Carrier composition for calcium-embedded emulsion helpful for improving bone mineral density and application thereof

Abstract

The invention discloses a calcium-embedded emulsion carrier composition helpful for improving bone mineral density and application of the calcium-embedded emulsion carrier composition. The carrier composition comprises a hydrophilic emulsifier and a colloid composition, the colloid composition comprises a colloid component A and a colloid component B, and the viscosity of an aqueous solution of the colloid component A with the mass fraction of 0.5% is 4000-4300 mPas under the condition of testing at 25 DEG C by adopting a 64 # rotor; and the viscosity of the aqueous solution of the colloid component B with the mass fraction of 0.5% is 800-900 mPas under the condition of 25 DEG C tested by a No.64 rotor. The carrier composition disclosed by the invention is applied to the calcium-embedded emulsion, so that the bioavailability of calcium is improved, and the bone mineral density is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food nutrition fortifier, in particular to a carrier composition for calcium-embedded emulsion for improving bone density and application thereof. BACKGROUND

[0002] With the continuous improvement of people's health awareness, dairy products, as an important source of nutrition in daily diet, 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 an important role in the health of bones and teeth. In the prior art, liquid dairy products such as milk, yogurt and milk beverages have become important carriers for calcium supplementation due to their convenience for drinking and absorption. However, due to the limited solubility of calcium in liquid systems, calcium often exists in the form of microsolubility or insolubility, which is easy to precipitate during storage, affecting the stability and taste of the product.

[0003] In addition, in order to improve the calcium content in dairy products, the prior art usually adds calcium salts such as calcium lactate, calcium carbonate and calcium phosphate to liquid dairy products to achieve calcium fortification. However, these calcium salts have poor dispersibility in liquid systems, which can lead to uneven distribution of calcium in dairy products, affecting product quality. At the same time, the absorption rate of some calcium salts in the gastrointestinal tract is low, which makes it difficult to fully exert the physiological function of calcium supplementation. Therefore, in the prior art, the poor dispersion stability of calcium in liquid dairy products, the low calcium loading efficiency and the limited calcium absorption promoting effect are important factors affecting the quality and nutritional effect of calcium fortified dairy products. In view of this, the present application is proposed. SUMMARY

[0004] The present application aims to provide a carrier composition for calcium-embedded emulsion for improving bone density and application thereof, which is beneficial to improve the stability of calcium-embedded emulsion for improving bone density, thereby improving the calcium loading efficiency and promoting calcium absorption.

[0005] The present application is achieved in the following manner: In a first aspect, the present application provides a carrier composition for calcium-embedded emulsion for improving bone density, which 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, which is tested by a No. 64 rotor at a rotation speed of 50 rpm and 25°C. The viscosity of a 0.5% mass fraction aqueous solution of the colloidal component B is 800-900 mPa•s, which is tested by a No. 64 rotor at a rotation speed of 50 rpm and 25°C.

[0006] In an optional embodiment, the hydrophilic emulsifier is selected from whey protein concentrate; and / or, the colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum; and / or, the mass ratio of the colloidal component A and the colloidal component B in the colloidal composition is 1: (0.1-10); and / or, the mass ratio of the hydrophilic emulsifier and the colloidal composition in the carrier composition is (1-30): 1.

[0007] In a second aspect, the present application provides a calcium-embedded emulsion for improving bone density, comprising a calcium source, a fat, a lipophilic emulsifier, and the carrier composition according to any one of the preceding embodiments. The viscosity of the calcium-embedded emulsion is 200-500 mPa·s, which is measured at 25°C and a rotational speed of 50 rpm using a No. 27 rotor.

[0008] In an optional embodiment, the calcium-embedded emulsion for improving bone density comprises a water phase and an oil phase embedded in the water phase, and the oil phase contains the calcium source.

[0009] In an optional embodiment, the calcium-embedded emulsion for improving bone density comprises a water phase and an oil phase embedded in the water phase, and the oil phase contains the calcium source. and / or, the calcium source comprises at least one of milk mineral salt, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate; and / or, the lipophilic emulsifier comprises fatty acid ester emulsifiers and / or phospholipids.

[0010] In an optional embodiment, the lipophilic emulsifier comprises one or more of glycerol monostearate, mono-, di-glycerol fatty acid ester, sucrose fatty acid ester, phospholipid, polyglycerol fatty acid ester, and diacetyl tartaric acid ester of mono-, di-glycerol.

[0011] In a third aspect, the present application provides a preparation method of the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments, comprising the following steps: heating and mixing the carrier composition in water to obtain a water phase; heating and mixing the calcium source with the fat and the lipophilic emulsifier to obtain an oil-solid phase; mixing the oil-solid phase with the water phase, and obtaining the calcium-embedded emulsion for improving bone density after emulsification.

[0012] In an optional embodiment, the mass fraction of the hydrophilic emulsifier in the aqueous phase is 1.5%-2.5%, the mass fraction of the gellan gum is 0.05%-0.5%, and the mass fraction of the xanthan gum is 0.05%-0.5%; 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).

[0013] In an optional embodiment, the temperature for heating and mixing the carrier composition in water is 40℃-80℃, and the time is 10 min-60 min; And / or, the temperature for heating and mixing the calcium source with the oil and the lipophilic emulsifier is 40℃-70℃, and the time is 10 min-60 min.

[0014] In an optional embodiment, the emulsification comprises high-speed shearing and homogenization performed in sequence, and the emulsification step further satisfies at least one of the following characteristics: a. The rotation speed of the high-speed shearing is 10000 rpm-20000 rpm; b. The time for high-speed shearing is 1 min-5 min; c. The pressure of the homogenization is 20 MPa-40 MPa.

[0015] In a fourth aspect, the present application provides a calcium-containing composition obtained by drying the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments.

[0016] In a fifth aspect, the present application provides a dairy product comprising the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments or the calcium-containing composition according to the preceding embodiment.

[0017] The present application has the following beneficial effects: The carrier composition in the present application is applied to the calcium-embedded emulsion, which can effectively reduce the particle size of the milk mineral salt calcium preparation and increase the specific surface area, thereby promoting the dispersion and dissolution of the calcium source in the digestive juice, and further realizing high bioavailability. Further, the carrier composition plays a role in the interface layer between the oil-solid phase and the aqueous phase, thereby enhancing the stability of the calcium source in the digestive environment, improving the contact efficiency thereof with the gastric juice, and facilitating the release of more Ca 2+ Further, part of the peptide-calcium combination can slow down the formation of calcium salt precipitation under the neutral conditions in the small intestine, thereby reducing the loss of calcium salt, further improving the bioavailability of calcium and bone density. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. Other related drawings can also be obtained by those of ordinary skill in the art without any creative effort.

