A method for preparing nano-aspartic acid chelated calcium to increase bone density

Abstract

This invention discloses a method for preparing nano-aspartic acid chelated calcium to increase bone density, belonging to the field of chelated calcium technology. The method uses L-aspartic acid and a calcium source as raw materials; after the reaction, it is subjected to alternating multi-stage filtration and multi-stage homogenization, followed by repeated processing using a high-pressure fluid nanomill. The parameters of the nano-aspartic acid chelated calcium are: bulk density 0.14-0.20 g / mL, solubility ≥8.0 g / 100 mL at 25℃, and hydrodynamic particle size D. 50 The nanometer size is 150-220 nm. Using L-aspartic acid and calcium hydroxide as raw materials, high-pressure fluid nanomilling technology is employed to precisely construct a stable five-membered ring molecular chelate structure combining coordination and covalent bonds, utilizing the principle of cavitation collapse. Calcium ions are encapsulated and chelated by amino acid molecules, exhibiting excellent water solubility, and no harmful ions are generated during the chelation process. This significantly improves calcium bioavailability and solves problems such as poor absorption, bloating, and constipation associated with traditional calcium supplements.

Description

Technical Field

[0001] This invention belongs to the field of chelated calcium technology, and specifically relates to a method for preparing nano-aspartic acid chelated calcium that increases bone density. Background Technology

[0002] Calcium is the most abundant mineral in the human body, with 99% found in bones and teeth and 1% in blood and soft tissues. It is an essential nutrient for life activities and a vital macroelement for maintaining bone health, nerve conduction, and physiological metabolism. Bones serve as a "reservoir" of calcium, maintaining bone density through a dynamic balance between osteoblasts and osteoclasts. Furthermore, calcium acts as a second messenger, activating over 300 enzymes and participating in cell signaling and energy metabolism. Abnormal calcium function is closely related to various diseases such as osteoporosis and cardiovascular disease.

[0003] Intestinal calcium absorption mainly occurs through two pathways: the transcellular pathway and the paracellular pathway, and these two pathways have a synergistic regulatory mechanism. Data from the Chinese Center for Disease Control and Prevention (CDC) in 2025 showed that the calcium intake of adults was only 288.3 mg / day, with 96.1% below the average daily requirement (EAR), and less than 10% reaching the adequate intake level (AI). The calcium deficiency rate among children and adolescents reached 95%, and the calcium deficiency rate among middle-aged and elderly people was as high as 98%, with average intake far below the recommended standard (800-1200 mg / day).

[0004] Many factors contribute to insufficient calcium intake, but the main ones are dietary structure and lifestyle, as well as physiological and environmental factors. An unbalanced diet is the core cause of insufficient calcium intake: insufficient dairy intake, refined staple foods leading to reduced whole grain intake, and high-salt, high-phosphorus diets (such as processed foods and carbonated drinks) accelerate calcium excretion in urine. Improper vegetable cooking methods (failure to blanch to remove oxalic acid) also reduce calcium absorption; for example, vegetables rich in oxalic acid, such as spinach and amaranth, have a calcium absorption rate of only about 5%. Furthermore, modern populations generally lack outdoor activities and sunlight exposure, leading to insufficient vitamin D synthesis—90% of the body's vitamin D is synthesized through ultraviolet radiation from the skin, and sedentary lifestyles and excessive sun protection significantly reduce calcium absorption. Simultaneously, aging and changes in physiological state significantly affect calcium metabolism; bone mass begins to decline annually after age 30-35, digestive and absorptive functions decline in the elderly, and the sharp drop in estrogen levels in postmenopausal women accelerates bone calcium loss. Unhealthy lifestyle habits, such as smoking, excessive drinking, caffeine intake, and lack of weight-bearing exercise and resistance training, can all exacerbate calcium deficiency by interfering with calcium absorption or inhibiting bone calcium deposition.

