Carrier composition for calcium-encapsulated emulsion useful for improving bone density and use thereof

Abstract

The application discloses a carrier composition for calcium-embedded emulsion which is helpful to improve bone density and application thereof. The carrier composition comprises a hydrophilic emulsifier and a colloidal composition, wherein the colloidal composition comprises a colloidal component A and a colloidal component B, the viscosity of a 0.5% mass fraction aqueous solution of the colloidal component A is 4000-4300 mPa·s at 25 DEG C by using a 64 rotor, and the viscosity of a 0.5% mass fraction aqueous solution of the colloidal component B is 800-900 mPa·s at 25 DEG C by using a 64 rotor. The carrier composition in the application is applied to calcium-embedded emulsion, so that the bioavailability of calcium is improved and the bone density is improved.

Description

Technical Field

[0001] This invention relates to the field of food fortification technology, and more specifically, to a calcium-encapsulated emulsion carrier composition that helps improve bone density and its application. Background Technology

[0002] With increasing health awareness, dairy products, as an important source of nutrition in daily diets, are widely used to supplement protein, vitamins, and minerals, such as calcium. Calcium is one of the essential minerals for the human body and plays a vital role in bone and teeth health. Currently, liquid dairy products such as milk, yogurt, and milk beverages are important carriers for calcium supplementation due to their ease of consumption and absorption. However, because calcium has limited solubility in liquid systems, it often exists in a slightly soluble or insoluble form, making it prone to precipitation during storage, affecting product stability and taste.

[0003] Furthermore, to increase the calcium content in dairy products, existing technologies typically employ the addition of calcium salts such as calcium lactate, calcium carbonate, and calcium phosphate to liquid dairy products for fortification. However, these calcium salts exhibit poor dispersibility in liquid systems, easily leading to uneven calcium distribution within the dairy products and affecting product quality. Simultaneously, some calcium salts have low absorption rates in the gastrointestinal tract, hindering their ability to fully exert their physiological function of calcium supplementation. Therefore, in existing technologies, the poor dispersion stability of calcium in liquid dairy products, low calcium loading efficiency, and limited calcium absorption promotion effects have become significant factors affecting the quality and nutritional efficacy of calcium-fortified dairy products. In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a carrier composition for calcium-embedded emulsions that helps improve bone density and its application, which is beneficial to improving the stability of calcium-embedded emulsions that help improve bone density, thereby improving calcium loading efficiency and promoting calcium absorption.

[0005] This invention is implemented as follows:

[0006] In a first aspect, the present invention provides a carrier composition for calcium-embedded emulsions that helps improve bone density, comprising, by weight parts: a hydrophilic emulsifier and a colloidal composition, wherein the colloidal composition contains colloidal component A and colloidal component B;

[0007] The viscosity of the colloidal component A aqueous solution with a mass fraction of 0.5% was tested using a No. 64 rotor at a speed of 50 rpm and a temperature of 25°C and the result was 4000-4300 mPa•s.

[0008] The viscosity of the colloidal component B aqueous solution with a mass fraction of 0.5% was tested using a No. 64 rotor at a speed of 50 rpm and a temperature of 25°C and the result was 800-900 mPa•s.

[0009] In an optional embodiment, the hydrophilic emulsifier is selected from whey protein concentrate;

[0010] And / or, the colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum;

[0011] And / or, the mass ratio of colloidal component A to colloidal component B in the colloidal composition is 1:(0.1-10).

[0012] And / or, the mass ratio of the hydrophilic emulsifier to the colloidal composition in the carrier composition is (1-30):1.

[0013] In a second aspect, the present invention provides a calcium-encapsulated emulsion that helps improve bone density, comprising a calcium source, an oil, a lipophilic emulsifier, and the carrier composition described in the foregoing embodiments;

[0014] The viscosity of the calcium-encapsulated emulsion was tested using a CC27 rotor at 50 rpm and 25°C, and the result was 200-500 mPa•s.

[0015] In an optional embodiment, the calcium-encapsulated emulsion that helps improve bone density contains 1% to 10% calcium source by mass, 5% to 15% oil by mass, 0.5% to 2% lipophilic emulsifier by mass, 1% to 3% hydrophilic emulsifier by mass, 0.05% to 0.5% gellan gum by mass, and 0.05% to 0.5% xanthan gum by mass.

[0016] In an optional embodiment, the calcium-encapsulated emulsion that helps improve bone density includes an aqueous phase and an oil phase encapsulated in the aqueous phase, wherein a calcium source is dispersed in the oil phase.

[0017] And / or, the calcium source includes at least one of milk mineral salts, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate;

[0018] And / or, the lipophilic emulsifier includes fatty acid ester emulsifiers and / or phospholipids.

[0019] In an optional embodiment, the lipophilic emulsifier includes one or more of glyceryl monostearate, mono- and diglycerides of fatty acids, sucrose fatty acid esters, phospholipids, polyglycerol fatty acid esters, and diacetyl tartaric acid mono- and diglycerides.

[0020] Thirdly, the present invention provides a method for preparing a calcium-embedded emulsion that helps improve bone density as described in any of the foregoing embodiments, comprising the following steps:

[0021] The carrier composition was heated and mixed in water to obtain an aqueous phase;

[0022] The calcium source is heated and mixed with the oil and a lipophilic emulsifier to obtain an oil-solid phase.

[0023] The oil-solid phase is mixed with the aqueous phase and then emulsified to obtain the calcium-encapsulated emulsion that helps improve bone density.

[0024] In an optional embodiment, the mass fraction of the hydrophilic emulsifier in the aqueous phase is 1.5%-2.5%, the mass fraction of gellan gum is 0.05%-0.5%, and the mass fraction of xanthan gum is 0.05%-0.5%.

[0025] And / or, the mass ratio of the calcium source to the oil is 1:(1~10);

[0026] And / or, the mass ratio of the oil-solid phase to the aqueous phase is (6~27):(94~73).

[0027] In an optional embodiment, the carrier composition is heated and mixed in water at a temperature of 40°C to 80°C for 10 min to 60 min.

[0028] And / or, the calcium source is heated and mixed with oils and lipophilic emulsifiers at a temperature of 40℃~70℃ for 10min~60min.

[0029] In an optional implementation, emulsification includes sequential high-speed shearing and homogenization, and the emulsification step further satisfies at least one of the following characteristics:

[0030] a. The rotational speed of the high-speed shearing is 10,000 rpm ~ 20,000 rpm;

[0031] b. The high-speed shearing time is 1 min to 5 min;

[0032] c. The pressure of the homogenizer is 20 MPa ~ 40 MPa.

[0033] Fourthly, the present invention provides a calcium-containing composition obtained by drying a calcium-encapsulated emulsion that helps improve bone density as described in any of the foregoing embodiments.

[0034] Fifthly, the present invention provides a dairy product comprising a calcium-encapsulated emulsion that helps improve bone density as described in any of the foregoing embodiments, or a calcium-containing composition as described in the foregoing embodiments.

