A composition for improving bone health, its use and products

Abstract

The application provides a composition for improving bone health and application and products thereof, and belongs to the technical field of nutritional compositions.The composition for improving bone health provided by the application is composed of 1-50 parts of OPN and 1-100 parts of breast milk oligosaccharides in terms of weight; the breast milk oligosaccharides are LNT or LNnT.The composition of the application can improve calcium absorption, enhance osteoblast activity, promote bone mineralization and growth, has a significant synergistic gain effect in improving bone health, is safe and mild in raw material, simple in preparation process, can be prepared into food, health products or medicines, and meets the bone maintenance demand.

Description

Technical Field

[0001] This invention belongs to the field of nutritional composition technology, specifically relating to a composition for improving bone health and its applications and products. Background Technology

[0002] Bone mineral density (BMD) is a core indicator for evaluating human skeletal health and measuring bone strength and quality. BMD levels directly determine bone structural stability, mechanical strength, and fracture resistance. Bone health throughout the entire lifespan is closely related to dynamic changes in BMD. Normal accumulation, maintenance, and metabolic balance of BMD are fundamental prerequisites for ensuring normal bone growth, development, and homeostasis. Abnormally low BMD and insufficient bone mass directly lead to a series of skeletal health problems, such as weak bone structure and decreased bone resilience.

[0003] The accumulation of bone mineral density (BMD) depends crucially on the process of human skeletal growth and development. Skeletal growth and development is a core physiological process throughout the entire human lifespan. The period from 0 to 18 years of age, specifically childhood and adolescence, is an irreversible critical window for linear bone growth, height increase, and peak bone mass accumulation. The state of skeletal development during this stage directly determines a child's final adult height and is directly related to the risk of osteoporosis and fragility fractures in adulthood. Simultaneously, after reaching peak bone mass at age 30, the body enters a phase of continuous bone loss. Pregnant women, perimenopausal women, and the middle-aged and elderly all face accelerated bone loss and a rapid decline in bone density, disrupting skeletal homeostasis and necessitating safe and long-term bone health intervention programs.

[0004] Long-term bone density loss and continuous bone loss can directly induce various bone metabolic diseases, among which osteoporosis is the most common metabolic bone disease in clinical practice. Its core pathological features are bone tissue microstructure degeneration, a significant decrease in bone quality and mass, ultimately leading to a significant increase in bone fragility, making it extremely prone to fragility fractures, and seriously affecting people's health and quality of life. Currently, drug therapy is an important part of osteoporosis treatment, with the core goal of stopping the progression of osteoporosis and intervening in and improving existing osteoporotic symptoms. Clinically used drugs for osteoporosis are mainly divided into two categories: drugs that inhibit bone resorption and drugs that promote bone formation. Drugs that inhibit bone resorption include calcium supplements, vitamin D, active vitamin D, calcitonin, bisphosphonates, estrogen, and isoflavones; drugs that promote bone formation include fluorides, anabolic steroids, parathyroid hormone, and isoflavones. However, existing clinical treatment drugs generally have certain toxic side effects and contraindications, making them unsuitable for daily bone maintenance and long-term intervention in the general population, severely limiting their applicability in various scenarios and to a limited number of people.

[0005] Currently, conventional techniques used both domestically and internationally to promote bone growth and development, improve slow growth, maintain bone health, increase bone density, and prevent osteoporosis are mainly divided into two major systems: clinical drug intervention and nutritional combination intervention. In the field of clinical intervention, for children with pathological growth retardation and idiopathic short stature, the routine treatment recommended by domestic and international clinical guidelines uses recombinant human growth hormone and gonadotropin-releasing hormone analogs as the core treatment methods, while calcium supplements combined with vitamin D serve as basic nutritional support. For osteoporosis, osteoarthritis, and other bone metabolism-related diseases, clinical practice routinely uses drugs that inhibit bone resorption, drugs that promote bone formation, and symptomatic treatments such as nonsteroidal anti-inflammatory drugs, analgesics, and antirheumatic drugs. Although these are effective, they are only suitable for treating pathological bone diseases and cannot be used for daily health maintenance.

