Gel microspheres co-loaded with iron and vitamin D as well as preparation method and application of gel microspheres

Abstract

The invention discloses gel microspheres co-loaded with iron and vitamin D as well as a preparation method and application of the gel microspheres, and belongs to the technical field of nutrient enrichment compounds. The preparation method disclosed by the invention comprises the following steps: swelling a whey protein isolate fiber solution and an artemisia desertorum polysaccharide solution, and mixing to obtain a mixed solution; then adding algal oil into the mixed solution, and emulsifying the obtained mixture to obtain O / W type composite emulsion; adding the O / W type composite emulsion into a FeSO4 solution A, mixing, and carrying out static oxidation to obtain composite emulsion gel; and extruding the composite emulsion gel into a FeSO4 solution B, and carrying out cross-linking curing to obtain the gel microspheres co-loaded with iron and vitamin D. According to the method disclosed by the invention, the obtained microspheres can keep a stable structure in a simulated gastric juice environment and realize gradient release of iron ions and vitamin D in simulated intestinal juice, and the oral bioavailability of the two nutrients is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of nutritional fortification compounds, and particularly relates to gel microspheres co-loaded with iron and vitamin D, and a preparation method and application thereof. Background Art

[0002] Compared with cow's milk, goat's milk has the following significant advantages: (1) Higher digestibility and absorption: goat's milk fat globules are smaller in size and rich in medium and short chain fatty acids, which are easy for human digestion and absorption, especially suitable for people with lactose intolerance; (2) Lower allergenicity: its α s1 - The low casein content can reduce the risk of allergies and has potential adaptability for some groups allergic to cow's milk protein; (3) It contains rich functional ingredients: such as lactoferrin, nucleotides and natural prebiotics, which have a positive effect on regulating intestinal health. However, although goat milk has good nutritional value in terms of protein, calcium and some vitamins, it is naturally deficient in two key nutrients: iron and vitamin D (Vit D). This defect seriously limits goat milk products from becoming a food that meets comprehensive nutritional needs, especially for special groups with higher nutritional needs such as athletes.

[0003] Iron is a core element that constitutes hemoglobin and myoglobin. It is responsible for the transport and storage of oxygen and is crucial for maintaining cellular energy metabolism, body vitality and immune function. Iron deficiency can lead to anemia, fatigue and cognitive decline, which is particularly significant in high-oxygen-demand groups such as athletes. It is one of the key factors in nutritional security. Vitamin D (Vitamin D, Vit D) mainly regulates calcium and phosphorus metabolism, significantly improves intestinal calcium absorption rate and promotes bone mineralization, while affecting muscle function and immune response. The vitamin D required by the human body mainly depends on skin sunlight synthesis, but due to climate, lifestyle and lack of food sources, athletes have a more urgent need for vitamin D supplementation due to heavy training loads and frequent indoor environments. Therefore, strengthening the intake and absorption of iron and vitamin D is an important issue that urgently needs to be addressed. At present, some sports supplements on the market have tried to improve the deficiency of iron and vitamin D through nutritional fortification, but the following problems still exist: (1) Technical challenges of iron fortification: Iron compounds (such as ferrous sulfate and ferrous pyrophosphate) may affect the flavor and stability of goat milk, resulting in metallic taste or oxidation problems; some iron fortifiers have low absorption rates and cannot effectively improve iron deficiency. (2) Technical challenges of vitamin D fortification: Fat-soluble vitamin D is difficult to disperse evenly in a water-based goat milk system and is prone to precipitation or aggregation; the lack of an efficient delivery system leads to low intestinal release and absorption rates. Summary of the Invention

[0004] The purpose of the present invention is to provide gel microspheres co-loaded with iron and vitamin D, and a preparation method and application thereof, so as to solve the technical problem of poor bioavailability of existing sports nutrition supplements.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a method for preparing gel microspheres co-loaded with iron and vitamin D, comprising the following steps:

[0007] The whey protein isolate fiber solution and the Artemisia sphaerocarpa polysaccharide solution are swollen and then mixed to obtain a mixed solution;

[0008] Subsequently, algae oil is added to the mixed solution, and the resulting mixture is emulsified to obtain an O / W composite emulsion;

[0009] The O / W composite emulsion was added to FeSO4 solution A and mixed, and static oxidation was performed to obtain a composite emulsion gel;

[0010] The composite emulsion gel is extruded into FeSO4 solution B for cross-linking and curing to obtain gel microspheres co-loaded with iron and vitamin D.