[0005] Currently, traditional calcium supplements are mainly inorganic calcium and organic acid calcium. Inorganic calcium includes calcium carbonate, calcium phosphate, and calcium sulfate, which are characterized by low solubility, dependence on gastric acid activation, and a tendency to cause gastrointestinal discomfort and constipation, as well as low bioavailability. Organic acid calcium includes calcium citrate and calcium lactate, which are characterized by lower irritation, but lower calcium content and higher cost. Therefore, developing a calcium supplement with high absorption, low irritation, and high utilization has become an inevitable trend, such as amino acid chelated calcium. Amino acid chelated calcium is generated by the reaction of calcium ions with amino acids, forming a coordination compound or chelate with a cyclic structure, and is known as a "third-generation" novel calcium elemental additive (supplement). It has advantages such as molecular absorption, independence from vitamin D, high stability, and low gastrointestinal irritation. In existing technologies, aspartic acid chelated calcium can be prepared using L-aspartic acid and a calcium source.

[0006] For example, patent application number CN118557539A discloses a calcium tablet containing calcium aspartate and its preparation method. The prepared calcium tablet has a good disintegration effect, shortens the disintegration time of existing calcium tablets, and the calcium tablet of the present invention can dissolve quickly in the body after disintegration, and the nutrients are easily absorbed, making it suitable for people of different age groups.

[0007] For example, patent application number CN115073315A discloses a method for synthesizing sodium aspartate and calcium aspartate, which uses aspartic acid and calcium carbonate to chelate calcium aspartate.

[0008] For example, patent application number CN111675627A discloses a method for preparing calcium aspartate, which uses calcium carbonate and calcium oxide to react with aspartic acid to prepare calcium aspartate, thus obtaining high-purity calcium aspartate.

[0009] Existing calcium aspartate preparation processes mostly use calcium chloride and calcium carbonate as calcium sources, which easily introduce impurity ions such as chloride and carbonate ions. This results in a product chelation rate that is generally lower than 85%, and the product is prone to precipitation and stratification during storage. Moreover, the purity is difficult to meet the high-end requirements of food additives GB 29226-2012. At the same time, existing processes mostly use conventional mechanical stirring and single homogenization, which cannot achieve precise control of nanoscale particle size. The average particle size of the product is mostly above 0.5μm, the solubility is only 2.5-3.0g / 100mL, and the bioavailability is less than 60%.

[0010] Patent application CN201520406611.6 discloses a high-efficiency production upgrade system for nano-amino acid chelated calcium, including a mixer, a water tank, a homogenizer, a storage tank, a filter, a nanomachine, a drying tower, and a hot air blower. The inlet of the water tank is connected to the outlet of the mixer via a water pump, the outlet of the water tank is connected to the inlet of the homogenizer via a pipe, the inlet of the storage tank is connected to the outlet of the homogenizer via a pipe, the outlet of the storage tank is connected to the inlet of the filter via a water pump, the inlet of the nanomachine is connected to the outlet of the filter via a pipe, the outlet of the nanomachine is connected to the inlet of the drying tower via a water pump, and the outlet of the hot air blower is connected to the inlet of the drying tower via a pipe. This patent uses a single homogenization and filtration process to treat the reaction solution, and finally uses a conventional nanomachine to treat the reaction solution before spraying. Although nano-sized amino acid chelated calcium was obtained, the applicant found that there is still room for further optimization (especially the chelation rate is low, less than 85%), to further improve solubility and further ensure the chelation rate. Summary of the Invention

[0011] This patent, through screening calcium sources and researching aspects such as calcium content, solubility, safety, absorption rate, and product cost, focuses on the development of chelated calcium. Based on the advantages of calcium-amino acid chelates, including stable chemical properties, high bioavailability, non-toxicity, non-irritation, and good palatability, a formula combining L-aspartic acid and calcium hydroxide was selected. Using high-pressure fluid nano-milling technology and the principle of cavitation collapse, calcium atoms are embedded between two L-aspartic acid molecules, allowing the two amino acid molecules to clamp one calcium atom like "crab claws," forming a super-stable chelate structure. This eliminates various interfering components in the gastrointestinal tract that hinder calcium absorption, making it a truly amino acid chelated calcium. As a new generation of calcium preparation, L-aspartic acid chelated calcium, especially nano-amino acid chelated calcium, dissolves rapidly in water, with a solubility 400 times that of edible calcium carbonate. It does not require gastric acid breakdown and is directly absorbed through the absorption channels of the small intestinal mucosa, achieving an absorption rate of over 90%, three times that of ordinary calcium sources. Therefore, nano-sized aspartic acid chelated calcium, as a representative of amino acid chelated calcium, is a good choice for daily calcium intake. The technical solution is as follows:

[0012] On one hand, this invention provides a nano-aspartic acid chelated calcium, prepared using L-aspartic acid and a calcium source as raw materials; after the reaction is completed, it is subjected to alternating multi-stage filtration and multi-stage homogenization, followed by repeated processing using a high-pressure fluid nanomill; the parameters of this nano-aspartic acid chelated calcium are: bulk density 0.14-0.20 g / mL, solubility at 25℃ ≥8.0 g / 100 mL, and hydrodynamic particle size D in solution. 50 The wavelength is 150-220 nm, the moisture content is ≤5.5%, and the pH value is 6.2-7.5.

[0013] High-pressure fluid nanomilling technology is a novel nanotechnology device. Using water as the medium, carrier, and kinetic energy carrier, it utilizes the principle of cavitation collapse to create an ultrasonic stress field within the machine, providing a unique environment for chemical reactions. Under the influence of external conditions such as light, heat, or ultrasound, the covalent bonds in aqueous solutions of amino acids undergo homolytic cleavage, forming unpaired electron clusters. Most free radicals are unstable and difficult to exist independently, tending to combine spontaneously to form stable molecules or react with ions and free radicals of other substances to form new, more stable molecules. An amino acid molecule contains an amino or nitrogen group on one side and an organic acid anion or carboxyl group on the other. When the forming medium is at a specific pH value and there is no interference from H+ ions, the carboxyl and amino groups on both sides can bind with metal ions at different positions to form chelates. High-pressure fluid nanomilling utilizes ultrasonic frequencies that reach or approach the molecular vibration frequency, enabling amino acid free radicals to rapidly chelate with calcium ions to form amino acid chelated calcium.

[0014] Among them, the nano-aspartic acid chelated calcium has a stable double five-membered ring chelate structure. The calcium ion is connected to the nitrogen atoms in the two L-aspartic acid molecules by coordination bonds and to the carboxyl oxygen atoms by covalent bonds.

[0015] The calcium source is selected from calcium hydroxide, calcium carbonate, calcium oxide, calcium chloride, calcium citrate, or calcium lactate, with calcium hydroxide being preferred. The molar ratio of L-aspartic acid to the calcium source is 2.6-3.5:0.8-1.2.

[0016] The nano-aspartic acid chelated calcium is obtained through the following steps: chelation reaction, primary filtration, primary homogenization, secondary filtration, secondary homogenization, tertiary filtration, high-pressure fluid nanomilling treatment, settling, supernatant filtration, and spray drying. Both primary and secondary homogenization utilize conventional homogenizers. The high-pressure fluid nanomilling treatment conditions are: pressure 80-120 MPa, time 60-90 min.

[0017] On the other hand, embodiments of the present invention also provide a method for producing nano-aspartic acid chelated calcium, the method comprising: S1 chelation reaction: Using L-aspartic acid and calcium source as raw materials, the chelation reaction is carried out in water with a weight of 2-5 times that of the raw materials. The reaction conditions are: temperature 40-65℃, pH value 6.2-7.5, and molar ratio of L-aspartic acid to calcium source of 2.6-3.5:0.8-1.2.

[0018] S2 Post-processing: After the reaction is complete, the mixture is alternately treated with multi-stage filtration and multi-stage homogenization, followed by repeated treatment with a high-pressure fluid nanomill, and finally spray-dried to obtain nano-aspartic acid chelated calcium. The conditions for high-pressure fluid nanomilling are: pressure 80-120 MPa, homogenization time 60-90 min. Preferably, in step S2, the homogenization pressure increases gradually, and the pore size of the filter decreases gradually.

[0019] The calcium source is selected from calcium hydroxide, calcium carbonate, calcium oxide, calcium chloride, calcium citrate, or calcium lactate, etc.

[0020] Preferably, the calcium source is selected from calcium hydroxide, and the molar ratio of L-aspartic acid to calcium hydroxide is 3.5:1.

[0021] Specifically, step S2 includes the following steps: after the reaction is completed, nano-aspartic acid chelated calcium is obtained by going through primary filtration, primary homogenization, secondary filtration, secondary homogenization, tertiary filtration, high-pressure fluid nano-milling treatment, settling, supernatant filtration and spray drying.