[0019] Figure 1 Preparation flowchart of calcium-embedded emulsion for improving bone density; Figure 2 Changes of uteruses (A) and serum estradiol concentrations (B) of the sham operation group and the model group; Figure 3 Body weight change trends of rats in different groups; Figure 4 24-hour blood calcium concentration changes of rats in different groups; Figure 5 Calcium absorption rates of rats in different groups; Figure 6 Calcium retention rates in vivo of rats in different groups; Figure 7 Bone calcium contents of rats in different groups; Figure 8 Femur lengths (A) and diameters (B) of rats in different groups; Figure 9 Maximum loads (A) and maximum stresses (B) of femurs and tibias of rats in different groups; Figure 10 Micro CT images of femurs of rats in different groups; Figure 11 Bone density trends of rats in different groups; Figure 12 Fecal water contents of rats in different groups; Figure 13 Intestinal ink migration rates of rats in different groups; Figure 14 Calcium-embedded emulsion sterilization stability test results of different xanthan gum addition ratios; Figure 15 Microstructures of calcium-embedded emulsion after sterilization stability test of different xanthan gum addition ratios; Figure 16 Centrifugal stability test results of calcium-embedded emulsion of different xanthan gum addition ratios; Figure 17 Freeze-thaw stability test results of calcium-embedded emulsion of different xanthan gum addition ratios; Figure 18 Lumisizer stability test results of calcium-embedded emulsion after freeze-thaw of different xanthan gum addition ratios; Figure 19 The results of the interfacial stability test of the calcium-embedded emulsion with different proportions of xanthan gum; Figure 20 The infrared spectra of the aqueous phase and calcium-embedded emulsion with different proportions of xanthan gum; Figure 21 The emulsion particle size (A) and zeta potential (B) of the calcium-embedded emulsion with different proportions of xanthan gum; Figure 22 The photos (A) and lumisizer curves (B) of the calcium-embedded emulsion with different proportions of xanthan gum after the lumisizer stability test; Figure 23 The graph of the relationship between viscosity and shear rate of the calcium-embedded emulsion with different proportions of xanthan gum; Figure 24 The graph of the relationship between oscillation frequency and G' and G'' of the calcium-embedded emulsion with different proportions of xanthan gum; Figure 25 The microstructure of whey protein concentrate, calcium-embedded emulsion with different proportions of xanthan gum, and milk mineral salt; Figure 26 The photos of the calcium-embedded emulsion with different carrier compositions after the lumisizer stability test; Figure 27 The photos of the calcium-embedded emulsion with different carrier compositions after the thermal stability test; Figure 28 The photos of the calcium-embedded emulsion with different carrier compositions after the lumisizer stability test; Figure 29 The results of the lumisizer stability test of the calcium-embedded emulsion with different carrier compositions; Figure 30 The photos of four colloidal solutions. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not specified in the embodiments, conventional conditions or manufacturer-recommended conditions are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0021] The carrier composition for the calcium-embedded emulsion provided in the embodiments of the present application is helpful to improve bone density and comprises, by weight fraction: the carrier composition comprises a hydrophilic emulsifier and a colloidal composition, the colloidal composition comprises a colloidal component A and a colloidal component B; The viscosity of the 0.5% by mass aqueous solution of the colloidal component A is 4000-4300 mPa·s, which is tested by using a 64 rotor at a rotation speed of 50 rpm and 25℃. The viscosity of the 0.5% by mass aqueous solution of the colloidal component B is 800-900 mPa·s, which is tested by using a 64 rotor at a rotation speed of 50 rpm and 25℃.

[0022] The carrier composition in the present application has a suitable viscosity, which is applied to the calcium-embedded emulsion, can effectively reduce the particle size of the milk mineral salt calcium preparation, increase the specific surface area, thereby promoting the dispersion and dissolution of the calcium source in the digestive juice, and further realizing high bioavailability. Further, the carrier composition plays a role in the interface layer of the oil-solid phase and the water phase, which enhances the stability of the calcium source in the digestive environment, improves the contact efficiency thereof with the gastric juice, and is beneficial to the release of more Ca 2+ Further, part of the peptide-calcium combination can slow down the formation of calcium salt precipitation under the neutral conditions in the small intestine, thereby reducing the loss of calcium salt, further improving the bioavailability of calcium and bone density.

[0023] It should be noted that in the present application, the viscosity of the aqueous solution of the colloidal component A and the aqueous solution of the colloidal component B is tested by using a Brookfield rotary viscometer, and the aqueous solution of the carrier composition is tested by using a TA Instruments rotary rheometer.

[0024] Exemplarily, in the carrier composition for the calcium-embedded emulsion which is helpful to improve the bone density, the hydrophilic emulsifier is selected from whey protein concentrate; and / or, the colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum; and / or, the mass ratio of the colloidal component A to the 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; 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.

[0025] The present application also provides a calcium-embedded emulsion which is helpful to improve the bone density, comprising a calcium source, a fat, a lipophilic emulsifier and the carrier composition as described in the foregoing embodiments. The viscosity of the calcium-embedded emulsion is 200-500 mPa•s, such as 200 mPa•s, 250 mPa•s, 300 mPa•s, 350 mPa•s, 400 mPa•s, 450 mPa•s, 500 mPa•s, preferably 300-500 mPa•s, and more preferably 400-500 mPa•s, as measured at 25°C and 50 rpm using a Brookfield DV-I+ viscometer with a #27 spindle.

[0026] The calcium-embedded emulsion for improving bone density includes the calcium source, the oil, the lipophilic emulsifier, the hydrophilic emulsifier, the gellan gum, and the xanthan gum, wherein the mass fraction of the calcium source is 1%-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; the mass fraction of the oil is 5%-15%, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%; the mass fraction of the lipophilic emulsifier is 0.5%-2%, such as 0.5%, 1%, 1.5%, 2%; the mass fraction of the hydrophilic emulsifier is 1%-3%, such as 1%, 1.5%, 2.0%, 2.5%, 3%; the mass fraction of the gellan gum is 0.05%-0.5%, such as 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%; and the mass fraction of the xanthan gum is 0.05%-0.5%, such as 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%.

[0027] Experiments show that the calcium-embedded emulsion for improving bone density can effectively improve the absorption efficiency and bioavailability of calcium, promote bone anabolism, and is less likely to form insoluble calcium soap with fatty acids during calcium supplementation, and almost does not cause adverse reactions such as constipation.