[0011] Furthermore, the pH values ​​of the whey protein isolate fiber solution and the Artemisia polysaccharide solution are both 3.8-4.2. The whey protein isolate fiber solution and the Artemisia polysaccharide solution are respectively obtained by mixing whey protein isolate fiber powder and Artemisia polysaccharide powder with deionized water;

[0012] The mass ratio of the Artemisia polysaccharide fiber powder to the whey protein isolate powder in the mixed solution is (7-19): (1-3).

[0013] Furthermore, in the mixture, the mass concentration of algae oil is 20% to 25%, and the mass concentration of Artemisia polysaccharide powder is 1.5% to 2%.

[0014] Furthermore, the emulsification is carried out in a shearing machine at 18000-19000 r / min for 3-4 minutes.

[0015] Furthermore, the final concentration of the whey protein isolate fiber in the O / W composite emulsion is 0.08% to 0.64%.

[0016] Furthermore, the molar concentration of Fe in the composite emulsion gel is 80 to 100 mM;

[0017] The concentration of the FeSO4 solution A is 1.0-1.5 mol / L;

[0018] The static oxidation time is 48 to 72 hours.

[0019] Furthermore, the concentration of the FeSO4 solution B is 1.0 to 1.5 mol / L;

[0020] The cross-linking and curing time is 3 to 4 hours.

[0021] Furthermore, the extrusion is performed using a syringe; the volume of the syringe is 1 to 1.2 mL, and the inner diameter of the needle connected to the syringe is 0.09 to 0.1 mm;

[0022] The pH value of the mixed solution is 3.8 to 4.2;

[0023] The cross-linking and curing step further includes a washing process.

[0024] The invention also discloses gel microspheres co-loaded with iron and vitamin D prepared by the preparation method.

[0025] The present invention also discloses the use of the above-mentioned gel microspheres co-loaded with iron and vitamin D in sports nutrition supplements. The total iron content of the sports nutrition supplement reaches 4.16±0.73%. In simulated gastrointestinal digestion, the iron ion release rate is greater than 98%, and the free fatty acid release rate is 42.23±2.61%.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses a preparation method of gel microspheres co-loaded with iron and vitamin D, which is based on the electrostatic interaction between whey protein isolate fiber and Artemisia sphaerocarbam polysaccharide to construct an O / W type composite emulsion, and further 2+ to Fe 3+ Controllable oxidative transformation of WPIF-Fe 2+ Chelation cross-linking and ASKP-Fe 3+ Chelation and cross-linking are performed to form a composite emulsion gel with a semi-interpenetrating network structure, which is finally prepared into gel microspheres. The method of the present invention enables the obtained microspheres to maintain structural stability in a simulated gastric fluid environment and achieve a gradient release of iron ions and vitamin D in simulated intestinal fluid, significantly improving the oral bioavailability of the two nutrients.

[0028] The present invention also discloses gel microspheres co-loaded with iron and vitamin D prepared by the above-mentioned preparation method. The gel microspheres co-loaded with iron and vitamin D prepared by the present invention can form a composite emulsion gel delivery system. The composite emulsion gel delivery system can simultaneously deliver hydrophilic iron and hydrophobic vitamin D. A typical semi-interpenetrating network exists in its structure. The system has the characteristics of high-density structure and small porosity. The obtained gel microspheres are spherical, smooth on the surface, and slightly yellow in color. This unique structure gives the system the two core functions of maintaining structural integrity in the gastric fluid environment, realizing gastric protection of nutrients and orderly dissociation through responsive swelling in the intestinal fluid environment, and has significant digestion lag effect and intestinal targeting. As a co-loaded and enhanced model for iron and vitamin D, the system can effectively overcome the problem of synergistic delivery of the two types of nutrients in goat milk powder, and has clear application potential in the field of nutritional fortification of goat milk products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 2 shows the particle size distribution, Zeta potential and storage stability of O / W composite emulsions obtained at different WPIF and ASKP concentration ratios.

