60 µg / 100g.

[0218] Suitablely, the nutritional composition comprises vitamin A in amounts of at least about 100 µg RE / 100g, at least about 200 µg RE / 100g, or at least about 300 µg RE / 100g based on dry weight. Suitablely, the nutritional composition contains vitamin A in amounts based on a dry weight of about 1000 µg RE / 100g or less, about 800 µg RE / 100g or less, about 600 µg RE / 100g or less, or about 400 µg RE / 100g or less. Suitablely, the nutritional composition contains vitamin A in amounts based on a dry weight of about 100 µg RE / 100g to about 1000 µg RE / 100g, about 200 µg RE / 100g to about 800 µg RE / 100g, about 300 µg RE / 100g to about 600 µg RE / 100g, or about 300 µg RE / 100g to about 400 µg RE / 100g.

[0219] Suitably, the nutritional composition comprises vitamin A in an amount of at least about 100 µg RAE / 100g, at least about 200 µg RAE / 100g, or at least about 300 µg RAE / 100g based on a dry weight. Suitably, the nutritional composition comprises vitamin A in an amount of at least about 1000 µg RAE / 100g or less, at least about 800 µg RAE / 100g or less, at least about 600 µg RAE / 100g or less, or at least about 400 µg RAE / 100g based on a dry weight.Vitamin A in an amount of 100g or less. Suitably, the nutritional composition contains vitamin A in an amount based on a dry weight of about 100µg RAE / 100g to about 1000µg RAE / 100g, about 200µg RAE / 100g to about 800µg RAE / 100g, about 300µg RAE / 100g to about 600µg RAE / 100g, or about 300µg RAE / 100g to about 400µg RAE / 100g.

[0220] Suitably, the nutritional composition contains vitamin D in an amount based on a dry weight of at least about 5µg / 100g, at least about 10µg / 100g, or at least about 15µg / 100g. Suitably, the nutritional composition contains vitamin D in an amount based on a dry weight of about 100µg / 100g or less, about 75µg / 100g or less, or about 50µg / 100g or less. Suitablely, the nutritional composition comprises vitamin D in amounts based on dry weight of about 5 µg / 100g to about 100 µg / 100g, about 10 µg / 100g to about 75 µg / 100g, or about 15 µg / 100g to about 50 µg / 100g.

[0221] Probiotics

[0222] The nutritional composition according to the invention may contain any suitable amount of probiotics.

[0223] Suitablely, the nutritional composition comprises one or more probiotics in total amounts based on dry weight of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at least about 107 cfu / 100g, or at least about 108 cfu / 100g, at least about 109 cfu / 100g, or at least about 1010 cfu / 100g. Suitablely, the nutritional composition comprises one or more probiotics in a total amount based on a dry weight of about 10¹² cfu / 100g or less, about 10¹¹ cfu / 100g or less, or about 10¹⁰ cfu / 100g or less. Suitablely, the nutritional composition comprises one or more probiotics in a total amount based on a dry weight of about 10⁶ cfu / 100g to about 10¹² cfu / 100g, about 10⁷ cfu / 100g to about 10¹¹ cfu / 100g, or about 10⁸ cfu / 100g to about 10¹⁰ cfu / 100g.

[0224] Suitably, the nutritional composition comprises a quantity of Bifidobacterium longum (e.g., Bifidobacterium infantis) based on a dry weight of at least about 10⁵ CFU / 100g, at least about 10⁶ CFU / 100g, at least about 10⁷ CFU / 100g, or at least about 10⁸ CFU / 100g, at least about 10⁹ CFU / 100g, or at least about 10¹⁰ CFU / 100g. Suitably, the nutritional composition comprises a quantity of Bifidobacterium longum (e.g., Bifidobacterium infantis) based on a dry weight of about 10¹² CFU / 100g or less, about 10¹¹ CFU / 100g or less, or about 10¹⁰ CFU / 100g or less. SuitablyThe nutritional composition comprises Bifidobacterium longum (e.g., Bifidobacterium infantis) in an amount based on a dry weight of about 10⁶ CFU / 100g to about 10¹² CFU / 100g, about 10⁷ CFU / 100g to about 10¹¹ CFU / 100g, or about 10⁸ CFU / 100g to about 10¹⁰ CFU / 100g.

[0225] Suitably, the nutritional composition comprises Bifidobacterium animalis (e.g., Bifidobacterium lactis) in an amount based on a dry weight of at least about 10⁵ CFU / 100g, at least about 10⁶ CFU / 100g, at least about 10⁷ CFU / 100g, or at least about 10⁸ CFU / 100g, at least about 10⁹ CFU / 100g, or at least about 10¹⁰ CFU / 100g. Suitable, the nutritional composition contains animal Bifidobacterium (e.g., Bifidobacterium lactis) in an amount based on about 10¹² CFU / 100g or less, about 10¹¹ CFU / 100g or less, or about 10¹⁰ CFU / 100g or less of dry weight. Suitable, the nutritional composition contains animal Bifidobacterium (e.g., Bifidobacterium lactis) in an amount based on about 10⁶ CFU / 100g to about 10¹² CFU / 100g, about 10⁷ CFU / 100g to about 10¹¹ CFU / 100g, or about 10⁸ CFU / 100g to about 10¹⁰ CFU / 100g of dry weight.