[0006] In the field of nutritional intervention, most mainstream bone growth and bone health products on the market use "calcium + vitamin D" as their core basic formula. Some products additionally add ingredients such as casein phosphopeptide, colostrum basic protein, lysine, and hydrolyzed egg yolk powder to help improve calcium absorption and supplement basic nutrition, thus having a certain effect on improving bone health. However, existing technologies and products on the market still have significant shortcomings. Currently, the industry lacks bone health formulas that are simplified, highly targeted, and have clear synergistic effects.

[0007] Relevant patent documents retrieved: This document, published in China (CN112890200A) on June 4, 2021, discloses a nutritional composition comprising OPN and specific human milk oligosaccharides, wherein the specific human milk oligosaccharides comprise at least one sialylated oligosaccharide and at least one N-acetylated oligosaccharide. In one specific embodiment, the specific human milk oligosaccharides comprise at least two or more of LDFT, 3'SL, LNT, 2-FL, and LNnT.

[0008] Relevant non-patent literature retrieved: The journal or book title is "The American Journal of Clinical Nutrition," and the article title is "Associations of human milk oligosaccharides and bioactive proteins within child growth and development among Malawian mother-infant dyads," volume number 113, publication date 2021.01.04. This article discloses the association between specific active breast milk proteins and human milk oligosaccharides and infant growth, motor and cognitive development.

[0009] Given the numerous shortcomings of existing technologies, such as significant drug side effects, limited applicable populations, single target, poor efficacy in improving non-pathological slow growth, and redundant formulations without clear synergistic effects, there is an urgent need in the industry to develop a novel active composition with a simplified formulation, high safety, clear synergistic effects, and long-term usability. This composition can simultaneously promote longitudinal bone growth, improve calcium absorption efficiency, accelerate bone mineralization, and regulate bone metabolism balance, thereby filling the current technological gap and addressing the core pain points in the field of promoting bone growth and development. Summary of the Invention

[0010] To address the above-mentioned shortcomings, the present invention provides a composition for improving bone health, its application, and products.

[0011] the term: In this invention, the term "osteopontin" (English name: Osteopontin, abbreviated as OPN) refers to a secretory phosphorylated glycoprotein rich in aspartic acid, serine, and glutamic acid. Osteopontin derived from the milk of various mammals, including human milk, is called milk-derived osteopontin, or simply lactopontin (LPN), also known as mammary osteopontin. The osteopontin described in this invention includes naturally extracted osteopontin, genetically engineered recombinant osteopontin, and its functional fragments or variants. As long as the functional fragment or variant retains the same or similar RGD sequence binding activity and bone metabolism regulatory activity as natural osteopontin, it falls within the protection scope of the osteopontin described in this invention. The osteopontin used in this invention was purchased from Arla, catalog number Lacprodan® OPN-10.

[0012] In this invention, the term "lacto-n-tetraose" (LNT) refers to a neutral functional oligosaccharide naturally present in breast milk. The LNT described in this invention includes naturally isolated and purified products, artificially synthesized products, products prepared through bio-fermentation, and their structural homologs. As long as the structural homolog retains the same or similar calcium absorption-promoting and bone development-regulating activities as natural LNT, it falls within the protection scope of the LNT described in this invention. The LNT used in this invention was purchased from Huangshan Tongxi Biotechnology Co., Ltd.

[0013] In this invention, the term "lacto-n-neotetraose" (English name: Lacto-N-neotetraose, abbreviation LNnT) refers to the neutral oligosaccharide active component naturally present in breast milk. The lacto-n-neotetraose described in this invention includes naturally extracted products, chemically synthesized products, microbial fermentation products, and their structural derivatives. As long as the structural derivative retains the same or similar osteoblast activation and bone growth promoting activities as natural lacto-n-neotetraose, it falls within the protection scope of the lacto-n-neotetraose described in this invention. The LNnT used in this invention was purchased from Huangshan Tongxi Biotechnology Co., Ltd.

[0014] In this invention, the term "lactodifucotetraose" (LDFT) is used as a comparative component and was purchased from Glycom.

