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 to promote vitamin K2 production in the intestines of a subject.

Description

[Technical Field]

[0001] 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 intestines of a subject. [Background technology]

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

[0003] However, over the past few decades, vitamin K2 intake has decreased significantly among parents and their descendants, leading to serious health consequences. Furthermore, even with sufficient intake, secondary vitamin K2 deficiency can occur in individuals with malabsorption or liver disease when vitamin K antagonists are used or after long-term use of glucocorticoid antibiotics.

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

[0005] To our surprise, we have found that vitamin K2 production in the gastrointestinal tract (e.g., via the conversion of vitamin K1) is promoted by prebiotics, such as bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), and / or cellooligosaccharides (COS).

[0006] In one embodiment, the present invention provides a prebiotic agent for use in the treatment and / or prevention of a target vitamin K2 deficiency.

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

[0008] In another embodiment, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in subjects requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a prebiotic agent to the subjects.

[0009] Prebiotics can treat or prevent vitamin K2 deficiency by promoting vitamin K2 production in the target gut. For example, prebiotics can treat or prevent vitamin K2 deficiency by promoting the de novo production of menaquinone-7 and / or the bioconversion of phylloquinone to menaquinone-4 in the target gut.

[0010] In another aspect, the present invention provides the use of a prebiotic agent to promote vitamin K2 production in the intestines of a subject.

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

[0012] The prebiotic agent may be any suitable prebiotic agent that promotes vitamin K2 production in the target intestine. Preferably, 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, fructooligosaccharides (FOS), galactooligosaccharides (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 cellooligosaccharides (COS).

[0013] In some embodiments, the prebiotic agent contains BMO in an amount of about 80% 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 sialyl oligosaccharide, at least one fucosyl oligosaccharide, and / or at least one N-acetyl oligosaccharide. Preferably, the at least one sialyl oligosaccharide is selected from the group consisting of 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), disial lacto-N-tetraose, and combinations thereof. In some embodiments, the at least one sialyl oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof. Preferably, at least one fucosyl oligosaccharide is 2'-fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), difucosyl lactose (diFL), lacto-N-fucopentaose-I (LNFP-I), lacto-N-fucopentaose-II (LNFP-II), lacto-N-fucopentaose-III (LNFP-III), lacto-N-fucopentaose-V ( The fucosyl oligosaccharide is selected from the group consisting of LNFP-V, lacto-neo-fucopentaose V (LNnFP-V), lacto-N-difucosylhexaose-I (LNDFH-1), lacto-N-neo-difucosylhexaose (LNnDFH), monofucosyl lacto-n-hexaose-III (MFNLH-III), difucosyl lacto-N-hexaose-a (DFLNHa), and combinations thereof. In some embodiments, at least one fucosyl oligosaccharide is 2'-fucosyl lactose (2'FL) and / or difucosyl lactose (diFL). Preferably, at least one N-acetyl oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof.In some embodiments, at least one N-acetyloligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof.

[0015] In some embodiments, the prebiotic agent contains or comprises cellooligosaccharides (COS).

[0016] Prebiotics can be administered to subjects in any preferred amount. Preferably, prebiotics are administered to subjects in a total amount of about 0.5 g / day to about 10 g / day. Preferably, BMO is administered to subjects in a total amount of about 0.5 g / day to about 10 g / day. Preferably, HMO is administered to subjects in a total amount of about 0.5 g / day to about 10 g / day. Preferably, cellooligosaccharides (COS) are administered to subjects 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 absorption in the intestinal mucosa and other organs. Prebiotics and vitamin K1 may be administered separately, simultaneously, or sequentially. In a preferred embodiment, prebiotics and vitamin K1 are administered simultaneously. Preferably, vitamin K1 is administered to the subject in amounts of about 5 μg / day to about 200 μg / day.

[0018] Prebiotics may be administered in combination with probiotics. Surprisingly, the inventors have found 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 separately, simultaneously, or sequentially. In a preferred embodiment, prebiotics and probiotics are administered simultaneously. The probiotic may contain any suitable probiotic. Preferably, the probiotic contains Lactobacillus rhamnosus, Bifidobacterium infantis, and / or Bifidobacterium lactis. Preferably, the probiotic contains about 10 6 cfu / day ~ approximately 10 12 The total dose of cfu / day is administered to the target individual.

[0019] Prebiotics (or combinations thereof) may be provided in any preferred form, for example, in the form of a composition. Prebiotics (or combinations thereof) may be provided in the form of a nutritional composition. Prebiotics (or combinations thereof) may be provided in the form of a medical food for clinical nutrition.

[0020] Preferably, the composition contains a total amount of prebiotics of about 0.5 g / 100 g to about 10 g / 100 g on a dry weight basis. Preferably, the composition contains an amount of vitamin K1 of about 5 μg / 100 g to about 200 μg / 100 g on a dry weight basis. Preferably, the composition contains about 10 6 cfu / 100g~approx. 10 12 Contains probiotics in the amount of cfu / 100g.

[0021] The subject may be any suitable subject. Preferably, the subject is human or animal. In a preferred embodiment, the subject is human. In some embodiments, the subject is a young person, adolescent, child, infant, or young child. In some embodiments, the subject is a child, infant, or young child. 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 may have or be at risk of dyslipidemia, diabetes mellitus, severe thalassemia™, cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease. In some embodiments, the subject may have or be at risk of reduced bone growth and / or bone strength. [Brief explanation of the drawing]

[0022] [Figure 1] The effects of human milk oligosaccharides (HMOs) on vitamin K2 production in a gut model were evaluated. The following groups were assessed: blank; single HMO (2FL at 1.3 g / L); HMO mix (2FL, DiFL, LNnT, LNT, and 6SL at 2.5 g / L); B. infantis (107 cfu / mL); single HMO + B. infantis; HMO mix + B. infantis. [Figure 2] Effects of oligosaccharides and probiotics on vitamin K2 production in an intestinal model: (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 mix of 6 HMOs); MM+B. infantis (10¹¹ cfu / g). [Figure 3-1]Effect of human milk oligosaccharides (HMOs) on vitamin K2 production in an intestinal 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); COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 x 10⁶ cfu / mL). (B) The following groups were evaluated (in a milk matrix in the presence of 4.5 × 10⁷ cfu / mL of L. rhamnosus LPR): Blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 × 10⁶ cfu / mL). (C) The following groups were evaluated (in milk matrix): LPR-free (no L. rhamnosus LPR added); and LPR-added (4.5 × 10⁷ cfu / mL L. rhamnosus LPR). [Figure 3-2] Effect of human milk oligosaccharides (HMOs) on vitamin K2 production in an intestinal 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); COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 x 10⁶ cfu / mL). (B) The following groups were evaluated (in a milk matrix in the presence of 4.5 × 10⁷ cfu / mL of L. rhamnosus LPR): Blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 × 10⁶ cfu / mL). (C) The following groups were evaluated (in milk matrix): LPR-free (no L. rhamnosus LPR added); and LPR-added (4.5 × 10⁷ cfu / mL L. rhamnosus LPR). [Figure 4]Preclinical Experiment Design: Growth Restriction Group (using vit.K1): To induce dietary restriction, the number of offspring mice per BALB / c mother was increased by 50% from D8 to D18. In both groups (normal and growth restriction groups), weaning was performed on D18. Subsequently, male and female mice were given free access to food for 30 days, with nutritional supplementation once daily via pipette feeding. [Figure 5] Figures 5A and 5B show the effects of Vit.K2AD on trabecular BV / TV and cortical BMD as evaluated by microcomputed tomography of the femur. As previously reported, trabecular and cortical microstructure were evaluated in the distal metaphysis and middiaphysis of the femur, respectively, using microcomputed tomography (μCT UCT35, Scanco Medical AG, Basserdorf, Switzerland).1 Briefly, isotropic 6 μm voxels were used to evaluate the trabecular and cortical bone regions. For the femoral trabecular region, 30 slices of bone beneath the distal growth plate were not considered in order to remove primary cancellous bone. 80 slices of secondary cancellous bone directly beneath were analyzed. Femoral cortical structure was evaluated using 60 consecutive CT slides in the mid-femur. Morphometric variables were calculated from binarized images using a direct 3D technique that does not depend on prior assumptions about the underlying structure.2 For the cancellous bone region, the BV / TV fraction (%) was evaluated. For the cortical bone in the central part of the femoral shaft, cortical bone mineral density (Ct.BMD) was evaluated. [Figure 6]Figures 6A and 6B show the effect of Vit.K2AD on femoral strength as evaluated by a three-point bending test. The three-point bending test was performed to examine the biomechanical properties of the femur as previously reported (CHTurner, DBBurr, Basic biomechanical measurements of bone: a tutorial, Bone, 1993, 14(4):595-608). The load was applied in compression mode at a nominal deformation rate of 0.08 mm / sec until fracture. The load-displacement curve was recorded, and the fracture force (Fmax(N) / AP(mm)) and fracture energy (Wmax(N) / AP(mm)), normalized by the anteroposterior diameter of the femur, were calculated. [Modes for carrying out the invention]

[0023] Herein, various preferred features and embodiments of the present invention are described by non-limiting examples. Those skilled in the art will understand that all features of the present invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0024] No reference to prior art documents in this specification should be considered an acknowledgment that such prior art is well known or that it forms part of a common general understanding in the art. All publications referenced herein are incorporated herein by reference.

[0025] As used herein, the words “comprises,” “comprising,” and similar words should not be interpreted as exclusive or exhaustive. In other words, they mean “including, but not limited to.” The terms “comprises,” “comprising,” etc., also include the term “consisting of.”

[0026] Unless otherwise specified, the implementation of the present invention will employ prior art that is within the capabilities of those skilled in the art. Such art is described in the literature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are commonly understood by those skilled in the art.

[0027] Numerical ranges include the numerical values ​​that define the range, and all percentages disclosed herein are weight / weight unless otherwise specified. As used herein, the term “about” means approximately, nearly, roughly, or near. When the term “about” is used with a number or range, the value or range is modified by expanding the upper and lower boundaries of the stated number(s). Generally, the terms “about” and “near” are used herein to adjust numbers by more than 10% above and less than the stated value.

[0028] Prebiotics The present invention provides a prebiotic agent for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0029] As used herein, the term “prebiotics” may refer to non-digestible components that benefit a subject by selectively stimulating the desired growth and / or activity of one or more microbial communities. A 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, cellooligosaccharides (COS), inulin, lactose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and β-glucans.

[0030] 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, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and β-glucan.

[0031] In some embodiments, the prebiotic agent is selected from one or more of bovine milk oligosaccharides (BMOs), human milk oligosaccharide (HMO) mixtures, and cellooligosaccharides (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 cellooligosaccharides (COS).

[0032] Bovine milk oligosaccharides The prebiotic agent used in the present invention may contain or consist of bovine milk oligosaccharide (BMO).

[0033] Oligosaccharides in bovine milk are formed in the mammary glands by the combination of monosaccharides glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine, fucose, and sialic acids N-acetylneuraminic acid and N-glycolylneuraminic acid. The composition (collection) of BMOs found in milk and colostrum has been analyzed in detail, and comprehensive studies typically identify 30 to 50 different structures (see, for example, Robinson, RC, 2019 Frontiers in nutrition, 6, p. 50).

[0034] Although bovine milk typically contains fewer oligosaccharide structures than human milk, the two share at least 10 common structures, including 3'-sialyl lactose and 6'-sialyl lactose, which constitute the majority of the BMO pool (see, for example, Robinson, RC, 2019. Frontiers in nutrition, 6, p. 50).

[0035] The prebiotic agent may contain BMO in an amount of approximately 50% or more by weight, approximately 60% or more by weight, approximately 70% or more by weight, approximately 80% or more by weight, approximately 85% or more by weight, approximately 90% or more by weight, approximately 95% or more by weight, or approximately 100% by weight, relative to the total weight of the prebiotic agent.