[0028] In an optional embodiment, the calcium-embedded emulsion for improving bone density includes an aqueous phase and an oil phase embedded in the aqueous phase, and the oil phase has the calcium source dispersed therein.

[0029] In an optional embodiment, the calcium source includes at least one of milk mineral salt, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate, wherein the milk mineral salt is a natural mineral concentrate extracted from milk, which is not a single salt, but a natural compound mainly composed of calcium and phosphorus, and also contains various other trace minerals, and is more beneficial to improve the acceptance of consumers when applied to dairy products.

[0030] In an optional embodiment, the lipophilic emulsifier includes a fatty acid ester emulsifier and / or a phospholipid.

[0031] In optional embodiments, the lipophilic emulsifier includes one or more of glyceryl monostearate, mono-, di-glyceride fatty acid ester, sucrose fatty acid ester, phospholipid, polyglyceryl fatty acid ester, and diacetyl tartaric acid ester of mono-, di-glyceride.

[0032] The embodiments of the present application also provide a calcium-containing composition obtained by drying the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments. Specifically, the drying method can be spray drying, freeze drying, or the like.

[0033] The embodiments of the present application also provide a method for preparing the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments, which comprises the following steps as shown in the figure: Figure 1 Heating and mixing the carrier composition in water to obtain an aqueous phase; Heating and mixing the calcium source, oil, and lipophilic emulsifier to obtain an oil-solid phase; Mixing the oil-solid phase and the aqueous phase, and then emulsifying to obtain the calcium-embedded emulsion for improving bone density.

[0034] In optional embodiments, the mass fraction of the hydrophilic emulsifier in the aqueous phase is 1.5%-2.5%, such as 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%, such as 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%, such as 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%.

[0035] In optional embodiments, the mass ratio of the calcium source to oil is 1: (1-10), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0036] In optional embodiments, the mass ratio of the oil-solid phase to the aqueous phase is (6-27):(94-73), such as 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, or 27:73. Increasing the proportion of the oil-solid phase is conducive to increasing the calcium content in the emulsion.​

[0037] In an alternative embodiment, the temperature for heating and mixing the carrier composition in water is 40-80℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃; and the time is 10-60min, such as 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min.

[0038] In an alternative embodiment, the temperature for heating and mixing the calcium source with the oil and the lipophilic emulsifier is 40-70℃, such as 40℃, 43℃, 46℃, 49℃, 52℃, 55℃, 58℃, 61℃, 64℃, 67℃, 70℃; and the time is 10-60min, such as 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min.

[0039] In an alternative embodiment, the emulsification comprises high-speed shearing and homogenization in sequence.

[0040] In an alternative embodiment, the rotation speed for high-speed shearing is 10000-20000rpm, such as 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, 17000rpm, 18000rpm, 19000rpm, 20000rpm.

[0041] In an alternative embodiment, the time for high-speed shearing is 1-5min, such as 1min, 1.4min, 1.8min, 2.2min, 2.6min, 3.0min, 3.4min, 3.8min, 4.2min, 4.6min, 5min.

[0042] In an alternative embodiment, the pressure for homogenization is 20-40MPa, such as 20MPa, 22MPa, 24MPa, 26MPa, 28MPa, 30MPa, 32MPa, 34MPa, 36MPa, 38MPa, 40MPa.

[0043] The present application also provides a dairy product comprising the calcium-embedded emulsion for improving bone density according to any one of the preceding embodiments or the calcium-containing composition according to the preceding embodiment.

[0044] The features and advantages of the present application will be further described in the following examples.

[0045] Example 1 The present embodiment provides a preparation method of a calcium-embedded emulsion for improving bone density, which specifically comprises the following steps: The carrier composition is mixed in water at 60℃ for 30 min to obtain an aqueous phase, wherein the mass fraction of whey protein concentrate (whey protein content 80%) is 2%, the mass fraction of gellan gum is 0.1%, and the mass fraction of xanthan gum is 0.5%; The milk mineral salt is mixed with oil and oil-soluble emulsifier monoglyceride fatty acid ester and diglyceride fatty acid ester at a mass ratio of 2:6:1 and heated to 55℃ to obtain an oil-solid phase; The oil-solid phase is mixed with the aqueous phase at a mass ratio of 1:5, and then high-speed shearing and homogenization are sequentially performed to obtain the calcium-embedded emulsion for improving bone density; wherein the rotation speed of the high-speed shearing is 15000 rpm, the time of the high-speed shearing is 3 min, and the pressure of the homogenization is 30 MPa.

[0046] Test Example 1: Calcium absorption efficacy evaluation The present application constructs an ovariectomy-induced rat osteoporosis model, systematically explores the influence of the calcium-embedded emulsion for improving bone density on calcium metabolism, bone microstructure and fecal excretion of rats, aims to clarify the calcium utilization mechanism from the absorption and function dual dimensions, provides a theoretical basis for calcium bioavailability regulation research, and lays a theoretical foundation for developing efficient and safe calcium nutrition fortification strategies.

[0047] 1. Experimental materials 1.1. Experimental animals The SPF 3-month-old female SD rats used in the present application are used as research objects, and a total of 80 rats are purchased from Beijing Huafukang Biosciences and Technologies Co., Ltd., license number: SCXK (jing) 2013-0038.

[0048] 1.2. Main reagents Feed is purchased from Beijing Huafukang Biosciences and Technologies Co., Ltd.; serum calcium, serum phosphorus, bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), type I collagen amino-terminal cross-linking peptide (NTX) and type I collagen carboxy-terminal cross-linking peptide (CTX) kits are purchased from Nanjing Jiancheng Biological Engineering Institute; type I procollagen amino-terminal peptide (PINP) and osteocalcin (BGP) kits are purchased from Beijing Solabio Technology Co., Ltd.

[0049] 2. Experimental method 2.1. Preparation of animal model After 1 week of adaptive feeding, 80 SPF SD female rats were randomly divided into two groups: sham operation group (Sham group, n=10) and model group (n=70). Sham operation group: the skin and peritoneum of the mouse were cut, part of the fat around the ovary was removed, and then sutured; model group: ovariectomy to establish an osteoporosis model.