[0015] In this invention, the term "3'-sialyllactose" (English name: 3'-Sialyllactose, abbreviation 3'-SL) is used as a comparative component and was purchased from Glycom.

[0016] In this invention, the term "2'-fucosyllactose" (English name: 2'-Fucosyllactose, abbreviation 2'-FL) is used as a comparative component and was purchased from Glycom.

[0017] The technical solution of this invention is as follows: On one hand, the present invention provides a composition for improving bone health, the composition comprising, by weight, 1-50 parts of OPN and 1-100 parts of human milk oligosaccharides; wherein the human milk oligosaccharides are LNT or LNnT.

[0018] Specifically, the composition contains, by weight, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, and 25-26. 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49 or 49-50 OPN copies.

[0019] Preferably, the composition contains 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15 or 15-16 parts of OPN by weight.

[0020] More preferably, the composition contains 10-11, 11-12, 12-13, 13-14, 14-15 or 15-16 parts of OPN by weight.

[0021] More preferably, the composition contains 16 parts by weight of OPN.

[0022] Specifically, the composition contains, by weight, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26. 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 50-51 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 71-72, 72-73, 73-74, 74-75, 75-76 76-77, 77-78, 78-79, 79-80, 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98, 98-99 or 99-100 parts of human milk oligosaccharides.

[0023] Preferably, the composition contains, by weight, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49 or 49-50 parts of human milk oligosaccharides.

[0024] More preferably, the composition contains, by weight, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49 or 49-50 parts of human milk oligosaccharides.

[0025] In another aspect, the present invention provides a method for preparing the composition described in any of the above claims, the method comprising mixing OPN and human milk oligosaccharides uniformly in parts by weight.

[0026] In another aspect, the present invention provides a food product comprising the composition described in any of the preceding claims.

[0027] Specifically, the food products mentioned include any one or more of the following: finished food products, semi-finished food products, food additives, and food supplements.

[0028] Specifically, the food products mentioned include infant food, children's food, adolescent food, adult food, or pet food.

[0029] Preferably, the infants and young children include infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0030] Specifically, the food products include, but are not limited to, any one or more of the following: candy, soy milk, yogurt, milk, juice, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria drinks, carbonated beverages, coffee, jelly, protein bars, energy bars, energy gels, and puffed foods.

[0031] Specifically, the food products also include any one or more of the following: nutritional additives, food-medicine homologous ingredients, and excipients acceptable in the food or health product industry.

[0032] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0033] Preferably, the medicinal and edible ingredients include cloves, star anise, sword bean, fennel, thistle, yam, hawthorn, purslane, black-striped snake, dried plum, papaya, hemp seed, bitter orange flower, Solomon's seal, licorice, angelica, ginkgo, white hyacinth bean, white hyacinth bean flower, longan pulp, cassia seed, lily, nutmeg, cinnamon, amla, bergamot, almond, sea buckthorn, oyster, fox nut, Sichuan pepper, red adzuki bean, donkey-hide gelatin, chicken gizzard lining, malt, kelp, jujube, monk fruit, apricot kernel, honeysuckle, green plum, houttuynia cordata, ginger, Japanese raisin tree fruit, wolfberry, gardenia, amomum villosum, sterculia lychnophora, poria cocos, citron, elsholtzia ciliata, peach kernel, mulberry. Leaves, mulberries, tangerine peel, platycodon root, alpinia oxyphylla, lotus leaves, radish seeds, lotus seeds, galangal, bamboo leaves, fermented soybean, chrysanthemum, chicory, yellow mustard seeds, polygonatum, perilla, perilla seeds, kudzu root, black sesame, black pepper, sophora japonica buds, sophora japonica flowers, dandelion, honey, torreya nuts, jujube seeds, fresh imperata root, fresh reed root, viper, tangerine peel, mint, coix seed, allium macrostemon, raspberry, patchouli, angelica, galangal, saffron, cardamom, turmeric, long pepper, codonopsis, cistanche, dendrobium officinale, American ginseng, astragalus, ganoderma, cornus officinalis, gastrodia elata, eucommia ulmoides leaves, rehmannia glutinosa, ophiopogon japonicus, asparagus cochinchinensis, and tangerine peel.