[0036] Human milk oligosaccharides (HMOs) The prebiotic agent used in the present invention may contain or consist of one or more human milk oligosaccharides (HMOs).

[0037] Many different types of HMOs are found in human milk and are typically based on combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine, and many different bonds between them. Almost all HMOs have a lactose moiety at the reducing end, and the non-reducing end portion is occupied by sialic acid and / or fucose (if present). HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosyl oligosaccharides).

[0038] Preferably, one or more HMOs may comprise at least one fucosyl oligosaccharide, at least one sialyl oligosaccharide, and / or at least one N-acetyl oligosaccharide. In some embodiments, one or more HMOs comprise or consist of at least one fucosyl oligosaccharide, at least one sialyl oligosaccharide, and at least one N-acetyl oligosaccharide. In some embodiments, one or more HMOs comprise or consist of 2'-fucosyl lactose (2'FL), difucosyl lactose (diFL), 6'-sialyl lactose (6'-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). In some embodiments, one or more HMOs include or consist of 2'-fucosyl lactose (2'FL), difucosyl lactose (diFL), 6'-sialyl lactose (6'-SL), 3'-sialyl lactose (3'-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT).

[0039] In some embodiments (for example, when the prebiotic agent contains BMO), the prebiotic agent contains at least one fucosyl oligosaccharide in an amount of about 0.5% to about 2% by weight relative to the total weight of the prebiotic agent. In some embodiments (for example, when the prebiotic agent contains BMO), the prebiotic agent contains at least one sialyl oligosaccharide in an amount of about 2% to about 6% by weight relative to the total weight of the prebiotic agent. In some embodiments (for example, when the prebiotic agent contains BMO), the prebiotic agent contains at least one N-acetyl oligosaccharide in an amount of about 1% to about 4% by weight relative to the total weight of the prebiotic agent.

[0040] In some embodiments (for example, when the prebiotic agent contains BMO), the prebiotic agent contains at least one fucosyl oligosaccharide in an amount of about 2% to about 6% by weight relative to the total weight of the prebiotic agent, and at least one N-acetyl oligosaccharide in an amount of about 1% to about 4% by weight relative to the total weight of the prebiotic agent.

[0041] In some embodiments (for example, when the prebiotic agent contains BMO), the prebiotic agent comprises at least one fucosyl oligosaccharide in about 2% to about 6% by weight relative to the total weight of the prebiotic agent, at least one sialyl oligosaccharide in about 0.5% to about 2% by weight relative to the total weight of the prebiotic agent, and at least one N-acetyl oligosaccharide in about 1% to about 4% by weight relative to the total weight of the prebiotic agent.

[0042] In other embodiments (for example, when the prebiotic does not contain BMO), the prebiotic contains at least one fucosyl oligosaccharide in about 30 to about 80% by weight, about 40 to about 80% by weight, or about 50 to about 70% by weight, based on the total weight of the prebiotic. In some embodiments (for example, when the prebiotic does not contain BMO), the prebiotic contains at least one sialyl oligosaccharide in about 10 to about 35% by weight, about 10 to about 30% by weight, or about 10 to about 25% by weight, based on the total weight of the prebiotic. In some embodiments (for example, when the prebiotic does not contain BMO), the prebiotic contains at least one N-acetyl oligosaccharide in about 10 to about 35% by weight, about 15 to about 30% by weight, or about 15 to about 20% by weight, based on the total weight of the prebiotic.

[0043] In some embodiments (for example, when the prebiotic does not contain BMO), the prebiotic comprises or consists of at least one fucosyl oligosaccharide in 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; and at least one N-acetyl oligosaccharide in 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.

[0044] In some embodiments (for example, when the prebiotic does not contain BMO), the prebiotic comprises or consists of at least one fucosyl oligosaccharide in 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; at least one sialyl oligosaccharide in 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; and at least one N-acetyl oligosaccharide in 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.

[0045] HMOs can be obtained by any suitable method. Suitable methods for synthesizing oligosaccharides are well known to those skilled in the art. For example, processes for producing oligosaccharides have been developed by microbial fermentation, enzymatic processes, chemical synthesis, or combinations thereof (see, for example, Zeuner et al., 2019. Molecules, 24(11), p. 2033).

[0046] Fucosyl oligosaccharide In some embodiments, the prebiotic agent comprises at least one fucosyl oligosaccharide.

[0047] Non-limiting examples of fucosyl oligosaccharides include 2'-fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), difucosyl lactose (diFL), lacto-N-fucopentaose, e.g., 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-I (LNDFH-1), lacto-N-neodifucosylhexaose Examples include ose (LNnDFH), fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose (e.g., fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II), monofucosyllacto-n-hexaose-III (MFNLH-III), difucosyllacto-N-hexaose I, difucolact-N-neohexaose, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, difucosyllacto-N-hexaose-a (DFLNHa), fucosyl-para-lacto-N-hexaose, tri-fucopara-lacto-N-hexaose I, and combinations thereof.

[0048] In a preferred embodiment, at least one fucosyl oligosaccharide includes 2'-fucosyl lactose (2'FL), which is typically the most common HMO naturally present in human breast milk.

[0049] In some embodiments, at least one fucosyl oligosaccharide is selected from the group consisting of 2'-fucosyl lactose (2'FL), difucosyl lactose (diFL), and combinations thereof. In some embodiments, at least one fucosyl oligosaccharide comprises or consists of 2'-fucosyl lactose (2'FL) and difucosyl lactose (diFL).

[0050] Fucosyl oligosaccharides can be obtained by any suitable method. For example, 2'FL can be produced by biotechnological methods using specific fucosyltransferases and / or fucosidases, either by using enzyme-based fermentation techniques (recombinant or natural enzymes) or microbial fermentation techniques. In the latter case, microorganisms can be manipulated to express or produce their respective natural enzymes and substrates. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose. diFL can be synthesized by enzymatic, biotechnological, and / or chemical processes.

[0051] sialyl oligosaccharide In some embodiments, the prebiotic agent comprises at least one sialyl oligosaccharide.

[0052] Non-exclusive examples of sialyl oligosaccharides include 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), disial lacto-N-tetraose, and combinations thereof.

[0053] In some embodiments, at least one sialyl oligosaccharide is selected from the group consisting of 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof. In some embodiments, at least one sialyl oligosaccharide includes or consists of 6'-sialyl lactose (6'-SL). In some embodiments, at least one sialyl oligosaccharide includes or consists of 6'-sialyl lactose (6'-SL) and 3'-sialyl lactose (3'-SL).

[0054] Sialyl oligosaccharides can be obtained by any preferred 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 by chromatography or filtration techniques. Alternatively, 3'-sialyl lactose (3'-SL) and / or 6'-sialyl lactose (6'-SL) can be produced by biotechnological methods using specific sialyltransferases or sialidases, neuraminidases, either by enzyme-based fermentation techniques (recombinant or natural enzymes), chemical synthesis, or microbial fermentation techniques. In the latter case, microorganisms can be manipulated to express or produce their respective natural enzymes and substrates. Single-microbial cultures or mixed cultures can be used. The formation of sialyl oligosaccharides can be initiated from acceptor substrates of any degree of polymerization (DP), DP=1 or greater. Alternatively, sialyl lactose can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid). Siaylic lactose is also commercially available from companies such as Kyowa Hakko Kogyo (Japan) or GeneChem (South Korea).

[0055] If a prebiotic contains 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL), it may be particularly beneficial if 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL) are included in the nutritional composition in a weight ratio of approximately 10:1 to approximately 1:10, for example, approximately 10:1 to approximately 2:1, approximately 8:1 to approximately 3:1, approximately 6:1 to approximately 3:1, approximately 5:1 to approximately 3:1, approximately 5:1 to approximately 4:1, or approximately 1:2 to approximately 1.5:1.

[0056] N-acetyloligosaccharide In some embodiments, the prebiotic agent comprises at least one N-acetyloligosaccharide.

[0057] Preferably, at least one N-acetyloligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, and combinations thereof. Non-limiting examples of N-acetyloligosaccharides include LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), and any combination thereof. Other examples include lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, isolact-N-octaose, para-lacto-N-octaose, and lacto-N-decaose.

[0058] In some embodiments, at least one N-acetyloligosaccharide is selected from the group consisting of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof. In some embodiments, at least one N-acetyloligosaccharide comprises or consists of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0059] N-acetyloligosaccharides can be obtained by any preferred method. For example, LNnT can be chemically synthesized by enzymatic transfer of sugar units from the donor to the acceptor using glycosyltransferase. Alternatively, LNnT can be prepared by chemically converting free or oligosaccharide-bound ketohexoses (e.g., fructose) to N-acetylhexosamine or N-acetylhexosamine-containing oligosaccharides. LNT can be synthesized by enzymatic, biotechnological, and / or chemical processes.

[0060] Cellobiose and cellooligosaccharides (COS) The prebiotic agent used in the present invention may contain or consist of cellobiose and / or cellooligosaccharides (COS).

[0061] Cellobiose is a disaccharide having the formula (C6H7(OH)4O)2O, 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. Preferably, cellobiose is in the form of free cellobiose or cellooligosaccharide.

[0062] In some embodiments, cellobiose is in the form of cellooligosaccharide (COS). Cellooligosaccharide may refer to an oligomer of β-glucose molecules having a β-1,4-link (e.g., about 2 to about 6 β-glucose molecules) and may mainly contain cellobiose. The prebiotic agent used in the present invention may contain or consist of cellooligosaccharide (COS). Studies have shown that COS may be a prebiotic (see, for example, Zhong, C., et al., 2020. Journal of agricultural and food chemistry, 68(32), pp.8557-8567).

[0063] Administration of prebiotics The subjects may be administered any suitable amount of prebiotics in any suitable form and via any suitable route of administration (e.g., via any form and route described herein).

[0064] Suitable doses of human oligosaccharides are described, for example, in 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.

[0065] Preferably, 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. Preferably, 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. Preferably, the prebiotic agent 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.

[0066] Preferably, BMO is administered to the subject in amounts of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Preferably, BMO is administered to the subject in amounts of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Preferably, BMO is administered to the subject in amounts 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.

[0067] Preferably, HMO is administered to the subject in amounts of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Preferably, HMO is administered to the subject in amounts of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Preferably, HMO is administered to the subject in amounts 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.

[0068] Preferably, COS is administered to the subject in amounts of at least about 0.5 g / day, at least about 1 g / day, or at least about 2 g / day. Preferably, COS is administered to the subject in amounts of about 10 g / day or less, about 8 g / day or less, or about 5 g / day or less. Preferably, COS is administered to the subject in amounts 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.

[0069] Combination therapy The prebiotic agent may be administered in combination with one or more further activators, mixtures, or compositions.

[0070] As used herein, “combination therapy” may mean a therapy comprising the administration of two or more activators (e.g., prebiotics, one or more vitamins, and / or probiotics), mixtures, or compositions.

[0071] The combination may be administered in any preferred form by any preferred route. Preferably, the combination is administered orally and / or enterally. In a preferred embodiment, the combination is administered orally. The combination may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the combination is administered simultaneously.

[0072] In one embodiment, the present invention provides a combination of a prebiotic agent, one or more vitamins (e.g., vitamin K1), and one or more probiotics for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0073] 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 pharmacopoeia for the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0074] In another embodiment, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, the method comprising administering to the subject an effective amount of a prebiotic agent, one or more vitamins (e.g., vitamin K1), and one or more probiotics in combination.

[0075] 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 to promote vitamin K2 production in the target intestine.

[0076] In another aspect, the present invention provides a method for promoting vitamin K2 production in the intestines of a subject, comprising administering to the subject an effective amount of a prebiotic agent, a combination of one or more vitamins (e.g., vitamin K1), and one or more probiotics.

[0077] Vitamins Prebiotics may be administered in combination with one or more vitamins. The prebiotics and one or more vitamins may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the prebiotics and one or more vitamins are administered simultaneously.