[0226] Suitably, the nutritional composition comprises *Lactobacillus rhamnosus* in an amount of at least about 105 cfu / 100g, at least about 106 cfu / 100g, at least about 107 cfu / 100g, or at least about 108 cfu / 100g, at least about 109 cfu / 100g, or at least about 1010 cfu / 100g based on a dry weight. Suitably, the nutritional composition comprises *Lactobacillus rhamnosus* in an amount of about 1012 cfu / 100g or less, about 1011 cfu / 100g or less, or about 1010 cfu / 100g or less based on a dry weight. Suitablely, the nutritional composition comprises *Lactobacillus rhamnosus* in amounts based on dry weight of about 10⁶ cfu / 100g to about 10¹² cfu / 100g, about 10⁷ cfu / 100g to about 10¹¹ cfu / 100g, or about 10⁸ cfu / 100g to about 10¹⁰ cfu / 100g.

[0227] Other Components

[0228] The nutritional composition of the present invention may contain a protein source, a carbohydrate source, and a lipid source. However, in some embodiments, particularly if the nutritional composition of the present invention is a supplement or fortifier, only lipids (or lipid sources) may be present.

[0229] The nutritional composition of the present invention may contain based on dry weight of about 100 kcal / 100g to about 1000 kcal / 100g, about 200 kcal / 100g to about 800 kcal / 100g, or about 400 kcal / 100g to about 600 kcal / 100g.

[0230] Protein

[0231] The nutritional composition according to the invention may contain a protein source. The amount of protein may be from about 1 g to about 4 g / 100 kcal or from about 1.5 g to about 3 g / 100 kcal.

[0232] Protein sources based on, for example, whey, casein, and mixtures thereof may be used, as well as plant-based (e.g., soybean-based) protein sources. With regard to whey protein of interest, the protein source may be based on acid whey or sweet whey or mixtures thereof, and may contain any desired proportions of α-lactalbumin and β-lactoglobulin. In some embodiments, the protein source is whey-based (i.e., more than 50% of the protein is derived from whey protein, such as 60% or 70%). The protein may be whole or hydrolyzed, or a mixture of whole and hydrolyzed proteins. The term "intact" means that the major part of the protein is intact, i.e., the molecular structure is not altered, for example, at least 80% of the protein is not altered, such as at least 85% of the protein is not altered, preferably at least 90% of the protein is not altered, and even more preferably at least 95% of the protein is not altered, such as at least 98% of the protein is not altered. In one specific embodiment, 100% of the protein is not altered.

[0233] The term "hydrolyzed" means, in the context of this invention, that the protein has been hydrolyzed or broken down into its constituent amino acids.

[0234] The protein may be completely hydrolyzed or partially hydrolyzed. If a hydrolyzed protein is required, the hydrolysis process can be carried out as needed and as is known in the art. For example, whey protein hydrolysates can be prepared by enzymatic hydrolysis of whey fractions in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This allows the degree of lysine blocking to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source.

[0235] In one embodiment, the protein of the composition is hydrolyzed, completely hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be 2 to 20, 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, 20, 40, 60, 80, or 90.

[0236] At least 70%, 80%, 85%, 90%, 95%, or 97% of the protein may be hydrolyzed. In one embodiment, 100% of the protein is hydrolyzed.

[0237] In one embodiment, the protein of the composition is plant-based protein.

[0238] Carbohydrates

[0239] The nutritional composition according to the invention may contain a carbohydrate source. The amount of carbohydrates may be about 5g to about20g / 100kcal or about 10g to about 15g / 100kcal.

[0240] Any carbohydrate source commonly found in nutritional compositions, such as lactose, sucrose (see specification 20 / 29 pages 23 CN 121175055 A saccharose), maltodextrin, starch, and mixtures thereof, may be used, but the preferred carbohydrate source is lactose.

[0241] Lipids

[0242] The nutritional compositions according to the invention may contain lipids and essential fatty acids. The amount of lipids may be about 1g to about 10g / 100kcal or about 2g to about 6g / 100kcal.

[0243] Non-limiting examples of lipids include: palm oil extract, high-oleic sunflower oil, high-oleic safflower oil, canola oil, fish oil, coconut oil, milk fat, and combinations thereof. It may be particularly advantageous if the composition contains fat in an amount of about 25 to about 30g / 100g dry weight of the composition. Non-limiting examples of essential fatty acids include linoleic acid (LA) and alpha-linolenic acid (ALA). The compositions of the present invention may also contain gangliosides, monosialotetrahexosylganglioside-3 (GM3) and disialialotetrahexosylganglioside-3 (GD3), and combinations thereof.

[0244] Other Components

[0245] The nutritional compositions of the present invention may also contain all vitamins and minerals considered essential for a daily diet and required in significant amounts. Minimum requirements for certain vitamins and minerals have been determined. Examples of minerals, vitamins, and other nutrients optionally present in the compositions of the present invention include vitamins B1, B2, B3, B6, B12, E, C, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are typically added in salt form. The presence and amount of specific minerals and other vitamins will vary depending on the target population. If necessary, the nutritional compositions of the present invention may contain emulsifiers and stabilizers, such as soybean, lecithin, monoglyceride citrate, and diglyceride citrate.

[0246] The nutritional compositions of the present invention may also contain other substances that may have beneficial effects, especially on bone health or bone development, such as lactoferrin, osteopontin, TGFbeta, sIgA, glutamine, nucleotides, nucleosides, etc.

