Methods for improving bone health using bovine milk exosome-enriched products and vitamin K2

Abstract

A method for improving bone formation in an individual comprises administering to the individual a bovine milk exosome-enriched product and vitamin K2. A method for reducing fracture risk or strengthening bone in an individual comprises administering to the individual a bovine milk exosome-enriched product and vitamin K2. A method for preventing or delaying the onset or progression of osteoporosis in an individual comprises administering to the individual a bovine milk exosome-enriched product and vitamin K2.

Description

[Technical Field]

[0001] The present invention relates to the use of bovine milk exosome enriched products and vitamin K2 to improve bone formation in an individual and / or to reduce the risk of fracture or strengthen bones in an individual, and also to the use of bovine milk exosome enriched products and vitamin K2 to prevent or delay the onset or progression of osteoporosis in an individual. [Background technology]

[0002] Bone is a tissue whose supportive function is achieved by the hardening of the extracellular matrix. Bone is composed of approximately 30% water, with the remainder consisting of various minerals (e.g., calcium salts) and various cell types, including osteoprogenitor cells, osteoblasts, osteocytes, bone-lining cells, and osteoclasts. Bone development is a lifelong process that involves the integration of multiple signaling pathways and requires the coordinated action of these cells.

[0003] Bones protect organs from mechanical forces, transmit force between different regions of the body, and anchor skeletal muscles. Bone is a metabolically active living tissue that serves as a reservoir for calcium, phosphorus, and carbonate ions. Bone also contributes to buffering changes in hydrogen ion concentration. Generally, in humans, approximately 90% of bone mass is acquired during childhood and adolescence. Therefore, optimizing bone growth early in life is crucial for preventing fractures and osteoporosis later in life.

[0004] In children, malnutrition is a major cause of short stature and growth retardation, ultimately leading to failure to grow long bones. A 2021 joint report by the World Bank, UNICEF, and WHO found that 22% of all children under the age of five worldwide suffered from linear growth restriction due to chronic malnutrition. In developing countries, this rate increased to approximately 35%. Failure to develop healthy bone tissue during childhood can affect height and cause serious problems later in life. Because bone mass declines with age throughout adulthood, the higher the bone mass achieved before adulthood, i.e., between the ages of 20 and 25, the better the prospects for good bone health. Improving peak bone mass before adulthood can reduce the risk of osteoporotic fractures in adulthood.

[0005] Bones undergo a constant remodeling process in which old bone is replaced by new bone. In the average human, approximately 5-10% of bone is renewed per year. This bone remodeling process occurs throughout life and is necessary for adapting the skeleton to mechanical use, promoting fracture healing, and maintaining calcium homeostasis. Calcium homeostasis largely reflects the balance between osteoblast activity (or bone formation) and osteoclast activity (bone resorption).

[0006] Osteoclasts are responsible for bone resorption. They are terminally differentiated multinucleated cells derived from mononuclear cells of the hematopoietic stem cell lineage. Osteoblasts, on the other hand, synthesize bone matrix by the deposition of organic matrix and subsequent mineralization. Osteoblasts are derived from multipotent bone marrow mesenchymal stem cells (MSCs). MSCs can differentiate into various tissue-specific cells, including osteoblasts, chondrocytes, and adipocytes. The commitment of MSCs to osteoblast differentiation requires the expression of specific genes.

[0007] In humans, longitudinal bone growth occurs rapidly during fetal life and early childhood, then gradually ceases during adolescence. Longitudinal bone growth, or the elongational process of endochondral ossification, is accomplished by the action of specialized cartilaginous structures known as growth plates located at the distal and proximal ends of long bones and vertebrae.

[0008] The growth plate is divided into five zones: the resting zone, the proliferative zone, the hypertrophic cartilage zone, the calcified cartilage zone, and the ossification zone. The resting zone is located at the top of the growth plate and is furthest from the ossification zone. It is composed of chondrocytes embedded in cartilage matrix. This zone is crucial for maintaining the structure of the growth plate and is sometimes referred to as the "stem cell" zone. Chondrocytes within the resting zone slowly divide to supply the adjacent proliferative zone. In the proliferative zone, chondrocytes organize into tandem columns and undergo rapid mitosis. Once these divisions begin, chondrocytes rapidly increase in size and begin to change their extracellular matrix, resulting in the hypertrophic cartilage zone. In the hypertrophic cartilage zone, the extracellular matrix is ​​resorbed, reducing to thin septa between the enlarged chondrocytes. In the adjacent calcified cartilage zone, hypertrophic chondrocytes undergo apoptosis, and the thin septa begin to calcify. Eventually, bone tissue appears in the ossification zone. The cavities left by dead chondrocytes are invaded by osteoprogenitor cells, which further differentiate into active osteoblasts, which are important for bone formation by depositing bone matrix on the mineralized cartilage matrix.

[0009] Osteoblasts are not only important for longitudinal bone growth, but also for bone apposition or bone remodeling. Bone apposition is a process that occurs in the diaphysis of long bones. This growth changes the shape and structure of the bone while increasing its width. Bone apposition occurs during bone length increase but continues even after adolescence, i.e., after about age 21, when growth plates close and longitudinal growth ceases. The mechanism of bone apposition depends on a balance between osteoblast and osteoclast activity, as osteoblasts add bone matrix to the outer surface of the bone and osteoclasts remove bone from the inner surface of the diaphyseal bone.

[0010] Longitudinal bone growth and remodeling is a complex process, and its success requires a balance between bone formation and bone resorption. While overall bone health can be actively promoted through proper nutrition and exercise, many factors can threaten bone growth and overall skeletal health. These factors include malnutrition, malabsorption, vitamin deficiencies (primarily vitamins K, D, and B), zinc and calcium deficiencies, the use of certain medications such as chemotherapy, long-term use of antibiotics or glucocorticoids, diseases such as type 1 diabetes, type 2 diabetes, and osteoporosis, and hormonal imbalances associated with aging (including menopause and postmenopause in women). Many of these factors can interfere with osteoblast differentiation and activity, thereby inhibiting normal bone growth and remodeling.