[0022] The conditions for primary filtration are: 100-300 mesh filter aperture; secondary filtration is: 100-300 mesh filter aperture; tertiary filtration is: 100-300 mesh filter aperture; supernatant filtration is: 300-500 mesh filter aperture; primary homogenization is: pressure 60-90 MPa, homogenization time 60-90 min; secondary homogenization is: pressure 80-120 MPa, homogenization time 60-90 min; settling time is 18-24 h (preferably 19-20 h); spray drying is: inlet air temperature 180-240℃ (preferably 200-220℃), outlet air temperature 115-135℃ (preferably 115-120℃).

[0023] Specifically, both primary filtration and supernatant filtration use pipeline filters, while secondary and tertiary filtration use circular sieve filters.

[0024] Preferably, see Figure 1 The present invention provides a method for producing nano-aspartic acid chelated calcium, the method comprising: (1) Chelation reaction: L-aspartic acid and calcium hydroxide are used as raw materials. The chelation reaction is carried out in water with a weight of 2-5 times that of the raw materials. The reaction conditions are: temperature 40-65℃, pH value 6.2-7.5, reaction time 30-60min, and molar ratio of L-aspartic acid to calcium hydroxide 2.6-3.5:0.8-1.2. After the reaction is completed, the reaction solution is sent to step (2).

[0025] (2) First-stage homogenization: First-stage filtration is performed using a 100-300 mesh (specifically 200 mesh) pipeline filter. The filtrate is then homogenized to obtain homogenate liquid one. The homogenization pressure is 60-90 MPa and the homogenization time is 60-90 min. Homogenate liquid one is then sent to step (3).

[0026] (3) Secondary homogenization: Use a 100-300 mesh (specifically 200 mesh) round sieve filter for secondary filtration. The filtrate is then homogenized to obtain homogenized liquid II. The secondary homogenization pressure is 80-120 MPa and the homogenization time is 60-90 min. Homogenized liquid II is then sent to step (4).

[0027] (4) High-pressure fluid nanomilling treatment: Three-stage filtration is carried out using a 100-300 mesh (specifically 300 mesh) round sieve filter. The filtrate is sent to the high-pressure fluid nanomill for repeated treatment to obtain homogenized liquid three. The treatment pressure is 80-120MPa and the treatment time is 60-90min. Homogenized liquid three is sent to step (5).

[0028] (5) Filtration: Let the settling tank stand for 18-24 hours, and filter the supernatant using a 300-500 mesh (specifically 500 mesh) pipeline filter; send the filtrate to step (6).

[0029] (6) Spray drying: The filtrate is spray dried. The conditions for spray drying are: inlet air temperature of 180-240℃ and outlet air temperature of 115-135℃.

[0030] On the other hand, see Figure 7 The present invention also provides a production system for nano-aspartic acid chelated calcium. The system includes a packaging device, a batching device, and a reactor, a primary pipeline filter, a primary homogenizer, a primary transfer tank, a secondary circular sieve filter, a secondary homogenizer, a secondary transfer tank, a tertiary circular sieve filter, a high-pressure fluid nanomill, a settling tank, a supernatant pipeline filter (specifically a pipeline filter), and a spray drying device, etc., connected in sequence by pipelines. The batching device is connected to the reactor (through pipelines, conveyor belts, chutes, negative pressure, etc.), and the spray drying device and the packaging device are connected in sequence (through pipelines, conveyor belts, chutes, negative pressure, etc.).

[0031] The present invention has the following advantages: (1) Green and safe raw materials: Calcium hydroxide is preferred as the calcium source, and the only reaction byproduct is water. No harmful impurities such as chloride ions and carbonate ions are introduced. The product purity is ≥99.0%, which meets the requirements of food-grade safe production. Furthermore, the aqueous phase is reacted at normal pressure, and the pH is maintained between 6.2 and 7.5, which is close to neutral. The calcium content is greater than 12%. The reaction conditions are mild, the equipment is simple, and the energy consumption is low, making it suitable for industrial production.