[0247] Preparation of the Composition

[0248] The compositions according to the present invention may be prepared by any known or other suitable means. For example, the nutritional composition may be perfected by blending a protein source with a carbohydrate source and a lipid source in appropriate proportions. If an emulsifier is used, it may be included in this stage. Vitamins and minerals may be added at this stage, but may also be added subsequently to prevent thermal degradation.Water (preferably reverse osmosis water or deionized water) can then be added and mixed to form a liquid mixture. The mixing temperature is preferably room temperature, but can also be higher. The liquid mixture can then be heat-treated to reduce the bacterial load. The mixture can then be homogenized.

[0249] If a powdered composition is required, the homogenized mixture is dried in a suitable drying device (such as a spray dryer or freeze dryer) and converted into a powder.

[0250] The method for manufacturing formulated foods is based on the concept that the product must be nutritionally adequate and microbiologically safe for consumption. Therefore, steps to eliminate or limit microbial growth are essential for the production method. The processing technology for each specific formulated food is the property of the manufacturer, but generally speaking, for powdered products, it involves preserving oil-in-water (o / w) emulsions by dehydration, or for ready-to-eat or concentrated liquid products, it involves sterilization. Powdered formulations can be produced by various methods, such as dry mixing of dehydrated components to form a homogeneous formulation, or hydrating and wet mixing of a mixture of macro-components (such as fats, proteins, and carbohydrates), and then evaporating and spray-drying the resulting mixture. A combination of the above two methods can be used, wherein a base powder is first prepared by wet mixing and spray-drying of all or some of the macro-components, and then the remaining components (including carbohydrates, minerals, vitamins, and other micronutrients) are dry-mixed to obtain the final formulation. Liquid formulations are provided in ready-to-eat form or as concentrated liquids, the latter requiring dilution with water, typically 1:1. The manufacturing methods for these products are similar to those used for manufacturing reconstituted milk. Specification 21 / 29 pages 24 CN 121175055 A

[0251] If it is necessary to produce liquid formulations, the homogenized mixture can be filled into a suitable container, preferably under aseptic conditions. However, the liquid composition can also be cooked in the container, and suitable equipment for such filling and cooking is commercially available.

[0252] Subject

[0253] The subject can be any suitable subject. Suitablely, the subject can be a mammal. In a preferred embodiment, the subject is a human. In other embodiments, the subject is an animal, preferably a pet. The pet can be an animal selected from dogs, cats, birds, fish, rodents (such as mice, rats and guinea pigs, rabbits), etc. In some embodiments, the pet is a dog. In some embodiments, the pet is a small dog breed.

[0254] In some embodiments, the subject is a teenager. The term "teenager" can refer to an individual who has not yet reached adulthood. In some embodiments, the subject is an adolescent or a child. The term "adolescent" can refer to an individual from the onset of puberty to adulthood. The term "child" can refer to an individual between birth and puberty.

[0255] In some embodiments, the subjects are infants, toddlers, or preschoolers. The term “infant” may refer to a subject aged about 0 to about 1 year. The term “toddler” may refer to a subject aged about 1 to about 3 years. The term “preschooler” may refer to a subject aged about 3 to about 10 years, about 3 to about 9 years, about 3 to about 8 years, about 3 to about 7 years, about 3 to about 6 years, or about 3 to about 5 years.

[0256] In some embodiments, the subjects are newborns. Newborns typically have low vitamin K levels and may be at risk of vitamin K deficiency bleeding (see, for example, Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p. 780). In some embodiments, the subjects are neonates.

[0257] In some embodiments, the subjects are about 5 years or younger, about 4 years or younger, about 3 years or younger, about 2 years or younger, or about 1 year or younger. In some embodiments, the subject's age is about 12 months or less, about 11 months or less, about 10 months or less, about 9 months or less, about 8 months or less, about 7 months or less, or about 6 months or less. In some embodiments, the subject's age is about 0.5 months or more. In some embodiments, the subject's age is about 0.5 months to about 6 months.

[0258] In some embodiments, the subject is an adolescent or an adult. In some embodiments, the subject is an adult. In some embodiments, the subject is an elderly person. In some embodiments, the subject is at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old.

[0259] The subject may have or may be at risk of vitamin K2 deficiency. The subject may have or may be at risk of primary vitamin K2 deficiency (i.e., insufficient vitamin K2 consumption). The subject may have or may be at risk of secondary vitamin K2 deficiency. Secondary deficiency can occur in people who consume adequate amounts but have malabsorption conditions such as cystic fibrosis or chronic pancreatitis, as well as in people with liver injury or liver disease. Secondary vitamin K deficiency can also occur in people who have been prescribed vitamin K antagonist drugs. Long-term use of antibiotics and long-term use of glucocorticoids can also affect vitamin K levels, particularly in children (see, for example, Kozio ł-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p. 78).

[0260] In some embodiments, the subject has dyslipidemia, diabetes, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or liver disease or is at risk of having these diseases.

[0261] In some embodiments, the subject has cystic fibrosis, inflammatory bowel disease (e.g., Crohn's disease).Disease) or ulcerative colitis), liver disease (e.g., chronic liver disease), long-term antibiotic use or long-term glucocorticoid use.

[0262] Subjects may have or may be at risk of reduced bone growth and / or bone strength. Vitamin K is required for the γ-carboxylation of osteocalcin in bone (see, for example, Hamidi, MS et al., Journal of Clinical Densitometry, 16(4), pp. 409-413).