[0011] Osteoporosis is an age-related pathological condition characterized by a loss of bone mineral content and bone mass. Approximately one in three women and one in five men over the age of 50 will experience an osteoporotic fracture. Age-related skeletal fragility, such as that seen in osteoporosis, may result from impaired bone formation due to an insufficient amount of osteoblasts. This lack of osteoblast numbers in the aging skeleton may be due, at least in part, to impaired differentiation of progenitor cells or the conversion of these progenitor cells to the adipocyte lineage. Therefore, developments aimed at enhancing osteoblast differentiation and activity are desirable to improve bone homeostasis and overall skeletal health in both adult and pediatric populations.

[0012] Previous treatments to combat disturbances to normal bone growth and remodeling have included calcium and vitamin D and K supplements to restore bone homeostasis and prevent the onset and progression of bone diseases such as osteoporosis. However, many factors still influence the balance of bone homeostasis.

[0013] In view of the above, there is an urgent need to develop novel, accessible technologies that can conveniently provide improved bone health in vivo in human and animal subjects. Summary of the Invention [Problem to be solved by the invention]

[0014] (Summary of the Invention) It is therefore an object of the present invention to provide a convenient means for improving bone health in individuals. [Means for solving the problem]

[0015] In one embodiment, the present invention is directed to a method for increasing bone formation in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0016] In another embodiment, the present invention is directed to a method for reducing fracture risk or strengthening bones in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0017] In another embodiment, the present invention is directed to a method for preventing or delaying the onset or progression of osteoporosis in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0018] Administration of bovine milk exosome-enriched products and vitamin K2 provides a convenient means for improving bone health and development. These and additional objects and advantages of the present invention will become more fully apparent in view of the following detailed description.

[0019] The drawings illustrate certain embodiments of the invention and are exemplary in nature and are not intended to limit the invention as defined by the claims. [Brief explanation of the drawings]

[0020] [Figure 1A] FIG. 1A shows the expression levels of Runx2 protein, a molecular marker of osteoblast differentiation, resulting from treatment with different effectors described in Example 2. [Figure 1B] FIG. 1B shows the expression levels of the osteoblast differentiation molecular marker LC3 protein as a result of treatment with the different effectors described in Example 2. [Figure 2A] FIG. 2A shows a Western blot of the osteoblast differentiation molecular marker Runx2 following treatment with different effectors as described in Example 2. [Figure 2B] FIG. 2B shows a Western blot of the osteoblast differentiation molecular marker LC3 following treatment with the different effectors described in Example 2. [Figure 3A] FIG. 3A shows a photomicrograph of the results of an Alizarin Red S (ARS) extracellular mineralization assay of control cells, as described in Example 3, showing calcium deposition after 12 days of incubation. [Figure 3B] FIG. 3B shows photomicrographs of the results of the ARS extracellular mineralization assay of cells from the exosome-treated group, demonstrating calcium deposition after 12 days of incubation, as described in Example 3. [Figure 3C] FIG. 3C shows photomicrographs of the results of an ARS extracellular mineralization assay of cells from the vitamin K2 treatment group, demonstrating calcium deposition after 12 days of incubation, as described in Example 3. [Figure 3D] FIG. 3D shows photomicrographs of the results of an ARS extracellular mineralization assay of cells from the vitamin K2 + exosome-treated group, demonstrating calcium deposition after 12 days of incubation, as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0021] Specific embodiments of the present invention are described herein. However, the present invention may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided to illustrate to those skilled in the art more specific features of certain embodiments of the present invention.

[0022] The terminology described herein is for the purpose of describing embodiments only and should not be construed as limiting the present disclosure in general. Unless otherwise specified or clearly indicated to the contrary by the context in which the reference is made, all references to singular features or limitations in this disclosure shall include the corresponding plural features or limitations, and vice versa. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably. Furthermore, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include their plural forms unless the context clearly dictates otherwise.

[0023] The terms "includes" or "including," when used in the specification or claims, are intended to have a similar meaning to the term "comprising," including additional elements or steps, in the same manner as the term "comprising" is interpreted when used as a transitional term in a claim. Furthermore, to the extent that the term "or" is used (e.g., A or B), this term is intended to mean "A or B, or both." When "only A or B, but not both," is intended, the phrase "only A or B, but not both" is used. Thus, the use of the term "or" herein is inclusive, not exclusive. When the terms "and" and "or" are used together, as in "A and / or B," this refers not only to A and B, but also to A or B.

[0024] All ranges and parameters, including but not limited to percentages, parts, and ratios disclosed herein, are understood to encompass all subranges contained therein, and all numerical values ​​between the endpoints. For example, a range stated as "1 to 10" should be considered to include all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less (e.g., 1 to 6.1, or 2.3 to 9.4), as well as each integer subsumed within that range (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10).

[0025] As used herein, any combination of method or process steps can be performed in any order unless otherwise stated or unless the contrary is clearly and specifically implied by the context in which the referenced combination is made.

[0026] All percentages are by weight unless otherwise indicated.

[0027] The term "exosome-enriched product," as used herein, unless otherwise specified, refers to a product containing bovine milk-derived exosomes, in which the exosomes have been substantially separated from other bovine milk components, such as lipids, cells, and debris, and have been enriched to a higher concentration than that found in bovine milk. Exosomes are small extracellular vesicles that represent a small percentage of the total content of milk. In certain embodiments, the exosome-enriched product is administered in the form of an exosome-enriched liquid or exosome-enriched powder. In certain embodiments, the exosome-enriched product also contains co-isolated milk solids. Bovine milk exosomes are extracellular membrane vesicles approximately 20-200 nm in diameter. These nano-sized structures contain several bioactive substances, including, but not limited to, enzymes and non-enzyme proteins (e.g., CD9, CD63, MHC-class II, lactadherin, TSG101, and Hsc70), nucleic acids (including abundant microRNAs (miRNAs) and messenger RNAs (mRNAs)), and lipids (e.g., phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingomyelin).