[0035] The present invention has the following beneficial effects:

[0036] The carrier composition described in this application, when applied to calcium-encapsulated emulsions, effectively reduces the particle size of the calcium mineral salt formulation and increases its specific surface area, thereby promoting the dispersion and dissolution of the calcium source in digestive fluids and achieving high bioavailability. Furthermore, the carrier composition, acting at the interface between the oil-solid phase and the aqueous phase, enhances the stability of the calcium source in the digestive environment, improves its contact efficiency with gastric juice, and facilitates the dissolution of more calcium. 2+ The release of calcium salts. Furthermore, some peptide-calcium conjugates may slow the formation of calcium salt precipitation under neutral conditions in the small intestine, thereby reducing calcium salt loss and further improving calcium bioavailability and bone density. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 A schematic diagram of the preparation process of calcium-embedded emulsions that help improve bone density;

[0039] Figure 2 Changes in uterine (A) and serum estradiol concentration (B) between the sham surgery group and the model group;

[0040] Figure 3 The trend of body weight change in rats of different groups;

[0041] Figure 4 Changes in serum calcium concentration in rats of different groups over 24 hours;

[0042] Figure 5 Calcium absorption rate in rats of different groups;

[0043] Figure 6 Calcium retention rate in rats of different groups;

[0044] Figure 7 Bone calcium content in rats of different groups;

[0045] Figure 8 Femur length (A) and diameter (B) of rats in different groups;

[0046] Figure 9 Maximum load (A) and maximum stress (B) of femur and tibia in different groups;

[0047] Figure 10 Micro CT images of the femur in different groups;

[0048] Figure 11Trends in bone mineral density in different groups of rats;

[0049] Figure 12 Water content in feces of rats in different groups;

[0050] Figure 13 Intestinal ink migration rate in rats of different groups;

[0051] Figure 14 Results of antibacterial stability tests for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0052] Figure 15 Microstructure of calcium-encapsulated emulsions with different xanthan gum addition ratios after sterilization stability test;

[0053] Figure 16 Centrifugal stability test results of calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0054] Figure 17 Freeze-thaw stability test results for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0055] Figure 18 Results of lumisizer stability tests after freeze-thaw cycles for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0056] Figure 19 Results of interfacial stability tests for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0057] Figure 20 Infrared spectra of aqueous phases and calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0058] Figure 21 Emulsion particle size (A) and zeta potential (B) of calcium-encapsulated emulsions with different xanthan gum addition ratios.

[0059] Figure 22 Photographs (A) and lumisizer curves (B) of calcium-encapsulated emulsions with different xanthan gum addition ratios after lumisizer stability tests.

[0060] Figure 23 A graph showing the relationship between viscosity and shear rate for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0061] Figure 24 Relationship between oscillation frequency and G' and G'' for calcium-encapsulated emulsions with different xanthan gum addition ratios;

[0062] Figure 25 Microstructure diagrams of whey protein concentrate, calcium-encapsulated emulsions with different xanthan gum addition ratios, and milk mineral salts.

[0063] Figure 26 Photographs after stability tests of different calcium-encapsulated emulsions lumisizers as carriers;

[0064] Figure 27 Photographs after thermal stability testing of calcium-embedded emulsions with different carriers;

[0065] Figure 28 Photographs after stability tests of different calcium-encapsulated emulsions lumisizers as carriers;

[0066] Figure 29 Stability test results of lumisizers with different calcium-encapsulated emulsions as carriers;

[0067] Figure 30 These are photographs of four colloidal solutions. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0069] This invention provides a carrier composition for calcium-embedded emulsions that helps improve bone density, comprising, by weight: a hydrophilic emulsifier and a colloidal composition, wherein the colloidal composition contains colloidal component A and colloidal component B;

[0070] The viscosity of the colloidal component A aqueous solution with a mass fraction of 0.5% was tested using a No. 64 rotor at a speed of 50 rpm and a temperature of 25°C and the result was 4000-4300 mPa•s.

[0071] The viscosity of the colloidal component B aqueous solution with a mass fraction of 0.5% was tested using a No. 64 rotor at a speed of 50 rpm and a temperature of 25°C and the result was 800-900 mPa•s.

[0072] The carrier composition in this application has a suitable viscosity. When applied to calcium-encapsulated emulsions, it effectively reduces the particle size of the calcium mineral salt formulation and increases its specific surface area, thereby promoting the dispersion and dissolution of the calcium source in digestive juices and achieving high bioavailability. Furthermore, the carrier composition acts at the interface between the oil-solid phase and the aqueous phase, enhancing the stability of the calcium source in the digestive environment and improving its contact efficiency with gastric juices, thus facilitating the dissolution of more calcium. 2+The release of calcium salts. Furthermore, some peptide-calcium conjugates may slow the formation of calcium salt precipitation under neutral conditions in the small intestine, thereby reducing calcium salt loss and further improving calcium bioavailability and bone density.

[0073] It should be noted that in this application, the viscosities of the aqueous solutions of colloidal component A and colloidal component B were tested using a Brookfield rotational viscometer, and the aqueous solution of the carrier composition was tested using a Thermo Fisher Hacker rotational rheometer.

[0074] For example, the hydrophilic emulsifier in the calcium-encapsulated emulsion carrier composition of this application that helps improve bone density is selected from whey protein concentrate;

[0075] And / or, the colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum;

[0076] And / or, the mass ratio of colloidal component A to colloidal component B in the colloidal composition is 1:(0.1-10), for example 1:0.1, 1:0.3, 1:0.5, 1:0.6, 1:0.9, 1:1.1, 1:1.2, 1:2.3, 1:3.4, 1:4.5, 1:5.6, 1:6.7, 1:7.8, 1:8.9, 1:10;

[0077] And / or, the mass ratio of the hydrophilic emulsifier to the colloidal composition in the carrier composition is (1-30):1, for example 1:1, 4.2:1, 7.4:1, 10.6:1, 13.8:1, 17:1, 20.2:1, 23.4:1, 26.6:1, 30:1.

[0078] This invention also provides a calcium-encapsulated emulsion that helps improve bone density, comprising a calcium source, an oil, a lipophilic emulsifier, and the carrier composition described in the foregoing embodiments;

[0079] The viscosity of the calcium-encapsulated emulsion was tested using a CC27 rotor at 50 rpm and 25°C. The viscosity was 200-500 mPa•s, for example, 200 mPa•s, 250 mPa•s, 300 mPa•s, 350 mPa•s, 400 mPa•s, 450 mPa•s, and 500 mPa•s, preferably 300-500 mPa•s, and more preferably 400-500 mPa•s.

[0080] In the calcium-encapsulated emulsion that helps improve bone density, the mass fraction of the calcium source is 1% to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; and the mass fraction of the oil is 5% to... 15%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%; lipophilic emulsifiers have a mass fraction of 0.5%-2%, for example, 0.5%, 1%, 1.5%, 2%; hydrophilic emulsifiers have a mass fraction of 1%~3%, for example, 1%, 1.5%, 2.0%, 2.5%, 3%; gellan gum has a mass fraction of 0.05%~0.5%, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%; xanthan gum has a mass fraction of 0.05%~0.5%, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%.

[0081] Experiments have shown that the calcium-encapsulated emulsion in this application, which helps improve bone density, can effectively improve calcium absorption efficiency and bioavailability, promote bone synthesis and metabolism, and during calcium supplementation, it is not easy to form insoluble calcium soaps with fatty acids, and it almost does not cause adverse reactions such as constipation.

[0082] In an optional embodiment, the calcium-encapsulated emulsion that helps improve bone density includes an aqueous phase and an oil phase encapsulated in the aqueous phase, wherein a calcium source is dispersed in the oil phase.

[0083] In an optional embodiment, the calcium source includes at least one of milk mineral salts, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate. Among them, milk mineral salts are natural mineral concentrates extracted from milk. They are not a single salt, but a natural complex with calcium and phosphorus as the main components, while also containing a variety of other trace minerals. Their application in dairy products is more conducive to improving consumer acceptance.

[0084] In an optional embodiment, the lipophilic emulsifier includes fatty acid ester emulsifiers and / or phospholipids.

[0085] In an optional embodiment, the lipophilic emulsifier includes one or more of glyceryl monostearate, mono- and diglycerides of fatty acids, sucrose fatty acid esters, phospholipids, polyglycerol fatty acid esters, and diacetyl tartaric acid mono- and diglycerides.

[0086] This invention also provides a calcium-containing composition, obtained by drying the calcium-encapsulated emulsion that helps improve bone density as described in any of the foregoing embodiments. Specifically, the drying method can be spray drying, freeze-drying, etc.