[0050] 2.2, Experimental animal grouping After ovariectomy, 70 rats were randomly divided into the following 7 groups (n=10): model group (OVX), positive control group (ALN, alendronate sodium, daily gavage amount (in terms of alendronate sodium) 1 mg / kg), calcium carbonate group (CaCO3, daily gavage amount (in terms of calcium element) 133.3 mg / kg), low-dose milk calcium group (MMS-L, milk mineral salt, daily gavage amount (in terms of calcium element) 66.65 mg / kg), high-dose milk calcium group (MMS-H, milk mineral salt, daily gavage amount (in terms of calcium element) 133.3 mg / kg), low-dose embedded milk calcium group (MFEs-L, calcium-embedded emulsion prepared in Example 1 to help improve bone density, daily gavage amount (in terms of calcium element) 66.65 mg / kg), and high-dose embedded milk calcium group (MFEs-H, calcium-embedded emulsion prepared in Example 1 to help improve bone density, daily gavage amount (in terms of calcium element) 133.3 mg / kg).

[0051] 2.3, Intervention and treatment of test substances After the model was successfully established, each group was intervened according to the corresponding test substance. The dosages were determined with reference to the “Chinese Dietary Nutrient Reference Intake (2023 Edition)” and “Guidelines for Health Food Function Test and Evaluation (2023 Edition)”.

[0052] 2.4, Calcium absorption test The calcium metabolism experiment was carried out 3 days before sacrifice. The rats were single-caged in a metabolic cage, and the daily food intake was accurately recorded. The feces and urine samples were collected within 12 hours. After acid digestion, the calcium content in the samples was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and the apparent calcium absorption rate and calcium retention rate were calculated: Calcium absorption rate (%) = (intake calcium-fecal calcium) / intake calcium x 100%; Calcium retention rate (%) = (intake calcium-fecal calcium-urine calcium) / intake calcium x 100%.

[0053] 2.5, Evaluation of intestinal function After the rats were fasted for 12 hours, 2 mL of ink was administered by gavage, and the rats were single-caged in a metabolic cage. The time of the first feces and the time of the first black feces were recorded, and the feces were collected to determine the dry weight and wet weight, and the water content of the feces was calculated. Moisture content of feces (%) = (wet weight - dry weight) / wet weight x 100%; After 30 min of gavage, the animals were sacrificed, and the small intestine was immediately removed. The ink propulsion distance and the total length of the small intestine were measured, and the propulsion rate was calculated: Ink propulsion rate (%) = ink propulsion distance / total length of small intestine x 100%.

[0054] 2.6, Bone density and bone microstructure analysis The distal end of the right femur was taken and scanned using Micro-CT. The scanning parameters were set as follows: voltage 90 kV, current 88 μA, field of view 86 mm, high resolution mode, scanning time 4 minutes, and filter thickness 0.1 mm. The bone trabecular microstructure parameters in the region of interest were reconstructed and quantitatively analyzed using the Analysis 12 software.

[0055] 2.7, Bone biomechanical property test The bone biomechanics was measured using a texture analyzer three-point bending probe. The right femur was placed flat on two supported plates, and the flat probe was positioned directly above the middle of the femur, 2 cm away from the femur, for three-point bending test measurement.

[0056] 2.8, Bone calcium content determination The femur samples were dried to constant weight, 10 mL of mixed digestion acid (nitric acid: perchloric acid = 4:1, v / v) was added, and the sample was heated on an electric hot plate until it was clear and transparent. After cooling, the volume was adjusted, and the calcium content was determined using ICP-OES after appropriate dilution.

[0057] 2.9, Determination of organ index The uterus, kidneys, liver and other organs were washed with physiological saline to remove bloodstains, dried and weighed, and the ratio of their weight to body weight was calculated: Uterus index (%) = uterus weight / rat weight x 100%; Kidney index (%) = double kidney weight / rat weight x 100%; Liver index (%) = liver weight / rat weight x 100%.

[0058] 2.10, Statistical analysis The experimental data were expressed as mean ± standard deviation (mean ± SD), and the Origin 2022 and GraphPad Prism 8.0 software were used for statistical analysis. One-way ANOVA was used for comparison among multiple groups, and the significance level was set as * P<0.05 (significant difference) and ** P<0.01 (extremely significant difference).

[0059] 3 Results and analysis 3.1, Modeling 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.

[0060] 3.2. Weight 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.

[0061] 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.

[0062] 3.3. Rat organ index 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.

[0063] 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.

[0064] Table 1. Organ Index of Rats

[0065] 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).

[0066] 3.4 Evaluation of Calcium Absorption 3.4.1 Blood calcium concentration 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.

[0067] 3.4.2 Calcium absorption rate and retention rate in rats like Figure 5As shown, compared with the sham group, the calcium absorption rate of the ovariectomized group of rats did not change significantly, indicating that ovariectomy itself did not have a significant negative impact on the calcium metabolism capacity of rats. After supplementing different calcium agents, the calcium absorption rate of each group showed an upward trend, among which the calcium absorption rate of the milk calcium (MMS) and S / O / W embedded milk calcium (MFEs) groups 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 increased, and the low-dose group (MMS-L) was more effective, 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, which proved that there may be no obvious dose-dependent relationship for milk calcium, and further increasing the dose may not necessarily enhance the calcium absorption effect after exceeding a certain threshold.

[0068] The calcium absorption rate of the MFEs-L group was 5.65% higher than that of the MFEs-H group, and the absorption rate of the MMS-L group was only 1.41% higher than that of the MMS-H group, which proved that embedding may improve the dispersibility of calcium ions in the intestinal lumen, alleviate the problem of dose-dependent absorption decline of calcium ions, and avoid over-saturation excretion. The calcium absorption rate of the MFEs group was higher than that of the MMS group as a whole, among which the MFEs-L group was significantly higher than the other groups, and the MFEs-H group also showed a further improvement trend. The embedded milk calcium improved the release and absorption of calcium in milk mineral salt by 8.05% compared with naked calcium, indicating that the S / O / W emulsion embedding technology helps to improve the release and absorption of calcium in milk mineral salt, and shows good potential in enhancing the calcium bioavailability of ovariectomized rats. This result is consistent with the conclusion of the previous study that the embedding system can improve the bioavailability of calcium.

[0069] As shown, Figure 6 Compared with the sham group (Sham), the calcium retention rate of the ovariectomized group (OVX) and the alendronate sodium positive control group (ALN) rats decreased. After supplementing different calcium sources, the calcium retention rate of each group showed an upward trend. Among them, the calcium retention rate of the MMS-L and MMS-H groups was significantly higher than that of the OVX group, indicating that milk calcium is more conducive to the absorption and retention of calcium than traditional calcium supplementation methods.

[0070] 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.