[0034] Preferably, the excipients include any one or more of fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic regulators, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0035] In another aspect, the present invention provides a health product comprising the composition described in any one of the above claims.

[0036] Specifically, the health products mentioned also include any one or more of the following: nutritionally acceptable nutritional additives, food-medicine homologous ingredients, and excipients acceptable in the food or health product fields.

[0037] Preferably, the dosage form of the health product includes any one or more of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0038] Specifically, the health products mentioned include health foods, which include, but are not limited to: candy, soy milk, yogurt, milk, fruit juice, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria drinks, carbonated beverages, coffee, jelly, protein bars, energy bars, energy gels, and puffed foods, any one or more of these.

[0039] In another aspect, the present invention provides a medicine comprising the composition described in any of the preceding claims.

[0040] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0041] Preferably, the pharmaceutically acceptable excipients are selected from any one or more of the following: fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic modifiers, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0042] Specifically, the dosage form of the drug includes gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0043] Preferably, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0044] Preferably, the gastrointestinal dosage forms include, but are not limited to, injectable dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0045] In another aspect, the present invention provides the use of the composition described in any of the above claims in the preparation of products for improving bone health.

[0046] Specifically, the bone health improvement products include, but are not limited to, any one or more of the following: calcium supplements, products that help improve bone density, products that promote bone growth and development, and medications for treating osteoporosis.

[0047] Preferably, the product includes food, health products, or medicine.

[0048] In another aspect, the present invention provides a method for improving bone health, the method comprising using any of the compositions, foods, health products or pharmaceuticals described above.

[0049] Specifically, the method includes administering an effective amount of the composition, food, health product, or medicine to the subject.

[0050] Preferably, the subject is a mammal.

[0051] More preferably, the subject is a human being.

[0052] The beneficial effects of this invention are as follows: This invention employs a binary compound system of OPN and a single neutral human milk oligosaccharide LNT or LNnT, resulting in a simplified formulation that differs from existing technologies that use complex compounding schemes of OPN with multiple sialylated and fucoidated human milk oligosaccharides. Individual application of any one of OPN, LNT, or LNnT only slightly promotes body length growth, with no significant improvement in bone mineralization, calcium absorption, or osteogenic activity. However, when combined within the weight limits specified in this invention, they produce a significant synergistic effect, significantly enhancing bone health-related activities compared to various hybrid human milk oligosaccharide compound systems. The composition of this invention can synergistically regulate bone metabolism through multiple pathways: significantly improving intestinal calcium ion absorption efficiency and increasing the body's calcium reserves; upregulating osteoblast alkaline phosphatase activity, accelerating bone mineralization and deposition, and increasing bone density; simultaneously promoting longitudinal bone growth and increasing the number of bone segments, achieving a triple effect of promoting growth, calcium absorption, and bone mineralization, thus comprehensively improving bone health. Attached Figure Description

[0053] Figure 1 Alizarin Red staining for zebrafish facial bones.

[0054] Figure 2 Alizarin red staining for zebrafish vertebrae.

[0055] Figure 3 Staining calcium ions in the intestines of zebrafish.

[0056] Figure 4 Alkaline phosphatase staining of the ventral skeleton of zebrafish head. Detailed Implementation

[0057] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0058] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0059] Example 1 A composition A composition comprising 1 part OPN and 100 parts LNT, prepared by mixing OPN and LNT evenly.

[0060] Example 2 A composition A composition comprising 50 parts of OPN and 1 part of LNT, prepared by mixing OPN and LNT evenly.

[0061] Example 3 A composition A composition comprising 1 part OPN and 50 parts LNT, prepared by mixing OPN and LNT evenly.

[0062] Example 4 A composition A composition comprising 16 parts OPN and 8 parts LNT is prepared by mixing OPN and LNT evenly.

[0063] Example 5 A composition A composition comprising 16 parts OPN and 8 parts LNnT is prepared by mixing OPN and LNnT evenly.

