Application of calcium-reinforced lamellar network structure emulsion in preparation of product for promoting calcium absorption and enhancing bone mineral density

Abstract

The invention discloses an application of a calcium-reinforced layered network structure emulsion in preparation of a product for promoting calcium absorption and enhancing bone mineral density, and aims to solve the technical bottlenecks of low absorption rate and insufficient bone targeting of a traditional calcium supplement. According to the emulsion, the insoluble calcium salt is uniformly dispersed in an oil phase by utilizing the density difference between the insoluble calcium salt and the oil phase, and is embedded into a layered network structure formed by proteoglycan. Hydrophobic molecules or hydrophobic chain segments spontaneously gather to form a hydrophobic region, and free diffusion of water molecules and water-soluble ions is effectively prevented, so that solubilization and stable dispersion of insoluble calcium salt are realized. The obtained emulsion has good processing stability and tolerance, the absorption rate of calcium is remarkably improved, and the bone mineral density is favorably enhanced. The system can be used as a calcium fortifying preparation to realize on-line addition, the process is simple and convenient, the system is suitable for various foods such as dairy products, biscuits, ice cream and plant milk, and preparation and application of high-calcium nutritious foods are realized.

Description

Technical Field

[0001] This invention belongs to the field of food colloids and food industry technology, specifically relating to the use of a calcium-enhanced layered network structure emulsion in the preparation of products that promote calcium absorption and enhance bone density. Background Technology

[0002] Calcium is the most abundant mineral element in the human body and an essential nutrient for maintaining normal physiological functions. Calcium accounts for approximately 1.5% to 2.0% of body weight, with about 99% distributed in bones and teeth, and the remainder found in blood, extracellular fluid, and soft tissues. It participates in various physiological processes such as nerve signal transmission, muscle contraction, and thrombin activation. The World Health Organization recommends a daily calcium intake of 800–1200 mg for adults; however, a large population worldwide still suffers from insufficient calcium intake, and the incidence of calcium deficiency and its associated metabolic diseases such as osteoporosis is on the rise.

[0003] The primary source of calcium is diet, but a regular diet often falls short of the body's calcium requirements, necessitating calcium supplementation. Commonly used calcium supplements include calcium carbonate, calcium lactate, and calcium gluconate. In recent years, milk mineral salts (also known as milk calcium) have been widely used as a novel calcium source in dairy products, functional beverages, and baked goods. Milk mineral salts are derived from milk and whey processing byproducts, primarily composed of calcium phosphate, and contain small amounts of protein, lactose, and various minerals. However, calcium salt supplements generally suffer from poor solubility, low bioavailability, and a tendency to cause gastrointestinal irritation. Furthermore, during liquid food processing, they are susceptible to precipitation or crystallization due to heat treatment, shear stress, and pH changes, leading to reduced system stability.

[0004] To improve the dispersibility and absorption efficiency of calcium salts in food systems, researchers have proposed various calcium delivery strategies such as chelation, encapsulation, and emulsification. However, existing technologies still suffer from shortcomings such as poor structural stability, low encapsulation efficiency, and insufficient processing adaptability, making it difficult to meet the needs of industrial applications. Especially in liquid systems, the aqueous environment easily leads to the reaction of insoluble calcium salts with external ions to form precipitates, affecting the calcium fortification effect. Therefore, there is an urgent need to develop a calcium-fortified emulsion system with good dispersibility and processing tolerance to improve calcium bioavailability and broaden its application in liquid foods. Summary of the Invention

[0005] The purpose of this invention is to provide a calcium-enhanced layered network structure emulsion for use in the preparation of products that promote calcium absorption and enhance bone density. By embedding calcium milk into the network structure, the hydrophobic barrier effect is utilized to achieve a solubilizing effect on poorly soluble substances. Moreover, the emulsion preparation is simple, low-energy, and easy to add online. It is high in calcium and rich in nutrients, and can be applied to dairy products, plant-based beverages, and functional beverages.

[0006] This invention is achieved through the following technical solution: The use of a calcium-fortified layered network emulsion in the preparation of high-calcium nutritional foods and bone-density-enhancing health products, the preparation method of the calcium-fortified layered network emulsion includes the following steps: (1) Network structure preparation: Dissolve the protein in deionized water, add a small amount of defoamer, and stir at 50 °C for 30 min. Add the polysaccharide to the water and stir at 70-80 °C for 30 min; add the polysaccharide solution to the protein solution in different proportions and stir and shear at 50 °C for 10-20 min to form a layered network structure of the proteoglycan solution.