[0030] Wherein: a-particle size distribution diagram of O / W composite emulsion obtained with different WPIF and ASKP concentration ratios; b-Zeta potential diagram of O / W composite emulsion obtained with different WPIF and ASKP concentration ratios; c-storage stability diagram of O / W composite emulsion obtained with different WPIF and ASKP concentration ratios;

[0031] Figure 2 Polarizing microscope and fluorescence microscope images of O / W composite emulsion;

[0032] Among them: a polarizing microscope image of O / W composite emulsion; b fluorescence microscope image of O / W composite emulsion;

[0033] Figure 3 Macroscopic image and SEM image of composite emulsion gel; Figure 4 The appearance and particle size distribution of gel microspheres co-loaded with iron and vitamin D;

[0034] Figure 5 The appearance and particle size distribution of gel microspheres co-loaded with iron and vitamin D;

[0035] Figure 6 Figure 2 is a diagram of the transcellular efficiency of iron ions and FFAs in gel microspheres co-loaded with iron and vitamin D;

[0036] Among them: a-transcellular efficiency diagram of iron ions in gel microspheres; b-transcellular efficiency diagram of FFAs in gel microspheres;

[0037] Figure 7This is a graph showing the changes in serum iron and FFAs over time after rats orally administered gel microspheres co-loaded with iron and vitamin D.

[0038] Among them: a-the change of serum iron ion concentration over time after rats orally administered gel microsphere samples; b-the change of serum FFAs concentration over time after rats orally administered gel microsphere samples. DETAILED DESCRIPTION

[0039] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0040] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0041] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0042] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0043] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0044] The present invention provides a method for preparing gel microspheres co-loaded with iron and vitamin D, comprising the following steps:

[0045] S1: Dissolve whey protein isolate (WPI) powder in deionized water, stir and mix to obtain a WPI solution, adjust the pH value to 2.0, heat in a 90°C water bath for 5 hours, cool to room temperature, and then vacuum freeze-dry to obtain whey protein isolate fiber (WPIF) powder;

[0046] S2: S2 dissolves coarse Artemisia sphaerosa gum powder in deionized water to obtain a 0.7% (w / v) solution. Magnetic stirring is performed at room temperature for 12 hours until fully swollen. The solution is then vacuum filtered and concentrated by rotary evaporation at 50°C and 0.1 MPa to 1 / 5 of its original volume. The resulting concentrate is dialyzed for 72 hours to fully remove small molecule impurities. The dialyzed solution is collected, concentrated by rotary evaporation a second time, and then freeze-dried under vacuum to obtain purified Artemisia sphaerosa polysaccharide (ASKP) powder.

[0047] S3: Prepare a solution of a certain concentration of ASKP and WPIF obtained in steps S1 and S2, adjust the pH to 4.0 after sufficient swelling, and mix them in a certain ratio so that the ratio of ASKP to Artemisia polysaccharide WPIF in the mixed solution is 19:1, 14:1, 9:1, 7:3 and 8:2. After sufficient mixing, add a certain amount of algae oil to make the final concentration of ASKP in the system 1.5%, the algae oil 20%, and the WPIF concentration of 0 as a control. Use a high-speed shearing machine to emulsify at 19000r / min for 3min to obtain an O / W type composite emulsion. According to the above ratios, the final concentrations of whey protein isolate fibers are 0%, 0.08%, 0.11%, 0.17%, 0.38% and 0.64% respectively;

[0048] S4: Add FeSO4 solution to the composite emulsion obtained in step S3, mix quickly and dispense into molds, and let it stand at room temperature for 72 hours to achieve Fe 2+ to Fe 3+ Oxidation of WPIF with Fe 2+ , ASKP and Fe 3+ Composite emulsion gel (CEG) was formed under the cross-linking mode, and the Fe concentration in the final system was 80 mM;

[0049] S5: The composite emulsion obtained in step S4 is squeezed and dripped into a high concentration FeSO4 solution (1.0 mol / L) using a syringe. 2+ Gradually transformed into Fe during continuous oxidation 3+ , cross-linked with WPIF and ASKP, and fully cured for 3 hours to form gel microspheres co-loaded with iron and vitamin D (CEGM). The collected gel microspheres were rinsed three times with ultrapure water to remove excess iron ions on the surface.