[0064] Comparative Example 1: A composition A composition comprising 6 parts OPN, 70 parts LDFT, 70 parts 3'-SL, 120 parts 2'-FL and 100 parts LNnT, prepared by mixing OPN, LDFT, 3'-SL, 2'-FL and LNnT uniformly.

[0065] Comparative Example 2: A composition A composition comprising 0.5 parts OPN and 150 parts LNT, prepared by mixing OPN and LNT evenly.

[0066] Comparative Example 3: A composition A composition comprising 0.5 parts OPN and 150 parts LNnT is prepared by mixing OPN and LNnT evenly.

[0067] Comparative Example 4: A composition A composition comprising 16 parts OPN and 8 parts LDFT, prepared by mixing OPN and LDFT evenly.

[0068] Comparative Example 5: A composition A composition comprising 16 parts OPN, 4 parts LDFT, and 4 parts 3'-SL, prepared by mixing OPN, LDFT, and 3'-SL evenly.

[0069] Experimental Example 1 1. Laboratory animals This experiment used wild-type AB strain zebrafish, all provided by Watersilver (International) Biotechnology Co., Ltd. Wild-type AB strain adult zebrafish were cultured in a recirculating aquaculture system according to GB / T 39649-2020, with a water temperature of 28±0.5℃, a photoperiod of 14:10h, pH 6.5-8.5, and conductivity of 500-800 µS / cm. Embryos were obtained through natural spawning, washed, and after removing any abnormal or unfertilized eggs, they were placed in fish embryo culture medium (containing CaCl2 294.0 mg / L, MgSO4·7H2O 123.3 mg / L, NaHCO3 63.0 mg / L, KCl 5.5 mg / L) and statically cultured at 28±0.5℃ in a constant temperature incubator. All animal experiments have been approved by the Department of Health of the Hong Kong Special Administrative Region of the People's Republic of China (License No.: DH / HT&A / 8 / 218Pt.524.10.2023 No. 23-86).

[0070] 2. Preparation of reagents used in the experiment (1) Tricaine solution: Weigh 200 mg of tricaine (analytical grade) and dissolve it in 48.95 mL of water to prepare the stock solution. Refrigerate and it is effective within 30 days. Dilute the stock solution to 0.04% with fish embryo culture medium and prepare fresh before use.

[0071] (2) Paraformaldehyde: Prepare a solution containing 4% paraformaldehyde and 0.8% sodium hydroxide (NaOH) with pH 7.2-7.4 using phosphate buffered saline (PBS) and refrigerate.

[0072] (3) PBST: Prepare 0.04% Triton X-100 in PBS solution and store at room temperature.

[0073] (4) Bleaching solution: Prepare a solution containing 3% H2O2 and 1% KOH with water, and prepare it fresh before use.

[0074] (5) Alizarin Red S solution: Weigh 50 mg of Alizarin Red S powder and dissolve it in 50 mL of 70% alcohol solution to prepare the stock solution, and store at room temperature. Dilute the stock solution 100 times with 70% alcohol to prepare the working solution.

[0075] (6) Clear solution 1: Prepare a solution containing 20% ​​glycerol and 0.25% KOH with water and store at room temperature.

[0076] (7) Clear solution 2: Prepare a solution containing 50% glycerol and 0.25% KOH with water and store at room temperature.

[0077] (8) Low calcium solution: It is prepared by dissolving CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4 and KH2PO4 in water, and the working solution concentration is 25μM.

[0078] (9) Na2S solution: The working solution concentration is 10 μM.

[0079] (10) ALP substrate solution: containing 0.5 mL of 10 mg / mL naphthol AS-TR phosphate / N,N-dimethylformamide solution, 50 mL of Tris-maleate buffer (pH 8.3) and 40 mg of nitrogen salt (Fix Blue).

[0080] 3. Sample to be tested The test samples were the compositions of Examples 1-5 or Comparative Examples 1-5, OPN, LNT, and LNnT. The experimental groups and experimental concentrations of the test samples are shown in Table 1.