[0071] 3.5 Evaluation of skeletal condition 3.5.1 Bone calcium content 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.

[0072] 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.

[0073] 3.5.2 Bone length and bone diameter like Figure 8 As 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.

[0074] like Figure 8As shown in FIG. 13B, the femur diameter of the Sham group represents the normal development state, and the diameter of the OVX group is significantly reduced, indicating that ovariectomy not only affects bone length, but also damages the transverse development of bone structure, which may lead to osteoporosis and mechanical property degradation. After the intervention of milk calcium and embedded milk calcium, the femur diameter of the MMS group and the MFEs group is increased compared with the OVX group, and the effect of the embedded group (MFEs) is more prominent. The average diameter of the MFEs group is increased by 0.02 mm compared with the MMS group. It is shown that the supplement of milk calcium, especially after embedding treatment, is helpful to improve the bone structure and bone density, so as to promote the recovery of femur morphology and mechanical function. The change trend of the MFEs-L and MFEs-H groups is also consistent with the previous study.

[0075] 3.5.3, bone biomechanics As shown in FIG. 13B, the femur diameter of the Sham group represents the normal development state, and the diameter of the OVX group is significantly reduced, indicating that ovariectomy not only affects bone length, but also damages the transverse development of bone structure, which may lead to osteoporosis and mechanical property degradation. After the intervention of milk calcium and embedded milk calcium, the femur diameter of the MMS group and the MFEs group is increased compared with the OVX group, and the effect of the embedded group (MFEs) is more prominent. The average diameter of the MFEs group is increased by 0.02 mm compared with the MMS group. It is shown that the supplement of milk calcium, especially after embedding treatment, is helpful to improve the bone structure and bone density, so as to promote the recovery of femur morphology and mechanical function. The change trend of the MFEs-L and MFEs-H groups is also consistent with the previous study. Figure 9

[0076] The maximum load reflects the biological strength of the bone to resist fracture, and the maximum stress is closely related to the bone mineral density and microstructure integrity. Both of them are key indicators for evaluating bone mechanical properties and osteoporosis risk. The results of the present application show that the milk calcium (MFEs) treated by S / O / W embedding can more effectively enhance the load capacity of the bone, and the mechanism may be related to the improvement of the bioavailability of calcium by embedding technology, and the promotion of the directional deposition and microstructure reconstruction of calcium in bone tissue. Therefore, MFEs has a significant advantage in improving osteoporosis and enhancing the mechanical properties of bone in ovariectomized rats, and has good application potential.

[0077] 3.5.4, Micro CT The three-dimensional image of the bone is obtained by three-dimensional reconstruction and other technologies, and the microstructure of the bone is quantitatively analyzed by metrology. As shown in FIG. 13C, the bone volume / tissue volume (BV / TV) of the OVX group is significantly lower than that of the Sham group, indicating that ovariectomy can cause bone loss and damage the bone microstructure. After the intervention of milk calcium and embedded milk calcium, the bone volume / tissue volume (BV / TV) of the MMS group and the MFEs group is increased compared with the OVX group, and the effect of the embedded group (MFEs) is more prominent. The bone volume / tissue volume (BV / TV) of the MFEs group is increased by 0.02 mm compared with the MMS group. It is shown that the supplement of milk calcium, especially after embedding treatment, is helpful to improve the bone structure and bone density, so as to promote the recovery of femur morphology and mechanical function. The change trend of the MFEs-L and MFEs-H groups is also consistent with the previous study. Figure 10 ​The three-dimensional reconstruction images of the femur of each group of mice are shown. The OVX group shows sparse, fine, and unevenly arranged trabecular bone, with large areas of non-trabecular bone marrow region, and the bone microstructure is obviously damaged. Compared with the model group, the bone microstructure damage of CaCO3, ALN, MMS and MFEs groups is reduced, which is manifested as more continuous connection, increased number and reduced intertrabecular space. Among them, the MFEs-L trabecular bone is arranged in an orderly and uniform manner, and the number of trabecular bone is increased. From the observation of the cross-sectional scan of the femur, the proportion of the middle black hollow area is obviously reduced, which is caused by the growth of the trabecular bone. It is proved that this system can effectively enhance the reconstruction and calcium deposition capacity of bone microstructure by promoting the absorption and utilization of calcium.

[0078] 3.5.5. Bone mineral density As shown in Figure 11 , the bone mineral density (BMD) of rats can directly reflect the strength of the skeleton, and the diagnosis of osteoporosis can be facilitated by checking the bone mineral density. Compared with the Sham group, the bone mineral density of the OVX group was significantly reduced, and the bone mineral density of the MMS-H and MFEs-L groups was significantly increased compared with the OVX group. The MMS-H group increased by 18.35%, and the MFEs-L group increased by 26.81%. The bone mineral density of the low-dose embedded milk calcium was better than that of the high-dose naked calcium, which proved that the embedding system was beneficial to the improvement of calcium absorption efficiency. In summary, the embedded milk calcium can enhance the bone mineral density of the model rats and improve the bone loss of the ovariectomized osteoporotic rats.

[0079] 3.6. Fecal index 3.6.1. Fecal water content As shown in Figure 12 , the fecal water content of the Sham group was at the basic level, and the water content of the OVX group was slightly higher than that of the Sham group, which proved that ovariectomy significantly increased the fecal water content of rats, which was due to the sharp decline in estrogen levels, leading to faster intestinal peristalsis, shorter transmission time, and increased fecal water content. The water content of the feces of the ALN and CaCO3 groups was significantly lower than that of the OVX group, indicating that long-term calcium supplementation under this condition may increase the mineral metabolism burden and the risk of constipation. The fecal water content of the MMS and MFEs groups showed a gradual upward trend, and the overall level was closer to that of the Sham group. Compared with the naked calcium of the same dose, the fecal water content of the embedded milk calcium was higher, which may be related to the polysaccharides and proteins in the loading system. It helps to maintain normal fecal water content and reduce the risk of constipation through improving the intestinal microenvironment and regulating calcium ion absorption and metabolism.

[0080] 3.6.2. Ink migration rate As shown in Figure 13As shown, compared with the sham operation, the ink migration rate of the ovariectomized rats was significantly reduced, and the ink migration rate was not significantly improved when CaCO3, high and low concentration milk calcium were supplemented; when low concentration of embedded milk calcium was supplemented, the intestinal migration rate was improved, and the migration rate could reach a level similar to that of the normal group. This may be due to the fact that the absorption effect of calcium after embedding is enhanced, restoring smooth muscle contraction; the wrapping wall material is beneficial to improve the diversity of intestinal flora, which may further promote intestinal peristalsis through metabolic products, so the embedded milk calcium is more helpful to intestinal peristalsis.