[0087] The present invention also provides a method for preparing a calcium-encapsulated emulsion that helps improve bone density as described in any of the foregoing embodiments, such as... Figure 1 As shown, it includes the following steps:

[0088] The carrier composition was heated and mixed in water to obtain an aqueous phase;

[0089] The calcium source was heated and mixed with oil and a lipophilic emulsifier to obtain an oil-solid phase.

[0090] The oil-solid phase is mixed with the aqueous phase and then emulsified to obtain the calcium-encapsulated emulsion that helps improve bone density.

[0091] In an optional embodiment, the mass fraction of the hydrophilic emulsifier in the aqueous phase is 1.5%-2.5%, for example 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%; the mass fraction of gellan gum is 0.05%-0.5%, for example 0.05%~0.5%, for example 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%; and the mass fraction of xanthan gum is 0.05%-0.5%, for example 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%.

[0092] In an optional embodiment, the mass ratio of the calcium source to the oil is 1:(1~10), for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0093] In an optional embodiment, the mass ratio of the oil-solid phase to the aqueous phase is (6~27):(94~73), for example, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:80, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73. Increasing the proportion of the oil-solid phase is beneficial for increasing the calcium content in the emulsion.

[0094] In an optional embodiment, the carrier composition is heated and mixed in water at a temperature of 40°C to 80°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C; and for a time of 10 min to 60 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0095] In an optional embodiment, the temperature for heating and mixing the calcium source with the oil and lipophilic emulsifier is 40°C to 70°C, for example, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, or 70°C; and the time is 10 min to 60 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0096] In an optional implementation, emulsification includes sequential high-speed shearing and homogenization.

[0097] In an optional embodiment, the high-speed shearing speed is 10,000 rpm to 20,000 rpm, for example, 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 rpm, 16,000 rpm, 17,000 rpm, 18,000 rpm, 19,000 rpm, or 20,000 rpm.

[0098] In an optional implementation, the high-speed shearing time is 1 min to 5 min, for example 1 min, 1.4 min, 1.8 min, 2.2 min, 2.6 min, 3.0 min, 3.4 min, 3.8 min, 4.2 min, 4.6 min, and 5 min.

[0099] In an optional embodiment, the homogenization pressure is 20MPa to 40MPa, for example 20MPa, 22MPa, 24MPa, 26MPa, 28MPa, 30MPa, 32MPa, 34MPa, 36MPa, 38MPa, or 40MPa.

[0100] The present invention also provides a dairy product comprising a calcium-encapsulated emulsion that helps improve bone density as described in any of the foregoing embodiments, or a calcium-containing composition as described in the foregoing embodiments.

[0101] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0102] Example 1

[0103] This embodiment provides a method for preparing a calcium-embedded emulsion that helps improve bone density, specifically including the following steps:

[0104] The carrier composition was mixed in water at 60°C for 30 min to obtain an aqueous phase, wherein the mass fraction of whey protein concentrate (whey protein content 80%) in the aqueous phase was 2%, the mass fraction of gellan gum was 0.1%, and the mass fraction of xanthan gum was 0.5%.

[0105] The emulsion mineral salt was mixed with oil and lipophilic emulsifiers mono- and diglycerides of fatty acids at a mass ratio of 2:6:1 and heated to 55°C to obtain an oil-solid phase.

[0106] The oil-solid phase and the aqueous phase were mixed at a mass ratio of 1:5, and then subjected to high-speed shearing and homogenization in sequence to obtain the calcium-embedded emulsion that helps improve bone density; wherein, the high-speed shearing speed was 15000 rpm; the high-speed shearing time was 3 min; and the homogenization pressure was 30 MPa.

[0107] Experimental Example 1: Evaluation of Calcium Absorption Efficacy

[0108] This application systematically investigates the effects of a calcium-encapsulated emulsion that helps improve bone density on calcium metabolism, bone microstructure, and fecal excretion in rats by constructing an ovariectomized induced rat osteoporosis model. The aim is to elucidate its calcium utilization-promoting mechanism from both absorption and functional dimensions, provide a theoretical basis for the study of calcium bioavailability regulation, and lay a theoretical foundation for the development of efficient and safe calcium fortification strategies.

[0109] 1. Experimental materials

[0110] 1.1 Laboratory Animals

[0111] The SPF-grade 3-month-old female SD rats used in this application were the research subjects, a total of 80 rats, all purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (jing) 2013-0038.

[0112] 1.2 Main Reagents

[0113] Feed was purchased from Beijing Huafukang Biotechnology Co., Ltd.; reagent kits for serum calcium, serum phosphorus, bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), type I collagen N-terminal cross-linked peptide (NTX) and type I collagen C-terminal cross-linked peptide (CTX) were purchased from Nanjing Jiancheng Bioengineering Institute; reagent kits for type I procollagen N-terminal propeptide (PINP) and osteocalcin (BGP) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0114] 2. Experimental Methods

[0115] 2.1 Preparation of animal models

[0116] Eighty SPF-grade female SD rats were randomly divided into two groups after one week of acclimatization: a sham-operated group (Sham group, n=10) and a model group (n=70). In the sham-operated group, the skin and peritoneum of the rats were cut open, some fat around the ovaries was removed, and the wound was sutured back together. In the model group, ovariectomy was performed to establish an osteoporosis model.

[0117] 2.2 Grouping of experimental animals

[0118] Seventy ovariectomized rats were randomly divided into the following seven groups (n=10): model group (OVX), positive control group (ALN, alendronate sodium, daily gavage dose (calculated as alendronate sodium) 1 mg / kg), calcium carbonate group (CaCO3, daily gavage dose (calculated as elemental calcium) 133.3 mg / kg), low-dose milk calcium group (MMS-L, milk mineral salt, daily gavage dose (calculated as elemental calcium) 66.65 mg / kg), and high-dose milk calcium group (MM). S-H, milk mineral salt, daily gavage dose (calculated as elemental calcium) of 133.3 mg / kg; low-dose encapsulated milk calcium group (MFEs-L, calcium-encapsulated emulsion prepared in Example 1 that helps improve bone density, daily gavage dose (calculated as elemental calcium) of 66.65 mg / kg); and high-dose encapsulated milk calcium group (MFEs-H, calcium-encapsulated emulsion prepared in Example 1 that helps improve bone density, daily gavage dose (calculated as elemental calcium) of 133.3 mg / kg).

[0119] 2.3. Intervention and treatment of test substance

[0120] After successful model establishment, each group received intervention according to the corresponding test substance. The dosage was determined with reference to the "Chinese Dietary Reference Intakes (2023 Edition)" and the "Technical Guidelines for Functional Testing and Evaluation of Health Foods (2023 Edition)".

[0121] 2.4 Calcium Absorption Test

[0122] Calcium metabolism experiments were conducted 3 days prior to sacrifice. Rats were housed individually in metabolic cages, and daily food intake was accurately recorded. Fecal and urine samples were collected over 12 hours. After acid digestion, the calcium content of the samples was determined using inductively coupled plasma optical emission spectrometry (ICP-OES), and the apparent calcium uptake and retention rates were calculated.

[0123] Calcium absorption rate (%) = (calcium intake - fecal calcium) / calcium intake × 100%;

[0124] Calcium retention rate (%) = (calcium intake - fecal calcium - urinary calcium) / calcium intake × 100%.

[0125] 2.5. Intestinal function evaluation

[0126] After fasting for 12 hours, rats were administered 2 mL of ink via gavage and housed individually in metabolic cages. The time of the first defecation and the time of the first black feces were recorded. Fecal samples were collected to determine dry and wet weights, and the fecal water content was calculated.