[0078] Vitamins are organic micronutrients necessary for the body to perform a certain range of normal functions, and include vitamin K1, vitamin A, vitamin D, vitamin C, folate, vitamin B3, vitamin B6, vitamin B12, and vitamin E. Preferably, one or more vitamins may include or consist of vitamin K1, vitamin K2, vitamin A, and / or vitamin D. In a preferred embodiment, one or more vitamins may include or consist of vitamin K1.

[0079] In one embodiment, the present invention provides a combination of a prebiotic agent and one or more vitamins for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0080] In another aspect, the present invention provides the 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.

[0081] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in subjects requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a prebiotic agent in combination with one or more vitamins to the subject.

[0082] In another aspect, the present invention provides the use of a combination of a prebiotic agent and one or more vitamins to promote vitamin K2 production in the target intestine.

[0083] In another aspect, the present invention provides a method for promoting vitamin K2 production in the intestines of a subject, comprising administering an effective amount of a prebiotic agent in combination with one or more vitamins to the subject.

[0084] Vitamin K1 In a preferred embodiment, 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 a preferred embodiment, the prebiotic agent and vitamin K1 are administered simultaneously.

[0085] In one embodiment, the present invention provides a combination of a prebiotic agent and vitamin K1 for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0086] In another aspect, the present invention provides the use of a combination of a prebiotic agent and vitamin K1 in the manufacture of a pharmaceutical product for treating and / or preventing a target vitamin K2 deficiency.

[0087] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a combination of a prebiotic agent and vitamin K1 to the subject.

[0088] In another aspect, the present invention provides the use of a combination of a prebiotic agent and vitamin K1 to promote vitamin K2 production in the target intestine.

[0089] In another aspect, the present invention provides a method for promoting vitamin K2 production in the intestines of a subject, comprising administering an effective amount of a combination of a prebiotic agent and vitamin K1 to the subject.

[0090] Vitamin K represents a family of lipid-soluble compounds that share a common chemical structure of 3-substituted 2-methyl-1,4-naphthoquinone. This family exists naturally in food as phylloquinone (vitamin K1) and menaquinone (vitamin K2). Vitamin K1 (phylloquinone) may have the following general formula:

[0091] [ka]

[0092] Vitamin K1 has a phytyl side chain and is typically the major form of dietary vitamin K, found in dark green leafy vegetables (e.g., spinach, lettuce, and other salad plants) and Brassica species.

[0093] Subjects may be administered any suitable amount of vitamin K1 in any suitable form and via any suitable route of administration (e.g., via any form and route described herein). Suitable doses of vitamin K1 are described, for example, in 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.

[0094] Preferably, 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. Preferably, 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. Preferably, vitamin K1 is administered to the subject at a dose 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 a dose of about 1 μg / kg / day.

[0095] Preferably, vitamin K1 is administered to the subject in amounts 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. Preferably, vitamin K1 is administered to the subject in amounts 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. Preferably, vitamin K1 is administered to the subject in amounts of approximately 5 μg / day to approximately 200 μg / day, approximately 10 μg / day to approximately 100 μg / day, approximately 15 μg / day to approximately 90 μg / day, approximately 20 μg / day to approximately 80 μg / day, approximately 25 μg / day to approximately 70 μg / day, or approximately 30 μg / day to approximately 60 μg / day.

[0096] Preferably, the prebiotic is administered to the subject at a dose of approximately 0.5 g / day to approximately 10 g / day, and vitamin K1 is administered to the subject at a dose of approximately 5 μg / day to approximately 200 μg / day.

[0097] Vitamin K2 While phylloquinone is a plant-derived vitamin K, menaquinone is a vitamin K quinone that can be obtained through 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, prebiotics promote the de novo production of menaquinone-7 in the target gut (e.g., by the gut microbiota). In some embodiments, prebiotics promote the bioconversion of phylloquinone to menaquinone-4 in the target gut (e.g., by the gut microbiota).

[0098] Vitamin K2 (menaquinone) may have the following general formula:

[0099] [ka]

[0100] Vitamin K2 is composed of various forms with different numbers (n) of isoprenyl units, where n can range from 4 to 13. Each form is indicated by the suffix (-n); for example, menaquinone-4 (abbreviated as MK-4) has four isoprene residues (n=4). MK-4 can be formed through the metabolic conversion of phylloquinone during the absorption of phylloquinone in the intestinal mucosa and other organs. Other menaquinones can be produced in the gastrointestinal tract by the gut microbiota.

[0101] The prebiotic may be administered in combination with vitamin K2. The prebiotic and vitamin K2 may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the prebiotic and vitamin K2 are administered simultaneously.

[0102] In one embodiment, the present invention provides a combination of a prebiotic agent and vitamin K2 for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0103] In another aspect, the present invention provides the use of a combination of a prebiotic agent and vitamin K2 in the manufacture of a pharmaceutical product for treating and / or preventing a target vitamin K2 deficiency.

[0104] In another aspect, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a combination of a prebiotic agent and vitamin K2 to the subject.

[0105] Subjects may be administered any suitable amount of vitamin K2 in any suitable form and via any suitable route of administration (e.g., via any form and route described herein). Suitable doses of vitamin K2 are described, for example, 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.

[0106] Preferably, vitamin K2 is administered to the subject in amounts of at least about 0.5 μg / kg / day, at least about 1 μg / kg / day, or at least about 2 μg / kg / day. Preferably, vitamin K2 is administered to the subject in amounts of about 10 μg / kg / day or less, about 7.5 μg / kg / day or less, or about 5 μg / kg / day or less. Preferably, vitamin K2 is administered to the subject in amounts of about 0.5 μg / kg / day to about 10 μg / kg / day, about 1 μg / kg / day to about 7.5 μg / kg / day, or about 2 μg / kg / day to about 5 μg / kg / day.

[0107] Preferably, vitamin K2 is administered to the subject in amounts 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. Preferably, vitamin K2 is administered to the subject in amounts 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. Preferably, vitamin K2 is administered to the subject in amounts of approximately 5 μg / day to approximately 200 μg / day, approximately 10 μg / day to approximately 100 μg / day, approximately 15 μg / day to approximately 90 μg / day, approximately 20 μg / day to approximately 80 μg / day, approximately 25 μg / day to approximately 70 μg / day, or approximately 30 μg / day to approximately 60 μg / day.

[0108] Vitamin A Vitamin A comprises a family of molecules containing 20 carbon atoms, comprising a methyl-substituted cyclohexenyl ring (beta-ionone ring) and a tetraene side chain having a hydroxyl group (retinol), an aldehyde group (retinal), a carboxylic acid group (retinoic acid), or an ester group (retinyl ester) at the 15th carbon atom. The term vitamin A may also include provitamin A carotenoids, which are food-derived precursors of retinol. Of the many carotenoids found in nature, several, including α-carotene, β-carotene, and β-cryptoxanthin, possess provitamin A nutritional activity.

[0109] The amount of vitamin A may be referred to in terms of retinol equivalents (RE) or retinol activity equivalents (RAE). For the food-derived provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin, the REs are set at 6 μg, 12 μg, and 12 μg, respectively. Using μgRAE, the vitamin A activity of provitamin A carotenoids is estimated to be half the vitamin A activity when μgRE is used. For the food-derived provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin, the RAEs are set at 12 μg, 24 μg, and 24 μg, respectively. (For example, see Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington (DC): National Academies Press (US); April 2001, Vitamin A.)

[0110] Subjects may be administered any suitable amount of vitamin A in any suitable form and via any suitable route of administration (e.g., via any form and route described herein). Suitable doses of vitamin A are described, for example, in 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.

[0111] Preferably, vitamin A is administered to the subject in amounts of at least about 100 μgRE / day, at least about 200 μgRE / day, or at least about 300 μgRE / day. Preferably, vitamin A is administered to the subject in amounts of about 1000 μgRE / day or less, about 800 μgRE / day or less, about 600 μgRE / day or less, or about 400 μgRE / day or less. Preferably, vitamin A is administered to the subject in amounts of about 100 μgRE / day to about 1000 μgRE / day, about 200 μgRE / day to about 800 μgRE / day, about 300 μgRE / day to about 600 μgRE / day, or about 300 μgRE / day to about 400 μgRE / day.

[0112] Preferably, vitamin A is administered to the subject in amounts of at least about 100 μgRE / day, at least about 200 μgRAE / day, or at least about 300 μgRAE / day. Preferably, vitamin A is administered to the subject in amounts of about 1000 μgRAE / day or less, about 800 μgRAE / day or less, about 600 μgRAE / day or less, or about 400 μgRAE / day or less. Preferably, vitamin A is administered to the subject in amounts of about 100 μgRAE / day to about 1000 μgRAE / day, about 200 μgRAE / day to about 800 μgRAE / day, about 300 μgRAE / day to about 600 μgRAE / day, or about 300 μgRAE / day to about 400 μgRAE / day.

[0113] Vitamin D Vitamin D is a group of lipid-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. Preferably, vitamin D includes or consists of vitamin D2 and vitamin D3. Preferably, vitamin D includes or consists of calcitriol.

[0114] Subjects may be administered any suitable amount of vitamin D in any suitable form and via any suitable route of administration (e.g., via any form and route described herein). Suitable doses of vitamin D are described, for example, in Greer, FR, 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.

[0115] Preferably, vitamin D is administered to the subject in amounts 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. Preferably, vitamin D is administered to the subject in amounts of about 100 μg / day or less, about 75 μg / day or less, or about 50 μg / day or less. Preferably, vitamin D is administered to the subject in amounts 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. Preferably, vitamin D is administered to the subject in amounts of about 15 μg / day.

[0116] Vitamin mixture The prebiotic may be administered in combination with a vitamin mixture. The prebiotic and the vitamin mixture may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the prebiotic and the vitamin mixture are administered simultaneously.

[0117] In one embodiment, the present invention provides a combination of a prebiotic agent and a vitamin mixture for use in the treatment and / or prevention of a target vitamin K2 deficiency.

[0118] In another aspect, the present invention provides the use of a combination of a prebiotic agent and a vitamin mixture in the manufacture of a pharmaceutical product for treating and / or preventing a target vitamin K2 deficiency.

[0119] In another embodiment, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a combination of a prebiotic agent and a vitamin mixture to the subject.

[0120] In another aspect, the present invention provides the use of a combination of a prebiotic agent and a vitamin mixture to promote vitamin K2 production in the target intestine.

[0121] As used herein, “vitamin mixture” may refer to a mixture of two or more vitamins. Any suitable vitamin mixture may be used.

[0122] In some embodiments, the vitamin mixture comprises or consists of vitamin K1, vitamin A, and vitamin D. Preferably, vitamin K1 is administered to the subject at a dose of about 5 μg / day to about 200 μg / day, vitamin A at a dose of about 100 μgRE / day to about 1000 μgRE / day, and vitamin D at a dose of about 2.5 μg / day to about 100 μg / day.

[0123] In other embodiments, the vitamin mixture comprises or consists of vitamin K2, vitamin A, and vitamin D. Preferably, vitamin K2 is administered to the subject at a dose of about 5 μg / day to about 200 μg / day, vitamin A at a dose of about 100 μgRE / day to about 1000 μgRE / day, and vitamin D at a dose of about 2.5 μg / day to about 100 μg / day.

[0124] In further embodiments, the present invention provides the use of a combination of a prebiotic agent and vitamin K2 for treating and / or preventing vitamin K2 deficiency in a subject.

[0125] In further embodiments, the present invention provides the use of a combination of a prebiotic agent and vitamin A for treating and / or preventing vitamin K2 deficiency in a subject.

[0126] In further embodiments, the present invention provides the use of a combination of a prebiotic agent and vitamin D for treating and / or preventing vitamin K2 deficiency in a subject.

[0127] In further embodiments, the present invention provides the use of a prebiotic agent in combination with vitamin K2, vitamin A, vitamin D, or any combination thereof, for treating and / or preventing vitamin K2 deficiency in a subject.