[0081] 4、Conclusion The present experimental example mainly evaluates the absorption of calcium embedded emulsion which is helpful to improve bone density. Ovariectomized rats are used as models, which are divided into Sham, OVX, ALN, CaCO3, milk calcium (high and low doses), and embedded milk calcium (high and low doses) 8 groups for evaluation. The indicators such as blood calcium, absorption retention rate, bone condition, and fecal index are analyzed from multiple dimensions. The results show that (1) in the 0-24 hour blood calcium concentration monitoring, the blood calcium growth rate of the MFEs group is significantly higher than that of other groups, which is 4.46% higher than that of the MMS group. The embedding system can promote the rapid dissolution and absorption of calcium; (2) in terms of calcium apparent absorption rate and in vivo retention rate, MFEs-L group performs particularly outstanding, and the absorption rate of MFEs group is 8.05% higher than that of MMS group. (3) Bone morphology and biomechanics analysis shows that after MFEs-L intervention, femoral bone density increases by 26.81%, bone diameter increases and mechanical strength increases by 25.94%, indicating that embedded milk calcium has the effect of relieving bone loss. (4) In terms of fecal metabolism indicators, embedded milk calcium group does not show a tendency of constipation, and the water content and shape of feces are within the normal range, which increases the lubrication of the intestinal tract, which may be related to the particle size distribution of the S / O / W system and the intestinal lubrication and calcium release regulation brought by lipids and hydrophilic colloids (such as polysaccharides and proteins).

[0082] In summary, the present experimental example demonstrates that calcium embedded emulsion which is helpful to improve bone density can improve the dispersibility and stability of milk calcium in the intestinal tract, increase the available concentration of calcium ions in the intestinal lumen, and further promote the uptake and transport of calcium by intestinal epithelial cells by improving the local environment of the intestinal tract and the efficiency of calcium transfer, thereby significantly improving the bioavailability of calcium and the deposition efficiency of the skeleton.

[0083] Test Example 2: Effect of the mass fraction of xanthan gum in the aqueous phase on the performance of calcium embedded emulsion which is helpful to improve bone density.

[0084] In this test example, the mass fraction of xanthan gum in the aqueous phase is adjusted based on Example 1, obtaining an aqueous phase with a mass fraction of xanthan gum of 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, and obtaining the corresponding calcium embedded emulsion which is helpful to improve bone density.

[0085] 1. Sterilization stability test 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 14 As 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.

[0086] 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.

[0087] 2. Centrifugal stability test 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.

[0088] 3. Freeze-thaw stability test 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 17As shown, freeze-thaw treatment can cause the emulsion to appear water separation and flocculation, which may be caused by whey protein denaturation or butter crystal structure changes, but the above phenomenon can be significantly improved with the increase of xanthan gum proportion.

[0089] LUMiSizer stability test was performed on the samples after freeze-thaw, and the parameters were set as follows: running temperature 25℃; sample amount 0.4 mL; rotation speed 4000 rpm; scanning profile line 255; time interval 60 s; total duration 255 min. The results are shown in Figure 18 As shown, with the increase of freeze-thaw times, the stability of the emulsion as a whole showed a downward trend. However, when the xanthan gum addition amount was ≥0.4%, the stability of the emulsion changed little after three freeze-thaw treatments, especially the emulsion prepared with 0.5% XG, which had no shift in LUMiSizer transmission curve after three freeze-thaw treatments, i.e. had good stability.

[0090] The present application proves that the addition of xanthan gum can effectively improve the freeze-thaw stability of the emulsion, which provides theoretical support for the application of calcium-embedded emulsion in ice cream and other frozen products.

[0091] 4. Interfacial stability test The oil-water interface is an important factor affecting the stability of the emulsion, and the smaller the oil-water interfacial tension, the more stable the interfacial layer. The interfacial tension of the water phase was tested, and the results are shown in Figure 19 As the proportion of XG increases, the interfacial tension between the water phase and the oil phase gradually decreases, which may be because polysaccharides promote the adsorption of emulsifiers on the oil-water interface and the formation of elastic interfacial films, thereby reducing the interfacial tension and improving the stability of the emulsion.

[0092] 5. Fourier infrared spectrum The Fourier infrared spectrum of the water phase with different xanthan gum contents, the corresponding calcium-embedded emulsion for improving bone density, and the milk mineral salt were tested, and the test results are shown in Figure 20 The entire infrared spectrum can be divided into a functional group region of 4000cm -1 -1300cm -1 and a fingerprint region of 1300cm -1 -600cm -1 The high-frequency region of 3000-3500cm -1 in the figure represents the stretching vibration and absorption of -OH, and compared with the spectrum of milk mineral salt, the characteristic peak value of the infrared spectrum of the calcium-embedded emulsion in this region moves, which is attributed to the change in the number of intermolecular hydrogen bonds, indicating that there is hydrogen bond interaction between the systems. Compared with the spectrum of milk mineral salt and water phase alone, the emulsion has a characteristic peak at 2925.72cm -1The peak height of left and right position changed significantly, corresponding to C-H stretching vibration, which might come from the fatty chain structure of oil phase or emulsifier. In 1600-1700 cm -1 (Amide I band) belongs to C-O stretching, 1500-1600 cm -1 (Amide II band) represents -NH bending vibration.

[0093] It was observed that after the water phase was prepared into calcium-embedded emulsion, the peak of Amide I band moved from 1653.35 cm -1 to 1746.81 cm -1 and 1743.21 cm -1 , which indicated that the protein secondary structure in the emulsion system might be slightly adjusted, and the rearrangement of hydrogen bond network affected the performance of -NH bending vibration. The main characteristic peak of milk mineral salt was 1412.54 cm -1 , representing symmetric COO- stretching, while in the calcium-embedded emulsion, the characteristic peak shifted to 1455.67 cm -1 . The peak intensity of milk mineral salt at 1031.55 cm -1 and 560.71 cm -1 decreased after the emulsion was formed, which corresponded to C-O stretching vibration and C-C=O in-plane bending vibration of fatty ketone, respectively. The broadening, overlapping and shifting of bands in FTIR spectrum caused by the enhancement of hydrogen bond further indicated that milk mineral salt could be well dispersed in the emulsion system, thereby improving the structural stability of the emulsion.