[0028] The term "intact exosomes", as used herein, unless otherwise specified, refers to exosomes in which the vesicle membrane has not been ruptured and / or otherwise degraded, and in which the endogenous cargo, i.e., bioactive agents, therapeutic agents (e.g., miRNAs), and / or other biomolecules originally present in the bovine milk exosomes, is retained in an active form.

[0029] In embodiments of the invention, the bovine milk exosome-enriched product comprises intact bovine milk exosomes. In specific embodiments, at least about 50% by weight of the exosomes in the bovine milk exosome-enriched product are intact. In other specific embodiments, at least about 55, 60, 70, 75, 80, 85, 90, or 95% by weight of the exosomes in the bovine milk exosome-enriched product are intact.

[0030] Bovine milk exosomes can be isolated from milk whey fractions or from other dairy fluids, such as cheese whey fractions. Bovine milk exosomes can be isolated by a variety of physical methods (e.g., ultracentrifugation at increasing speeds, membrane ultrafiltration, and / or size exclusion chromatography) and / or chemical methods (e.g., using polymers to precipitate bovine milk exosomes in an incubation step). Surprisingly, most of these procedures tend to co-purify the exosomes with other dairy components (i.e., casein and other whey proteins). The isolation process yields a fraction enriched in bovine milk exosomes, which can then undergo further processing, such as freeze-drying or spray-drying, to produce a powder containing bovine milk exosomes for further use.

[0031] In certain embodiments of the invention, the bovine milk exosome-enriched product comprises at least 0.001% exosomes by weight. In other specific embodiments, the bovine milk exosome-enriched product comprises at least about 0.001, 0.01, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% exosomes by weight. In additional specific embodiments of the invention, the bovine milk exosome-enriched product comprises at least 10% exosomes by weight. In further embodiments, the bovine milk exosome-enriched product comprises at least about 10% exosomes per gram. 8 exosomes, which are measured using a nanotracking procedure. Briefly, nanoparticle tracking analysis (NTA) can be used to determine the diameter and concentration of exosomes. The principle of NTA is based on the characteristic movement of nanosized particles in solution, which follows Brownian motion. When the particles are illuminated with a laser, their trajectories in a defined volume are recorded by a camera used to capture the scattered light. The Stokes-Einstein equation is used to determine the size of each tracked particle. This technique makes it possible to determine particle concentration in addition to particle size.

[0032] In a more particular embodiment, the bovine milk exosome-enriched product used in the present invention comprises about 10 8 ~about 10 15 In yet a further particular embodiment, the exosome-enriched product comprises about 10 exosomes per gram of exosome-enriched product. 9 ~about 10 13 In another particular embodiment, the exosome-enriched product comprises at least about a three-fold increase in the number of exosomes compared to a raw whey-containing bovine milk fraction. In even more particular embodiments, the exosome-enriched product comprises a three- to fifty-fold increase in the number of exosomes compared to a raw whey-containing bovine milk fraction, such as cheese whey.

[0033] The term "vitamin K2," as used herein, unless otherwise specified, refers to a fat-soluble vitamin also known as menaquinone (referred to as MK-n, where n indicates the number of isoprenoid units) with a variable side chain length of 4 to 15 isoprene units. Typically, vitamin K2 has an average of 7 isoprenoid units and is referred to as MK-7. Vitamin K2 is commercially available through various extraction and purification processes.

[0034] The term "mesenchymal stem cells" or "MSCs" refers to multipotent bone marrow cells that can differentiate into a variety of tissue-specific cells, such as osteoblasts, chondrocytes, and adipocytes.

[0035] Runx2 (Runt-related transcription factor 2) is an early osteoblast differentiation marker. Runx2 can induce bone matrix protein gene expression and mineralization in osteoblasts in vitro. Certain factors or diseases, such as long-term glucocorticoid use, may increase the incidence of osteoporosis and suppress osteogenic differentiation of MSCs, for example, by antagonizing Runx2. Runx2 is considered a master osteogenic transcription factor and is essential for the expression of osteogenic differentiation genes. In an embodiment of the present invention, the activity of differentiated osteoblasts is evaluated by the expression level of Runx2.

[0036] LC3 (microtubule-associated protein 1 light chain 3-alpha) is a late osteoblast differentiation marker. LC3 is a protein required for autophagy-induced differentiation and mineralization of MC3T3-E1 osteoblasts. Lack of autophagy in osteoblasts may reduce mineralization activity. In an embodiment of the present invention, differentiated osteoblasts are assessed by LC3 expression levels.

[0037] The Alizarin Red S (ARS) assay is an anthraquinone dye used to visualize and assess extracellular calcium deposition in cell cultures. In an embodiment of the present invention, the mineralization activity of differentiated osteoblasts is assessed by the ARS assay.

[0038] The term "nutritional composition," as used herein, unless otherwise specified, refers to nutritional liquids and nutritional powders, the latter of which may be reconstituted with or otherwise mixed with a liquid to form a nutritional liquid suitable for oral consumption by a human. The nutritional liquid may be prepared in a ready-to-drink (RTD) form or may be reconstituted from a powder as described.

[0039] In embodiments of the present invention, vitamin K2 and the exosomes of the bovine milk exosome-enriched product may be administered separately or in combination. The combination may be prepared by stirring, mixing, shaking, or otherwise combining the vitamin K2 and the exosomes, where care is taken to maintain the exosomes substantially in an intact form.

[0040] In certain embodiments of the present invention, vitamin K2 is loaded into the exosomes of the bovine milk exosome-enriched product. In other certain embodiments, vitamin K2 is a component separated from the exosomes, i.e., vitamin K2 is not loaded into the exosomes of the bovine milk exosome-enriched product.