[0128] Probiotics Prebiotics may be administered in combination with one or more probiotics. The prebiotic and one or more probiotics may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the prebiotic and one or more probiotics are administered simultaneously.

[0129] As used herein, the term “probiotics” may refer to any component containing a sufficient number of viable microorganisms to alter the target gut microbiota (see, for example, Hill, C., et al., 2014. Nature reviews Gastroenterology & hepatology, 11(8), p. 506). Preferably, 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). Examples of probiotic microorganisms include Bifidobacterium, Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and Escherichia coli.

[0130] In one embodiment, the present invention provides a combination of a prebiotic agent and one or more probiotics for use in the treatment and / or prevention of vitamin K2 deficiency in a subject.

[0131] In another aspect, the present invention provides the use of a combination of a prebiotic agent and one or more probiotics in the manufacture of a medicament for treating and / or preventing vitamin K2 deficiency in a subject.

[0132] In another embodiment, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, the method comprising administering an effective amount of a prebiotic agent and one or more probiotic agents to the subject.

[0133] In another aspect, the present invention provides the use of a combination of a prebiotic agent and one or more probiotics to promote vitamin K2 production in the target intestine.

[0134] In another aspect, the present invention provides a method for promoting vitamin K2 production in the intestines of a subject, comprising administering an effective amount of a prebiotic agent and a combination of one or more probiotic agents to the subject.

[0135] One or more probiotics can increase vitamin K2 production. Surprisingly, the inventors have found that probiotics can promote endogenous vitamin K2 production through the production of precursors.

[0136] In some embodiments, one or more probiotics include or consist of Lactobacillus caseibacillus, Bifidobacterium, Lactobacillus, and / or Rimosilactobacillus. In some embodiments, one or more probiotics include or consist of Lactobacillus rhamnosus, Bifidobacterium longum, and / or Bifidobacterium animalis.

[0137] Lactobacillus rhamnosus In some embodiments, one or more probiotics include or consist of Lactobacillus rhamnosus.

[0138] Lacticaseibacillus rhamnosus (also known as Lactobacillus rhamnosus) is a Gram-positive, short-rod bacterium, a homofermentative facultative anaerobe, non-spore-forming, and often observed in a chain-like form. Lactobacillus rhamnosus GG (LGG) is one of the most widely used probiotic strains. Its various health benefits have been well-established (see, for example, Segers, ME and Lebeer, S., 2014. Microbial cell factories, 13(1), pp. 1-16). L. rhamnosus may promote the endogenous production of vitamin K2 through the production of precursors.

[0139] In some embodiments, one or more probiotics include or consist of Lactobacillus rhamnosus LPR.

[0140] Bifidobacterium longum In some embodiments, one or more probiotics include or consist of Bifidobacterium longum.

[0141] Bifidobacterium longum is a bacterium found in the human digestive tract. In 2002, three previously considered separate species of Bifidobacterium—B. infantis, B. longum, and B. suis—were merged into a single species named B. longum, encompassing the respective biotypes of infantis, longum, and suis (Sakata, S., et al., 2002. International Journal of Systematic and Evolutionary Microbiology, 52(6), pp.1945-1951).

[0142] In some embodiments, one or more probiotics include 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).

[0143] In some embodiments, one or more probiotics include or consist of Bifidobacterium infantis. In some embodiments, one or more probiotics include or consist of Bifidobacterium infantis LMG 11588 or its derivatives (e.g., R0033, which is reported to the U.S. Food and Drug Administration as generally recognized as safe (GRAS)) (see, for example, Duboux, S., et al., 2022. Microorganisms, 10(2), p.203). B. Infantis may promote the endogenous production of vitamin K2 through the production of precursors.

[0144] Bifidobacterium animalis In some embodiments, one or more probiotics include or consist of Bifidobacterium animalis.

[0145] Bifidobacterium animalis is a bacterium of the genus Bifidobacterium that can be found in the large intestine of most mammals, including humans. Bifidobacterium animalis and Bifidobacterium lactis were previously described as two different species. Currently, both are considered B. animalis, including the subspecies Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis (see Masco, L., et al, 2004. International Journal of Systematic and Evolutionary Microbiology, 54(4), pp. 1137-1143).

[0146] In some embodiments, the one or more probiotics comprise or consist of Bifidobacterium animalis ssp. lactis (also known as Bifidobacterium lactis). For example, the bacterial strain Bifidobacterium lactis HN019 has been studied for various traits important for its ability to function as a probiotic (see, for example, Sanders, M.E., 2006. Journal of clinical gastroenterology, 40(9), pp. 776-783).

[0147] Administration of Probiotics The subject can be administered any suitable amount of the probiotics in any suitable form via any suitable route of administration (e.g., via any of the forms and any of the routes described herein).

[0148] Preferably, the one or more probiotics are at least about 10 5 cfu / day, at least about 10 6 cfu / day, at least about 10 7 cfu / day, at least about 10 8 cfu / day, at least about 10 9 cfu / day or at least about 10 10The total dose administered to the subject is cfu / day. Preferably, one or more probiotics are administered in approximately 10 12 cfu / day or less, approximately 10 11 Less than or equal to 10 cfu / day 10 The total dose administered to the subject is less than or equal to cfu / day. Preferably, one or more probiotics are administered in approximately 10 doses. 6 cfu / day ~ approximately 10 12 cfu / day, approximately 10 7 cfu / day ~ approximately 10 11 cfu / day or approximately 10 8 cfu / day ~ approximately 10 10 The total dose of cfu / day is administered to the target individual.

[0149] Preferably, Lactobacillus rhamnosus is present in at least about 10 5 cfu / day, at least about 10 6 cfu / day, at least about 10 7 cfu / day, at least about 10 8 cfu / day, at least about 10 9 cfu / day, or at least about 10 10 The subject is administered a dose of cfu / day. Preferably, Lactobacillus rhamnosus is administered at a dose of approximately 10 12 cfu / day or less, approximately 10 11 Less than or equal to 10 cfu / day 10 The target is administered a dose of less than cfu / day. Preferably, Lactobacillus rhamnosus is administered at approximately 10 6 cfu / day ~ approximately 10 12 cfu / day, approximately 10 7 cfu / day ~ approximately 10 11 cfu / day or approximately 10 8 cfu / day ~ approximately 10 10 The patient is administered cfu / day.

[0150] Preferably, Bifidobacterium longum (e.g., B. infantis) is present at least about 10 5 cfu / day, at least about 10 6 cfu / day, at least about 10 7 cfu / day, at least about 10 8 cfu / day, at least about 10 9cfu / day, or at least about 10 10 The subject is administered a dose of cfu / day. Preferably, Bifidobacterium longum (e.g., B. infantis) is administered at a dose of approximately 10 12 cfu / day or less, approximately 10 11 Less than cfu / day, or about 10 10 The target is administered a dose of less than cfu / day. Preferably, Bifidobacterium longum (e.g., B. infantis) is administered at a dose of approximately 10 6 cfu / day ~ approximately 10 12 cfu / day, approximately 10 7 cfu / day ~ approximately 10 11 cfu / day or approximately 10 8 cfu / day ~ approximately 10 10 The patient is administered cfu / day.

[0151] Preferably, Bifidobacterium animalis (e.g., B. lactis) is present at least about 10 5 cfu / day, at least about 10 6 cfu / day, at least about 10 7 cfu / day, at least about 10 8 cfu / day, at least about 10 9 cfu / day, or at least about 10 10 The subject is administered a dose of cfu / day. Preferably, Bifidobacterium animalis (e.g., B. lactis) is administered at a dose of approximately 10 12 cfu / day or less, approximately 10 11 Less than cfu / day, or about 10 10 The target is administered a dose of cfu / day or less. Preferably, Bifidobacterium animalis (e.g., B. lactis) is administered at a dose of approximately 10 6 cfu / day ~ approximately 10 12 cfu / day, approximately 10 7 cfu / day ~ approximately 10 11 cfu / day or approximately 10 8 cfu / day ~ approximately 10 10 The patient is administered cfu / day.

[0152] Preferably, the prebiotic is administered to the subject in an amount of approximately 0.5 g / day to approximately 10 g / day, and one or more probiotics are administered in an amount of approximately 10 6 cfu / day ~ approximately 10 12 The total dose of cfu / day is administered to the target individual.

[0153] Preferably, prebiotics are administered to the subject at a dose of approximately 0.5 g / day to approximately 10 g / day, vitamin K1 is administered to the subject at a dose of approximately 5 μg / day to approximately 200 μg / day, and one or more probiotics are administered at a dose of approximately 10 6 cfu / day ~ approximately 10 12 The total dose of cfu / day is administered to the target individual.

[0154] composition Preferably, the prebiotic (or a combination thereof) is in the form of a composition. The composition may contain the combination in any therapeutically effective amount.

[0155] The composition may be any type of composition that can incorporate the combination, for example, a food or beverage product, an animal feed product, a composition in the form of a nutritional supplement for humans or animals, or a pharmaceutical composition. The composition may be in the form of a solid (e.g., powder), a liquid, or a semi-liquid. This combination may be in the form of a food composition, a pet food composition, a beverage, a nutritional formula, a nutritional supplement, or nutraceuticals.

[0156] Food and beverage products include all products intended for oral consumption by humans for the purpose of providing nutrition and / or pleasure. For example, food and beverage products may be nutritional compositions, such as nutritional compositions for young children. Examples of food and beverage products include dairy products, such as milk products or yogurt, soups, sauces, sweets and savory snacks, powdered beverages, and cereal products.

[0157] In some embodiments, the prebiotic (or combination thereof) is in the form of a nutritional composition, a medical food for clinical nutrition, or a supplement.

[0158] In some embodiments, the prebiotic (or combination thereof) is in the form of a nutritional composition. As used herein, “nutritional composition” may mean a composition that provides nutrition to a subject. This nutritional composition is usually administered orally or intravenously and typically comprises a lipid source or fat source and a protein source.

[0159] In some embodiments, prebiotics (or combinations thereof) are in the form of medical foods for clinical nutrition. As used herein, “medical foods for clinical nutrition” is also known as “Foods for Special Medical Purposes (FSMPs)” and refers to special foods designed to help satisfy the nutritional or dietary needs of persons living with a disease, disability, or medical condition that prevents them from achieving adequate nutritional intake temporarily or permanently from or through changes in their regular diet.

[0160] In some embodiments, the prebiotic (or combination thereof) is in the form of an infant formula. In this case, the infant formula may be a premature 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.

[0161] In some embodiments, the prebiotic (or combination thereof) is in the form of growing-up milk. As used herein, the term “growing-up milk” (or GUM) refers to a milk formula product given to children aged 1 year and older. Growing-up milk is generally a diet-based beverage adapted to the specific nutritional requirements of young children (e.g., children approximately 1 to 3 years of age). Growing-up milk is also known as “young child formula” or “infant milk.”

[0162] In some embodiments, the prebiotic (or combination thereof) is in the form of a milk formula. As used herein, the term “milk formula” can refer to, for example, a food intended for the nutrition of childhood, which can provide, for example, a sole source or supplemental nutrition for children about three years of age or older. In some embodiments, the milk formula is Glow Milk.

[0163] In some embodiments, the composition is in powder form and is reconstituted with an aqueous medium (e.g., water) before administration. In other embodiments, the composition is in a ready-to-administer liquid form (e.g., a ready-to-feed formula).

[0164] In another embodiment, the prebiotic (or combination thereof) is in the form of a supplement. As used herein, “supplement” or “dietary supplement” may be used to complement the nutrition of a subject (typically so, but dietary supplements may also be added to any type of composition intended to be ingested by the subject). If the composition is a supplement, such composition may be provided in the form of a unit dose. Supplements typically exist in the form of a liquid, gel, powder, or tablet or capsule. Powdered supplements typically include supplements that will be dissolved in water or milk, or sprinkled on food or beverages. Such supplements are intended to provide additional nutrition and / or health benefits to the subject ingesting the supplement. Supplements may be used to provide nutrition and / or health benefits to humans and animals.

[0165] 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-up milk fortifier.