[0127] Fecal moisture content (%) = (wet weight - dry weight) / wet weight × 100%;

[0128] The animals were euthanized 30 minutes after gavage, and the small intestine was immediately removed. The distance the ink was propelled and the total length of the small intestine were measured, and the propulsion rate was calculated.

[0129] Ink propulsion rate (%) = ink propulsion distance / total length of small intestine × 100%.

[0130] 2.6 Bone mineral density and bone microstructure analysis

[0131] The distal right femur was scanned using Micro-CT with the following parameters: voltage 90 kV, current 88 μA, field of view 86 mm, high resolution mode, scan time 4 minutes, and filter thickness 0.1 mm. The accompanying analysis software (Analysis 12) was used to perform three-dimensional reconstruction and quantitative analysis of the trabecular microstructure parameters within the region of interest.

[0132] 2.7 Bone biomechanical performance testing

[0133] Bone biomechanics was measured using a texture analyzer with a three-point bending probe. The right femur was placed flat on two supporting horizontal plates, and the flat probe was positioned directly above the middle of the femur, 2 cm away from the femur, to perform a three-point bending test.

[0134] 2.8 Determination of bone calcium content

[0135] The femoral bone sample was dried to constant weight, and 10 mL of mixed digestive acid (nitric acid: perchloric acid = 4:1, v / v) was added. The sample was heated on a hot plate until it became clear and transparent. After cooling, the sample was brought to a final volume and diluted appropriately before the calcium content was determined by ICP-OES.

[0136] 2.9 Organ Index Measurement

[0137] Wash the uterus, kidneys, liver, and other organs with saline solution to remove blood stains, dry them, and weigh them. Calculate the ratio of each organ's weight to the body weight.

[0138] Uterine index (%) = Uterine mass / Rat weight × 100%;

[0139] Kidney index (%) = (Weight of both kidneys / Weight of rat) × 100%;

[0140] Liver index (%) = liver mass / rat weight × 100%.

[0141] 2.10 Statistical Analysis

[0142] Experimental data are expressed as mean ± standard deviation (mean ± SD) and statistical analysis was performed using Origin 2022 and GraphPadPrism 8.0 software. One-way ANOVA was used for comparisons among multiple groups, with significance levels set at * P < 0.05 (significant difference) and ** P < 0.01 (extremely significant difference).

[0143] 3 Results and Analysis

[0144] 3.1 Modeling

[0145] After successful bilateral ovariectomy in rats, estrogen levels decreased significantly, leading to changes in uterine morphology and structure. Dissection at the end of the experiment revealed significant uterine atrophy in the ovariectomized group compared to the sham-operated group. Figure 2 The results showed that changes in postoperative serum estradiol (E2) levels served as an important marker for successful model establishment. Compared with the sham-operated group, serum estradiol levels in rats in each ovariectomy group were significantly lower ( P <0.01). The above results indicate that the osteoporosis animal model was successfully established, meeting the requirements for subsequent intervention experiments with the test substance.

[0146] 3.2. Weight

[0147] In this application, changes in rat body weight were recorded during model establishment and treatment with the test substance. Rats were weighed weekly to continuously monitor their overall health. Results are as follows: Figure 3 As shown, the body weight of rats in the ovariectomized group was significantly increased compared to the sham-operated group (Sham). P The value was <0.05%, a phenomenon associated with decreased estrogen levels, reduced insulin sensitivity, and enhanced hepatic gluconeogenesis and lipogenesis after ovariectomy, further confirming the successful establishment of the osteoporosis model. No significant toxic reactions or growth inhibition were observed in any of the experimental groups, indicating that the test substance has good in vivo safety under experimental conditions.

[0148] The weight gain rates for MMS-L and MMS-H were 7.81% and 4.79%, respectively, while those for MFEs-L and MFEs-H were 12.11% and 8.23%, respectively. The overall weight gain of the encapsulated milk calcium (MFEs) group was higher than that of the unencapsulated milk calcium group (MMS). Specifically, the weight gain rate of the MFEs-L group was 3.88% higher than that of the MFEs-H group, and the weight gain rate of the low-dose encapsulated milk calcium was as high as 7.32% compared to the high-dose naked calcium (MMS-H). These results indicate that encapsulation treatment helps improve the bioavailability of calcium from milk mineral salts, promoting bone growth and overall development in rats. It also demonstrates that calcium bioavailability is not simply proportional to the supplementation dose; in practical applications, the dosage form and dose effect must be comprehensively considered to rationally select calcium fortification strategies.

[0149] 3.3. Rat organ index

[0150] As shown in Table 1, ovariectomy in rats resulted in decreased estrogen secretion, leading to significant atrophy of the fallopian tubes and uterus, and a marked reduction in organ weight. Compared to the sham-operated group, the uterine index of the ovariectomized rats was significantly lower. P <0.05). After intervention, the uterine index of each MMS group and MFEs group did not show significant changes compared with the OVX group, indicating that the calcium-embedded emulsion, which helps improve bone density, did not have a significant stimulating effect on uterine tissue.

[0151] Regarding hepatic and renal safety, compared with the OVX group, there were no significant changes in the renal index of each MMS and MFEs treatment group, and no significant difference was found in the liver index of the low-dose embedded calcium emulsion group (MFEs-L). This indicates that the calcium-embedded emulsion that helps improve bone mineral density did not cause abnormal changes in liver and kidney function-related indicators, reflecting that the delivery system has low toxicity and good biosafety, providing in vivo safety evidence for its further application in promoting calcium absorption.

[0152] Table 1. Organ Index of Rats

[0153]

[0154] Data are expressed as mean ± standard deviation (mean ± SD) for 6 rats in each group. #p<0.05, ##p<0.01: difference compared with the normal group (# indicates statistical significance, ## indicates extremely statistical significance); *p<0.05, **p<0.01: difference compared with the model group (* indicates statistical significance, ** indicates extremely statistical significance).

[0155] 3.4 Evaluation of calcium absorption

[0156] 3.4.1 Blood calcium concentration

[0157] like Figure 4 As shown, this application compared the dynamic changes in serum calcium concentration in rats over 0–24 hours after daily gavage administration of calcium carbonate (CaCO3), milk calcium (MMS), and milk calcium encapsulated in a S / O / W system (MFEs), all at a dose of 133.3 mg / kg of elemental calcium. The results showed that serum calcium concentration in all groups increased over 0–6 hours, reaching a peak at hour 6. The encapsulated MFEs group exhibited the fastest rate of increase in serum calcium, with a peak concentration significantly higher than the other two groups: 4.46% higher than the MMS group and 7.26% higher than the CaCO3 group. These results indicate that the S / O / W encapsulation system improves the solubility and release characteristics of milk calcium, making it more readily absorbed in the intestines and thus enabling it to enter the bloodstream more quickly, thereby improving calcium bioavailability.

[0158] 3.4.2 Calcium absorption rate and retention rate in rats

[0159] like Figure 5 As shown, compared with the sham group, the calcium absorption rate of ovariectomized rats did not change significantly, indicating that ovariectomy itself did not have a significant negative impact on the calcium metabolism capacity of rats. After supplementation with different calcium agents, the calcium absorption rate of all groups showed an increasing trend. Among them, the calcium absorption rate of milk calcium (MMS) and milk calcium embedded in S / O / W (MFEs) was higher than that of the calcium carbonate group. Compared with the Sham group and the OVX group, the calcium absorption rate of the MMS group was significantly improved, and the effect of the low-dose group (MMS-L) was more obvious, increasing by 11.27%, indicating that milk calcium has an advantage in promoting calcium absorption. There was no significant difference in absorption rate between the MMS-L (59.61%) group and the MMS-H (58.20%) group, proving that there may not be a significant dose-dependent relationship for milk calcium. After exceeding a certain threshold, further increasing the dose may not necessarily enhance the calcium absorption effect.