[0007] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then glyceryl monostearate was added and dissolved at 70 °C for 10 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase was added to the proteoglycan solution and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 200-300 bar. After two homogenizations, an emulsion with a milky aroma was obtained.

[0008] Further, in step (1), the concentration of the protein solution is 1~4 wt%, the concentration of the polysaccharide is 0.01~0.5 wt%, and the mass ratio of protein to polysaccharide is 1:1-1:5.

[0009] Furthermore, during the dissolution process of the colloid in step (1), the temperature is measured with a thermometer in advance to ensure that the temperature is 70 ℃ (±5 ℃) to prevent the colloid from clumping in the low-temperature aqueous solution, making it difficult to disperse and affecting the formation of the network structure. After the polysaccharide is dissolved, there are no white undissolved particles visible to the naked eye, and the solution is slightly transparent.

[0010] Furthermore, in step (2), the proportion of glyceryl monostearate added to the butter is 0.1-1 wt%, ensuring that the glyceryl monostearate is completely dissolved, the oil becomes clear and transparent, without crystal particles, and changes the interfacial characteristics of the oil phase; the milk mineral salt is completely dispersed in the oil phase without agglomeration, and stirring is kept as much as possible before mixing and shearing, and the uniformity is judged according to a 40-mesh sieve.

[0011] Further, in step (2), the milk calcium is added to the oil emulsion and dispersed by ultrasonication at 800 W for 10-15 min. Ultrasonication is used to embed the milk calcium into the treated oil phase, thereby improving its dispersibility. If the ultrasonic power is too low (<800 W), the milk calcium will be unevenly dispersed, the system will be unstable, and precipitation will be easy; if the ultrasonic rate is too high (>800 W), it will easily damage the structure of the treated oil phase, leading to milk calcium precipitation and system instability.

[0012] Further, in step (3), the mass ratio of the solid dispersed phase to the proteoglycan solution is 1:9-4:6. The proteoglycan solution needs to be preheated to 50 °C to prevent the treated oil phase from crystallizing in the proteoglycan solution and affecting the dispersion of milk calcium; after mixing, rapid shearing causes the inner hydrophobic molecules or hydrophobic segments to spontaneously aggregate and form hydrophobic regions, preventing premature release of calcium salts and improving their targeting and stability in the digestive tract; the outer layer forms hydrophilic regions, thereby solving the problem of poor dispersion stability of insoluble calcium salts in liquid foods such as dairy products and functional beverages.

[0013] The above-prepared emulsion is used in dairy products, plant-based beverages, functional beverages, and other foods and health products that enhance bone density.

[0014] Beneficial effects This application provides the use of a calcium-fortified layered network emulsion in the preparation of products that promote calcium absorption and enhance bone density. By embedding a poorly soluble calcium salt with altered crystal structure into the network structure, the calcium milk is uniformly dispersed in the aqueous phase, forming an emulsion with good physical stability and tolerance to processing environments. Compared to calcium milk and calcium carbonate, the calcium-fortified layered network emulsion increases blood calcium levels by 4.46%, increases the concentration of calcium ions in the intestinal lumen, promotes calcium uptake and transport by intestinal epithelial cells, increases bone diameter and mechanical strength by 25.94%, and enhances bone density by 26.81%, significantly improving calcium bioavailability and demonstrating a clear effect on promoting calcium absorption. Furthermore, it can be used as a calcium fortification or bone density-enhancing ingredient, allowing for online addition to liquid foods and application in calcium-fortified foods and health products. Attached Figure Description