[0166] In another embodiment, the prebiotic (or combination thereof) is in the form of a pharmaceutical product. Examples of pharmaceutical products include drops, syrups, powders, tablets, or capsules intended to treat or prevent an adverse medical condition in a person in need.

[0167] The prebiotic (or combination thereof) may be in the form of animal food products or animal nutritional supplements. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, and birds.

[0168] Prebiotics The nutritional composition according to the present invention may contain any suitable amount of prebiotics.

[0169] Preferably, the nutritional composition contains a prebiotic agent in a total amount 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 on a dry weight basis. Preferably, the nutritional composition contains a prebiotic agent in a total amount of about 10% by weight or less, about 8% by weight or less, or about 5% by weight or less on a dry weight basis. Preferably, the nutritional composition contains a prebiotic agent in a total amount of about 0.5% to about 10% by weight, about 1% to about 8% by weight, or about 2% to about 5% by weight on a dry weight basis.

[0170] Preferably, the prebiotic agent contains BMO in an amount of about 80% to about 100% by weight relative to the total weight of the prebiotic agent. Preferably, the nutritional composition contains a total amount of BMO 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 on a dry weight basis. Preferably, the nutritional composition contains a total amount of BMO of about 10% by weight or less, about 8% by weight or less, or about 5% by weight or less on a dry weight basis. Preferably, the nutritional composition contains a total amount of BMO of about 0.5% to about 10% by weight, about 1% to about 8% by weight, or about 2% to about 5% by weight on a dry weight basis.

[0171] Preferably, the nutritional composition contains a total amount of HMO 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 on a dry weight basis. Preferably, the nutritional composition contains a total amount of HMO of about 10% by weight or less, about 8% by weight or less, or about 5% by weight or less on a dry weight basis. Preferably, the nutritional composition contains a total amount of HMO of about 0.5% to about 10% by weight, about 1% to about 8% by weight, or about 2% to about 5% by weight on a dry weight basis.

[0172] Preferably, the nutritional composition contains at least one fucosyl oligosaccharide in an amount of about 0.05% to about 3% by weight, about 0.1% to about 2% by weight, or about 0.2% to about 1.5% by weight on a dry weight basis. Preferably, the nutritional composition contains at least one sialyl oligosaccharide in an amount of about 0.01% to about 2% by weight, about 0.05% to about 1.5% by weight, or about 0.07% to about 1% by weight on a dry weight basis. Preferably, the nutritional composition contains at least one N-acetyl 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 on a dry weight basis.

[0173] Preferably, the nutritional composition contains 2'FL 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 (the concentration may refer to the concentration after the composition has been reconstituted, for example, with water).

[0174] Preferably, the nutritional composition contains fucosyl oligosaccharides (e.g., 2'FL and / or diFL) 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 (the concentration may refer to the concentration after the composition has been reconstituted with, for example, water). In a particular embodiment, the nutritional composition contains a total of about 0.2 g / L to about 1.8 g / L of fucosyl oligosaccharides.

[0175] Preferably, the nutritional composition contains sialyl oligosaccharides (e.g., 3'-sialyl lactose (3'-SL) and / or 6'-sialyl lactose (6'-SL)) 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 g / L (the concentration may refer to the concentration after the composition has been reconstituted with, for example, water). In a particular embodiment, the nutritional composition contains a total of about 0.12 g / L to about 0.4 g / L of sialyl oligosaccharides.

[0176] Preferably, the nutritional composition contains N-acetyloligosaccharides (e.g., LNT and / or LNnT) 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, about 0.2 g / L to about 0.4 g / L, or about 0.3 g / L (the concentration may refer to the concentration after the composition has been reconstituted with, for example, water).

[0177] Preferably, the nutritional composition is At least one sialyl oligosaccharide in a total amount of approximately 0.05 g / L to approximately 0.75 g / L, approximately 0.05 g / L to approximately 0.5 g / L, approximately 0.1 g / L to approximately 0.3 g / L, or approximately 0.1 g / L to approximately 0.4 mg / L; At least one fucosyl oligosaccharide in a total amount of approximately 0.1 g / L to 4 g / L, approximately 0.1 g / L to 3.5 g / L, approximately 0.15 g / L to 3 g / L, approximately 0.2 g / L to 2.5 g / L, approximately 0.3 g / L to 2 g / L, approximately 0.4 g / L to 2 g / L, or approximately 0.5 g / L to 2 g / L; and / or It contains at least one N-acetyloligosaccharide in a total amount of approximately 0.05 g / L to approximately 0.5 g / L, approximately 0.1 g / L to approximately 0.5 g / L, or approximately 0.2 g / L to approximately 0.4 g / L.

[0178] Preferably, the nutritional composition contains COS in an amount 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 on a dry weight basis. Preferably, the nutritional composition contains COS in an amount of about 10% by weight or less, about 8% by weight or less, or about 5% by weight or less on a dry weight basis. Preferably, the nutritional composition contains COS in an amount of about 0.5% to about 10% by weight, about 1% to about 8% by weight, or about 2% to about 5% by weight on a dry weight basis.

[0179] Vitamins The nutritional composition according to the present invention may contain any suitable amount of vitamins.

[0180] Preferably, the nutritional composition contains 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 on a dry weight basis. Preferably, the nutritional composition contains vitamin K1 in an amount 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 on a dry weight basis. Preferably, the nutritional composition contains vitamin K1 in amounts of approximately 5 μg / 100g to approximately 200 μg / 100g, approximately 10 μg / 100g to approximately 100 μg / 100g, approximately 15 μg / 100g to approximately 90 μg / 100g, approximately 20 μg / 100g to approximately 80 μg / 100g, approximately 25 μg / 100g to approximately 70 μg / 100g, or approximately 30 μg / 100g to approximately 60 μg / 100g.

[0181] Preferably, the nutritional composition contains vitamin K2 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 on a dry weight basis. Preferably, the nutritional composition contains vitamin K2 in an amount 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 on a dry weight basis. Preferably, the nutritional composition contains vitamin K2 in amounts of approximately 5 μg / 100g to approximately 200 μg / 100g, approximately 10 μg / 100g to approximately 100 μg / 100g, approximately 15 μg / 100g to approximately 90 μg / 100g, approximately 20 μg / 100g to approximately 80 μg / 100g, approximately 25 μg / 100g to approximately 70 μg / 100g, or approximately 30 μg / 100g to approximately 60 μg / 100g.

[0182] Preferably, the nutritional composition contains vitamin A in an amount of at least about 100 μgRE / 100g, at least about 200 μgRE / 100g, or at least about 300 μgRE / 100g on a dry weight basis. Preferably, the nutritional composition contains vitamin A in an amount of about 1000 μgRE / 100g or less, about 800 μgRE / 100g or less, about 600 μgRE / 100g or less, or about 400 μgRE / 100g or less on a dry weight basis. Preferably, the nutritional composition contains vitamin A in amounts of approximately 100 μgRE / 100g to approximately 1000 μgRE / 100g, approximately 200 μgRE / 100g to approximately 800 μgRE / 100g, approximately 300 μgRE / 100g to approximately 600 μgRE / 100g, or approximately 300 μgRE / 100g to approximately 400 μgRE / 100g on a dry weight basis.

[0183] Preferably, the nutritional composition contains 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 on a dry weight basis. Preferably, the nutritional composition contains vitamin A in an amount of about 1000 μg RAE / 100g or less, about 800 μg RAE / 100g or less, about 600 μg RAE / 100g or less, or about 400 μg RAE / 100g or less on a dry weight basis. Preferably, the nutritional composition contains vitamin A in amounts of approximately 100 μg RAE / 100g to approximately 1000 μg RAE / 100g, approximately 200 μg RAE / 100g to approximately 800 μg RAE / 100g, approximately 300 μg RAE / 100g to approximately 600 μg RAE / 100g, or approximately 300 μg RAE / 100g to approximately 400 μg RAE / 100g on a dry weight basis.

[0184] Preferably, the nutritional composition contains vitamin D in an amount of at least about 5 μg / 100g, at least about 10 μg / 100g, or at least about 15 μg / 100g on a dry weight basis. Preferably, the nutritional composition contains vitamin D in an amount of about 100 μg / 100g or less, about 75 μg / 100g or less, or about 50 μg / 100g or less on a dry weight basis. Preferably, the nutritional composition contains vitamin D in an amount 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 on a dry weight basis.

[0185] Probiotics The nutritional composition according to the present invention may contain any suitable amount of probiotics.

[0186] Preferably, the nutritional composition contains one or more probiotics, at least about 10% on a dry weight basis. 5 cfu / 100g, at least about 10 6 cfu / 100g, at least about 10 7 cfu / 100g, or at least about 10 8 cfu / 100g, at least about 10 9 cfu / 100g, or at least about 10 10 Contains in total amount of cfu / 100g. Preferably, the nutritional composition is about 10 by dry weight. 12 cfu / 100g or less, approximately 10 11 cfu / 100g or less, approximately 10 10 It contains one or more probiotics in a total amount of cfu / 100g or less. Preferably, the nutritional composition is about 10 by dry weight. 6 cfu / 100g~approx. 10 12 cfu / 100g, approx. 10 7 cfu / 100g~approx. 10 11 cfu / 100g, or approximately 10 8 cfu / 100g~approx. 10 10 Contains one or more probiotics in total amount per 100g of cfu.

[0187] Preferably, the nutritional composition contains at least about 10% on a dry weight basis.5 cfu / 100 g, at least about 10 6 cfu / 100 g, at least about 10 7 cfu / 100 g, or at least about 10 8 cfu / 100 g, at least about 10 9 cfu / 100 g, or at least about 10 10 contains Bifidobacterium longum (e.g., B. infantis) in an amount of about 10 cfu / 100 g. Preferably, the nutritional composition is about 10 12 cfu / 100 g or less, about 10 11 cfu / 100 g or less, about 10 10 contains Bifidobacterium longum (e.g., B. infantis) in an amount of about 10 cfu / 100 g or less. Preferably, the nutritional composition is about 10 6 cfu / 100 g to about 10 12 cfu / 100 g, about 10 7 cfu / 100 g to about 10 11 cfu / 100 g, or about 10 8 cfu / 100 g to about 10 10 contains Bifidobacterium longum (e.g., B. infantis) in an amount of about 10 cfu / 100 g.

[0188] Preferably, the nutritional composition contains at least about 10 5 cfu / 100 g, at least about 10 6 cfu / 100 g, at least about 10 7 cfu / 100 g, or at least about 10 8 cfu / 100 g, at least about 10 9 cfu / 100 g, or at least about 10 10 contains Bifidobacterium animalis (e.g., B. lactis) in an amount of about 10 cfu / 100 g. Preferably, the nutritional composition is about 10 12 cfu / 100 g or less, about 10 11 cfu / 100 g or less, about 10 10 contains Bifidobacterium animalis (e.g., B. lactis) in an amount of about 10 cfu / 100 g or less. Preferably, the nutritional composition is about 10 6cfu / 100g~approx. 10 12 cfu / 100g, approx. 10 7 cfu / 100g~approx. 10 11 cfu / 100g, or approximately 10 8 cfu / 100g~approx. 10 10 Contains Bifidobacterium animalis (e.g., B. lactis) at a cfu / 100g level.

[0189] Preferably, the nutritional composition has at least about 10% on a dry weight basis. 5 cfu / 100g, at least about 10 6 cfu / 100g, at least about 10 7 cfu / 100g, or at least about 10 8 cfu / 100g, at least about 10 9 cfu / 100g, or at least about 10 10 Contains Lactobacillus rhamnosus at a cfu / 100g. Preferably, the nutritional composition is about 10 by dry weight. 12 cfu / 100g or less, approximately 10 11 cfu / 100g or less, approximately 10 10 Contains Lactobacillus rhamnosus in amounts of less than cfu / 100g. Preferably, the nutritional composition is about 10 by dry weight. 6 cfu / 100g~approx. 10 12 cfu / 100g, approx. 10 7 cfu / 100g~approx. 10 11 cfu / 100g, or approximately 10 8 cfu / 100g~approx. 10 10 Contains Lactobacillus rhamnosus at a cfu / 100g level.