[0160] The calcium absorption rate in the MFEs-L group was 5.65% higher than that in the MFEs-H group, while the absorption rate in the MMS-L group was only 1.41% higher than that in the MMS-H group. This demonstrates that encapsulation may have improved the dispersibility of calcium ions in the intestinal lumen, alleviating the dose-dependent decline in calcium absorption and preventing supersaturation excretion. The overall calcium absorption rate in the MFEs group was higher than that in the MMS group, with the MFEs-L group showing a significantly higher rate than the other groups. The MFEs-H group also showed a further increasing trend, with encapsulated milk calcium increasing by 8.05% compared to naked calcium. This indicates that S / O / W emulsion encapsulation technology helps improve the release and absorption of calcium from milk mineral salts, showing good potential in enhancing calcium bioavailability in ovariectomized rats. These results are consistent with previous studies on the conclusion that encapsulation systems can improve calcium bioavailability.

[0161] like Figure 6As shown, compared with the sham-operated group (Sham), the calcium retention rates of rats in the ovariectomized group (OVX) and the alendronate sodium positive control group (ALN) were both decreased. After supplementation with different calcium sources, the calcium retention rates of all groups showed an increasing trend. Among them, the calcium retention rates of the MMS-L and MMS-H groups were significantly higher than those of the OVX group, indicating that milk calcium is more conducive to calcium absorption and retention than traditional calcium supplementation methods.

[0162] The calcium retention rates in the MFEs-L and MFEs-H groups were significantly higher than those in the Sham and OVX groups, with the MFEs-L group showing a significantly higher retention rate than the MMS-L and MMS-H groups. Specifically, the calcium retention rate in the MFEs-L group was 5.42% higher than that in the MFEs-H group, while the retention rate in the MMS-L group was only 0.56% higher than that in the MMS-H group. This demonstrates that calcium-encapsulated emulsions, which help improve bone mineral density, increase the in vivo calcium retention rate. This system can effectively enhance the dispersibility of calcium ions in the gastrointestinal tract, reduce their aggregation, thereby improving calcium bioavailability and absorption efficiency, ultimately promoting calcium retention in the body. The changing trends of calcium retention rates in each MFE group are consistent with previous research findings, further validating the positive effect of encapsulation treatment on enhancing the potency of calcium fortifiers.

[0163] 3.5 Evaluation of skeletal condition

[0164] 3.5.1 Bone calcium content

[0165] like Figure 7 As shown, compared with the sham group, the ovariectomized rats had significantly lower bone calcium content, indicating a sharp decrease in estradiol levels and accelerated bone calcium loss after ovariectomy. This change may be closely related to bone metabolism disorders caused by decreased estrogen levels.

[0166] After supplementation with different calcium sources, bone calcium content in all groups showed varying degrees of recovery. Among them, the high-dose encapsulated milk mineral salt group (MFEs-H) showed a significantly higher bone calcium content (14.15%) than the OVX group, and was also higher than the equivalent doses of ordinary milk calcium (MMS) and calcium carbonate (CaCO3) groups. The results indicate that S / O / W encapsulation treatment improves the dispersibility and solubility of milk calcium in the gastrointestinal tract, increases calcium bioavailability, and thus promotes the deposition and accumulation of calcium ions in bone tissue.

[0167] 3.5.2 Bone length and bone diameter

[0168] like Figure 8As shown in Figure A, the femur length in the sham-operated group (Sham) was used as a normal baseline. The femur length in the ovariectomized group was significantly shorter than that in the Sham group, indicating that ovariectomy inhibited the longitudinal growth of the rat femur, possibly related to the decreased osteogenic activity caused by estrogen deficiency. After supplementation with milk mineral salts, the femur length in both the MMS and MFEs groups recovered compared to the OVX group, indicating that milk mineral salts can promote the longitudinal growth of bones in ovariectomized rats to some extent. Among them, the low-dose embedded group (MFEs-L) showed a more significant effect in improving femur length, with an average increase of 0.19 mm compared to the same dose of unembedded naked calcium (MMS-L), indicating that the bioavailability of calcium was improved after S / O / W embedding treatment, which is more conducive to the recovery of bone structure.

[0169] like Figure 8 As shown in Figure B, the femur diameter in the Sham group represents the normal developmental state, while the diameter in the OVX group is significantly reduced, suggesting that ovariectomy not only affects bone length but also impairs the transverse development of bone structure, potentially leading to osteoporosis and decreased mechanical properties. After intervention with milk calcium and embedded milk calcium, the femur diameter in both the MMS and MFEs groups increased compared to the OVX group, with the embedded group (MFEs) showing a more significant effect, with an average diameter increase of 0.02 mm compared to the MMS group. This indicates that milk calcium supplementation, especially after embedding, helps improve bone structure and increase bone density, thereby promoting the recovery of femur morphology and mechanical function. The trends in the MFEs-L and MFEs-H groups are also consistent with previous studies.

[0170] 3.5.3 Bone Biomechanics

[0171] like Figure 9 As shown, the biomechanical properties of the femur and tibia were measured, and the maximum load and maximum stress in the ovariectomy group were significantly lower than those in other experimental groups, indicating that ovariectomy leads to a severe decline in bone mechanical properties. Compared with the OVX group, the maximum load of the rat femur increased by 25.94% after MFEs-L intervention; MFEs-L increased by 9.69% compared with MMS-L. Among them, the maximum load of the low-dose embedding group (MFEs-L) was even significantly higher than that of the sham surgery group (Sham), and the maximum load and maximum stress of other groups were basically restored to or better than the level of the normal control group.

[0172] Maximum load reflects the biological strength of bone against fracture, while maximum stress is closely related to bone mineral density and microstructural integrity; both are key indicators for evaluating bone mechanical properties and osteoporosis risk. The results of this application indicate that milky calcium (MFEs) embedded in S / O / W shells can more effectively enhance bone load-bearing capacity. The mechanism may be related to the embedding technique improving calcium bioavailability and promoting directional calcium deposition and microstructural reconstruction in bone tissue. Therefore, MFEs show significant advantages in improving osteoporosis and enhancing bone mechanical properties in ovariectomized rats, and have good application potential.

[0173] 3.5.4 Micro CT

[0174] Three-dimensional images of bones are obtained through techniques such as 3D reconstruction, and quantitative analysis of the bone's microstructure is performed. For example... Figure 10 The image shows three-dimensional reconstructions of the femurs of mice in each group. In the OVX group, the trabeculae were sparse, fine, and unevenly arranged, with large areas of trabecular bone marrow absent, indicating significant damage to the bone microstructure. Compared to the model group, the CaCO3, ALN, MMS, and MFEs groups showed reduced bone microstructure damage, specifically manifested as more continuous trabecular connections, increased trabecular number, and reduced trabecular spacing. Among these, the MFEs-L group exhibited a more uniform and orderly arrangement of trabeculae, with an increased number of trabeculae. Cross-sectional scans of the femur showed a significant reduction in the proportion of the central black hollow area, which is attributed to trabecular growth. This demonstrates that this system may effectively enhance bone microstructure reconstruction and calcium deposition by promoting calcium absorption and utilization.

[0175] 3.5.5 Bone mineral density

[0176] like Figure 11 As shown, bone mineral density (BMD) in rats directly reflects bone strength, and BMD examination can help diagnose osteoporosis. Compared with the Sham group, the OVX group showed significantly lower BMD. The MMS-H and MFEs-L groups showed significantly higher BMD than the OVX group, with the MMS-H group showing an increase of 18.35% and the MFEs-L group showing an increase of 26.81%. The low-dose encapsulated milk calcium showed better BMD than the high-dose naked calcium, demonstrating that this encapsulation system is beneficial for improving calcium absorption efficiency. In conclusion, encapsulated milk calcium can enhance bone mineral density in model rats and improve bone loss in ovariectomized osteoporotic rats.