[0015] Figure 1 Microstructure diagrams of calcium milk and calcium milk dispersed in a network structure emulsion for Comparative Example 1 and Example 1; Figure 2 This is a microstructure diagram of the emulsion based on a hydrophobic barrier network structure in Example 2. Figure 3 Visual representation of Comparative Example 5 and Examples 1 and 2 after centrifugation for 4.25 h; Figure 4 This is a visual diagram and a microstructure diagram of the thermal stability of the network structure emulsion based on hydrophobic barrier in Example 3; Figure 5The freeze-thaw stability results of the hydrophobic barrier network structure emulsions in Comparative Example 6 and Example 4 are shown. Figure 6 The images show samples of calcium-fortified emulsions applied to yogurt in Comparative Examples 3, 4, and Example 5. Figure 7 The rheological properties and calcium bioavailability of yogurts in Comparative Examples 3, 4 and Example 5; Figure 8 The blood calcium absorption of Comparative Example 1, Comparative Example 2 and Example 1; Figure 9 Bone density for Comparative Example 1, Comparative Example 2 and Example 1. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0017] Example 1 (1) Dissolve 3 wt% whey protein in 96.999 g deionized water, add 0.001 wt% defoamer, and stir at 50°C for 30 min. Add 0.2 wt% gellan gum to 99.8 g water, stir at 70°C for 30 min, mix the protein solution and polysaccharide solution at a 1:1 mass ratio, and shear at 10000 rpm for 5 min to form a proteoglycan solution with a layered network structure.

[0018] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10-15 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 1:9 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0019] Example 2 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.1 wt% gellan gum and 0.4 wt% xanthan gum were added to 99.9 g and 99.6 g water, respectively, and stirred at 70 ℃ for 30 min. The gellan gum solution and xanthan gum solution were mixed at a mass ratio of 1:2 and stirred at 50 ℃ for 10 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1 and sheared at 10000 rpm for 5 min to form a layered network structure proteoglycan solution.

[0020] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10-15 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 2:8 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0021] Example 3 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.1 wt% gellan gum and 0.3-0.5 wt% xanthan gum were added to 99.9 g and 99.5-99.7 g water, respectively, and stirred at 70 ℃ for 30 min. The gellan gum solution and xanthan gum solution were mixed at a mass ratio of 1:2 and stirred at 50 ℃ for 10 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1 and sheared at 10000 rpm for 5 min to form a layered network structure proteoglycan solution.

[0022] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10-15 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 2:8 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0023] (4) The calcium-fortified emulsion was pasteurized (63 °C, 30 min) and subjected to high temperature short-time treatment (90 °C, 3 min), and then immediately cooled to room temperature after heat treatment.

[0024] Example 4 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.1 wt% gellan gum and 0.5 wt% xanthan gum were added to 99.9 g and 99.5 g water, respectively, and stirred at 70 ℃ for 30 min. The gellan gum solution and xanthan gum solution were mixed at a mass ratio of 1:2 and stirred at 50 ℃ for 10 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1 and sheared at 10000 rpm for 5 min to form a layered network structure proteoglycan solution.

[0025] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10-15 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 2:8 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0026] (4) The freshly prepared emulsion was frozen in a -20°C freezer for 24 h, and then thawed at 25°C for 2 h. Physical stability was measured and appearance was analyzed. The freeze-thaw cycle was repeated 3 times.

[0027] Example 5 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.1 wt% gellan gum and 0.5 wt% xanthan gum were added to 99.9 g and 99.5 g water, respectively, and stirred at 70 ℃ for 30 min. The gellan gum solution and xanthan gum solution were mixed at a mass ratio of 1:4 and stirred at 50 ℃ for 10 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1 and sheared at 10000 rpm for 5 min to form a layered network structure proteoglycan solution.

[0028] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 2:8 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0029] (4) Mix 5 g of calcium-fortified emulsion with 90 g of milk and 4.9 g of white sugar thoroughly, add 0.1 g of coagulating yogurt starter (Streptococcus thermophilus and Lactobacillus bulgaricus = 1:1), mix thoroughly to disperse, put into a fermentation box, place in a constant temperature fermentation chamber at 42 ℃ for 8 h, take out a portion of the sample to measure the pH value, and ensure that the pH value is around 4.2 to reach the fermentation endpoint. Take out the yogurt, cool it to room temperature, and put it in the refrigerator at 4 ℃ for 24 h for post-fermentation.

[0030] Comparative Example 1 Milk mineral salt raw materials purchased from Fonterra Ltd. on the market.

[0031] Comparative Example 2 Food-grade calcium carbonate purchased from Zhengzhou Shuangteng Industrial Co., Ltd.