[0050] Preferably, in S1, the prepared WPI solution is 2% (w / v), and the pH value of the solution is adjusted to 2.0 with 4M hydrochloric acid.

[0051] Preferably, in S2, the dialysis uses a dialysis bag of 8000-14000Da.

[0052] Preferably, in S5, the syringe is 1 mL and is connected to a needle with an inner diameter of 0.09 mm.

[0053] More specifically, the following steps are included:

[0054] S1: WPI powder was dissolved in deionized water and stirred to obtain a 2% (w / v) WPI solution. The pH value was adjusted to 2.0 with 4M hydrochloric acid, and the solution was heated in a 90°C water bath for 5 h. The solution was cooled to room temperature and then freeze-dried in a vacuum oven to obtain WPIF powder.

[0055] S2: Dissolve the coarse Artemisia sphaerocarpa gum powder in deionized water to obtain a 0.7% (w / v) solution, magnetically stir at room temperature for 12 h until fully swollen, then vacuum filter, and concentrate the filtrate by rotary evaporation at 50°C and 0.1 MPa to 1 / 5 of the original volume. The resulting concentrate is dialyzed continuously for 72 h using an 8000-14000 Da dialysis bag to fully remove small molecular impurities; collect the dialyzed solution, concentrate it by rotary evaporation twice, and vacuum freeze-dry to obtain purified ASKP powder;

[0056] S3 prepares a solution of a certain concentration of ASKP and WPIF obtained in steps S1 and S2, adjusts the pH to 4.0 after sufficient swelling, and mixes them in a certain ratio so that the ratio of ASKP to WPIF in the mixed solution is 19:1, 14:1, 9:1, 7:3 and 8:2. After sufficient mixing, add a certain amount of algae oil to make the final concentration of ASKP in the system 1.5%, the algae oil is 20%, and the WPIF concentration is 0 as a control. Use a high-speed shearing machine to emulsify at 19000r / min for 3 minutes to obtain an O / W type composite emulsion. According to the above ratio, the final concentrations of WPIF are 0%, 0.08%, 0.11%, 0.17%, 0.38% and 0.64% respectively;

[0057] S4 Add FeSO4 solution to the composite emulsion obtained in step S3, quickly mix and dispense into molds, and let it stand at room temperature for 72 hours to achieve Fe 2+ to Fe 3+ Oxidation of WPIF with Fe 2+ , ASKP and Fe 3+ Composite emulsion gel (CEG) was formed under the cross-linking mode, and the Fe concentration in the final system was 80 mM;

[0058] S5: The composite emulsion obtained in step S4 was squeezed and dripped into the high concentration FeSO4 solution (1.0 mol / L) using a 1 mL syringe connected to a needle with an inner diameter of 0.09 mm. 2+ Gradually transformed into Fe during continuous oxidation 3+ , cross-linked with WPIF and ASKP, and fully cured for 3 hours to form composite emulsion gel microspheres (CEGM). The collected gel microspheres were rinsed three times with ultrapure water to remove excess iron ions on the surface.

[0059] The present invention also discloses the use of the above-mentioned gel microspheres co-loaded with iron and vitamin D in sports nutritional supplements. The oral nutritional supplement has a total iron content of 4.16±0.73%, an iron ion release rate of greater than 98%, and a free fatty acid (FFAs) release rate of approximately 42.23±2.61% in simulated gastrointestinal digestion.

[0060] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0061] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by weight, "part" indicates parts by weight, and "ratio" indicates weight ratio.

[0062] Example 1

[0063] A method for preparing gel microspheres co-loaded with iron and vitamin D comprises the following steps:

[0064] ASKP and WPIF were prepared into solutions of varying concentrations. After sufficient swelling, the pH was adjusted to 4.0, resulting in final WPIF concentrations of 0%, 0.08%, 0.11%, 0.17%, 0.38%, and 0.64%. After thorough mixing, algae oil was added to achieve a final ASKP concentration of 1.5% and algae oil concentration of 20%. Emulsification was performed using a high-speed shearing machine at 19,000 rpm for 3 minutes to produce O / W composite emulsions of varying proportions. These emulsions were then extruded using a 1 mL syringe with a 0.09 cm inner diameter needle and dripped into a 1.0 mol / L FeSO₄ solution. Cross-linking and curing were performed for 3 hours, followed by washing to obtain gel microspheres.