[0263] This invention is applicable to children who are premature or have low birth weight or experience intrauterine growth retardation, or infants and young children who suffer from growth retardation due to malnutrition or experience diseases (such as Crohn's disease and / or celiac disease and / or cancer), or infants and young children treated with drugs that cause malabsorption, anorexia and / or metabolic bone disease (such as chemotherapy drugs and / or corticosteroids). In some embodiments, the subject is preterm or has low birth weight or has experienced intrauterine growth retardation, or has intrauterine malnutrition or suffers from developmental delay. The invention is also applicable to subjects who are at risk of bone disease, have a family history of bone disease, or have experienced at least one, preferably several, fractures.

[0264] In some embodiments, the subject suffers from and / or is suffering from growth retardation. Growth retardation may be defined as a “height-for-age” value that is less than two standard deviations from the median of the WHO child growth criteria (see, for example, De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pp. 12-26).

[0265] In some embodiments, the subject suffers from and / or is suffering from growth slowing. The term “growth slowing” may describe a pattern of weight gain that is slower than expected for age and sex in children and other adolescents (see, for example, King, C. and Davis, T., 2010. European journal of clinical nutrition, 64(1), pp. S11-S13). In some embodiments, the subject suffers from and / or is suffering from growth retardation and / or slowed growth due to malnutrition or experiencing conditions such as anorexia, Crohn's disease, and / or celiac disease. In some embodiments, the subject suffers from and / or is suffering from growth retardation and / or slowed growth due to treatment with medications (such as chemotherapy drugs and / or corticosteroids) that cause malabsorption, anorexia, and / or metabolic bone disease.

[0266] In some embodiments, the subject is preterm or has low birth weight or experiences intrauterine growth retardation. The term "preterm infant" may refer to an infant born at a gestational age of less than 37 weeks. The term "low birth weight infant" may refer to an infant born with a live birth weight of less than 2,500g.

[0267] Possible factors inducing vitamin K deficiency in newborns include poor placental transfer of vitamin K, immature gut microbiota, low vitamin K content in breast milk and significant inter-individual variability, poor intestinal absorption of vitamin K, and low activity of vitamin K cyclooxygenase (see, for example, Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p. 780).

[0268] Methods for treating and / or preventing vitamin K2 deficiency

[0269] Prebiotics (or combinations thereof) may be used for treating and / or preventing vitamin K2 deficiency.

[0270] In one aspect, the present invention provides prebiotics for treating and / or preventing vitamin K2 deficiency in a subject.

[0271] In another aspect, the present invention provides the use of prebiotics in the manufacture of medicaments for treating and / or preventing vitamin K2 deficiency in a subject.

[0272] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering an effective amount of a prebiotic to the subject.

[0273] The inventors have surprisingly discovered that vitamin K2 production in the gastrointestinal tract (e.g., via the conversion of vitamin K1) is promoted by a prebiotic, which includes, for example, bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), and / or fiber oligosaccharides (COS). Prebiotics (or combinations thereof) can treat and / or prevent vitamin K2 deficiency by promoting vitamin K2 production in the subject's gut. For example, prebiotics can treat and / or prevent vitamin K2 deficiency by promoting the de novo production of methylnaphthoquinone-7 and / or the bioconversion from phylloquinone to methylnaphthoquinone-4 in the subject's gut.

[0274] Vitamin K2 deficiency can be diagnosed by any suitable method known in the art. For example, vitamin K deficiency can lead to reduced blood clotting, and in severe cases, can lead to reduced clotting, increased bleeding, and increased prothrombin time. The diagnosis of vitamin K deficiency bleeding is usually indicated by prolonged activated partial prothrombin kinase time (APTT) and prothrombin time (PT), for example, an international normalized ratio (INR) of PT ≥4 or a value greater than 4 times the normal value in the presence of normal platelet count and fibrinogen content (see, for example, Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p. 780).

[0275] Methods for enhancing bone growth and / or bone strength or preventing bone loss and decreased bone strength. Therefore, treating and / or preventing vitamin K2 deficiency in subjects can enhance bone growth and / or bone strength in subjects or prevent bone loss and decreased bone strength (see, for example, Hamidi,MS et al., Journal of Clinical Densitometry, 16(4), pp. 409-413.

[0276] In the context of this invention, the term “enhancing bone growth and / or bone strength, or limiting / preventing bone loss” may specifically refer to one or more of the following physiological processes: bone catch-up growth, bone mass gain, peak bone mass optimization, promoting bone formation, promoting bone anabolism, promoting bone mineralization, increasing bone mineral density and microstructure, regulating bone biomechanical properties and regulating the ratio of bone formation and / or bone resorption, maintaining bone mass, and reducing bone resorption.

[0277] As used herein, “bone quality” may refer to aspects of bone composition and structure that contribute to bone strength, independent of bone mineral density. These include bone turnover, microstructure, mineralization, microdamage, and the composition of the bone matrix and minerals. Methods for measuring bone quality are known in the art.

[0278] As used herein, “enhancing bone growth and / or strength” may refer to supporting normal bone growth and / or strength, for example, during childhood and adolescence. Support for normal bone growth and / or strength results in normal bone anatomy and physiology. Appropriate methods and parameters for determining bone growth and strength will be known to those skilled in the art (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469(8), pp. 2128–2138). Suitablely, normal bone growth and / or strength can be determined using one or more bone parameters selected from the following: trabecular bone volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medial and lateral diameters, anteroposterior diameters, bone ultimate force (FMax), and bone stiffness. In some embodiments, one or more bone parameters selected from the following are used to determine normal bone growth and / or strength: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax). Appropriate methods for determining these parameters are available to those skilled in the art.