[0032] Comparative Example 3 Mix 95 g of milk and 4.9 g of white sugar thoroughly, add 0.1 g of set-type yogurt starter (Streptococcus thermophilus and Lactobacillus bulgaricus = 1:1), mix thoroughly to disperse, put into a fermentation box, and place in a constant temperature fermentation chamber at 42 ℃ for 8 hours. Take out a sample to measure the pH value. Ensure that the pH value is around 4.2, which means that the fermentation has reached the endpoint. Take out the yogurt, cool it to room temperature, and put it in the refrigerator at 4 ℃ for 24 hours to ripen.

[0033] Comparative Example 4 Mix 1 g of milk calcium with 94 g of milk and 4.9 g of white sugar thoroughly. Add 0.1 g of set-type yogurt starter (Streptococcus thermophilus and Lactobacillus bulgaricus = 1:1), mix thoroughly to disperse, and put into a fermentation box. Place in a constant temperature fermentation chamber at 42 ℃ for 8 hours. Take out a sample to measure the pH value. Ensure that the pH value is around 4.2, which is the end of fermentation. Take out the yogurt, cool it to room temperature, and refrigerate it at 4 ℃ for 24 hours to ripen.

[0034] Comparative Example 5 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.2 wt% gellan gum was added to 99.8 g water, and the mixture was stirred at 70 ℃ for 30 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1, and the mixture was sheared at 10000 rpm for 5 min to form a layered network structure of proteoglycan solution.

[0035] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed to form a solid dispersed phase. (3) Emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 1:9 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0036] Comparative Example 6 (1) Network structure preparation: 3 wt% whey protein was dissolved in 96.999 g deionized water, 0.001 wt% defoamer was added, and the mixture was stirred at 50 ℃ for 30 min. 0.1 wt% gellan gum was added to 99.9 g water and stirred at 70 ℃ for 30 min. The protein solution and polysaccharide solution were mixed evenly at a mass ratio of 1:1 and sheared at 10000 rpm for 5 min to form a layered network structure of proteoglycan solution.

[0037] (2) Calcium milk embedded in the oil phase: The butter was melted at 70 °C, and then 1 wt% glyceryl monostearate was added and dissolved at 70 °C for 10 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase and the proteoglycan solution were mixed at a mass ratio of 2:8 and sheared at 12,000 rpm for 3 min to obtain a crude emulsion. The crude emulsion was then homogenized at 250 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

[0038] (4) The freshly prepared emulsion was frozen in a -20°C freezer for 24 h, and then thawed at 25°C for 2 h. Physical stability was measured and appearance was analyzed. The freeze-thaw cycle was repeated 3 times.

[0039] I. Physicochemical Properties Testing Microstructure testing: The microstructure of the emulsions prepared in Examples 1 and 2 was analyzed using a confocal laser scanning microscope (CLSM) according to the method described. Rhodamine B, Nile Red, and Nile Blue A were used to stain the calcium, oil phase, and proteins of the emulsion, respectively, with excitation wavelengths of 543 nm, 488 nm, and 635 nm. The stained samples were vortexed for 10 s, equilibrated for 12 h, magnified 60 times under an oil immersion microscope, and the obtained microscopic images were then processed and analyzed using software.

[0040] Cryo-scanning electron microscopy: After freezing a 2.0 μL sample in liquid nitrogen, it was transferred to a cryogenic preparation chamber under vacuum. The pre-frozen sample was fractured, sublimated (-80 °C, 1.3 × 10−6 mbar, 8 min), sputter-coated with gold (5 mA, 90 s), and then the microstructure of the sample was observed using field emission scanning electron microscopy (FE-SEM) at -140 °C.

[0041] Apparent viscosity: The apparent viscosity of the samples was determined using a Haake RheoStress rheometer through continuous shear tests. Shear viscosity, parameters: rotor model CC27 DG / Ti, shear rate 0.1-100 s⁻¹. -1 The test was conducted over a period of 60 seconds, with 65 data points at a test temperature of 25 ℃. The test was repeated three times.

[0042] Physical stability: The stability of the samples was analyzed using a LUMiSizer stability analyzer (LUM GmbH, Berlin, Germany). The test parameters were as follows: temperature 25℃; injection volume 0.4 mL; rotation speed 4000 rpm; spectral acquisition interval 60 s; total acquisition time 4.25 h.