[0065] Example 2

[0066] A method for preparing a gel co-loaded with iron and vitamin D comprises the following steps:

[0067] ASKP and WPIF were prepared into solutions of varying concentrations. After sufficient swelling, the pH was adjusted to 4.0, resulting in final WPIF concentrations of 0%, 0.08%, 0.11%, 0.17%, 0.38%, and 0.64%. After thorough mixing, algae oil was added to achieve a final ASKP concentration of 1.5% and algae oil concentration of 20%. Emulsification was performed using a high-speed shearing machine at 19,000 rpm for 3 minutes to produce O / W composite emulsions of varying proportions. These emulsions were then added to a 1.0 mol / L FeSO₄ solution and allowed to oxidize for 72 hours to yield gels.

[0068] Test Example 1

[0069] The Mastersizer 2000 laser particle size analyzer was used to characterize the droplet size distribution characteristics of the O / W composite emulsion obtained with different ASKP and WPIF ratios in Example 1. The sample needs to be pretreated before analysis: the emulsion is placed in a centrifuge tube, diluted with 10 times the volume of deionized water, and gently vortexed to ensure the uniformity of the dispersion system and eliminate the interference of particle agglomeration. The optical parameters are set to a dispersed phase refractive index of 1.54, a continuous phase refractive index of 1.33, and an absorbance coefficient compensation value of 0.01 to ensure the accuracy of the particle size analysis data. The Zeta potential of the emulsion was determined using a Zetasizer Nano-ZS nanoparticle size and potential analyzer. Before the test, the emulsion was gently shaken to ensure uniformity, the measurement temperature was maintained at 25°C, the refractive index was set to 1.33, the dispersed phase refractive index to 1.47, the absorbance index to 0.01, and the detection wavelength to 633nm. At the same time, the storage stability of the emulsion was observed macroscopically, and photographed and recorded. The results are as follows Figure 1 shown.

[0070] Depend on Figure 1It can be seen that the addition of different concentrations of WPIF has a significant effect on the particle size distribution, Zeta potential and storage stability of the composite emulsion. After adding WPIF, the emulsion particle size is greatly reduced, and as the WPIF concentration increases, the emulsion particle size distribution range gradually decreases. Studies have shown that due to the special size and morphology of WPIF, it can undergo irreversible adsorption at the oil-water interface and assemble to form an interfacial film with high electrostatic repulsion. At the same time, its intermolecular hydrogen bonds and van der Waals forces can effectively prevent the droplet from coalescing by enhancing the rigidity of the interfacial film. The absolute value of the Zeta potential of all samples is greater than 50mV. When the WPIF concentration is between 0.08% and 0.17%, the Zeta potential of the emulsion decreases significantly, indicating that the electrostatic repulsion increases, effectively preventing the droplet from agglomerating; when the WPIF concentration is greater than 0.17%, the electrostatic repulsion weakens and the aggregation tendency increases. The six groups of samples were stored at 4°C for 20 days. It was found that the emulsions without WPIF showed obvious stratification and poor stability. The five groups of emulsions with WPIF were relatively stable, evenly dispersed, and no stratification occurred. This may be because the irreversible adsorption of WPIF at the oil-water interface hindered the aggregation of oil droplets, effectively improving the stability of the emulsion.

[0071] Test Example 2

[0072] The polarized light mode of an optical microscope was used to identify the monorefractive and birefringent properties of the emulsion interface and to observe the arrangement of protein fibers at the emulsion interface. Fluorescence microscopy was used to analyze the interface structure of the O / W composite emulsion droplets obtained by different ratios of ASKP and WPIF in Example 1. The aqueous phase and the oil phase were fluorescently labeled with Nile blue (0.1%, w / v) and Nile red (0.1%, w / v), and two-color fluorescence visualization was achieved under 633nm and 488nm excitation light. Image analysis software was used to collect and store images. The results are shown in Figure 2. Figure 2 shown.