[0094] 6. Particle size and zeta potential of emulsion In order to further improve the calcium loading capacity and study the effect of viscosity effect on the stability of solubilization and delivery of poorly soluble bioactive ingredients, the particle size and zeta potential of calcium-embedded emulsion system were determined by Nano-ZS90 laser particle size analyzer. In order to avoid multiple scattering effect, the sample was diluted 400 times, and the refractive index of continuous phase and dispersed phase was set to 1.33 and 1.45, respectively. Each sample was measured in triplicate. The results are shown in Figure 21 It was found that the particle size and absolute value of zeta potential of calcium-embedded emulsion with different xanthan gum ratios increased with the increase of xanthan gum ratio, and the zeta potential tended to be stable when the xanthan gum ratio was 0.2%. With the increase of xanthan gum ratio, the negative charge in the system increased, and the negative charge between the side chains of xanthan gum and the interaction between the side chain un-polymer skeleton significantly improved the physical stability of calcium-embedded emulsion.

[0095] 7. Physical stability The physical stability of the calcium-embedded emulsion which helps to improve bone density was determined by LUMiSizer stability analyzer, and the parameters were set as follows: running temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; scanning profile line 255; time interval 60 s; total duration 255 min. The LUMiSizer stability analysis results are shown in Figure 22 As shown in the figure, when the calcium-embedded emulsion is only stabilized by gellan gum, there is obvious creaming phenomenon in the system, and with the increase of the proportion of xanthan gum, the phenomenon is significantly improved, and it can be seen that when the proportion of xanthan gum is 0.4%, the physical stability of the calcium-embedded emulsion system is good.

[0096] 8. Rheological properties With the increase of shear rate, the apparent viscosity of each sample showed a downward trend, as shown in Figure 23 , which may be due to the destruction of the spatial network structure of the emulsion system. Under a certain shear rate, the apparent viscosity of the calcium-embedded emulsion increased with the increase of the proportion of xanthan gum, and the thickening effect of the emulsion gradually increased, which may be an important factor to improve the stability of high calcium-embedded emulsion.

[0097] With the increase of oscillation frequency, the G' and G'' of the calcium-embedded emulsion with different proportions of xanthan gum showed an upward trend, as shown in Figure 24 , and G' and G'' were proportional to the proportion of xanthan gum. Within the frequency range of 0.1-100 Hz, the G' of the calcium-embedded emulsion was higher than the G'', which indicated that the emulsion was relatively stable under the influence of different frequencies, mainly in the form of elastic behavior.

[0098] 9. Microstructure The microstructure of the calcium-embedded emulsion which helps to improve bone density was observed by laser confocal microscope. Specifically, the S phase, O phase and W phase of the calcium-embedded emulsion sample were dyed with rhodamine B, Nile red and Nile blue A respectively, and the corresponding excitation wavelengths were 543 nm, 488 nm and 635 nm respectively. After dyeing, the sample was vortexed for 10 s and equilibrated for 12 h, and then observed under an oil lens with a magnification of 60 times by a confocal laser scanning microscope (CLSM) (FV 3000, Olympus Corporation, Japan). The obtained microscopic images were processed and analyzed by software, as shown in Figure 25 , with the increase of the proportion of xanthan gum, the emulsion system became more uniform. The sample without xanthan gum showed significant aggregation of milk mineral salt, and there were phenomena such as large oil droplet size, close to each other and unclear boundary, indicating that the coalescence degree between emulsion droplets was relatively high, which may be related to the weak electrostatic repulsion and low hydrophobicity between emulsion droplets. When the amount of xanthan gum added was 0.4%, the above phenomena were significantly improved.

[0099] In combination with the effect of xanthan gum addition on emulsion potential, the absolute value of calcium-embedded emulsion zeta-potential increased with increasing xanthan gum proportion, and the electrostatic interaction was enhanced. At the same time, the viscosity effect of the emulsion system was enhanced with increasing xanthan gum addition, which might be an important reason for improving the stability of the emulsion and the dispersibility of the poorly soluble mineral salt.

[0100] Test Example 3: Effect of the content of each component of the carrier composition in the water phase on the performance of the calcium-embedded emulsion that helps to improve bone density.

[0101] This test example is based on Example 1, in which whey protein concentrate, xanthan gum, and gellan gum in the water phase are replaced or the mass fraction of the components is adjusted, to obtain the corresponding calcium-embedded emulsion that helps to improve bone density, and the following tests are performed.

[0102] 1. LUMI stability analysis The physical stability of the calcium-embedded emulsion that helps to improve bone density is determined using a LUMiSizer stability analyzer, with the following parameters: running temperature 25°C; sample volume 0.4 mL; rotation speed 4000 rpm; scanning contour line 255; time interval 60 s; total duration 255 min. The sample photo after LUMiSizer stability analysis is shown in Figure 26 Under the condition of the same whey protein concentrate concentration as in Example 1, the amount of xanthan gum (GG) and gellan gum (XG) is adjusted, and under the conditions of 0.1% GG-0.4% XG and 0% GG-0.7% XG, the system does not undergo demulsification.

[0103] 2. Thermal stability analysis The calcium-embedded emulsion that helps to improve bone density is subjected to high-temperature treatment at 95°C for 5 min, and the results are shown in Figure 27 The colloid formulation can improve the thermal stability of the calcium-embedded emulsion system to some extent. Under the condition of the same whey protein concentrate concentration as in Example 1, the amount of xanthan gum and gellan gum is adjusted, and when the colloid formulation proportion is 0.1% GG-0.6% XG, the thermal denaturation degree of the emulsion is significantly improved. Similarly, in the emulsion system stabilized by pure xanthan gum, the thermal denaturation degree of the emulsion prepared by 0% GG-0.7% XG is higher than that of the emulsion prepared by 0% GG-0.5% XG.

[0104] 3. LUMiSizer analysis As shown in Figure 28The results showed that the high milk mineral content and the increase of the proportion of gellan gum in the carrier were the main reasons for the separation of the calcium-embedded emulsion system. The LUMiSizer stability analyzer was used to determine the physical stability of the calcium-embedded emulsion which improved the bone density. The parameters were set as follows: running temperature 25℃; sample volume 0.4 mL; rotation speed 4000 rpm; scanning profile line 255; time interval 60 s; total time 255 min. From the LUMiSizer analysis results, WPC and WPI had little difference in the effect on the system, and sodium caseinate as a hydrophilic emulsifier was not ideal.