[0279] Regular nutritional supply of the composition according to the invention can also be used to prevent bone loss that occurs with aging and / or to protect osteocytes during bone aging.

[0280] In one embodiment, the composition is used to i) improve bone quality; ii) prevent or treat conditions related to an imbalance in the relationship between bone formation and bone resorption.

[0281] The term "reduction / inhibition of bone resorption" according to the invention refers to the inhibition of osteoclast-mediated destructive activity against bone tissue. To demonstrate that supplying the composition inhibits bone resorption in humans or animals, those skilled in the art can measure urinary excretion of deoxypyridinium as described in the examples; a reduction in deoxypyridinium expression reflects inhibition of bone resorption.

[0282] Administering the composition according to the invention to an animal organism simultaneously induces stimulation of bone formation and inhibition of bone resorption; the overall increase in bone mineralization and thus the overall increase in bone density are the result of the induction of both mechanisms.

[0283] To determine whether a subject presents with a state of osteopenia and therefore requires administration of the composition according to the invention, those skilled in the art will be able to refer in particular to the World Health Organization (WHO) report entitled "Assessment of fracture risk and its application in postmenopausal osteoporosis screening" (WHO Technical Series-843) of 1994.

[0284] The composition according to the invention is also designed for individuals exhibiting symptoms of bone defects or who may have bone defects, i.e., an imbalance in the relationship between bone formation and bone resorption, which, if the imbalance persists, induces osteopenia. The composition according to the invention is also designed for individuals exhibiting symptoms of bone defects caused by fractures, surgery, or dental diseases.

[0285] Specifically, the composition is designed for the prevention or treatment of diseases selected from osteoporosis, Paget's disease, bone loss or osteolysis observed near prostheses, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, periodontitis, or osteoarthritis.

[0286] As mentioned above, many conditions associated with imbalances in bone metabolism (such as osteoporosis) develop gradually over a long period and require long-term treatment. Therefore, their prevention or treatment can be carried out by periodically supplying the composition according to the invention, preferably in the form of a nutritional composition.

[0287] Similarly, periodic nutritional supply of the composition according to the invention to an individual, human, or animal makes it possible to produce high bone density and elevated peak bone mass by stimulating bone formation when these individuals reach adulthood.

[0288] Periodic nutritional supply of the composition according to the invention can also be used to prevent bone loss that occurs with aging (which may lead to osteoporosis) and / or to protect osteocytes during bone aging.

[0289] Therefore, in humans and other mammals, a variety of disorders are associated with abnormal metabolism of bone resorption and bone formation, leading to metabolic or bone remodeling imbalances.

[0290] Among the pathological conditions associated with bone metabolic imbalances, conditions or diseases such as osteoporosis, Paget's disease, bone loss or osteolysis observed near prostheses, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, and periodontal disease are particularly mentioned. Some conditions or diseases of bone metabolism may be caused by prolonged immobility (e.g., long-term hospitalization) or even after a period of weightlessness. Among the conditions associated with abnormal bone resorption, the most common is osteoporosis, which is most commonly observed in postmenopausal women. Osteoporosis is a…Systemic skeletal diseases are characterized by reduced bone mass and deterioration of bone microstructure, which are associated with increased bone fragility and susceptibility to fractures.

[0291] Since osteoporosis (as with other conditions associated with bone loss) constitutes a chronic condition, its prevention and treatment must be planned long-term.

[0292] It is generally accepted that early treatment must be preferred because the two key phases of bone capital are: the growth phase, which achieves maximum bone mass (peak bone mass); and aging, which regulates the rate of bone loss. Therefore, prevention of osteoporosis is no longer limited to older individuals.

[0293] Furthermore, in humans and animals, there are many conditions characterized by the need to increase bone formation. For example, in the case of fractures, it is necessary to stimulate bone growth in order to accelerate complete bone repair. This need also exists in periodontal disease, bone metastasis, osteolytic disease, and conditions requiring the repair of connective tissue, such as for the healing or regeneration of cartilage defects or trauma. Stimulation of bone growth is also required in primary and secondary hyperparathyroidism, as well as in osteoporosis associated with diabetes and osteoporosis associated with glucocorticoids.

[0294] Methods for promoting catch-up growth

[0295] Treatment and / or prevention of vitamin K2 deficiency in subjects can thus promote catch-up growth.

[0296] As used herein, “catch-up growth” can refer to a high rate of growth exceeding the normal age limit for at least one year after a brief period of growth inhibition, and can be complete or incomplete (see, for example, Wit, J.M. and Boersma, B., 2002. Journal of Pediatric Endocrinology and Metabolism, 15, pp. 1229-1242).

[0297] Appropriate methods and parameters for determining catch-up growth are known to those skilled in the art. Appropriately, high speed or high standard deviation scores can be used to determine catch-up growth (see, for example, Frongillo, EA, Leroy, JL and Lapping, K., 2019. Advances in Nutrition, 10(3), pp. 372-379 and Desmond, C. and Casale, D., 2017. Pl1, 12(12), p. 0189135). Specification 25 / 29 pages 28 CN 121175055 A

[0298] In some embodiments, catch-up growth is determined as an absolute term of linear growth (i.e., a reduction in high deficiency from the mean of a healthy reference population). In some embodiments, catch-up growth is determined as a relative linear growth (i.e., an improvement in age-high z-score and / or passing the -2SD or -1SD cutoff point).