[0041] In one embodiment of the invention, the single agitation step is performed by vortexing for about 60 minutes, about 40-80 minutes, or about 50-70 minutes. In certain embodiments of the invention, the vortexing is performed at 500 rpm or greater, about 500-1300 rpm, about 600-1200 rpm, about 1000-1200 rpm, or about 1200 rpm.

[0042] In certain embodiments of the present invention, the bovine milk exosome enriched product and vitamin K2 are administered to a subject, wherein the subject may be a human adult, elderly adult, or pediatric individual.

[0043] The term "pediatric subject," as used herein, unless otherwise specified, refers to an infant, child, or adolescent individual (up to about 20 years of age). In certain embodiments of the invention, a pediatric subject is a child up to about 18 years of age, up to about 15 years of age, up to about 10 years of age, up to about 5 years of age, up to about 1 year of age, up to about 6 months of age, or up to about 3 months of age.

[0044] The term "adolescent," as used herein, unless otherwise specified, refers to a pediatric subject between about 10 and 20 years of age, which typically corresponds to the onset of physiologically normal adolescence and ends around the time of adoption of adult identity and behavior. Pediatric subjects under about 10 years of age are considered "childhood" individuals.

[0045] In certain embodiments of the present invention, bovine milk exosome-enriched products and vitamin K2 are administered to pediatric subjects.

[0046] The term "adult subject," as used herein, refers to an individual who is at least about 20 years of age.

[0047] The term "older adult," as used herein, refers to an adult subject who is at least about 50 years of age.

[0048] In certain embodiments of the invention, the bovine milk exosome-enriched product and vitamin K2 are administered to an adult subject. In embodiments of the invention, the adult subject is at least about 30 years old, at least about 40 years old, at least about 50 years old, at least about 60 years old, or at least about 70 years old, between about 40 and 80 years old, or between about 50 and 70 years old.

[0049] In certain embodiments of the invention, adult subjects include adults at least about 40 years of age, at least about 50 years of age, at least about 60 years of age, at least about 65 years of age, about 50-80 years of age, about 60-70 years of age, or about 63-64 years of age.

[0050] The term "bone development," as used herein, refers to the continuous process by which old bone is replaced by new bone through a balance between bone formation (via osteoblast activity) and bone resorption (via osteoclast activity) that maintains bone homeostasis. Osteoblasts are specialized cells derived from mesenchymal cells that synthesize bone matrix. Osteoclasts are cells that degrade bone and contribute to bone remodeling.

[0051] The term "osteogenesis," as used herein, unless otherwise specified, refers to the portion of bone development in which osteoblast activity is stimulated. Osteoblasts add bone matrix to the outer surface of bone by depositing an organic matrix and mineralizing it. Osteogenesis can refer to both longitudinal growth of bone and appositional growth of bone. Most bone formation occurs during childhood and adolescence during normal mammalian development. However, bone formation continues throughout life to promote bone repair, for example, bone repair of fractures, through the same action of osteoblasts. Therefore, the term osteogenesis can also include bone repair.

[0052] The term "bone resorption," as used herein, unless otherwise specified, refers to the portion of bone development in which osteoclast activity is stimulated. Osteoclasts remove bone from the inner surface of the diaphysis of long bones by degrading the bone matrix through phagocytosis.

[0053] The term "fracture," as used herein, unless otherwise specified, refers to a partial or complete break in a bone. Fracture can also include a crack in a bone.

[0054] The term "osteoporosis," as used herein, unless otherwise specified, refers to a condition in which bones become weak and / or fragile. In osteoporosis, bone homeostasis is not maintained because osteoblast activity, which creates new bone, does not keep up with osteoclast activity, which removes old bone.

[0055] In one embodiment, the present invention is directed to a method for increasing bone formation in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0056] In another embodiment, the present invention is directed to a method for reducing fracture risk or strengthening bones in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0057] In another embodiment, the present invention is directed to a method for preventing or delaying the onset or progression of osteoporosis in an individual, comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

[0058] All methods disclosed herein can also be considered as uses of bovine milk exosome-enriched products and vitamin K2 for improving bone health as described.

[0059] In certain embodiments, the present invention is directed to a medicament for use in improving bone formation in an individual, the medicament comprising a bovine milk exosome-enriched product and vitamin K2.

[0060] In another embodiment, the present invention is directed to a medicament for use in reducing fracture risk or strengthening bones in an individual, the medicament comprising a bovine milk exosome-enriched product and vitamin K2.

[0061] In yet another embodiment, the present invention is directed to a medicament for use in preventing or delaying the onset or progression of osteoporosis in an individual, the medicament comprising a bovine milk exosome-enriched product and vitamin K2.

[0062] In particular embodiments of the invention, the dosage of the exosome-enriched product is about 0.01 to about 30 g per day. More particularly, the dosage of the exosome-enriched product may be about 0.1 to about 30 g, about 0.1 to about 15 g, or about 1 to about 15 g per day. In certain embodiments of the invention, the dosage of the exosome-enriched product is administered according to the properties of the exosome-enriched product described above, and in particular, bovine milk exosome-enriched product is administered at a dosage of about 10 g per gram of exosome-enriched product. 8 ~about 10 15 Contains exosomes.

[0063] In certain embodiments of the present invention, the dosage of vitamin K2 follows the Recommended Daily Allowance (RDA) for vitamin K. For example, the RDA for vitamin K is 120 micrograms (mcg) per day for adult men, 90 mcg per day for adult women, 30 mcg per day for children aged 1-3 years, 55 mcg per day for children aged 4-8 years, 60 mcg per day for children aged 9-13 years, and 75 mcg per day for adolescents aged 14-18 years. In one embodiment, vitamin K2 is administered at a dose of about 0.001 to about 0.3 mg or about 0.03 to about 0.12 mg per day. In yet another embodiment, vitamin K2 is administered at a dose of about 0.03 mg, 0.055 mg, 0.06 mg, 0.075 mg, 0.09 mg, or 0.12 mg per day.