[0043] II. Animal Experiments 2.1 Preparation of animal models Eighty SPF-grade female SD rats were randomly divided into two groups after one week of acclimatization: a sham-operated 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.

[0044] 2.2 Grouping of experimental animals Seventy ovariectomized rats were randomly divided into the following seven groups (n=10): model group, calcium carbonate group (comparative example 2), comparative example 1 (low-dose milk calcium group (MMS-L), high-dose milk calcium group (MMS-H)), and example 1 (low-dose embedded milk calcium group (MFEs-L) and high-dose embedded milk calcium group (MFEs-H)). The calcium intake of the low-dose group rats was 66.65 mg / d, and the calcium intake of the high-dose group rats was 133.3 mg / d.

[0045] 2.3 Bone mineral density and bone microstructure analysis 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.

[0046] from Figure 1 As can be seen in the upper right corner, the calcium milk forms irregular crystalline aggregates, while the calcium-fortified emulsion of Example 1 exhibits a layered network structure. When both are dissolved in water simultaneously, it is observed that the calcium milk remains aggregated after dissolving in water. However, after treatment, the calcium-fortified emulsion of Example 1 disperses evenly in water, indicating that this technology has a solubilizing effect on poorly soluble substances.

[0047] Figure 2 The results show that the layered network of the protein-gellan gum / xanthan gum ternary complex can anchor oil droplets in the pores through the hydrophobic phase resistance effect, inhibiting droplet movement and aggregation, thereby forming an emulsion with smaller droplet size and better stability.

[0048] Figure 3 As can be seen, compared with Comparative Example 5, significant water separation occurred after 4.25 h of centrifugation at 4000 rpm, while Examples 1 and 2 showed a stable state without stratification. The results indicate that the addition of glyceryl monostearate can improve the interfacial properties and physical stability of the emulsion by forming a stable adsorption layer at the oil-water interface, reducing interfacial tension, and enhancing the viscoelasticity of the interfacial film.

[0049] Figure 4 The appearance of the emulsion after heat treatment under different conditions is shown. No changes were observed in the appearance of the emulsion after pasteurization (63℃ for 30 min) and sterilization (90℃ for 3 min). The addition of xanthan gum to the emulsion protects the proteins from heat and provides sufficient electrostatic repulsion and spatial resistance to prevent the emulsion from being affected by the degree of heating.

[0050] Figure 5To verify the extent of damage to the calcium-fortified emulsion caused by freeze-thaw treatment, LUMISizer stability tests were performed on Comparative Example 6 and Example 4. Under strong centrifugal force, the sample in Comparative Example 6 underwent phase separation. Compared to Comparative Example 6, the transmission curve of Example 4 did not shift after three freeze-thaw treatments, indicating better freeze-thaw stability. This is mainly due to the dense gel network formed by whey protein-gellan gum / xanthan gum. This network not only reduces droplet flocculation and aggregation by restricting droplet flow during freezing, but also protects droplets from internal pressure changes and shrinkage that may occur during freezing. Simultaneously, xanthan gum increases the viscosity of the emulsion, thereby inhibiting the desorption of the WPC-GG / XG complex during thawing, limiting ice crystal penetration into the oil phase, and contributing to enhanced oil-water interfacial film integrity.

[0051] Figure 6 As can be seen, compared with Comparative Example 3, some water was separated in Comparative Example 4, and the appearance of Example 5 is similar to that of Comparative Example 3. Figure 7 The results showed that all three yogurt samples exhibited a trend of decreasing overall viscosity with increasing shear rate, demonstrating a pseudoplastic flow state characterized by shear thinning. At the same shear rate, Example 5 showed the highest viscosity. This is mainly due to the embedding of tiny oil droplets or solid particles in the protein network within the emulsion, acting as "physical cross-linking points" and enhancing gel strength. Simultaneously, the emulsifier binds with casein to form a composite interfacial film, enhancing the elasticity of the gel network. With increasing shear rate, the apparent viscosities of Example 5 and Comparative Example 4 became identical, indicating the presence of similar rheological properties. Compared to Comparative Examples 3 and 4, the calcium bioavailability of Example 5 was improved by 22% and 43%, respectively.