[0081] Table 1

[0082] 4. Zebrafish growth promotion test Healthy zebrafish, 5 days post-fertilization, were randomly selected and tested in 96-well plates, with one zebrafish treated in each well (experimental group). The test samples were administered in aqueous solution, with a blank control group included. Each well contained 0.2 mL of solution, and each group had 24 zebrafish. The zebrafish were cultured in a 28℃±1℃ incubator until 10 days post-fertilization. On days 6, 7, and 8, they were fed paramecium for 1 hour, with the solution containing the corresponding test sample replaced after each feeding. The embryos were anesthetized with tricaine working solution for 2 minutes and photographed under a microscope using standardized parameters. The body length (in pixels) of each zebrafish was measured using software such as ImageJ. Statistical results are expressed as Mean ± SEM. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance. The results are shown in Table 2. Table 2

[0083] Note: Compared with the blank control group, *p<0.05, **p<0.01, ****p<0.0001.

[0084] 5. Zebrafish calcium density enhancement test Healthy, 5-day-old post-fertilization zebrafish were randomly selected for testing in 96-well plates, with one zebrafish treated in each well (experimental group). The test samples were administered in aqueous solution, with a blank control group included. Each well contained 0.2 mL of solution, and each group consisted of 24 zebrafish. The zebrafish were cultured at 28℃±1℃ for 9 days post-fertilization. On days 6, 7, and 8, they were fed paramecium for 1 hour, with the solution containing the corresponding test sample replaced after each feeding. The zebrafish in each test group were fixed in paraformaldehyde for at least 1 hour, then treated with PBST three times for 2 minutes each time, followed by treatment with bleach solution for 10 minutes. After 5 minutes of PBST treatment, the zebrafish were treated with 50% ethanol for 5 minutes. Finally, they were gently stained with Alizarin Red S solution for 1 hour. After rinsing three times with purified water, the zebrafish were treated with clarifier 1 for 15 min, clarifier 2 for 10 min, and then with PBST for 3 min. The facial bones of the zebrafish were then photographed under a microscope using uniform imaging parameters. The stained area (in pixels) of the facial bones of each zebrafish was measured using analysis software such as ImageJ. Statistical results are expressed as Mean ± SEM. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance. The measurement results are as follows: Figure 1 As shown in Table 3: Table 3

[0085] Note: There was no significant difference in ns between the control group and the control group in the table. *p<0.05, ***p<0.001, ****p<0.0001.

[0086] 6. Zebrafish vertebral column number and length enhancement test Healthy, 5-day-old post-fertilization zebrafish were randomly selected for testing in 96-well plates, with one zebrafish treated in each well (experimental group). The test samples were administered in aqueous solution, with a blank control group included. Each well contained 0.2 mL of solution, and each group consisted of 24 zebrafish. The zebrafish were cultured at 28℃±1℃ for 9 days post-fertilization. On days 6, 7, and 8, they were fed paramecium for 1 hour, with the solution containing the corresponding test sample replaced after each feeding. The zebrafish in each test group were fixed in paraformaldehyde for at least 1 hour, then treated with PBST three times for 2 minutes each time, followed by treatment with bleach solution for 10 minutes. After 5 minutes of PBST treatment, the zebrafish were treated with 50% ethanol for 5 minutes. Finally, they were gently stained with Alizarin Red S solution for 1 hour. After rinsing three times with purified water, the zebrafish were treated with clarifying solution 1 for 15 min, clarifying solution 2 for 10 min, and then with PBST for 3 min. The facial skeletons of the zebrafish were then photographed under a microscope using uniform imaging parameters. The number of chromatic segments and the length (in pixels) of the vertebrae of each zebrafish were measured using analysis software such as ImageJ. Statistical results are expressed as Mean ± SEM. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance. The measurement results are as follows: Figure 2 As shown in Table 4: Table 4