[0190] Other ingredients The nutritional composition of the present invention may contain a protein source, a carbohydrate source, and a lipid source. However, in some embodiments, particularly when the nutritional composition of the present invention is a supplement or fortifier, only lipids (or a lipid source) may be present.

[0191] The nutritional composition of the present invention may contain approximately 100 kcal / 100g to approximately 1000 kcal / 100g, approximately 200 kcal / 100g to approximately 800 kcal / 100g, or approximately 400 kcal / 100g to approximately 600 kcal / 100g on a dry weight basis.

[0192] protein The nutritional composition according to the present invention may contain a protein source. The amount of protein may be about 1g to about 4g per 100kcal, or about 1.5g to about 3g per 100kcal.

[0193] For example, protein sources based on whey, casein, and mixtures thereof can be used in the same way as, for example, soy-based plant protein sources. With respect to whey protein, the protein source may be based on acidic whey, sweet whey, or mixtures thereof, and may contain α-lactalbumin and β-lactoglobulin in any desired proportion. In some embodiments, the primary protein source is whey (i.e., more than 50%, for example more than 60% or more than 70% of the protein is derived from whey protein). The protein may be in an intact state, a hydrolyzed state, or a mixture of intact and hydrolyzed proteins. The term "intact" means that the main part of the protein is intact, i.e., the molecular structure has not changed, for example, at least 80% of the protein has not changed, for example at least 85% of the protein has not changed, preferably at least 90% of the protein has not changed, and more preferably at least 95% of the protein has not changed, for example at least 98% of the protein has not changed. In certain embodiments, the protein remains completely unchanged.

[0194] In the context of this invention, the term "hydrolyzed" means that a protein has been hydrolyzed, or broken down into its constituent amino acids.

[0195] The protein may be either completely hydrolyzed or partially hydrolyzed. If a hydrolyzed protein is required, the hydrolysis process may be carried out as desired, as is known in the art. For example, a hydrolyzed whey protein can be prepared by enzymatically hydrolyzing a whey fraction in one or more steps. It has been found that if the whey fraction used as a raw material is substantially lactose-free, the lysine blackage that the protein undergoes during the hydrolysis process is significantly reduced. This can reduce the degree of lysine blackage from about 15% by weight of the total lysine to less than 10% by weight of the total lysine, and for example, about 7% by weight of lysine significantly improves the nutritional value of the protein source.

[0196] In a particular 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, or 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, greater than 20, greater than 40, greater than 60, greater than 80, or greater than 90.

[0197] At least 70%, 80%, 85%, 90%, 95%, or 97% of the protein may be hydrolyzed. In certain embodiments, 100% of the protein is hydrolyzed.

[0198] In one particular embodiment, the protein of the composition is a plant protein.

[0199] carbohydrates The nutritional composition according to the present invention may contain a carbohydrate source. The amount of carbohydrates may be about 5g to about 20g per 100kcal, or about 10g to about 15g per 100kcal.

[0200] Any carbohydrate source commonly found in nutritional compositions, such as lactose, sucrose, saccharose, maltodextrin, starch, or mixtures thereof, may be used, but one preferred carbohydrate source is lactose.

[0201] Lipids The nutritional composition according to the present invention may contain lipids and essential fatty acids. The amount of lipids may be about 1g to about 10g per 100kcal, or about 2g to about 6g per 100kcal.

[0202] 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. Compositions may be particularly beneficial if they contain fat in an amount of about 25 to about 30 g / 100 g by 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 further contain gangliosides, monosialoganglioside-3 (GM3) and disialoganglioside-3 (GD3), and combinations thereof.

[0203] Other ingredients The nutritional compositions of the present invention may also contain all vitamins and minerals understood to be essential in a daily diet 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 compositions of the present invention include vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin E, vitamin 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 usually added in salt form. The presence and amount of certain minerals and other vitamins will vary depending on the target population. If necessary, the nutritional compositions of the present invention may also contain emulsifiers and stabilizers, such as soy, lecithin, and mono- and diglyceride citrate esters.

[0204] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, particularly on bone health or bone development, such as lactoferrin, osteopontin, TGFβ, slgA, glutamine, nucleotides, and nucleosides.

[0205] Preparation of composition The compositions according to the present invention can be prepared by any known or other suitable method. For example, a nutritional composition may be proposed by blending a protein source with a carbohydrate source and a lipid source in suitable proportions. If used, an emulsifier may be included at this point. Vitamins and minerals may be added at this point, but they can usually be added later to avoid thermal decomposition. Water, preferably reverse-osmotic water or deionized water, can then be added and mixed to form a liquid mixture. The mixing temperature is preferably room temperature, but may be higher. The liquid mixture can then be heat-treated to reduce the bacterial content. The mixture may then be homogenized.

[0206] If it is desired to produce a powdered composition, the homogenized mixture is dried in a suitable drying apparatus, such as a spray dryer or freeze dryer, to produce a powder.

[0207] The processes used in formula manufacturing are based on the concept that the product must be nutritionally appropriate and microbiologically safe for consumption. Therefore, steps to eliminate or limit microbial growth are central to the manufacturing process. While the processing techniques for each specific formula are proprietary to the manufacturer, they generally involve preservation of oil-in-water (o / w) emulsions by dehydration in the case of powder products, or sterilization in the case of ready-to-feed or concentrated liquid products. Powdered formulas can be manufactured using various processes, such as dry blending dehydrated components to form a uniform formula, or hydrating and wet mixing a mixture of macro components, such as fat components, protein components, and carbohydrate components, and then evaporating and spray-drying the resulting mixture. A combination of the above two processes may also be used, where the base powder is produced by first wet-mixing and spray-drying all or some of the macro components, and then dry-blending the remaining components, including carbohydrates, minerals, vitamins, and other micronutrients, to create the final formula. Liquid formulas are available in ready-to-feed form or as concentrates that typically need to be diluted with water at a 1:1 ratio. The manufacturing processes used for these products are similar to those used for the production of recycled milk.

[0208] If it is desirable to manufacture a liquid formula, the homogenized mixture may be filled into a suitable container, preferably aseptically. However, the liquid composition may be retorted in the container, and suitable equipment for this type of filling and retorting is commercially available.

[0209] subject The subject may be any suitable subject. Preferably, the subject may 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 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 breed of dog.

[0210] In some embodiments, the subjects are young people. The term “young people” may refer to individuals who have not yet reached adulthood. In some embodiments, the subjects are adolescents or children. The term “adolescents” may refer to individuals during the period from the onset of puberty to adulthood. The term “children” may refer to individuals in the stage between birth and puberty.

[0211] In some embodiments, the subjects are infants, toddlers, or young children. The term “infant” may refer to subjects approximately 0 to 1 year old. The term “toddler” may refer to subjects approximately 1 to 3 years old. The term “young children” may refer to subjects approximately 3 to 10 years old, 3 to 9 years old, 3 to 8 years old, 3 to 7 years old, 3 to 6 years old, or 3 to 5 years old.

[0212] In some embodiments, the subjects are neonates. Neonatals typically have low levels of vitamin K and may be at risk of bleeding due to vitamin K deficiency (see, for example, Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p. 780). In some embodiments, the subjects are neonates.

[0213] In some embodiments, the target age is approximately 5 years or younger, approximately 4 years or younger, approximately 3 years or younger, approximately 2 years or younger, or approximately 1 year or younger. In some embodiments, the target age is approximately 12 months or younger, approximately 11 months or younger, approximately 10 months or younger, approximately 9 months or younger, approximately 8 months or younger, approximately 7 months or younger, or approximately 6 months or younger. In some embodiments, the target age is approximately 0.5 months or older. In some embodiments, the target age is approximately 0.5 months to approximately 6 months.

[0214] In some embodiments, the subjects are young people or adults. In some embodiments, the subjects are adults. In some embodiments, the subjects are elderly. In some embodiments, the subjects are 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.

[0215] The subjects may have or be at risk of vitamin K2 deficiency. They may have primary vitamin K2 deficiency, i.e., insufficient vitamin K2 intake, or be at risk of it. They may have or be at risk of secondary vitamin K2 deficiency. Secondary deficiency can occur in individuals with malabsorption, such as those with cystic fibrosis or chronic pancreatitis, and those with liver damage or disease, even with adequate intake. Secondary vitamin K deficiency can also occur in individuals prescribed vitamin K antagonists. Long-term antibiotic use and long-term glucocorticoid use can also affect vitamin K levels, particularly in children (see, e.g., Kozil-Kozakowska, A. and Maresz, K., 2022. Children, 9(1), p.78).

[0216] In some embodiments, subjects have or are at risk of having dyslipidemia, diabetes mellitus, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or liver disease.

[0217] In some embodiments, the subjects include 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.

[0218] The subjects may have or be at risk of reduced bone growth and / or bone strength. Vitamin K is required for the gamma-carboxylation of osteocalcin in bone and may be necessary for the formation of strong bone (see, for example, Hamidi, MS, et al., Journal of Clinical Densitometry, 16(4), pp. 409-413).

[0219] The present invention is particularly suitable for children who have suffered from growth retardation due to being premature, having a low birth weight, having experienced intrauterine growth restriction, or having experienced malnutrition or disease, such as Crohn's disease and / or celiac disease and / or cancer, or children who have been treated with drugs that cause malabsorption, anorexia and / or metabolic bone disease, such as chemotherapy drugs and / or corticosteroids. In some embodiments, the subject was premature, had a low birth weight, had experienced intrauterine growth restriction, was malnourished in utero, or suffered from growth retardation. The present invention is also suitable for subjects who are at risk of bone disease, have a family history of bone disease, or have already experienced at least one, preferably multiple, fracture episodes.

[0220] In some embodiments, the subjects suffer from and / or stunted growth. Stunted growth may be defined as having an "age-specific height" value less than two standard deviations of the WHO Child Growth Standards median (see, for example, De Onis, M. and Branca, F., 2016. Maternal & Child Nutrition, 12, pp. 12-26).

[0221] In some embodiments, subjects suffer from and / or faltering growth. The term “faltering growth” can describe a pattern in children and other adolescents in which weight gain is slower than expected for their age and sex (e.g., King, C. and Davis, T., 2010. European Journal of Clinical Nutrition, 64(1), pp. S11-S13). In some embodiments, subjects suffer from and / or faltering growth due to malnutrition and a history of disease, such as anorexia, Crohn's disease and / or celiac disease. In some embodiments, subjects suffer from and / or faltering growth due to treatment with drugs that cause malabsorption, anorexia and / or metabolic bone disease, such as chemotherapy drugs and / or corticosteroids.

[0222] In some embodiments, subjects were premature, low birth weight, or experienced intrauterine growth restriction. The term “premature” may refer to an infant born at a gestational age of less than 37 weeks. The term “low birth weight” may refer to an infant with a birth weight of less than 2500g.

[0223] Factors that may lead to vitamin K deficiency in newborns include insufficient vitamin transfer through the placenta, immature gut microbiota, low and significant individual differences in vitamin K content in breast milk, poor absorption of vitamin K in the intestines, and low activity levels of vitamin K epoxide reductase (e.g., Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p.780).

[0224] Methods for treating and / or preventing vitamin K2 deficiency Prebiotics (or combinations with prebiotics) may be used to treat and / or prevent vitamin K2 deficiency.

[0225] In one embodiment, the present invention provides a prebiotic agent for use in the treatment and / or prevention of a target vitamin K2 deficiency.

[0226] In another aspect, the present invention provides the use of prebiotics in the manufacture of pharmaceuticals for treating and / or preventing a target vitamin K2 deficiency.

[0227] In another embodiment, the present invention provides a method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a prebiotic agent to the subject.