[0177] 3.6 Fecal index

[0178] 3.6.1 Fecal moisture content

[0179] like Figure 12As shown, fecal water content in the Sham group was at baseline, while the OVX group had slightly higher water content, demonstrating that ovariectomy significantly increased fecal water content in rats. This is attributed to the sharp decline in estrogen levels, leading to accelerated intestinal peristalsis and shortened transit time, resulting in increased fecal water content. In the ALN and CaCO3 groups, fecal water content was significantly lower than in the OVX group, suggesting that long-term calcium supplementation under these conditions may increase the burden of mineral metabolism and the risk of constipation. In the MMS and MFE groups, fecal water content showed a gradual increasing trend, and overall levels were closer to those of the Sham group. Compared to encapsulated milk calcium, the same dose of naked calcium resulted in higher fecal water content after encapsulation. This may be related to the polysaccharides and proteins in the loading system, which help maintain normal fecal water content and reduce the risk of constipation by improving the intestinal microenvironment and regulating calcium ion absorption and metabolism.

[0180] 3.6.2 Ink migration rate

[0181] like Figure 13 As shown, compared with sham surgery, the intestinal ink migration rate of ovariectomized rats was significantly reduced. Supplementation with CaCO3 and (high and low) concentrations of milk calcium did not significantly increase the ink migration rate. However, supplementation with low concentrations of encapsulated milk calcium increased the intestinal migration rate, bringing it to a level similar to that of the normal group. This may be because encapsulation enhances calcium absorption and restores smooth muscle contraction; the encapsulation material helps improve intestinal flora diversity and may further promote intestinal peristalsis through metabolites. Therefore, supplementation with encapsulated milk calcium is more conducive to intestinal peristalsis.

[0182] 4. Conclusion

[0183] This experiment mainly evaluated the absorption of calcium-encapsulated emulsions that help improve bone density. Using ovariectomized rats as a model, eight groups were divided into Sham, OVX, ALN, CaCO3, milk calcium (high and low doses), and encapsulated milk calcium (high and low doses) for evaluation. The results were analyzed from multiple dimensions, including rat blood calcium, absorption and retention rate, bone condition, and fecal index. The results showed that (1) in the 0–24 hour blood calcium concentration monitoring, the blood calcium growth rate of the MFEs group was significantly higher than that of other groups, and the MFEs group increased by 4.46% compared with the MMS group, indicating that the encapsulation system can promote the rapid dissolution and absorption of calcium; (2) in terms of apparent calcium absorption rate and in vivo retention rate, the MFEs-L group performed particularly well, with the absorption rate of the MFEs group being 8.05% higher than that of the MMS group; (3) skeletal morphology and biomechanical analysis showed that after MFEs-L intervention, femoral bone density increased by 26.81%, bone diameter increased and mechanical strength increased by 25.94%, indicating that the encapsulated milk calcium has the effect of alleviating bone loss. (4) In terms of fecal metabolic indicators, no constipation was observed in the group with encapsulated calcium milk. The water content and morphology of their feces were within the normal range, which increased intestinal lubrication. This may be related to the particle size distribution of the emulsion in the S / O / W system and the intestinal lubrication and calcium release regulation brought about by lipids and hydrophilic colloids (such as polysaccharides and proteins).

[0184] In conclusion, this experiment demonstrates that calcium-encapsulated emulsions, which help improve bone density, can enhance the dispersibility and stability of calcium in the intestine, increase the accessible concentration of calcium ions in the intestinal lumen, and may further promote the uptake and transport of calcium by intestinal epithelial cells by improving the local intestinal environment and calcium transfer efficiency, thereby significantly improving calcium bioavailability and bone deposition efficiency.

[0185] Experimental Example 2: The effect of xanthan gum mass fraction in aqueous phase on the properties of calcium-encapsulated emulsions that help improve bone mineral density.

[0186] Based on Example 1, this experimental example adjusted the mass fraction of xanthan gum in the aqueous phase to obtain aqueous phases with xanthan gum mass fractions of 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, respectively, and obtained corresponding calcium-encapsulated emulsions that help improve bone density.

[0187] 1. Sterilization stability test

[0188] The above-mentioned calcium-encapsulated emulsions, which help improve bone density, were subjected to sterilization stability tests using a water bath. The results are as follows: Figure 14As shown, after pasteurization at 63℃ for 30 min, the emulsion showed no instability and maintained a good appearance. The physical stability of the calcium-embedded emulsion, which helps improve bone mineral density, was determined using a LUMiSizer stability analyzer. The parameters were: operating temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; 255 scan lines; time interval 60 s; total duration 255 min. The LUMiSizer test results were similar to those of the untreated emulsion. After high-temperature sterilization at 90℃ for 3 min, flocculation and water separation were clearly visible to the naked eye, but these phenomena improved with increasing xanthan gum content. Furthermore, the emulsions with 0% and 0.1% xanthan gum (XG) addition showed irregular upper boundaries in the lower portion after LUMiSizer testing, which is likely due to thermal denaturation of the emulsion.

[0189] The sterilization stability of calcium-embedded emulsions was tested using a water bath. The S, O, and W phases of the samples after the sterilization stability test were stained with Rhodamine B, Nile Red, and Nile Blue A, respectively, at excitation wavelengths of 543 nm, 488 nm, and 635 nm. The stained samples were vortexed for 10 s and equilibrated for 12 h. The microscopic images were then processed and analyzed using a confocal laser scanning microscope (CLSM) (FV 3000, Olympus Corporation, Japan) under an oil immersion lens at 60x magnification. Figure 15 As shown, calcium-encapsulated emulsions with a low xanthan gum content exhibit poor stability during heat treatment, with larger and more unevenly distributed droplets and severe aggregation. With increasing xanthan gum content, the bactericidal stability of the emulsion is significantly improved, and the microstructure of the emulsion after heat treatment is optimized.

[0190] 2. Centrifugal stability test

[0191] The stability of the calcium-encapsulated emulsion was tested by centrifugation at 10,000 rpm for 20 minutes, and the results are as follows: Figure 16 As shown, with the increase of xanthan gum ratio, the precipitation at the bottom of the emulsion gradually decreased, and no other significant instability was observed except for precipitation.

[0192] 3. Freeze-thaw stability test

[0193] The freeze-thaw stability of calcium-encapsulated emulsions was tested at -20°C. Specifically, the freshly prepared emulsion was frozen at -20°C for 24 h, and then thawed at 25°C for 2 h, completing one freeze-thaw cycle. The samples after freeze-thaw treatment are shown below. Figure 17 As shown, freeze-thaw treatment can cause water separation and flocculation in the emulsion, which may be caused by the denaturation of whey protein in the emulsion or changes in the crystal structure of butter. However, these phenomena can be significantly improved with the increase of xanthan gum ratio.

[0194] The freeze-thawed samples were subjected to LUMiSizerr stability testing with the following parameters: operating temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; 255 scan profiles; time interval 60 s; total duration 255 min. The results are as follows: Figure 18 As shown, the overall stability of the emulsion decreases with increasing freeze-thaw cycles. However, when the xanthan gum content is ≥0.4%, the stability of the emulsion changes little after three freeze-thaw cycles. In particular, the emulsion prepared with 0.5% XG did not show any shift in the LUMiSizer transmission curve after three freeze-thaw cycles, indicating good stability.

[0195] This application confirms that the addition of xanthan gum can effectively improve the freeze-thaw stability of emulsions, which provides theoretical support for the application of calcium-encapsulated emulsions in frozen products such as ice cream.