[0064] In certain embodiments, the bovine milk exosome-enriched product and vitamin K2 are administered to an individual at least once daily, hi certain embodiments, the bovine milk exosome-enriched product and vitamin K2 are administered to an individual about 1-2 times per day.

[0065] In certain embodiments, the bovine milk exosome-enriched product and vitamin K2 are administered to an individual at least once daily, or about once or twice daily, for a period of at least three consecutive days, at least one week, at least 12 consecutive days, at least two weeks, at least three weeks, or at least four weeks.

[0066] In some aspects of the invention, the vitamin K2 and bovine milk exosome-enriched product are included in a nutritional composition, which further comprises protein, carbohydrate, and / or fat.

[0067] Vitamin K2 is included in the nutritional composition in an amount sufficient to provide a desired vitamin K2 content, e.g., a Recommended Daily Allowance. Thus, in one embodiment, the nutritional composition includes vitamin K2 in an amount sufficient to provide at least about 30 micrograms (0.03 mg) of vitamin K2 per serving, e.g., an 8 oz / 237 ml serving.

[0068] In other specific embodiments, the nutritional composition comprises at least about 0.0000004%, at least about 0.000025%, at least about 0.00005%, at least about 0.0001%, or at least about 0.0002% by weight of vitamin K2, based on the weight of the nutritional composition. In specific embodiments, the nutritional composition comprises from about 0.0000004% to about 0.00005%, or from about 0.000025% to about 0.0002% by weight of vitamin K, based on the weight of the nutritional composition.

[0069] In other specific embodiments, the nutritional composition comprises about 0.001 to about 30% by weight, about 0.001 to about 10% by weight, about 0.001 to about 5% by weight, about 0.001 to about 1% by weight, about 0.01 to about 10% by weight, about 0.01 to about 5% by weight, about 0.01 to about 1% by weight, about 0.1 to about 10% by weight, about 0.1 to about 5% by weight, about 0.1 to about 1% by weight, about 1 to about 10% by weight, or about 1 to about 5% by weight of the exosome-enriched product, based on the weight of the nutritional composition. In specific embodiments, the nutritional composition comprises about 0.001 to about 30% by weight of vitamin K2-loaded exosomes, based on the weight of the nutritional composition.

[0070] In another particular embodiment, the nutritional composition comprises dietary fiber, including oat fiber, soy fiber, and / or corn fiber, human milk oligosaccharides (HMO), maltodextrin, corn maltodextrin, organic corn, corn syrup, sucralose, cellulose gel, cellulose gum, gellan gum, inositol, carrageenan, fructooligosaccharides, hydrolyzed starch, glucose polymers, corn syrup solids, rice-derived carbohydrates, sucrose, glucose, lactose, honey, sugar alcohols, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, gum arabic (gum arabic), and the like. arable), sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, acacia gum, chitosan, arabinogalactin, glucomannan, xanthan gum, alginic acid, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucan, psyllium, inulin, corn starch, or a combination of two or more thereof.

[0071] The nutritional composition may comprise carbohydrates in an amount of about 5% to about 75% by weight of the nutritional composition. More particularly, the carbohydrates may be present in an amount of about 5% to about 70% by weight of the nutritional composition, including about 5% to about 65%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 25%, about 10% to about 65%, about 20% to about 65%, about 30% to about 65%, about 40% to about 65%, about 40% to about 70%, or about 15% to about 25% by weight of the nutritional composition.

[0072] In another specific embodiment, the nutritional composition comprises one or more fats including palm oil, fractionated palm oil, soybean oil, soybean lecithin, corn oil, safflower oil, high oleic sunflower oil, palm olein, canola oil monoglycerides, lecithin, medium chain triglycerides, linoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, olive oil, high gamma linolenic acid (GLA) safflower oil, palm oil, palm kernel oil, canola oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oils, transesterified oils, structured lipids, or a combination of two or more thereof.

[0073] The nutritional composition may include fat in an amount of about 0.5% to about 30% by weight of the nutritional composition. More particularly, fat may be present in an amount of about 0.5% to about 10%, about 1% to about 30% by weight of the nutritional composition, including about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 3% to about 30%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 10% to about 20% by weight of the nutritional composition.

[0074] In another particular embodiment, the nutritional composition comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, milk protein concentrate, organic milk protein concentrate, milk protein isolate, milk protein hydrolysate, skim milk powder, condensed skim milk, soy protein concentrate, isolated soy protein, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, collagen protein, collagen protein isolate, L-carnitine, L-lysine, The composition may comprise one or more proteins, including taurine, lutein, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, collagen protein, collagen protein isolate, meat protein, potato protein, chickpea protein, canola protein, mung bean protein, guinea fowl protein, amaranth protein, chia protein, hemp protein, flaxseed protein, earthworm protein, insect protein, one or more amino acids and / or metabolites thereof, or a combination of two or more thereof.

[0075] The one or more amino acids, which may be described as free amino acids, may be any amino acids known for use in nutritional products. The amino acids may be naturally occurring or synthetic. In certain embodiments, the one or more amino acids and / or their metabolites comprise one or more branched-chain amino acids or their metabolites. Examples of branched-chain amino acids include arginine, glutamine, leucine, isoleucine, and valine. In another specific embodiment, the one or more branched-chain amino acids or their metabolites comprise α-hydroxy-isocaproic acid (HICA, also known as leuic acid), ketoisocaproic acid (KIC), β-hydroxy-β-methylbutyrate (HMB), and combinations of two or more thereof.

[0076] The nutritional composition may comprise protein in an amount of about 1% to about 30% by weight of the nutritional composition. More particularly, the protein may be present in an amount of about 1% to about 25% by weight of the nutritional composition, including about 1% to about 20%, about 2% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 10%, about 10% to about 25%, or about 10% to about 20% by weight of the nutritional composition. Even more particularly, the protein comprises about 1% to about 5% by weight of the nutritional composition or about 20% to about 30% by weight of the nutritional composition.