[0299] Examples

[0300] The invention is further described with reference to the following examples. It should be understood that the invention protected by the claims is not intended to be limited in any way by these examples.

[0301] Example 1 - Promoting Vitamin K2 Production in the Gastrointestinal Tract

[0302] To assess the intestinal production of vitamin K2, the Human Microbial Ecosystem Simulator (SHIME®) was used (see Van de Wiele, T. et al., 2015. The Impact of Food Bioactives on Health: in vitro and ex vivo models, pp. 305-317). SHIME assays typically consist of colonic fermentation of selected amounts of the test compound under simulated conditions representing the gastrointestinal tract of the subject. The production of methylnaphthoquinone-7 was determined by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS / MS).

[0303] For this set of experiments, a two-stage batch system simulating upper gastrointestinal (upper GIT, stomach, and small intestine) and colonic conditions was used as a simplified SHIME® system. In these studies, milk-based infant beverages containing minerals appropriate for the age group, also known as infant formula milk, were used.

[0304] To simulate the absorption process that occurs in the infant's small intestine, a dialysis method was applied using a cellulose membrane with a 14 kDa cutoff. By introducing the small intestinal suspension into the dialysis membrane, molecules such as digested amino acids, sugars, micronutrients, and minerals were gradually removed from the upper gastrointestinal matrix.

[0305] Furthermore, during the 1-hour incubation period, the pH was gradually decreased from 5.5 to 3.0 during the gastric incubation to simulate the infant's gastric pH. Additionally, a fixed pH of 4.5 was implemented during the first 30 minutes of the small intestinal incubation (duodenum) to ensure optimal absorption of available minerals. In the subsequent 145-minute small intestinal phase (jejunum + ileum), pH 7 was introduced. The milk matrix, after exposure to gastric and small intestinal conditions, was transferred to the colonic chamber containing an infant fecal sample.

[0306] Fresh feces were collected from 12-month-old infant donors. A fecal suspension was prepared and mixed with a preservative. At the start of short-term colonic incubation, the test component (see below) was added to a sugar-depleted nutrient medium containing basal nutrients present in the colon (e.g., host-derived glycine, such as mucin).

[0307] In the first set of experiments, the following groups (test components) were evaluated: blank; single HMO (1.3 g / L 2FL); HMO mixture (2.5 g / L 2FL, DiFL, LNnT, LNT, and 6SL); Bifidobacterium infantis (107 cfu / ml); single HMO + Bifidobacterium infantis; HMO mixture + Bifidobacterium infantis. The results are shown in Figure 1. Compared with the negative control, the HMO mixture caused methylnaphthoquinone-The yield of methylnaphthoquinone-7 increased by 65%. Compared with the negative control, the addition of Bifidobacterium infantis further increased the yield of methylnaphthoquinone-7 by 163%.

[0308] In the second group of experiments, the following groups (test components) were evaluated (in the absence of milk matrix): blank; and HMO + BMO (total 7.2 g / L). The composition of HMO and BMO is shown in the table below: Specification 26 / 29 pages 29 CN 121175055 A

[0309]

[0310] The results are shown in Figure 2A. Compared with the negative control, the mixture of BMO and HMO increased the yield of methylnaphthoquinone-7 by 29%. It was further shown that the milk matrix containing a mixture of six HMOs significantly increased the yield of methylnaphthoquinone-7, and the addition of Bifidobacterium infantis further increased the yield (see Figure 2B).

[0311] In the last set of experiments, the following groups were evaluated (in the milk matrix, with or without Lactobacillus rhamnosus LPR): blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); and COS (2025 mg / L) + β-glucan (675 mg / L) + Bifidobacterium lactis (4.5 × 10⁶ cfu / ml). The results (with or without Lactobacillus rhamnosus LPR) are shown in Figures 3A and 3B, respectively. In the absence of LPR, COS increased MK-7 production by 178%. In the presence of LPR, the addition of Bifidobacterium lactis further increased MK-7 production by 22%.

[0312] Furthermore, when all groups were combined, it was shown that the addition of Lactobacillus rhamnosus LPR (4.5 × 10⁷ cfu / ml) further increased vitamin K2 production in the gastrointestinal tract by approximately 40% (see Figure 3C).

[0313] Example 2 - Preclinical Experiment Results

[0314] The preclinical experiment setup is shown in Figure 4. From D8 to D18, the number of pups per BALB / c mother was increased by 50% to induce food restriction. In both groups (normal and slowed growth), weaning occurred at D18. Male and female mice were then allowed to eat freely and were given daily nutritional supplementation via pipette feeding for 30 days.

[0315] As can be seen from Figures 5A and 5B, vitamin K2AD promotes bone quality by improving trabecular bone volume (BV / TV ratio) (trending increase) and cortical bone mineral density (Ct.BMD) (significant increase).

[0316] In addition, Figures 6A and 6B demonstrate that vitamin K2AD enhances bone strength by increasing the force (Fmax) and energy (Wmax) required to reach the femoral failure point.

[0317] Embodiments

[0318] Various preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs (sections).

[0319] 1. A prebiotic agent used to treat and / or prevent vitamin K2 deficiency in a subject.

[0320] 2. A prebiotic for the purpose according to any of the preceding paragraphs, wherein the prebiotic is administered in combination with vitamin K1.