[0087] Note: There was no significant difference in ns between the control group and the control group in the table. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0088] 7. Zebrafish calcium ion absorption enhancement test Healthy, 5-day-old post-fertilization zebrafish were randomly selected for testing in 96-well plates, with one zebrafish treated in each well (experimental group). The test samples were administered in water solution, and a blank control group was also included. Each well contained 0.2 mL of solution, with a total of 20 zebrafish per group. After incubation at 28℃±1℃ for 7 days, each group was fed paramecium for 2 hours, followed by replacement with fresh solution containing the corresponding test sample. Exposure continued until day 8. The fish were then transferred to 1.5 mL centrifuge tubes and incubated at 28℃±1℃ for staining with a calcium ion probe in the dark for 1 hour. For the positive control group, zebrafish were incubated in 10 μM Na₂S solution for 30 minutes before staining, followed by staining and photographing. All test reagents were prepared using low-calcium solutions. Twelve zebrafish were randomly selected and photographed under a laser confocal microscope. The fluorescence intensity of calcium ions in the intestines of each zebrafish was measured using analysis software such as ImageJ, and the relative fluorescence intensity (RFU) was calculated. Statistical results are expressed as Mean ± SEM. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance. The measurement results are as follows: Figure 3 As shown in Table 5: Table 5

[0089] Note: There was no significant difference in ns between the control group and the control group in the table. *p<0.05, ***p<0.001, ****p<0.0001.

[0090] 8. Zebrafish Alkaline Phosphatase (ALP) Test Healthy, 5-day-old post-fertilization zebrafish were randomly selected for testing in 96-well plates, with one zebrafish treated in each well (experimental group). The test samples were administered in aqueous solution, with a blank control group included. Each well contained 0.2 mL of solution, and each group consisted of 24 zebrafish. The zebrafish were cultured in a 28℃±1℃ incubator until 9 days post-fertilization. On days 6, 7, and 8, they were fed paramecium for 1 hour, with the solution containing the corresponding test sample replaced after each feeding. Each group of zebrafish was fixed in paraformaldehyde for at least 1 hour, then washed three times with distilled water for 15 minutes each time. They were then treated in 0.05 M Tris-maleate buffer (pH 8.3) for 1 hour, followed by treatment in ALP substrate chromogenic solution at room temperature in the dark for 1 hour. Finally, the zebrafish were bleached in a 10% hydrogen peroxide / 1% potassium hydroxide mixture until their eyes lightened. The zebrafish were then positioned with their heads tilted upwards. Then, the zebrafish heads were photographed under a stereomicroscope using the same settings on the ventral side. The stained area (in pixels) of the ventral bone of each zebrafish head was measured using analysis software such as ImageJ. Statistical results are expressed as Mean ± SEM. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance. The measurement results are as follows: Figure 4 As shown in Table 6: Table 6

[0091] Note: There was no significant difference in ns between the control group and the blank control group in the table, **p<0.01, ***p<0.001.

[0092] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for improving bone health, characterized in that, The composition comprises 1-50 parts by weight of OPN and 1-100 parts by weight of human milk oligosaccharides; the human milk oligosaccharides are LNT or LNnT.

2. The composition according to claim 1, characterized in that, The composition consists of 1-50 parts by weight of OPN and 1-50 parts by weight of human milk oligosaccharides.

3. The composition according to claim 1, characterized in that, The composition consists of 1-16 parts by weight of OPN and 8-50 parts by weight of human milk oligosaccharides.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The preparation method includes mixing OPN and human milk oligosaccharides evenly according to their weight proportions.

5. A food or health product, characterized in that, The food or health product described herein comprises the composition according to any one of claims 1-3.

6. The food or health product according to claim 5, characterized in that, The food or health product also includes any one or more of the following: nutritional additives, food-medicine homologous ingredients, and excipients acceptable in the food or health product field.

7. The food or health product according to claim 6, characterized in that, The nutritional additives include one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

8. A medicine, characterized in that, The medicine comprises the composition according to any one of claims 1-3.

9. The pharmaceutical product according to claim 8, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

10. The use of the composition according to any one of claims 1-3 in the preparation of a product for improving bone health, characterized in that, The bone health improvement products include any one or more of the following: calcium supplements, products that help improve bone density, products that promote bone growth and development, and medications for treating osteoporosis.

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