[0077] The concentrations and relative amounts of protein, carbohydrate, and fat sources in exemplary nutritional compositions can vary widely depending, for example, on the particular dietary needs of the intended user. In particular embodiments, the nutritional compositions include a protein source in an amount of about 2% to about 20% by weight, a carbohydrate source in an amount of about 5% to about 30% by weight, and a fat source in an amount of about 0.5% to about 10% by weight, based on the weight of the nutritional composition, and more particularly, such compositions are in liquid form. In another particular embodiment, the nutritional compositions include a protein source in an amount of about 10% to about 25% by weight, a carbohydrate source in an amount of about 40% to about 70% by weight, and a fat source in an amount of about 5% to about 20% by weight, based on the weight of the nutritional composition, and more particularly, such compositions are in powder form.

[0078] In one embodiment, the nutritional composition is a liquid nutritional composition and comprises about 1 to about 15% by weight of protein, about 0.5 to about 10% by weight of fat, and about 5 to about 30% by weight of carbohydrates, based on the weight of the nutritional composition.

[0079] In another embodiment, the nutritional composition is a powdered nutritional composition and comprises about 10 to about 30% by weight of protein, about 5 to about 15% by weight of fat, and about 30% to about 65% by weight of carbohydrates, based on the weight of the nutritional composition.

[0080] The nutritional composition may also contain one or more ingredients to modify the physical, chemical, aesthetic, or processing properties of the nutritional composition, or to function as additional nutritional ingredients. Non-limiting examples of additional ingredients include preservatives, emulsifiers (e.g., lecithin), buffers, sweeteners, including artificial sweeteners (e.g., saccharin, aspartame, acesulfame K, sucralose), colorants, flavorings, thickeners, stabilizers, etc. The nutritional composition may also contain additional vitamins and minerals, for example, in amounts that comply with the recommended daily allowance.

[0081] In particular embodiments, the nutritional compositions have a neutral pH, i.e., a pH of about 6 to 8, or more particularly about 6 to 7.5. In even more particular embodiments, the nutritional compositions have a pH of about 6.5 to 7.2, or more particularly about 6.8 to 7.1.

[0082] The nutritional composition may be formed using any technique known in the art. In one embodiment, the nutritional composition may be formed by (a) preparing an aqueous solution containing protein and carbohydrate; (b) preparing an oil blend containing fat-soluble components; and (c) mixing the aqueous solution and oil blend together to form an emulsified liquid nutritional composition. The bovine milk exosome-enriched product and vitamin K2 may be added at any point in the process as desired, for example, to the aqueous solution or the emulsified blend. The bovine milk-derived exosome-enriched product and vitamin K2 may also be dry-blended in powder form with one or more dry ingredients, for example, for combined addition to a liquid composition or when a powdered nutritional product is desired.

[0083] In certain embodiments, the nutritional composition is administered in powder form. In another specific embodiment, the nutritional composition is administered in liquid form. The nutritional composition may be administered to an individual in any form. When the nutritional composition is a powder, for example, a single serving size is about 40 g to about 60 g, such as 45 g, 48.6 g, or 50 g, and is administered as a powder or reconstituted with about 1 ml to about 500 ml of liquid.

[0084] When the nutritional composition is in liquid form, for example, reconstituted from a powder or manufactured as a ready-to-drink product, a single serving may range from about 1 ml to about 500 ml, including about 110 ml to about 500 ml, about 110 ml to about 417 ml, about 120 ml to about 500 ml, about 120 ml to about 417 ml, about 177 ml to about 417 ml, about 207 ml to about 296 ml, about 230 ml to about 245 ml, about 110 ml to about 237 ml, about 120 ml to about 245 ml, about 110 ml to about 150 ml, and about 120 ml to about 150 ml. In certain embodiments, a single serving is about 1 ml, about 100 ml, about 225 ml, about 237 ml, or about 500 ml.

[0085] As indicated above, the present invention provides methods for improving bone formation, reducing fracture risk, strengthening bones, and preventing or delaying the onset or progression of osteoporosis in an individual, comprising administering a bovine milk exosome-enriched product and vitamin K2.

[0086] The following examples illustrate various embodiments of the present invention. [Example]

[0087] Example 1: Method for preparing exosome-enriched products This example describes a method for preparing an exosome-enriched product from cheese whey, which was provided by adding rennet enzyme to bovine milk, resulting in the enzymatic coagulation of casein to produce sweet cheese whey.

[0088] Approximately 10 per gram of exosome-enriched product 8 ~about 10 15An exosome-enriched product containing intact bovine milk-derived exosomes was prepared by cascade membrane filtration. First, 1,000 L of sweet cheese whey was processed using tandem multiple ceramic filtration steps. The first microfiltration (MF) step used a membrane with a molecular weight cutoff of 1.4 μm to produce a first retentate R1 and a first permeate P1. The first permeate P1 was then subjected to an ultrafiltration (UF) step with a molecular weight cutoff of 0.14 μm to produce a second retentate R2 and a second permeate P2. Approximately 5 volumes of water were added to 1 volume of the second retentate R2, and the diluted retentate was then passed through a 0.14 μm UF membrane to again remove at least a portion of the lactose and minerals. The resulting retentate R3 was then combined with an equal volume of water and diafiltered using a 10 kDa membrane to produce a fourth retentate R4. The fourth retentate, R4, was diluted with five times its volume of water and diafiltered a second time using a 10 kDa membrane to obtain concentrated retentate, R5. The lactose-free exosome-enriched product, R5, was pasteurized at 72°C for 15 seconds to ensure microbial stability, yielding pasteurized exosome-enriched product, R6. The pasteurized exosome-enriched product, R6, was evaporated at approximately 65°C to increase the solids content to 17-18% and then spray-dried at 185°C / 85°C to obtain the exosome-enriched spray-dried product.