[0073] Depend on Figure 2 (a) It can be seen from the polarized light microscope image that the edge of the CE emulsion droplet is darker in color, and WPIF does not exhibit birefringence at the interface, indicating that it presents an isotropic random arrangement at the interface. Figure 2 (b) Fluorescence microscopy image shows a clear oil-water interface in the emulsion, where ASKP and WPIF exist as a complex. This indicates that the CE emulsion exhibits electrostatic attraction, allowing WPIF to intersperse within the ASKP and form a random arrangement at the interface.

[0074] Test Example 3

[0075] The macroscopic appearance of the gel obtained by the final oxidation in Example 2 was observed and photographed. The microscopic morphology of the gel was characterized by SEM. The emulsion gel was immersed in liquid nitrogen and quenched to obtain a cross-section sample. After vacuum freeze-drying, the natural cross-section gel block was fixed to the stage with conductive glue and metal-plated using an ion sputtering instrument. Finally, the gel network structure was observed by high-resolution microscopy under an accelerating voltage of 15kV. The results are shown in Figure 2. Figure 3 shown.

[0076] Depend on Figure 3 It can be seen that the emulsion gel obtained after iron ion crosslinking has a smooth surface and is elastic. From the scanning electron microscope image, it can be observed that CEG has a small, uniform and dense mesh structure. The average pore size of CEG is measured to be 8.93±1.53μm. For CEG, the system pH is 4.0, and there is electrostatic attraction. 2+ , ASKP and Fe 3+ Under cross-linking, a semi-interpenetrating gel network is formed in which WPIF is interspersed. Therefore, the cross-linking strengthening effect promotes the densification of the gel microstructure, and the porosity is significantly reduced and evenly distributed.

[0077] Test Example 4

[0078] The macroscopic appearance of the gel microspheres obtained by curing in Example 1 was observed and photographed. The particle size of 100 fresh latex gel microspheres was measured using ImageJ software, and the average size was calculated after Gaussian distribution fitting. Figure 4 shown.

[0079] Depend on Figure 4 As can be seen from the appearance, all microspheres are slightly yellow, regular spherical, uniform in size, good sphericity, and smooth on the surface. The average diameter of CEGM is 1.76±0.01mm.

[0080] Test Example 5

[0081] Simulated gastric fluid stage: Dissolve 2g / L NaCl and 7mL / L HCl in ultrapure water, adjust the pH to 2.0, add 3.2g pepsin, and mix thoroughly to prepare simulated gastric fluid (SGF). Subsequently, 0.5g of the gel microspheres cured in Example 1 were placed in 20mL of SGF and incubated at 37°C, 100rpm for 2h. Simulated intestinal fluid stage: After the simulated gastric fluid stage, the simulated gastric fluid was replaced with simulated intestinal fluid (SIF), and then the simulated intestinal digestion experiment was performed. The SIF preparation was as follows: KCl (0.514 g / L), KH2PO4 (0.1224 g / L), NaHCO3 (7.14 g / L), MgCl2·6H2O (0.067 g / L), CaCl2(H2O)2 (0.088 g / L), NaCl (0.02 g / L), trypsin (0.1 g / L), and bile salts (0.025 g / L). All reagents were dissolved in ultrapure water, adjusted to pH 7.0 with 0.1 M NaOH, and incubated at 37°C, 100 rpm, for 4 h. The digestion process was photographed and recorded. Figure 5 shown.

[0082] Depend on Figure 5 It can be seen that the microspheres maintain a stable structure in SGF, but gradually swell and dissociate after being transferred to SIF. 3+ The pH responsiveness of the gel structure is related to that the structure is stable under gastric fluid conditions and does not swell, while it gradually swells and dissociates under intestinal fluid conditions, indicating that the composite emulsion gel microspheres of the present invention have good digestion lag and intestinal targeting.