[0196] 4. Interface stability test

[0197] The oil-water interface is a crucial factor affecting emulsion stability; the lower the interfacial tension, the more stable the interfacial layer. Interfacial tension tests were performed on the aqueous phase, and the results are as follows: Figure 19 As the proportion of XG increases, the interfacial tension between the aqueous and oil phases gradually decreases. This may be because polysaccharides promote the adsorption of emulsifiers at the oil-water interface and the formation of elastic interfacial films, thereby reducing interfacial tension and improving emulsion stability.

[0198] 5. Fourier transform infrared spectroscopy

[0199] Fourier transform infrared spectroscopy was performed on aqueous phases with different xanthan gum contents, corresponding calcium-encapsulated emulsions that help improve bone density, and emulsion mineral salts. The test results are as follows: Figure 20 As shown. The entire infrared spectrum can be divided into 4000 cm⁻¹. -1 -1300cm -1 The functional group area and 1300cm -1 -600cm -1 The fingerprint area. (3000-3500cm in the image) -1 The high-frequency region represents the stretching vibrations and absorption of -OH groups. Compared to the spectra of emulsion mineral salts, the characteristic peak values ​​in this region of the infrared spectrum of calcium-embedded emulsions shift. This shift is attributed to a change in the number of intermolecular hydrogen bonds, indicating the presence of hydrogen bond interactions within the system. Compared to the individual spectra of emulsion mineral salts and aqueous phases, the emulsion shows a higher peak value at 2925.72 cm⁻¹. -1 Significant changes in peak height at the left and right positions correspond to CH stretching vibrations, possibly originating from the aliphatic chain structure of the oil phase or emulsifier. The peak height is between 1600-1700 cm⁻¹. -1 (Amide I band) belongs to CO2 tensile strength, 1500-1600 cm-1 (Amide II band) represents the -NH bending vibration.

[0200] Observations revealed that after the calcium-encapsulated emulsion was prepared in aqueous phase, the peak of amide I decreased from 1653.35 cm⁻¹. -1 They were moved to 1746.81cm respectively. -1 and 1743.21cm -1 This indicates that the secondary structure of proteins in the emulsion system may have undergone slight adjustments, and the rearrangement of the hydrogen bond network affected the behavior of the -NH bending vibration. The main characteristic peak of the emulsion mineral salts is 1412.54 cm⁻¹. -1 This indicates symmetrical COO-stretching, while in calcium-embedded emulsions, the characteristic peak shifts to 1455.67 cm⁻¹. -1 Milky mineral salts at 1031.55 cm⁻¹ -1 and 560.71cm -1 The peak intensity at a certain point decreases after emulsion formation, corresponding to the CO stretching vibration and the in-plane bending vibration of the aliphatic ketone (CC=O), respectively. The enhancement of hydrogen bonding leads to band broadening, overlap, and shifting in the FTIR spectrum, further indicating that the emulsion mineral salts can be well dispersed in the emulsion system, thereby improving the structural stability of the emulsion.

[0201] 6. Emulsion particle size, zeta potential

[0202] To further improve calcium loading, the influence of viscosity on the stability of solubilized delivery of poorly soluble active ingredients was investigated. The particle size and potential of the calcium emulsion encapsulation system were measured using a Nano-ZS90 laser particle size analyzer. To avoid multiscattering effects, the samples were diluted 400-fold, and the refractive indices of the continuous and dispersed phases were set to 1.33 and 1.45, respectively. Each sample was measured in triplicate. The results are as follows: Figure 21 As shown, the study found that the particle size and absolute value of zeta potential of calcium-encapsulated emulsions with different xanthan gum ratios both increased with the increase of xanthan gum ratio. When the xanthan gum ratio was 0.2%, the zeta potential tended to be stable. With the increase of xanthan gum ratio, the negative charge in the system increased. The negative charge between xanthan gum side chains and the interaction between the side chain nonpolymer backbone significantly improved the physical stability of calcium-encapsulated emulsions.

[0203] 7. Physical stability

[0204] The physical stability of calcium-embedded emulsions that help improve bone mineral density was determined using a LUMiSizer stability analyzer. The parameters were set as follows: operating temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; 255 scan profiles; time interval 60 s; total duration 255 min. The LUMiSizer stability analysis results are as follows: Figure 22As shown, when the calcium-encapsulated emulsion is stabilized solely by gellan gum, the system exhibits significant emulsification. This phenomenon is significantly improved with the increase of xanthan gum content, indicating that the calcium-encapsulated emulsion system has good physical stability when the xanthan gum content is 0.4%.

[0205] 8. Rheological properties

[0206] With increasing shear rate, the apparent viscosity of all samples showed a decreasing trend, such as... Figure 23 As shown, this may be due to the disruption of the spatial network structure of the emulsion system. At a certain shear rate, the apparent viscosity of the calcium-encapsulated emulsion increases with the increase of xanthan gum content, and the viscosity-enhancing effect of the emulsion gradually strengthens. This may be an important factor in improving the stability of high-load calcium-encapsulated emulsions.

[0207] With increasing oscillation frequency, the G' and G'' of calcium-encapsulated emulsions with different xanthan gum addition ratios all showed an increasing trend, such as... Figure 24 As shown, G' and G'' are directly proportional to the xanthan gum addition ratio. In the frequency range of 0.1-100Hz, the G' of the calcium-encapsulated emulsion is higher than that of G'', indicating that the emulsion remains relatively stable under different frequency influences and mainly exhibits elastic behavior.

[0208] 9. Microstructure

[0209] The microstructure of calcium-embedded emulsions, which can improve bone density, was observed using laser confocal microscopy. Specifically, the S, O, and W phases of the calcium-embedded emulsion samples were stained with Rhodamine B, Nile Red, and Nile Blue A, respectively, at excitation wavelengths of 543 nm, 488 nm, and 635 nm. After staining, the samples were vortexed for 10 s and equilibrated for 12 h. The microscopic images were then processed and analyzed using a confocal laser scanning microscope (CLSM) (FV 3000, Olympus Corporation, Japan) under an oil immersion lens at 60x magnification. Figure 25 As shown, the emulsion system became more homogeneous with increasing xanthan gum content. In the sample without xanthan gum, significant aggregation of emulsion mineral salts was observed, along with large, closely spaced oil droplets and indistinct boundaries, indicating a relatively high degree of droplet aggregation. This may be related to the weak electrostatic repulsion and low hydrophobicity between the droplets. These phenomena were significantly improved when the xanthan gum content was 0.4%.

[0210] In conjunction with the effect of xanthan gum addition on emulsion potential, the absolute value of zeta potential in calcium-encapsulated emulsions increases with the increase of xanthan gum ratio, indicating enhanced electrostatic interaction. At the same time, the viscosity effect of the emulsion system is enhanced with the increase of xanthan gum addition. These factors may be important reasons for improving emulsion stability and dispersibility of insoluble emulsion mineral salts.

[0211] Experimental Example 3: The effect of the content of each component of the carrier composition in the aqueous phase on the performance of calcium-embedded emulsions that help improve bone density.

[0212] Based on Example 1, this experimental example replaced whey protein concentrate, xanthan gum, and gellan gum in the aqueous phase or adjusted the mass fraction of the components to obtain a calcium-encapsulated emulsion that helps improve bone density, and conducted the following tests.

[0213] 1. LUMI Stability Analysis

[0214] The physical stability of calcium-embedded emulsions that help improve bone mineral density was determined using a LUMiSizer stability analyzer. The parameters were set as follows: operating temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; 255 scan profiles; time interval 60 s; total duration 255 min. The sample images after LUMiSizer stability analysis are shown below. Figure 26 As shown, under the same whey protein concentrate concentration as in Example 1, the amounts of xanthan gum (GG) and gellan gum (XG) were adjusted. Under the conditions of 0.1%GG-0.4%XG and 0%GG-0.7%XG, no demulsification occurred in the system.