[0105] 5. Adjustment of carrier composition components The foregoing study confirmed that the stability effect of whey protein concentrate on the system was better than that of casein. The present application also attempted to replace whey protein with other reagents: for example Figure 29 As shown, 4% Cas (sodium caseinate) and 2% WPC-2% Cas were used to replace the whey protein concentrate in Example 1, but the emulsion prepared in this way was extremely difficult to homogenize and was prone to block the homogenizer, so whey protein was more optimal as a carrier composition; in addition, mono- and diglycerides of fatty acids (monoglyceride) as an oleophilic emulsifier had the effect of stabilizing the oil and water phase system, but during the dissolution process, the oil phase temperature had to be raised to 70℃ to dissolve.

[0106] Test Example 4 In this test example, the viscosities of 0.25% xanthan gum aqueous solution, 0.25% guar gum aqueous solution, 0.25% pectin aqueous solution and 0.25% carrageenan aqueous solution were tested at 25℃, and the test results are shown in Table 1. Figure 30 As shown, the experiments confirmed that the viscosities of the four solutions from large to small were xanthan gum aqueous solution, guar gum aqueous solution, carrageenan aqueous solution and pectin aqueous solution.

[0107] Test Example 5 In this test example, the viscosities of 0.5% gellan gum aqueous solution, 0.5% xanthan gum aqueous solution, 0.5% guar gum aqueous solution, 0.5% locust bean gum aqueous solution, 0.5% pectin aqueous solution and 0.5% carrageenan aqueous solution were tested at 25℃ according to GB / T 10247-2008, and the test conditions and results are shown in Table 2. Gellan gum and xanthan gum together as a carrier can obtain a calcium-embedded emulsion with high bioavailability, while guar gum, locust bean gum, pectin and carrageenan alone or together with gellan gum or xanthan gum as a carrier cannot obtain a calcium-embedded emulsion with high bioavailability, especially the bone density and bone calcium content of rats under the same conditions are far less than those of Example 1.

[0108] Table 2

[0109] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. The present application can have various changes and modifications for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A carrier composition for calcium-encased emulsion for improving bone density, characterized by comprising: The carrier composition comprises a hydrophilic emulsifier and a colloidal composition comprising a colloidal component A and a colloidal component B; The viscosity of the colloidal component A aqueous solution with a mass fraction of 0.5% is 4000-4300 mPa·s, which is tested by using a No. 64 rotor at a rotation speed of 50 rpm and 25°C; The viscosity of the colloidal component B aqueous solution with a mass fraction of 0.5% is 800-900 mPa·s, which is tested by using a No. 64 rotor at a rotation speed of 50 rpm and 25°C.

2. The carrier composition for calcium-embedded emulsion for the improvement of bone density according to claim 1, wherein The hydrophilic emulsifier is selected from whey protein concentrate; And / or, the colloidal component A is selected from gellan gum, and the colloidal component B is selected from xanthan gum; And / or, the mass ratio of the colloidal component A to the colloidal component B in the colloidal composition is 1:(0.1-10); And / or, the mass ratio of the hydrophilic emulsifier to the colloidal composition in the carrier composition is (1-30):

1.

3. A calcium-encased emulsion that helps improve bone density, characterized in that, The calcium-embedded emulsion comprises a calcium source, oil, a lipophilic emulsifier, and the carrier composition of claim 1 or 2; The viscosity of the calcium-embedded emulsion is 200-500 mPa·s, which is tested by using a No. cc27 rotor at a rotation speed of 50 rpm and 25°C.

4. The calcium-embedded emulsion for improving bone density according to claim 3, wherein In the calcium-embedded emulsion for improving bone density, the mass fraction of the calcium source is 1%-10%, the mass fraction of the oil is 5%-15%, the mass fraction of the lipophilic emulsifier is 0.5%-2%, the mass fraction of the hydrophilic emulsifier is 1%-3%, the mass fraction of the gellan gum is 0.05%-0.5%, and the mass fraction of the xanthan gum is 0.05%-0.5%; And / or, the calcium-embedded emulsion for improving bone density comprises an aqueous phase and an oil phase embedded in the aqueous phase, and the oil phase comprises oil in which the calcium source is dispersed; And / or, the calcium source comprises at least one of milk mineral salt, calcium carbonate, calcium citrate, calcium lactate, and calcium gluconate; And / or, the lipophilic emulsifier comprises a fatty acid ester emulsifier and / or a phospholipid.

5. The calcium-embedded emulsion for helping to improve bone density according to claim 3 or 4, characterized by, The lipophilic emulsifier comprises one or more of glycerol monostearate, mono-, di-glycerol fatty acid ester, sucrose fatty acid ester, phospholipid, polyglycerol fatty acid ester, and diacetyl tartaric acid mono-, di-glycerol ester.

6. A method of preparing the calcium-entrapped emulsion for improving bone density according to any one of claims 3 to 5, wherein the method comprises the steps of: (a) mixing the calcium compound, the oil, the surfactant, and the water to prepare a mixture; (b) homogenizing the mixture; and (c) sterilizing the mixture. The method comprises the following steps: The carrier composition is heated and mixed in water to obtain an aqueous phase; The calcium source is heated and mixed with the oil and the lipophilic emulsifier to obtain an oil-solid phase; The oil-solid phase is mixed with the aqueous phase, and after emulsification, the calcium-embedded emulsion for improving bone density is obtained.

7. The method for preparing a calcium-encapsulated emulsion that helps improve bone density according to claim 6, characterized in that, The mass fraction of the hydrophilic emulsifier in the aqueous phase is 1.5%-2.5%, the mass fraction of the gellan gum is 0.05%-0.5%, and the mass fraction of the xanthan gum is 0.05%-0.5%; 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 temperature for heating and mixing the carrier composition in water is 40°C-80°C, and the time is 10 min-60 min; And / or, the temperature for heating mixing the calcium source with the oil and the lipophilic emulsifier is 40℃-70℃, and the time is 10min-60min.

8. The method for preparing a calcium-encapsulated emulsion that helps improve bone density according to claim 6, characterized in that, The emulsification comprises high-speed shearing and homogenization performed in sequence, and the emulsification step further satisfies at least one of the following characteristics: a. the rotation speed of the high-speed shearing is 10000rpm-20000rpm; b. the time of the high-speed shearing is 1min-5min; c. the pressure of the homogenization is 20MPa-40MPa.

9. A calcium-containing composition, characterized in that, The calcium-embedded emulsion for improving bone density according to any one of claims 3-5 after drying.

10. A dairy product, characterized in that, The calcium-embedded emulsion for improving bone density according to any one of claims 3-5 or the calcium-containing composition according to claim 9.

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