[0052] like Figure 8 As shown, the dynamic changes in serum calcium concentration in rats administered with the same calcium ion content via gavage (Comparative Example 1, MMS), calcium carbonate (Comparative Example 2, CaCO3), and calcium-fortified emulsions (Example 1, MFEs) were compared over 0-24 hours. The results showed that serum calcium concentration in all groups increased over 0-6 hours, reaching a peak at hour 6. Example 1 showed the fastest rate of increase in serum calcium, with a peak concentration significantly higher than Comparative Examples 1 and 2. These results indicate that the solubility and release characteristics of milk calcium are improved after treatment, making it more easily absorbed in the intestines and thus enabling it to enter the bloodstream more quickly, thereby improving calcium bioavailability.

[0053] Figure 9Three-dimensional images of the skeleton were obtained using techniques such as 3D reconstruction, and quantitative analysis of the bone microstructure was performed on the 3D reconstructed images of the femurs of each group of mice. In the model 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 with the model group, the positive control group, comparative examples 1 and 2, and Example 1 showed reduced bone microstructure damage, specifically manifested as more continuous trabecular connections, increased number of trabeculae, and reduced intertrabecular gaps. In Example 1, the low-dose trabeculae showed a more uniform and orderly arrangement, 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 the system may effectively enhance bone microstructure reconstruction and calcium deposition by promoting calcium absorption and utilization.

Claims

1. The use of a calcium-fortified layered network structure emulsion in the preparation of high-calcium nutritional foods and bone-density-enhancing health products, characterized in that, A method for preparing a calcium-reinforced layered network structure emulsion includes the following steps: (1) Network structure preparation: Dissolve the protein in deionized water, add a small amount of defoamer, and stir at 50 °C for 30 min; add the polysaccharide to water and stir at 70-80 °C for 30 min; add the polysaccharide solution to the protein solution in different proportions and stir and shear at 50 °C for 10-20 min to form a layered network structure of the proteoglycan solution. (2) Calcium milk embedded in the oil phase: The butter was melted at 70-75 ℃, and then glyceryl monostearate was added and dissolved at 70 ℃ for 10 min. Calcium milk powder was added to the treated butter at a calcium content of 1.5 g / 100 ml. The sample was sonicated to form a light yellow cream, so that the calcium milk was evenly dispersed and a solid dispersed phase was formed. (3) Hydrophobic barrier emulsion: The solid dispersed phase was added to the proteoglycan solution and sheared at 12,000 rpm for 3 min to obtain a crude emulsion; then the crude emulsion was homogenized at 200-300 bar, and after two homogenizations, an emulsion with a milky aroma was obtained.

2. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that, In the preparation method of calcium-reinforced layered network structure emulsion, the polysaccharide in step (1) is dissolved at a temperature of 70 ± 5 ℃.

3. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that... In the preparation method of calcium-reinforced layered network structure emulsion, the concentration of proteoglycan solution in step (1) is 1~4 wt%, and the concentration of polysaccharide is 0.01~0.5 wt%; the mass ratio of protein to polysaccharide is 1:1-1:

5.

4. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that, In the preparation method of calcium-reinforced layered network structure emulsion, the proportion of glyceryl monostearate added to butter in step (2) is 0.1~1 wt%.

5. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that, Preparation method of calcium-reinforced layered network structure emulsion: (2) Add calcium milk to the oil emulsion and disperse it under ultrasonic 800 W for 10-15 min.

6. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that, In the preparation method of calcium-reinforced layered network structure emulsion, after the emulsion mineral salt is completely dispersed in the oil phase in step (2), it can pass through a 40-mesh sieve.

7. The use of the calcium-fortified layered network structure emulsion according to claim 1 in the preparation of high-calcium nutritional foods and bone density-enhancing health products, characterized in that, In the preparation method of calcium-reinforced layered network structure emulsion, step (3) has a mass ratio of solid dispersed phase to proteoglycan solution of 1:9-4:6, and the proteoglycan solution is preheated to 50 °C.

8. The use of a calcium-fortified layered network structure emulsion according to any one of claims 1-7 in the preparation of high-calcium nutritional foods and bone-density-enhancing health products, characterized in that, The dosage forms of the high-calcium nutritional foods and bone density-enhancing health products include oral liquids, soft capsules, and / or the foods include dairy products, biscuits, and plant-based milks.

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