[0089] Example 2: In vitro cell model for monitoring osteoblast differentiation markers This example describes the surprising effect of a combination of bovine milk exosomes and vitamin K2 on osteoblast differentiation and activity when using mouse MC3T3-E1 cells, which are immature osteoblasts and are widely used as a model of immature bone precursor cell lines that have the ability to differentiate into osteoblasts.

[0090] The cells used were assessed for stimulated osteoblast activity via extracellular matrix mineralization and for induction of expression of osteoblast differentiation markers Runx2 and LC3.

[0091] To initiate the assay, MC3T3-E1 preosteoblasts were grown in MEM (minimum essential medium) culture medium supplemented with 10% (v / v) FBS (fetal bovine serum), 4 nM glutamine, 100 units / ml penicillin, and 0.1 mg / ml streptomycin in an atmosphere of 5% CO and 95% humidity. For osteogenic differentiation, these preosteoblasts were cultured at 3 × 10 in 24-well plates. 4 Cells were seeded at a density of 100 cells / well in complete medium for 1 day, and the medium was then replaced with MEM containing 10% (v / v) FBS, 4 nM glutamine, 100 units / ml penicillin, 0.1 mg / ml streptomycin, 10 nM β-glycerophosphate, and 50 μg / ml L-ascorbic acid.

[0092] The expression of differentiation markers Runx2 and LC3 was analyzed by Western blot after treatment with different effectors, namely bovine milk exosomes and / or vitamin K2, in differentiation medium.

[0093] Specifically, three groups of MC3T3-E1 preosteoblastic cells were treated: Group 1 was treated with 1 μM vitamin K2; Group 2 was treated with 15 μg / ml bovine milk exosomes; Group 3 was treated with a combination of 1 μM vitamin K2 and 15 μg / ml bovine milk exosomes; and Group 4 was included as an untreated control group.

[0094] After treatment with effectors, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (supplemented with phosphatase and protease inhibitors), 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM egtazic acid (EGTA), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Total proteins were denatured by heating the cells at 95°C for 5 min, and 20 μg of protein was loaded onto a 10% acrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were then transferred to nitrocellulose membranes and immunoblotted using specific antibodies (Runx2-Ref:1L7F, Cell Signaling, Inc., and LC3-Ref:4108, Cell Signaling, Inc.). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalizer.

[0095] Figure 1A shows the Runx2 protein expression levels obtained after 3 days of incubation for all four groups. As shown, Group 3, in which MC3T3-E1 preosteoblasts were incubated with a combination of vitamin K2 and bovine milk exosomes, showed a statistically significant increase in Runx2 osteogenic differentiation compared with Groups 1 and 2, which were treated with vitamin K2 and bovine milk exosomes separately, respectively. Group 3 also showed increased Runx2 expression (22.3%) compared with the control Group 4. These results demonstrate that the effect of the combination of bovine milk exosomes and vitamin K2 on Runx2 expression is striking, exceeding that of either of these effectors alone and also exceeding that of the control group. These results are also shown in the Western blot in Figure 2A.

[0096] Figure 1B shows the LC3 protein expression levels obtained after 7 days of incubation for all four groups. As shown, Group 3, in which MC3T3-E1 preosteoblasts were incubated with a combination of vitamin K2 and bovine milk exosomes, showed a statistically significant increase in LC3 osteogenic differentiation compared to Groups 1 and 2, which were treated with vitamin K2 and bovine milk exosomes separately, respectively. Group 3 also showed increased LC3 expression (123.2%) compared to the control Group 4. These results demonstrate that the effect of the combination of bovine milk exosomes and vitamin K2 on LC3 expression is also surprising, exceeding either of these effectors alone and the control group. These results are also shown in the Western blot in Figure 2B.

[0097] Example 3: In vitro cell model for monitoring calcium deposition In addition to the analysis of Runx2 and LC3 molecular markers in Example 2, cell groups 1 to 3 described in Example 2 were treated in the presence of differentiation medium for 12 days. Every 3 days, the differentiation medium with effectors was replaced with fresh differentiation medium and effectors. Group 4 in Example 2 was also maintained as a control group.

[0098] After 12 days of incubation, the supernatants were removed from all three treatment groups, and the cells were washed twice with PBS and fixed with 2% paraformaldehyde at room temperature for 20 minutes. Calcium nodules in all three groups were stained with Alizarin Red (2003999, MERCK) at 37°C for 1 hour. The calcium nodules were then observed under a microscope, and representative photomicrographs were taken for each group.

[0099] The resulting micrographs are shown in Figures 3A-3D. Figure 3A shows calcium deposition in control Group 4. Figure 3B shows calcium deposition in Group 2 treated with bovine milk exosomes. Figure 3C shows calcium deposition in Group 1 treated with vitamin K2. Figure 3D shows calcium deposition in Group 3 treated with both bovine milk exosomes and vitamin K2.

[0100] The ARS assay allows visualization of calcium deposits produced by activated osteoblasts. While Figures 3A-3D all show abundant calcium deposits in the micrographs, Figure 3D, which shows the results for Group 3 treated with both effectors, vitamin K2 and bovine milk exosomes, surprisingly induced enhanced extracellular matrix mineralization, with calcium deposits far greater than those in the other three groups (treatment groups 1-2 and control group 4). This indicates that the combination of vitamin K2 and bovine milk exosomes not only promotes an unexpected level of osteoblast differentiation (based on the results in Example 2), but also surprisingly increases osteoblast mineralization activity (based on increased calcium deposits).

[0101] The combined surprising results of Examples 2 and 3 demonstrate that the combination of vitamin K2 and bovine milk exosomes represents an unexpected treatment that increases osteoblast differentiation and activity, resulting in improved bone formation, reduced fracture risk, and prevention or delay of the onset or progression of osteoporosis in individuals.