[0321] 3. A prebiotic for the purpose according to paragraph 2, wherein the prebiotic and vitamin K1 are administered alone, simultaneously, or sequentially, preferably wherein the prebiotic and vitamin K1 are administered simultaneously.

[0322] 4. A prebiotic for the purpose according to any of the preceding paragraphs, wherein the prebiotic is administered in combination with a probiotic agent.

[0323] 5. A prebiotic for the purpose according to paragraph 4, wherein the prebiotic and the probiotic agent are administered alone, simultaneously, or sequentially, preferably wherein the prebiotic and the probiotic agent are administered simultaneously.

[0324] 6. A prebiotic agent for the purpose according to paragraph 4 or 5, wherein the prebiotic agent comprises *Bacillus rhamnosus*, *Bifidobacterium infantis*, and / or *Bifidobacterium lactis*.

[0325] 7. A prebiotic agent for the purpose according to any of the preceding paragraphs, wherein the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructose oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan.

[0326] 8. A prebiotic agent for the purpose according to any of the preceding paragraphs, wherein the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), a mixture of human milk oligosaccharides (HMO), and cello-oligosaccharides (COS).

[0327] 9. A prebiotic agent for the purpose according to any of the preceding paragraphs, wherein the prebiotic agent comprises about 80% by weight to about 100% by weight of BMO relative to the total weight of the prebiotic agent.

[0328] 10. A prebiotic agent for the purpose according to any of the preceding paragraphs, wherein the prebiotic agent comprises at least one sialylated oligosaccharide, at least one fucoidylated oligosaccharide, and / or at least one N-acetylated oligosaccharide, or is composed of the like.

[0329] 11. A prebiotic agent for the purpose according to paragraph 10, wherein the at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide, and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof.

[0330] 12. A prebiotic agent for the purpose according to paragraph 10 or 11, wherein the at least one fucoidan is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof.Oligosaccharides are selected from the group consisting of: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), and lactose-neofofucopentose V (LNnFP). The fucosylated oligosaccharides are 2'-fucosylated lactose (2'FL) and / or difucosylated lactose (diFL).

[0331] 13. A prebiotic agent for the purpose according to any one of paragraphs 10 to 12, wherein the at least one N-acetylated oligosaccharide is selected from the group consisting of: N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof, preferably wherein the at least one N-acetylated oligosaccharide is selected from lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof.

[0332] 14. A prebiotic for the purpose described in any of the preceding paragraphs, wherein the prebiotic comprises:

[0333] (a) at least one sialylated oligosaccharide at about 0.5% to about 2% by weight relative to the total weight of the oligosaccharide mixture;

[0334] (b) at least one fucoidylated oligosaccharide at about 2% to about 6% by weight relative to the total weight of the oligosaccharide mixture; and / or

[0335] (c) at least one N-acetylated oligosaccharide at about 1% to about 4% by weight relative to the total weight of the oligosaccharide mixture.

[0336] 15. A prebiotic for the purpose described in any of the preceding paragraphs, wherein the prebiotic comprises or is composed of fiber oligosaccharides (COS).

[0337] 16. A prebiotic for the purpose described in any of the preceding paragraphs, wherein the prebiotic is administered to the subject in a total dose of about 0.5 g / day to about 10 g / day. Instructions for Use, pages 28 / 29, CN 121175055 A

[0338] 17. A prebiotic preparation for the purpose described in any of the preceding paragraphs, wherein vitamin K1 is administered to the subject at an amount of about 5 µg / day to about 100 µg / day.

[0339] 18. A prebiotic preparation for the purpose described in any of the preceding paragraphs, wherein the probiotic preparation is administered at an amount of about 106 cfu / The subject is administered a total amount of approximately 10¹² cfu / day.

[0340] 19. A prebiotic for the purpose according to any of the preceding paragraphs, wherein the prebiotic is provided in the form of a nutritional composition.

[0341] 20. A prebiotic for the purpose according to any of the preceding paragraphs, wherein the prebiotic is provided in the form of a medical food product for clinical nutrition.

[0342] 21. A prebiotic for the purpose according to paragraph 19 or 20, wherein the composition comprises the prebiotic in a total amount based on approximately 0.5 g / 100 g to approximately 10 g / 100 g dry weight.

[0343] 22. A prebiotic for the purpose according to any one of paragraphs 19 to 21, wherein the composition comprises vitamin K1 in an amount based on approximately 5 µg / 100 g to approximately 100 µg / 100 g dry weight.

[0344] 23. A prebiotic preparation for the purpose according to any one of paragraphs 19 to 22, wherein the composition comprises a probiotic preparation in an amount based on about 106 cfu / 100g to about 1012 cfu / 100g dry weight.

[0345] 24. A prebiotic preparation for the purpose according to any of the preceding paragraphs, wherein the subject is an infant, toddler, or child.

[0346] 25. A prebiotic preparation for the purpose according to any of the preceding paragraphs, wherein the subject has vitamin K2 deficiency or is at risk of developing the disease.

[0347] 26. A prebiotic preparation for the purpose according to any of the preceding paragraphs, wherein the subject has dyslipidemia, diabetes, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease or is at risk of developing the disease.

[0348] 27. A prebiotic agent for the purpose described in any of the preceding paragraphs, wherein the subject suffers from reduced bone growth and / or bone strength or is at risk of developing the aforementioned condition.

[0349] 28. A prebiotic agent for the purpose described in any of the preceding paragraphs, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting the production of vitamin K2 in the gut of the subject.