[0083] Test Example 6

[0084] Caco-2 cell monolayer transport characteristics: Take an appropriate amount of sample after simulated gastrointestinal digestion and dissolve it in DMEM medium to prepare a certain concentration of digestion solution sample, filter it with a 0.22μm filter membrane to sterilize it and set aside. 5) were seeded in the upper chamber (AP) of a Transwell, and 1.5 mL of culture medium was added to the basolateral chamber (BL). Cells were cultured at 37°C, with medium changes every 48 hours for the first 7 days and daily thereafter. Prior to transwell studies, cell monolayer compactness was assessed by sodium fluorescein (FD4) permeation measurement. 500 μL of buffer containing 50 μg / mL FD4 was added to the AP side, and 1.5 mL of phenol red-free DMEM was added to the BL side. The cells were incubated at 37°C for 3 hours. At 1, 2, and 3 hours, 100 μL of aliquots were collected from the BL side for measurement and supplemented with the appropriate amount of culture medium. Fluorescence intensity of the samples was measured at 490 nm and 515 nm using a microplate reader. The transport and uptake of iron ions and FFAs in the latex gel microspheres were measured under conditions of intact and compact Caco-2 cell monolayers. The constructed Transwell model was then removed from the incubator, the cell culture medium in the upper and lower reservoirs was aspirated, and the cells were washed two to three times with sterile PBS buffer. Then, 1500 μL of PBS buffer was added to the lower chamber, and 500 μL of microsphere sample simulating digestion solution was added to the upper chamber. After maintaining the stability of the gas-liquid interface, the osmotic device was transferred to a 37°C cell culture incubator for transmembrane transport experiments. The culture dish was removed at 0, 1, 2, 4, 6, 8, 10, and 12 hours, and 200 μL of the lower chamber solution was collected. The same volume of PBS was added to the lower chamber after each sample was taken. The TEER value was measured before each sampling to evaluate the effect of the sample digestion solution on the integrity of the cell monolayer. The iron content was determined by the o-phenanthroline colorimetric method, the FFAs content was determined by the free fatty acid kit, and the transcellular efficiency of iron ions and FFAs was calculated. The results are shown in the figure below. Figure 6 shown.

[0085]

[0086] Depend on Figure 6 (a) It can be seen that from 0 to 4 hours, the transcellular efficiency of iron ions in each group of samples showed an overall trend of rapid growth. At 4 hours, the CEGM group reached 8.23±0.74%, and the Free group reached 3.24±0.08%. Then it gradually showed a slow increase from 4 to 8 hours, and remained basically stable from 8 to 12 hours. The final transcellular efficiency of iron ions in the samples was: 10.78±0.68% in the CEGM group and 4.44±0.60% in the Free group. Comparing the two groups of samples, the CEGM group showed a higher iron ion transport rate and significantly enhanced transcellular membrane ability. Iron ions were able to be transported and absorbed more through intestinal cells, while only a smaller amount of iron ions were transported in the Free group, showing a lower absorption efficiency. It can be seen that free iron ions are obviously not easily digested and absorbed by the body. Figure 6(b) It can be seen that from 0 to 6 hours, the transcellular efficiency of FFAs in each group of samples showed an overall trend of rapid growth. At 6 hours, the CEGM group reached 20.77±1.03%, and the Free group reached 8.81±0.73%. Then, it gradually increased slowly from 6 to 12 hours, and the final transcellular efficiency of FFAs in the samples was: 25.92±0.34% in the CEGM group and 11.04±0.78% in the Free group. Comparing the two groups of samples, CEGM also showed a higher FFA transport rate, and FFAs were able to be transported and absorbed more through the intestine, while only a smaller amount of FFAs was transported in the Free group, indicating that free FFAs are also not easily digested and absorbed by the body. This further demonstrates that the sports nutrition supplement prepared by the present invention can effectively encapsulate and deliver iron ions and FFAs, and significantly improve the transport and absorption efficiency of iron ions and FFAs by intestinal epithelial cells.

[0087] Test Example 7

[0088] The experiment was conducted using 6-week-old male Sprague-Dawley (SD) rats with a body weight of 200±20g. The iron intake dose of rats was determined to be 1.26mg / kg·bw. A Control (blank) group, a Free (free iron ions and FFAs) group, and a Niferex (commercially available polysaccharide iron complex) group were also set up. Blood samples were collected from the tail vein at 0, 1, 2, 4, 6, 8, 12, and 24h after administration. During the blood collection process, the first two drops of blood were discarded, and the remaining blood samples were placed in anticoagulant tubes containing EDTA and EP tubes, respectively. After the blood samples in the EP tubes were allowed to stand at room temperature for 30min, they were centrifuged at 4°C and 10,000r / min for 15min. The supernatant was collected as serum and stored in a 4°C refrigerator for later use. The serum iron and free fatty acid levels were quantitatively detected using a serum iron assay kit (A039-1-1 colorimetric method) and a free fatty acid kit (colorimetric method). The results are shown in the figure. Figure 7 shown.