[0102] In summary, the present invention provides methods for improving bone health by increasing osteoblast differentiation and activity through the administration of vitamin K2 and bovine milk exosome-enriched products. Such methods are particularly useful for the efficient and / or large-scale production of various nutritional compositions for growing children undergoing long bone growth and older adult individuals prone to osteoporosis.

[0103] The specific embodiments and examples described herein are illustrative only and do not limit the invention as defined by the claims. Moreover, the invention is illustrated by the description of these embodiments, and while the embodiments have been described in considerable detail, it is not intended that such description restrict or in any way limit the scope of the appended claims to such details. Additional advantages and modifications will be readily apparent to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details, representative compositions and processes, or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

Claims

1. 1. A method for improving bone formation in an individual, the method comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

2. 1. A method for reducing fracture risk or strengthening bones in an individual, the method comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

3. 1. A method for preventing or delaying the onset or progression of osteoporosis in an individual, the method comprising administering to the individual a bovine milk exosome-enriched product and vitamin K2.

4. 3. The method of claim 1 or 2, wherein the individual is a pediatric subject.

5. The method of claim 2 or 3, wherein the individual is an adult subject.

6. 6. The method of claim 5, wherein the adult subject is about 40 years of age or older, or greater than about 50 years of age, about 40-80 years of age, or about 50-70 years of age.

7. 7. The method of any one of claims 1 to 6, wherein the bovine milk exosome-enriched product is administered in the form of an exosome-enriched liquid.

8. 7. The method of any one of claims 1 to 6, wherein the bovine milk exosome-enriched product is administered in the form of an exosome-enriched powder.

9. 9. The method of any one of claims 1 to 8, wherein the bovine milk exosome enriched product comprises at least 0.001% by weight of bovine milk exosomes.

10. The bovine milk exosome-enriched product contains approximately 10 ８ ~about 10 １５ The method of any one of claims 1 to 9, comprising exosomes.

11. 11. The method of any one of claims 1 to 10, wherein the bovine milk exosome enriched product comprises intact bovine milk-derived exosomes.

12. 12. The method of claim 11, wherein at least about 50% by weight of the exosomes in the bovine milk exosome-enriched product are intact.

13. 13. The method of claim 11 or 12, wherein at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95% by weight of the exosomes in the bovine milk exosome-enriched product are intact.

14. 14. The method of any one of claims 1 to 13, wherein the exosome-enriched product is administered to the individual at a dose of about 0.01 to about 30 g per day.

15. 15. The method of any one of claims 1 to 14, wherein vitamin K2 is administered to the individual at a dose of about 0.001 to about 0.3 mg per day.

16. 16. The method of any one of claims 1 to 15, wherein the bovine milk exosome enriched product and vitamin K2 are administered to the individual at least once daily.

17. 17. The method of any one of claims 1-16, wherein the bovine milk exosome-enriched product and vitamin K2 are administered to the individual at least once daily for at least about 3 consecutive days, at least about 7 consecutive days, or at least about 12 consecutive days.

18. 18. The method of any one of claims 1 to 17, wherein the exosome-enriched product and vitamin K2 are administered to the individual in a nutritional composition comprising protein, carbohydrate, and / or fat.

19. 20. The method of claim 19, wherein the nutritional composition comprises from about 0.001 to about 30% by weight of the exosome-enriched product, based on the weight of the nutritional composition.

20. 21. The method of claim 19 or 20, wherein the nutritional composition comprises at least about 0.0000004 wt.%, at least about 0.000025 wt.%, at least about 0.00005 wt.%, at least about 0.0001 wt.%, or at least about 0.0002 wt.% vitamin K2, based on the weight of the nutritional composition.

21. 22. The method of claim 20 or 21, wherein the nutritional composition is administered in the form of a liquid having a serving size in the range of 110 mL to 500 mL.

22. 22. The method of claim 20 or 21, wherein the nutritional composition is administered in the form of a powder having a serving size in the range of about 40-60 g.

23. The nutritional composition comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, milk protein concentrate, organic milk protein concentrate, milk protein isolate, milk protein hydrolysate, skim milk powder, condensed skim milk, soy protein concentrate, isolated soy protein, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, collagen protein, collagen protein isolate, L-carnitine, L-lysine, taurine, lutein, rice protein 24. The method of any one of claims 19 to 23, comprising one or more proteins including rice protein concentrate, rice protein isolate, rice protein hydrolysate, faba bean protein concentrate, faba bean protein isolate, faba bean protein hydrolysate, collagen protein, collagen protein isolate, meat protein, potato protein, chickpea protein, canola protein, mung bean protein, guinea fowl protein, amaranth protein, chia protein, hemp protein, linseed protein, earthworm protein, insect protein, one or more amino acids and / or metabolites thereof, or a combination of two or more thereof.

24. 25. The method of any one of claims 19 to 24, wherein the nutritional composition comprises one or more fats including coconut oil, fractionated coconut oil, soybean oil, soybean lecithin, corn oil, safflower oil, high oleic sunflower oil, palm olein, canola oil monoglycerides, lecithin, medium chain triglycerides, linoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, olive oil, high gamma linolenic acid (GLA) safflower oil, palm oil, palm kernel oil, canola oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oils, transesterified oils, structured lipids, or a combination of two or more thereof.

25. The nutritional composition comprises dietary fiber, human milk oligosaccharides (HMO), maltodextrin, corn maltodextrin, organic corn, corn syrup, sucralose, cellulose gel, cellulose gum, gellan gum, inositol, carrageenan, fructooligosaccharides, hydrolyzed starch, glucose polymers, corn syrup solids, rice-derived carbohydrates, sucrose, glucose, lactose, honey, sugar alcohols, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, and arabic.

26. The method of any one of claims 19 to 25, further comprising one or more carbohydrates comprising: gum, sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, acacia gum, chitosan, arabinoglactin, glucomannan, xanthan gum, alginic acid, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucan, psyllium, inulin, corn starch, or a combination of two or more thereof.

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