[0032] (2) Stable and efficient structure: The double five-membered ring chelate structure is formed by high pressure fluid nano-grinding technology, with a chelation rate of ≥95%. Calcium ions are not easily dissociated in the gastrointestinal tract, avoiding mucosal irritation of traditional calcium supplements and without side effects such as bloating and constipation.

[0033] (3) Excellent absorption performance: nanoscale particle size (D 50 =180nm) makes the product solubility ≥8.0g / 100mL at 25℃, which is more than 3 times that of ordinary aspartic acid calcium; it can be directly absorbed through the intestinal amino acid channel without the need for vitamin D, and the bioavailability is ≥90%, which is 3 times that of calcium carbonate.

[0034] (4) Clear efficacy: According to zebrafish tests, the product has a bone density enhancement effect of 14%, which is better than commercially available calcium citrate (11%) and calcium carbonate (4%), and can effectively improve bone and joint health. Attached Figure Description

[0035] Figure 1 This is a process flow diagram for the preparation of nano-aspartic acid chelated calcium; Figure 2 It is an FT-IR infrared spectrum; Figure 3 This is a volume-weighted particle size distribution curve of DLS. Figure 4 It is a scanning electron microscope image; Figure 5 This is a comparison of the fluorescence intensity of bone formation in zebrafish after treatment, based on the evaluation results of the bone density enhancement effect. Figure 6 This is a typical image of osteogenic fluorescence intensity in zebrafish after processing to enhance bone density evaluation results; Figure 7 This is a schematic diagram of the production system for nano-aspartic amino acid chelated calcium. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Example 1 Example 1 discloses a method for producing nano-aspartic acid chelated calcium. L-aspartic acid and calcium hydroxide are weighed at a molar ratio of 3.5:1, and purified water is added in an amount three times the total mass of the raw materials. The mixture is then added to a reaction vessel and stirred. The temperature is raised to 55°C, and the pH of the reaction system is controlled at 7.0. The reaction is carried out for 45 minutes. The reaction solution is filtered through a 200-mesh pipe filter and then subjected to primary homogenization at a pressure of 60 MPa for 70 minutes. The homogenized solution is then filtered through a 200-mesh circular sieve and subjected to secondary homogenization at a pressure of 120 MPa for 70 minutes. After further filtration through a 300-mesh circular sieve, the solution is subjected to tertiary homogenization using a high-pressure fluid nanomill at a pressure of 100 MPa for 70 minutes. The homogenized solution is transferred to a settling tank and allowed to stand for 20 hours. The supernatant is then filtered through a 500-mesh pipe filter. Finally, the solution is spray-dried at an inlet air temperature of 210°C and an outlet air temperature of 120°C to obtain white powdery nano-aspartic acid chelated calcium.

[0038] According to the test results, the product of this embodiment has an average particle size of 535.8nm, a pH value of 7.0, a solubility of 8.2g / 100mL at 25℃, a moisture content of 4.8%, a bulk density of 0.17g / mL, and a bone density enhancement effect of 14%, with no diarrhea / bloating.

[0039] To verify that the product of this invention is a nano-aspartic acid chelated calcium that increases bone density, the product of Example 1 was subjected to FT-IR infrared spectroscopy, particle size determination, safety toxicology evaluation, and zebrafish testing. The specific details of each test item are as follows: I. FT-IR Infrared Spectroscopy Detection 1. Detection Method The infrared absorption spectrum of the nano-aspartic acid chelate prepared in Example 1 was tested using a FT-IR Fourier transform infrared spectrometer to characterize its chemical structure.

[0040] 2. Test Results and Analysis The structure of the synthesized aspartic acid chelated calcium sample was characterized by Fourier transform infrared spectroscopy (FT-IR), and the results are as follows: Figure 2 As shown. The sample is at 3410.11 cm. -1 and 3144.78cm -1 A broad and strong absorption peak appears at this point, which is attributed to the protonated amino group (-NH3). + The NH stretching vibration of the molecule and the OH stretching vibration of the intermolecular hydrogen bond; 1591.90 cm⁻¹ -1 and 1403.67cm -1 The characteristic peaks at 1700 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of the carboxylate (-COO-), indicating that the carboxyl group of aspartic acid has combined with calcium ions to form an ionic coordination structure, and no free carboxylic acid is present at 1700 cm⁻¹. -1Feature absorption in the vicinity. 1487.21cm -1 1352.82–1228.66cm -1 and fingerprint area (<1000cm) -1 The multiple absorption peaks of the sample correspond to the amino bending vibration, CH / CN stretching vibration, and Ca-O coordination bond vibration, respectively, which are consistent with the theoretical structural characteristics of calcium chelated from aspartic amino acids, confirming that the sample is the target product and has good purity.