[0228] The inventors have surprisingly found that vitamin K2 production in the gastrointestinal tract (e.g., via the conversion of vitamin K1) is promoted by prebiotics, including, for example, bovine milk oligosaccharides (BMOs), human milk oligosaccharides (HMOs), and / or cellooligosaccharides (COS). Prebiotics (or combinations with prebiotics) can treat and / or prevent vitamin K2 deficiency by promoting vitamin K2 production in the target intestine. For example, prebiotics can treat and / or prevent vitamin K2 deficiency by promoting the de novo production of menaquinone-7 and / or the bioconversion of phylloquinone to menaquinone-4 in the target intestine.

[0229] Vitamin K2 deficiency can be diagnosed by any suitable method known in the art. For example, vitamin K deficiency can lead to a reduction in blood clotting, and in severe cases, can lead to a decrease in clotting, an increase in bleeding, and an extension of the prothrombin time. The diagnosis of vitamin K deficiency bleeding is generally indicated by an extension of the activated partial thromboplastin time (APTT) and prothrombin time (PT), for example, the PT international normalized ratio (INR) ≥ 4, or a value more than 4 times the normal value in the presence of normal platelet counts and fibrinogen levels (e.g., Araki, S. and Shirahata, A., 2020. Nutrients, 12(3), p. 780).

[0230] Enhance bone growth and / or bone strength, or Bone loss and decreased bone density A method of prevention. By treating and / or preventing vitamin K2 deficiency in a subject, it is possible to enhance bone growth and / or bone strength in the subject or prevent bone loss and a decrease in bone strength (see, for example, Hamidi, M.S., et al., Journal of clinical densitometry, 16(4), pp. 409 - 413).

[0231] In the context of the present invention, the term "enhance bone growth and / or bone strength, or limit / prevent bone loss" particularly refers to one or more of the following physiological processes: catch-up growth of bone, acquisition of bone mass, optimization of peak bone mass, promotion of bone formation, promotion of bone assimilation, promotion of bone mineralization, increase in bone mineral density and microstructure, regulation of the biomechanical properties of bone, and regulation of the ratio of bone formation and / or bone resorption, bone mass maintenance, and reduction of bone resorption.

[0232] As used herein, "bone quality" may refer to properties related to bone composition and structure that contribute to bone strength independent of bone mineral density. Bone quality includes bone metabolic turnover, microstructure, mineralization, microdamage, and bone matrix and mineral composition. Methods for measuring bone quality are known in the art.

[0233] As used herein, "improving bone growth and / or strength" can refer to, for example, assisting normal bone growth and / or bone strength during childhood and adolescence. Assisting normal bone growth and / or bone strength can result in normal bone anatomical structure and physiological function. Suitable methods and parameters for measuring bone growth and bone strength are known to those skilled in the art (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469(8), pp. 2128-2138). Preferably, normal bone growth and / or strength can be evaluated using one or more bone parameters 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, ultimate force (FMax) of bone, and bone stiffness. In some embodiments, normal bone growth and / or strength is evaluated using one or more bone parameters selected from bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and ultimate force (FMax) of bone. Suitable methods for measuring these parameters are available to those skilled in the art.

[0234] Regular nutritional supplementation with the compositions of the present invention is also useful for preventing bone loss that occurs with aging and / or for protecting bone cells during the process of bone aging.

[0235] In one embodiment, such a composition is for use in i) improving bone quality and ii) preventing or treating disorders associated with an imbalance in the relationship between bone formation and bone resorption.

[0236] "Reduction / inhibition of bone resorption" means, according to the present invention, inhibition of the activity of osteoclasts in destroying bone tissue. To confirm that supplementation with the composition inhibits bone resorption in humans or animals, those skilled in the art can measure the urinary excretion of deoxypyridinoline as described in the examples. A decrease in the expression of deoxypyridinoline reflects inhibition of bone resorption.

[0237] When the composition of the present invention is administered to animals, it simultaneously induces stimulation of bone formation and inhibition of bone resorption. As a result of these two mechanisms being induced, bone mineralization and, consequently, overall bone density increase.

[0238] To determine whether a subject exhibits a state of bone loss and consequently requires supplementation with the composition according to the present invention, those skilled in the art can, in particular, refer to the 1994 World Health Organization (WHO) report entitled “Assessment of fracture risk and its application to screening for post-menopausal osteoporosis” (WHO Technical Series-843).

[0239] The compositions according to the present invention are also designed for individuals exhibiting bone deficit symptoms, or individuals susceptible to bone deficits, i.e., imbalances in the relationship between bone formation and bone resorption. If such imbalance persists, it leads to a decrease in bone mass. The compositions according to the present invention are also designed for individuals exhibiting bone deficiency symptoms resulting from fractures, surgery, or dental diseases.

[0240] In particular, the compositions are designed to prevent or treat diseases selected from osteoporosis, Paget's disease, bone loss or osteolysis observed around prostheses, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, periodontal disease, or osteoarthritis.

[0241] As mentioned above, many disorders related to imbalances in bone metabolism, such as osteoporosis, progress gradually over a long period of time and require long-term treatment. Therefore, their prevention or treatment can be carried out by regular supplementation of the compositions according to the present invention, preferably in the form of nutritional compositions.

[0242] Similarly, regular nutritional supplementation of human or animal individuals with the compositions of the present invention will, as a result, enable the production of high bone density and high peak bone mass by stimulating bone formation when these individuals reach adulthood.

[0243] Regular nutritional supplementation with the composition of the present invention is useful for preventing age-related bone loss (which can lead to osteoporosis) and / or for protecting bone cells during the bone aging process.

[0244] Therefore, in humans and other mammals, a wide variety of disorders are associated with abnormal metabolism of bone resorption and bone formation, leading to imbalances in metabolism or bone remodeling.

[0245] Among the pathological disorders associated with imbalances in bone metabolism, particularly noteworthy disorders or diseases include osteoporosis, Paget's disease, bone loss or osteolysis observed around prostheses, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, and periodontal disease. Some bone metabolic disorders or diseases may develop after prolonged immobilization, such as long-term hospitalization, or even after a period of weightlessness. Of the disorders associated with abnormal bone resorption, the most common is osteoporosis, and the symptoms of osteoporosis are most frequently observed primarily in postmenopausal women. Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and deterioration of the microstructure of bone tissue, accompanied by increased bone fragility and fracture susceptibility.

[0246] Osteoporosis, like other disorders associated with bone loss, constitutes a chronic condition; therefore, its prevention and treatment must be planned over the long term.

[0247] It is now recognized that early treatment is preferable because there are two important periods regarding bone capital: the growth period when peak bone mass is achieved, and the aging period when the rate of bone loss is regulated. Therefore, the prevention of osteoporosis should no longer be limited to the elderly.

[0248] Furthermore, in humans and animals, there are many conditions characterized by the need to increase bone formation. For example, in the case of fractures, bone growth needs to be stimulated to promote complete bone repair. Stimulation of bone growth is also necessary for periodontal disease, metastatic bone disease, osteolytic disease, and conditions requiring the repair of connective tissue, such as scar formation or regeneration related to cartilage defects or trauma. Stimulation of bone growth is also necessary in primary and secondary hyperparathyroidism, as well as in diabetes-related osteoporosis and glucocorticoid-related osteoporosis.

[0249] Methods to promote catch-up growth Treating and / or preventing vitamin K2 deficiency in the subject may thereby promote catch-up growth.

[0250] As used herein, "catch-up growth" may refer to a height growth rate exceeding the normal range for the age over a period of at least one year following a temporary growth suppression period, and this growth may be complete or incomplete (see, for example, Wit, J., and Boersma, B., 2002. Journal of Pediatric Endocrinology and Metabolism, 15, pp. 1229-1242).

[0251] Suitable methods and parameters for assessing catch-up growth are known to those skilled in the art. Preferably, catch-up growth can be assessed using the rate of height increase or the height standard deviation score (see, for example, 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).

[0252] In some embodiments, catch-up growth is evaluated in absolute terms of linear growth (i.e., the deficit in height compared to the healthy reference population average is reduced). In some embodiments, catch-up growth is evaluated in relative terms of linear growth (i.e., the age-specific height z-score is improved and / or exceeds the -2SD or -1SD cutoff point).

Example

[0253] The present invention will be further described by reference to the following examples. It will be understood that the claimed invention is in no way intended to be limited by these examples.

[0254] Example 1 - Promotion of vitamin K2 production in the digestive tract To evaluate the intestinal production of vitamin K2, a simulator of the human microbiota ecosystem (SHIME®) was used (Van de Wiele, T., et al., 2015 The Impact of Food Bioactives on Health: in vitro and ex vivo models, pp. 305-317). The SHIME assay typically consists of the colonic fermentation of a selected test compound at a dosage under simulated conditions representing the digestive tract of the subject. The production of menaquinone-7 was measured by supercritical fluid chromatography tandem mass spectrometry (SFC-MS / MS).

[0255] In these experiments, a two-stage batch system mimicking the conditions of the upper gastrointestinal tract (upper GIT, stomach, and small intestine) and colon was used as a simplified SHIME® system. A bovine milk-based infant formula, also known as infant formula milk, containing age-appropriate minerals, was used in these studies.

[0256] To simulate the absorption process occurring in the infant's small intestine, a dialysis approach was applied using a cellulose membrane with a 14 kDa cutoff. By introducing the small intestinal suspension into the dialysis membrane, digested molecules such as amino acids, sugars, micronutrients, and minerals were gradually removed from the upper gastrointestinal matrix.

[0257] Furthermore, the pH was gradually decreased from 5.5 to 3.0 during a one-hour incubation in the stomach to simulate the pH of an infant's stomach. During the first 30 minutes of small intestinal incubation (duodenum), the pH was maintained at 4.5 to optimize the absorption of available minerals. The pH was then induced to 7 during the subsequent 145-minute small intestinal phase (jejunum + ileum). After exposure to gastric and small intestinal conditions, the milk matrix was transferred to a colonic compartment containing infant fecal samples.

[0258] Fresh fecal material was collected from 12-month-old infant donors. Fecal suspensions were prepared and mixed with protective agents. At the start of short-term colon incubation, the test components (see below) were added to a sugar-depleted nutrient medium containing essential nutrients present in the colon (e.g., host-derived glycans such as mucin).

[0259] In the first experimental set, the following groups (test components) were evaluated: Single HMO (1.3 g / L 2 FL); HMO mix (2.5 g / L 2 FL, DiFL, LNnT, LNT, and 6 SL); B. infantis (10 7cfu / mL); single HMO + B. infantis; HMO mix + B. infantis. 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 the second experimental set, 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:

[0260] [Table 1]

[0261] The results are shown in Figure 2A. The BMO and HMO mixture increased menaquinone-7 production by 29% compared to the negative control. A milk matrix containing a mixture of six HMOs significantly increased menaquinone-7 production, and the addition of B. infantis further increased production (see Figure 2B).

[0262] In the final set of experiments, the following groups were evaluated (in the absence or presence of L. rhamnosus LPR, in a milk matrix): Blank; COS (2700 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L); COS (2025 mg / L) + β-glucan (675 mg / L) + B. lactis (4.5 × 10⁻¹⁰) 6 (cfu / mL). Results in the absence and presence of L. rhamnosus LPR are shown in Figures 3A and 3B, respectively. In the absence of LPR, COS increased MK-7 production by 178%. The addition of B. lactis in the presence of LPR further increased MK-7 production by 22%.

[0263] Furthermore, when all groups are combined, Lactobacillus rhamnosus LPR (4.5 × 10 7 The addition of cfu / mL was shown to further increase vitamin K2 production in the gastrointestinal tract by approximately 40% (see Figure 3C).

[0264] Example 2 - Preclinical Experiment Results The preclinical experiment was set up as shown in Figure 4. The number of pups per BALB / c mother mouse was increased by 50% from D8 to D18 to induce dietary restriction. In both groups (normal group and growth retardation group), weaning was performed on D18. Subsequently, male and female mice were given free access to food for 30 days, with nutritional supplementation once a day via pipette feeding.