[0089] Depend on Figure 7 (a) It can be seen that the serum iron content of rats in the Control group showed a trend of gradual decrease over time. After oral administration of CEGM, the serum iron content increased rapidly and reached a peak of 5.73 mg / L, while the serum iron content of the Free group increased slowly and the concentration was low, indicating that the emulsion gel can promote the rapid absorption of iron ions in the body. Compared with the Niferex group, the digestion and absorption trends of the two groups were similar. Finally, after a single oral administration, the absorption efficiency of iron ions in each group of samples by rats was CEGM>Niferex>Free>Control group. Figure 7(b) It can be seen that the serum FFAs content of rats in the Control group gradually decreased with time. One hour after gavage with the CEGM sample, the serum FFAs content of rats began to increase rapidly and reached a peak at 4 to 6 hours, and then slowly decreased and tended to balance. By comparison, it was found that the serum FFAs content of the CEGM group increased faster and had the highest peak value, which shows that it was absorbed more rapidly in the body. The Free group increased slowly and the serum FFAs content increased less. Finally, after a single oral administration, the absorption efficiency of FFAs in each group of samples by rats was CEGM>Free>Control group. The results show that the sports nutrition supplement prepared by the present invention can effectively encapsulate and deliver iron ions and FFAs and be efficiently absorbed by the body.

[0090] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing gel microspheres co-loaded with iron and vitamin D, characterized in that: The following steps are involved: The whey protein isolate fiber solution and the Artemisia sphaerocarpa polysaccharide solution are swollen and then mixed to obtain a mixed solution; Subsequently, algae oil is added to the mixed solution, and the resulting mixture is emulsified to obtain an O / W composite emulsion; The O / W composite emulsion was added to FeSO4 solution A and mixed, and static oxidation was performed to obtain a composite emulsion gel; The composite emulsion gel is extruded into FeSO4 solution B for cross-linking and curing to obtain gel microspheres co-loaded with iron and vitamin D.

2. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The pH values ​​of the whey protein isolate fiber solution and the Artemisia polysaccharide solution are both 3.8-4.

2. The whey protein isolate fiber solution and the Artemisia polysaccharide solution are obtained by mixing whey protein isolate fiber powder and Artemisia polysaccharide powder with deionized water, respectively. The mass ratio of the Artemisia polysaccharide fiber powder to the whey protein isolate powder in the mixed solution is (7-19): (1-3).

3. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: In the mixture, the mass concentration of algae oil is 20% to 25%, and the mass concentration of Artemisia polysaccharide powder is 1.5% to 2%.

4. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The emulsification is carried out in a shearing machine at 18000-19000 r / min for 3-4 minutes.

5. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The final concentration of the whey protein isolate fiber in the O / W type composite emulsion is 0.08% to 0.64%.

6. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The molar concentration of Fe in the composite emulsion gel is 80 to 100 mM; The concentration of the FeSO4 solution A is 1.0-1.5 mol / L; The static oxidation time is 48 to 72 hours.

7. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The concentration of the FeSO4 solution B is 1.0-1.5 mol / L; The cross-linking and curing time is 3 to 4 hours.

8. The method for preparing gel microspheres co-loaded with iron and vitamin D according to claim 1, characterized in that: The extrusion is performed using a syringe; the volume of the syringe is 1 to 1.2 mL, and the inner diameter of the needle connected to the syringe is 0.09 to 0.1 mm; The pH value of the mixed solution is 3.8 to 4.2; The cross-linking and curing step further includes a washing process.

9. A gel microsphere co-loaded with iron and vitamin D, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the gel microspheres co-loaded with iron and vitamin D according to claim 9 in sports nutrition supplements, characterized in that: The total iron content of the sports nutritional supplement reaches 4.16±0.73%. In simulated gastrointestinal digestion, the iron ion release rate is greater than 98%, and the free fatty acid release rate is 42.23±2.61%.

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