[0041] II. Particle Size Distribution and Micromorphology Detection 1. Testing instruments and methods Particle size distribution was determined using a nanoparticle size and Zeta potential analyzer based on dynamic light scattering (DLS). Before testing, the sample was diluted with ultrapure water to a suitable concentration, ultrasonically dispersed for 3-5 minutes to fully disperse the particles and remove air bubbles, and then transferred to the sample cell. Three parallel measurements were performed on the sample at 25℃. The hydrodynamic particle size was calculated based on the Stokes-Einstein equation. The instrument's software recorded and output the Z-Average average particle size, polydispersity index (PDI), volume-weighted particle size distribution, and D0. 10 D 50 D 90 Characteristic particle size parameters.

[0042] The microstructure of the microspheres was observed using scanning electron microscopy (SEM). The microsphere powder to be tested was placed on a sample stage coated with conductive adhesive, loose particles were removed by purging, and the microspheres were then plated with gold using a vacuum sputtering apparatus before testing. Instrument operating conditions: accelerating voltage 3.00 kV, working distance 8.4 mm, signal mode secondary electron imaging. Sample morphology images were captured sequentially at 100× and 300× magnification. Particle morphology, integrity, and surface condition were observed using an image scale, and the microsphere particle size range was statistically analyzed using image processing software.

[0043] 2. Test Results and Analysis The microstructure and particle size distribution of the samples were systematically characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS). SEM results showed that the samples were mainly composed of micron-sized spherical particles, with a particle size distribution at 300 nm. At magnification, the surface appears relatively smooth. DLS testing results show that the volume-weighted particle size distribution of the sample in solution exhibits a single-peak characteristic, with the main peak located at 180.1 nm and a median diameter D... 50 For 180nm, D 10 With D 90The values ​​of 152 nm and 213 nm, respectively, indicate that the hydrodynamic particle size of the main particles is concentrated between 150-220 nm, exhibiting good uniformity. However, the intensity-weighted Z-mean particle size is 535.8 nm, and the polydispersity index (PdI) is 0.879, showing a significant deviation from the volume distribution results. This phenomenon can be attributed to the presence of a very small number of large aggregates in the solution: since the scattering signal intensity of DLS is proportional to the sixth power of the particle size, the signal of a small number of aggregates is significantly amplified, thereby increasing the intensity-weighted average particle size and the PdI value. Furthermore, there is an order-of-magnitude difference between the micron-sized particles observed by SEM and the nano-sized particles measured by DLS. This is mainly due to significant agglomeration and secondary aggregation of the sample during the drying process, forming micron-sized spherical aggregates; while DLS measures the original hydrodynamic particle size of the dispersed particles in the solution. Comprehensive analysis indicates that the main particles of the sample exist in nanoscale form in the solution and easily agglomerate to form micron-sized microspheres after drying.

[0044] III. Calcium Content Detection (a) Detection methods The calcium content of the nano-aspartic acid chelated calcium prepared in Example 1 was determined according to GB 29226 standard. (1) Determination method: In this method, a small amount of magnesium ions are used as an auxiliary indicator. That is, magnesium ions form a stable complex with chrome black T to indicate the color change. Titration is performed with disodium ethylenediaminetetraacetate (EDTA) standard titration solution. The calcium content is calculated based on the amount of disodium ethylenediaminetetraacetate (EDTA) standard titration solution consumed. (2) Reagents and materials: trihexanolamine, magnesium sulfate solution 120 g / L, ammonia-ammonium chloride buffer pH=10.0, disodium ethylenediaminetetraacetate (EDTA) standard titration solution c(EDTA)=0.05 mol / L, chrome black T indicator solution: 5 g / L. (3) Analytical steps: Take 2 drops of magnesium sulfate solution, add 15 mL of ammonia-ammonium chloride buffer, add 2 drops of Eriochrome Black T indicator solution, and titrate with EDTA standard solution until pure blue; take another 0.35 g of sample, accurate to 0.0002 g, add 30 mL of water to dissolve it, add 5 drops of triethanolamine, and combine it with the magnesium sulfate solution titrated above, and titrate with EDTA standard solution until the color changes from purple-red to pure blue, which is the endpoint. Perform a blank experiment at the same time. (4) Calculation of calcium content: The mass fraction ω of calcium (calculated as Ca) content is calculated according to the following formula:

[0045] Each group was measured in triplicate, and the average value was taken.