[0350] 29. A prebiotic agent for the purpose described in any of the preceding paragraphs, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting the de novo production of methylnaphthoquinone-7 and / or the biotransformation from phylloquinone to methylnaphthoquinone-4 in the gut of the subject.

[0351] 30. Use of a prebiotic agent in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0352] 31. A method for treating and / or preventing vitamin K2 deficiency in a subject in need, the method comprising administering an effective amount of a prebiotic agent to the subject.

[0353] 32. Use of prebiotics in promoting vitamin K2 production in the gut of subjects.

[0354] 33. The use according to paragraph 32, wherein the prebiotic promotes the de novo production of methylnaphthoquinone-7 and / or the biotransformation from phylloquinone to methylnaphthoquinone-4 in the intestine of the subject.

[0355] Although the invention has been described by way of example, it should be understood that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, if known equivalents exist for specific features, such equivalents should be incorporated as expressly mentioned in this specification. Instruction manual page 29 / 29, page 32, CN 121175055 A, Figure 1; Instruction manual figure 1 / 6, page 33, CN 121175055 A, Figure 2; Instruction manual figure 2 / 6, page 34, CN 121175055 A, Figure 3; Instruction manual figure 3 / 6, page 35, CN 121175055 A, Figure 3 (continued), Figure 4; Instruction manual figure 4 / 6, page 36, CN 121175055 A, Figure 5; Instruction manual figure 5 / 6, page 37, CN 121175055 A, Figure 6; Instruction manual figure 6 / 6, page 38, CN 121175055 A

Claims

1. A prebiotic agent used to treat and / or prevent vitamin K2 deficiency in a subject.

2. The prebiotic agent for the stated purpose according to claim 1, wherein the prebiotic agent further comprises a vitamin mixture to promote the production of vitamin K2 in the intestine of the subject.

3. The prebiotic preparation for the stated purpose according to claim 1 or 2, wherein the vitamin mixture comprises vitamins K2, A and / or D.

4. A prebiotic for the purpose according to any of the preceding claims, wherein the prebiotic is administered in combination with vitamin K1.

5. A prebiotic agent for the stated purpose according to any of the preceding claims, wherein the prebiotic agent is administered in combination with a probiotic agent.

6. The prebiotic agent for the stated purpose according to claim 5, wherein the probiotic agent comprises Lactobacillus rhamnosus, Bifidobacterium infantis, and / or Bifidobacterium lactis.

7. A prebiotic agent for the stated purpose according to any of the preceding claims, wherein the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cello-oligosaccharides (COS), inulin, lactose, fructose oligosaccharides (FOS), galacto-oligosaccharides (GOS), and β-glucan, preferably wherein the prebiotic agent is selected from one or more of the following: a mixture of bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), and cello-oligosaccharides (COS).

8. A prebiotic for the purpose according to any of the preceding claims, wherein the prebiotic comprises an amount of BMO in a quantity of about 80% to about 100% by weight relative to the total weight of the prebiotic.

9. A prebiotic agent for the stated purpose according to any preceding claim, wherein the prebiotic agent comprises or is composed of HMOs, preferably wherein the prebiotic agent comprises or is composed of at least one sialylated oligosaccharide, at least one fucoidylated oligosaccharide, and / or at least one N-acetylated oligosaccharide, more preferably wherein: (a) The at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide, and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof; (b) The at least one fucosylated oligosaccharide is selected from the group consisting of: 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3FL), difucosylated lactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), lactose-neofofucopentose V (… LNnFP-V), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neo-difucosylhexose (LNnDFH), monofucosyllactose-N-hexose-III (MFNLH-III), difucosyllactose-N-hexose-a (DFLNHa), and combinations thereof, preferably wherein the at least one fucosylated oligosaccharide is 2'-fucosyllactose (2'FL) and / or difucosyllactose (diFL); and / or (c) The at least one N-acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof, preferably wherein the at least one N-acetylated oligosaccharide is selected from lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), and combinations thereof.

10. A prebiotic agent for the stated purpose according to any of the preceding claims, wherein the prebiotic agent comprises or is composed of COS.

11. A prebiotic for the purpose according to any of the preceding claims, wherein the prebiotic is administered to the subject in a total amount of about 0.5 g / day to about 10 g / day.

12. A prebiotic preparation for the stated purpose according to any preceding claim, wherein vitamin K1 is administered to the subject at an amount of about 5 µg / day to about 100 µg / day, and / or wherein a probiotic preparation is administered at an amount of about 10 µg / day. 6 CFU / day to approximately 10 12 The total amount of CFU / day was administered to the subject.

13. A prebiotic for the stated purpose according to any of the preceding claims, wherein the prebiotic is provided in the form of a nutritional composition.

14. A prebiotic agent for the purpose according to any of the preceding claims, wherein the subject is an infant, a toddler, or a child.

15. A prebiotic for the use according to any of the preceding claims, wherein the subject has dyslipidemia, diabetes, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease or is at risk of having said diseases, and / or wherein the subject has reduced bone growth and / or bone strength or is at risk of having said diseases.

16. A prebiotic agent for the stated purpose according to any of the preceding claims, wherein the prebiotic agent enhances bone growth, bone mineralization and / or bone strength of the subject, or limits / prevents bone loss and bone strength of the subject.

17. A prebiotic agent for the stated purpose according to any of the preceding claims, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting the production of vitamin K2 in the intestine of the subject.

18. Use of prebiotics in promoting vitamin K2 production in the gut of subjects.

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