[0215] 2. Thermal stability analysis

[0216] High-temperature treatment at 95°C for 5 minutes was performed on calcium-embedded emulsions that help improve bone density. The results were as follows: Figure 27 As shown, the compounding of colloids can improve the thermal stability of calcium-encapsulated emulsion systems to a certain extent. Under the same whey protein concentrate concentration as in Example 1, adjusting the amounts of xanthan gum and gellan gum to a compounding ratio of 0.1% GG-0.6% XG significantly increased the degree of thermal denaturation of the emulsion. Similarly, in emulsion systems where xanthan gum alone is used as a colloidal stabilizer, the degree of thermal denaturation of the emulsion prepared with 0% GG-0.7% XG is higher than that of the emulsion prepared with 0% GG-0.5% XG.

[0217] 3. LUMiSizer Analysis

[0218] like Figure 28As shown, the high content of emulsion minerals and the increased proportion of gellan gum in the carrier are the main reasons for the water separation phenomenon in the calcium-embedded emulsion system. The physical stability of the calcium-embedded emulsion, which helps improve bone mineral density, was determined using a LUMiSizer stability analyzer. The parameters were set as follows: operating temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; 255 scan profiles; time interval 60 s; total duration 255 min. The LUMiSizer analysis results showed that WPC and WPI had similar effects on the system, and sodium caseinate was not an ideal hydrophilic emulsifier.

[0219] 5. Adjustment of carrier composition components

[0220] The aforementioned studies have confirmed that whey protein concentrate has a better stabilizing effect on the system than casein. This application also explored other reagents to replace whey protein, such as... Figure 29 As shown, 4% Cas (sodium caseinate) and 2% WPC-2% Cas were used to replace the whey protein concentrate in Example 1. However, the emulsion prepared in this way is extremely difficult to homogenize and is prone to clogging the homogenizer. Therefore, whey protein is a better choice as a carrier composition. In addition, mono- and diglyceride fatty acid esters (monoglycerides) are used as lipophilic emulsifiers to stabilize the oil and aqueous phase systems. However, during the dissolution process, the oil phase temperature needs to be raised to 70°C to dissolve.

[0221] Test Example 4

[0222] In this experiment, the viscosity of xanthan gum aqueous solution, guar gum aqueous solution, pectin aqueous solution, and carrageenan aqueous solution with a mass fraction of 0.25% was tested at 25°C. The test results are as follows: Figure 30 As shown, the experiment confirmed that the viscosity of the four solutions, from largest to smallest, is xanthan gum aqueous solution, guar gum aqueous solution, carrageenan aqueous solution, and pectin aqueous solution.

[0223] Experimental Example 5

[0224] In this experimental example, the viscosity of 0.5% gellan gum aqueous solution, 0.5% xanthan gum aqueous solution, 0.5% guar gum aqueous solution, 0.5% saffron gum aqueous solution, 0.5% pectin aqueous solution and 0.5% carrageenan aqueous solution were tested at 25℃ according to the national standard GB / T 10247-2008. The test conditions and results are shown in Table 2. Among them, gellan gum and xanthan gum, when used as carriers, can obtain calcium-encapsulated emulsions with high bioavailability. However, the bioavailability of calcium-encapsulated emulsions obtained by using guar gum, saffron gum, pectin and carrageenan alone or in combination with gellan gum or xanthan gum as carriers is far lower than that of Example 1. In particular, the bone density and bone calcium content of rats under the same conditions are far lower than those of Example 1.

[0225] Table 2

[0226]

[0227] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A calcium-encapsulated emulsion that helps improve bone density, characterized in that, This includes compositions of calcium sources, oils, lipophilic emulsifiers, and carriers; The viscosity of the calcium-encapsulated emulsion was tested using a CC27 rotor at 50 rpm and 25°C, and the result was 200-500 mPa. s; The carrier composition includes a hydrophilic emulsifier and a colloidal composition, wherein the colloidal composition contains colloidal component A and colloidal component B; Using a No. 64 rotor at 50 rpm and 25°C, the viscosity of a 0.5% (w / w) aqueous solution of the colloidal component A was measured to be 4000-4300 mPa. s; Using a No. 64 rotor at 50 rpm and 25°C, the viscosity of a 0.5% (by mass) aqueous solution of the colloidal component B was tested and found to be 800-900 mPa. s; The colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum; The mass ratio of the hydrophilic emulsifier to the colloidal composition in the carrier composition is (1-30):1; In the calcium-encapsulated emulsion that helps improve bone density, the mass fraction of the calcium source is 2% to 10%. The hydrophilic emulsifier is selected from whey protein concentrate; The mass fraction of gellan gum is 5 / 6000, and the mass fraction of xanthan gum is 20 / 6000 to 25 / 6000.

2. The calcium-encapsulated emulsion for improving bone density according to claim 1, characterized in that, In the calcium-encapsulated emulsion that helps improve bone density, the mass fraction of oil is 5%~15%, the mass fraction of lipophilic emulsifier is 0.5%~2%, the mass fraction of hydrophilic emulsifier is 1%~3%, the mass fraction of gellan gum is 5 / 6000, and the mass fraction of xanthan gum is 20 / 6000~25 / 6000. And / or, the calcium-encapsulated emulsion that helps improve bone density includes an aqueous phase and an oil phase encapsulated in the aqueous phase, the oil phase including an oil in which a calcium source is dispersed; And / or, the calcium source includes at least one of milk mineral salts, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate; And / or, the lipophilic emulsifier includes fatty acid ester emulsifiers and / or phospholipids.

3. The calcium-encapsulated emulsion for improving bone density according to claim 1, characterized in that, The lipophilic emulsifier includes one or more of mono- and diglycerides of fatty acids, sucrose fatty acid esters, phospholipids, polyglycerol fatty acid esters, and diacetyl tartaric acid mono- and diglycerides.

4. A method for preparing a calcium-encapsulated emulsion that helps improve bone density as described in any one of claims 1-3, characterized in that, Includes the following steps: The carrier composition was heated and mixed in water to obtain an aqueous phase; The calcium source is heated and mixed with the oil and a lipophilic emulsifier to obtain an oil-solid phase. The oil-solid phase is mixed with the aqueous phase and then emulsified to obtain the calcium-encapsulated emulsion that helps improve bone density.

5. The method for preparing a calcium-encapsulated emulsion that helps improve bone density according to claim 4, characterized in that, The aqueous phase contains 1.5%-2.5% hydrophilic emulsifier, 0.1% gellan gum, and 0.4%-0.5% xanthan gum by mass. And / or, the mass ratio of the calcium source to the oil is 1:(1~10); And / or, the mass ratio of the oil-solid phase to the aqueous phase is (6~27):(94~73); And / or, the carrier composition is heated and mixed in water at a temperature of 40°C to 80°C for a time of 10 min to 60 min; And / or, the calcium source is heated and mixed with oils and lipophilic emulsifiers at a temperature of 40℃~70℃ for 10min~60min.

6. The method for preparing a calcium-encapsulated emulsion that helps improve bone density according to claim 5, characterized in that, Emulsification includes sequential high-speed shearing and homogenization, and the emulsification step also satisfies at least one of the following characteristics: a. The rotational speed of the high-speed shearing is 10,000 rpm to 20,000 rpm; b. The high-speed shearing time is 1 min to 5 min; c. The pressure of the homogenizer is 20MPa~40MPa.

7. A calcium-containing composition, characterized in that, It is obtained by drying the calcium-encapsulated emulsion that helps improve bone density as described in any one of claims 1-3.

8. A dairy product, characterized in that, Includes the calcium-encapsulated emulsion that helps improve bone density as described in any one of claims 1-3 or the calcium-containing composition as described in claim 7.

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