[0265] As can be seen from Figures 5A and 5B, vitamin K2AD promoted bone quality by improving trabecular bone volume (BV / TV ratio) (increasing trend) and cortical bone mineral density (Ct.BMD) (significant increase).

[0266] Furthermore, Figures 6A and 6B demonstrate that vitamin K2AD enhanced bone strength by increasing both the force (Fmax) and energy (Wmax) required to reach the fracture point of the femur.

[0267] Embodiment Various preferred features and embodiments of the present invention are described below with reference to the numbered paragraphs.

[0268] 1. A prebiotic agent for use in the treatment and / or prevention of vitamin K2 deficiency in the subject.

[0269] 2. The prebiotic agent for use as described in paragraph 1, wherein the prebiotic agent is administered in combination with vitamin K1.

[0270] 3. The prebiotic agent for use according to paragraph 2, wherein the prebiotic agent and vitamin K1 are administered separately, simultaneously, or sequentially, preferably simultaneously.

[0271] 4. A prebiotic agent for use as described in any one of paragraphs 1 to 3, wherein the prebiotic agent is administered in combination with a probiotic agent.

[0272] 5. The prebiotic agent for use according to paragraph 4, wherein the prebiotic agent and the probiotic agent are administered separately, simultaneously, or sequentially, preferably simultaneously.

[0273] 6. A prebiotic for use according to either paragraph 4 or 5, wherein the probiotic comprises Lactobacillus rhamnosus, Bifidobacterium infantis, and / or Bifidobacterium lactis.

[0274] 7. A prebiotic agent for use as described in any one of paragraphs 1 to 6, wherein the prebiotic agent is selected from one or more of the following: bovine milk oligosaccharide (BMO), human milk oligosaccharide (HMO), cellobiose, cellooligosaccharide (COS), inulin, lactose, fructooligosaccharide (FOS), galactooligosaccharide (GOS), and β-glucan.

[0275] 8. A prebiotic agent for use according to any one of paragraphs 1 to 7, wherein the prebiotic agent is selected from one or more of bovine milk oligosaccharides (BMO), human milk oligosaccharide (HMO) mixtures, and cellooligosaccharides (COS).

[0276] 9. A prebiotic agent for use according to any one of paragraphs 1 to 8, wherein the prebiotic agent contains BMO in an amount of about 80% to about 100% by weight relative to the total weight of the prebiotic agent.

[0277] 10. A prebiotic agent for use according to any one of paragraphs 1 to 9, wherein the prebiotic agent comprises or consists of at least one sialyl oligosaccharide, at least one fucosyl oligosaccharide, and / or at least one N-acetyl oligosaccharide.

[0278] 11. The prebiotic agent for use as described in paragraph 10, wherein the at least one sialyl oligosaccharide is selected from the group consisting of 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), disial lacto-N-tetraose, and combinations thereof, preferably the at least one sialyl oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof.

[0279] 12. The above-mentioned at least one fucosyl oligosaccharide is 2'-fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), difucosyl lactose (diFL), lacto-N-fucopentaose-I (LNFP-I), lacto-N-fucopentaose-II (LNFP-II), lacto-N-fucopentaose-III (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose-V (LNnFP-V), lacto-N-difucosylhexaose A prebiotic agent for use as described in paragraph 10 or 11, selected from the group consisting of -I (LNDFH-1), lacto-N-neo-difucosylhexaose (LNnDFH), monofucosyl lacto-n-hexaose-III (MFNLH-III), difucosyl lacto-N-hexaose-a (DFLNHa) and combinations thereof, preferably wherein at least one of the fucosyl oligosaccharides is 2'-fucosyl lactose (2'FL) and / or difucosyl lactose (diFL).

[0280] 13. A prebiotic agent for use according to any one of paragraphs 10 to 12, wherein the at least one N-acetyloligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof, preferably the at least one N-acetyloligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof.

[0281] 14. The prebiotic agent is (a) at least one sialyl oligosaccharide in an amount of about 0.5% to about 2% by weight relative to the total weight of the oligosaccharide mixture; (b) At least one fucosyl oligosaccharide in an amount of about 2% to about 6% by weight relative to the total weight of the oligosaccharide mixture; and / or (c) A prebiotic agent for use according to any one of paragraphs 1 to 13, comprising about 1% to about 4% by weight of at least one N-acetyloligosaccharide based on the total weight of the oligosaccharide mixture.

[0282] 15. A prebiotic agent for use according to any one of paragraphs 1 to 14, wherein the prebiotic agent contains or consists of cellooligosaccharide (COS).

[0283] 16. A prebiotic agent for use as described in any one of paragraphs 1 to 15, wherein the prebiotic agent is administered to the subject in a total amount of approximately 0.5 g / day to approximately 10 g / day.

[0284] 17. A prebiotic agent for use as described in any one of paragraphs 1 to 16, wherein vitamin K1 is administered to the subject in an amount of approximately 5 μg / day to approximately 100 μg / day.

[0285] 18. The aforementioned probiotic agent is approximately 10 6 cfu / day ~ approximately 10 12A prebiotic agent for use as described in any one of paragraphs 1 to 17, administered to the subject in a total amount of cfu / day.

[0286] 19. The prebiotic agent for use according to any one of paragraphs 1 to 18, wherein the prebiotic agent is provided in the form of a nutritional composition.

[0287] 20. The prebiotic agent for use as described in any one of paragraphs 1 to 19, wherein the prebiotic agent is provided in the form of a medical food for clinical nutrition.

[0288] 21. A prebiotic agent for use according to either paragraph 19 or 20, wherein the composition comprises a total amount of the prebiotic agent in about 0.5 g / 100 g to about 10 g / 100 g on a dry weight basis.

[0289] 22. A prebiotic agent for use according to any one of paragraphs 19 to 21, wherein the composition contains vitamin K1 in an amount of about 5 μg / 100g to about 100 μg / 100g on a dry weight basis.

[0290] 23. The composition is approximately 10% on a dry weight basis. 6 cfu / 100g~approx. 10 12 A prebiotic for use as described in any one of paragraphs 19-22, containing a probiotic in an amount of cfu / 100g.

[0291] 24. A prebiotic agent for use as described in any one of paragraphs 1 to 23, wherein the subject is an infant, toddler, or young child.

[0292] 25. A prebiotic agent for use as described in any one of paragraphs 1 to 24, for which the subject has or is at risk of vitamin K2 deficiency.

[0293] 26. A prebiotic agent for use as described in any one of paragraphs 1 to 25, for which the subject has or is at risk of having dyslipidemia, diabetes mellitus, severe thalassemia™, cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease.

[0294] 27. A prebiotic agent for use as described in any one of paragraphs 1 to 26, wherein the subject has or is at risk of having reduced bone growth and / or bone strength.

[0295] 28. A prebiotic agent for use according to any one of paragraphs 1 to 27, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting vitamin K2 production in the intestine of the subject.

[0296] 29. A prebiotic agent for use according to any one of paragraphs 1 to 28, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting the de novo production of menaquinone-7 and / or the bioconversion of phylloquinone to menaquinone-4 in the intestine of the subject.

[0297] 30. Use of prebiotics in the manufacture of pharmaceuticals for the treatment and / or prevention of vitamin K2 deficiency in subjects.

[0298] 31. A method for treating and / or preventing vitamin K2 deficiency in a subject requiring treatment and / or prevention of vitamin K2 deficiency, comprising administering an effective amount of a prebiotic agent to the subject.

[0299] 32. Use of prebiotics to promote vitamin K2 production in the target intestine.

[0300] 33. The use according to paragraph 32, wherein the prebiotic agent promotes the de novo production of menaquinone-7 and / or the bioconversion of phylloquinone to menaquinone-4 in the intestine of the subject.

[0301] While the present invention has been described using examples, it should be understood that modifications and alterations can be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist for certain features, such equivalents are incorporated as if they were specifically referred to herein.

Claims

1. A prebiotic agent for use in the treatment and / or prevention of vitamin K2 deficiency in the target population.

2. A prebiotic agent for use according to claim 1, further comprising a vitamin mixture for promoting vitamin K2 production in the target intestine.

3. A prebiotic agent for use according to any one of claims 1 or 2, wherein the vitamin mixture comprises vitamin K2, vitamin A and / or vitamin D.

4. The prebiotic agent for use according to any one of claims 1 to 3, wherein the prebiotic agent is administered in combination with vitamin K1.

5. A prebiotic agent for use according to any one of claims 1 to 4, 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 is selected from one or more of bovine milk oligosaccharides (BMO), human milk oligosaccharides (HMO), cellobiose, cellooligosaccharides (COS), inulin, lactose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and β-glucan, and preferably the prebiotic agent is selected from one or more of bovine milk oligosaccharides (BMO), a mixture of human milk oligosaccharides (HMO), and cellooligosaccharides (COS), as described in any one of claims 1 to 6.

8. A prebiotic agent for use according to any one of claims 1 to 7, wherein the prebiotic agent contains BMO in an amount of about 80% to about 100% by weight relative to the total weight of the prebiotic agent.

9. The prebiotic agent contains or comprises HMO, preferably the prebiotic agent contains or comprises at least one sialyl oligosaccharide, at least one fucosyl oligosaccharide and / or at least one N-acetyl oligosaccharide, more preferably (a) The at least one sialyl oligosaccharide is selected from the group consisting of 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl lacto-N-tetraose b (LSTb), sialyl lacto-N-tetraose c (LSTc), disial lacto-N-tetraose, and combinations thereof, preferably the at least one sialyl oligosaccharide is selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and combinations thereof; (b) The at least one fucosyl oligosaccharide is 2'-fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), difucosyl lactose (diFL), lacto-N-fucopentaose-I (LNFP-I), lacto-N-fucopentaose-II (LNFP-II), lacto-N-fucopentaose-III (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose-V (LNnFP-V), lacto- Selected from the group consisting of N-difucosylhexaose-I (LNDFH-1), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyllacto-n-hexaose-III (MFNLH-III), difucosyllacto-N-hexaose-a (DFLNHa) and combinations thereof, preferably, at least one fucosyloligosaccharide is 2'-fucosyllactose (2'FL) and / or difucosyllactose (diFL); and / or (c) The prebiotic agent for use according to any one of claims 1 to 8, wherein the at least one N-acetyloligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof, preferably the at least one N-acetyloligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof.

10. The prebiotic agent for use according to any one of claims 1 to 9, wherein the prebiotic agent contains or consists of COS.

11. A prebiotic agent for use according to any one of claims 1 to 10, wherein the prebiotic agent is administered to the subject in a total amount of approximately 0.5 g / day to approximately 10 g / day.

12. Vitamin K1 is administered to the subject in an amount of approximately 5 μg / day to approximately 100 μg / day, and / or a probiotic is administered in an amount of approximately 10 ６ cfu / day ~ approximately 10 １２ A prebiotic agent for use according to any one of claims 1 to 11, administered to the subject in a total amount of cfu / day.

13. A prebiotic agent for use according to any one of claims 1 to 12, wherein the prebiotic agent is provided in the form of a nutritional composition.

14. A prebiotic agent for use according to any one of claims 1 to 13, wherein the subject is an infant, toddler, or child.

15. A prebiotic agent for use according to any one of claims 1 to 14, wherein the subject has or is at risk of having dyslipidemia, diabetes mellitus, severe thalassemia (TM), cystic fibrosis (CF), inflammatory bowel disease (IBD), or chronic liver disease, and / or the subject has or is at risk of having reduced bone growth and / or bone strength.

16. A prebiotic agent for use according to any one of claims 1 to 15, wherein the prebiotic agent enhances bone growth, bone mineralization and / or bone strength, or limits / prevents bone loss and bone strength in the subject.

17. A prebiotic agent for use according to any one of claims 1 to 16, wherein the prebiotic agent treats and / or prevents vitamin K2 deficiency by promoting vitamin K2 production in the intestines of the subject.

18. Use of prebiotics to promote vitamin K2 production in the target intestines.

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