[0046] (II) Results and Analysis The calcium content detection results of aspartic amino acid chelated calcium are shown in Table 1 below: Table 1 Calcium content test results

[0047] As shown in Table 1 above, the chelated calcium content of the present invention is 12.2%, which meets the requirements of GB 29226 standard.

[0048] IV. Safety Toxicological Evaluation Tests and Results Analysis of the Product of this Invention Acute oral toxicity pretest (Kurt's method) was conducted according to GB 15193.3-2014; bacterial reverse mutation test was performed according to GB 15193.4-2014; mammalian erythrocyte micronucleus test was performed according to GB 15193.5-2014; and mouse spermatogonia or spermatocyte chromosomal aberration test was performed according to GB 15193.8-2014. The results of the bacterial reverse mutation test (50, 158, 500, 1580, 5000 g / plate and 8, 40, 200, 1000, 5000 g / plate) were all negative. The results of the mammalian erythrocyte micronucleus test and the mouse spermatocyte chromosomal aberration test (female: 0.74, 1.47, 2.94 g / kg BW; male: 0.79, 1.58, 3.16 g / kg BW) were also negative. Acute oral toxicity pretest (Kurt's method) results showed that nano-aspartic acid chelated calcium had an oral LD50 of 50% in mice of both sexes. 50 The doses were 5.89 g / kg BW for female mice and 6.33 g / kg BW for male mice, and were classified as practically non-toxic according to the acute toxicity (LDs0) dose classification.

[0049] V. Zebrafish Experiment The following are the details of the zebrafish experiment: Positive control of the present invention: (1) Efficacy of enhancing bone density (calcium supplementation): Alendronate sodium tablets (hereinafter referred to as alendronate sodium), white tablets, the solvent is ultrapure water.

[0050] Animals used in this invention: Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water; conductivity 450-550μS / cm; pH 6.2-5.5; hardness 50-100mg / L CaCO3). They were bred and provided by our company's aquarium. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management met the requirements of the international AAALAC certification (certification number: 001458). The IACUC ethics review number is: IACUC-2025-202508120040-01.

[0051] The instruments, consumables, and reagents used in the zebrafish experiments of this invention are as follows: dissecting microscope; CCD camera; motorized focusing continuous zoom fluorescence microscope; precision electronic balance; 6-well plate; digital display horizontal decolorizing shaker; methylcellulose (Shanghai Aladdin Biochemical Technology Co., Ltd.); 4% tissue cell fixative (Beijing Solarbio Science & Technology Co., Ltd.); Alizarin Red (Shanghai Maclean Biochemical Technology Co., Ltd.); anhydrous magnesium chloride (Shanghai Aladdin Biochemical Technology Co., Ltd.); potassium hydroxide (Sinopharm Chemical Reagent Co., Ltd.); glycerol (Shanghai Aladdin Biochemical Technology Co., Ltd.); anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.); hydrogen peroxide (Shanghai Aladdin Biochemical Technology Co., Ltd.); PBS phosphate buffer (dry powder) (Biosharp); dexamethasone (Shanghai Aladdin Biochemical Technology Co., Ltd.); dimethyl sulfoxide (Sinopharm Chemical Reagent Co., Ltd.).

[0052] The detection method of this invention includes: Firstly, the maximum detectable concentration (MTC) of the bone mineral density enhancement (calcium supplementation) efficacy was determined: Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered in water (concentrations shown in Table 2), and a normal control group and a model control group were also set up. Each beaker had a volume of 20 mL. Except for the normal control group, all other experimental groups were administered dexamethasone in water to establish a zebrafish osteoporosis model. After treatment at 28℃ for 2 days, the solution was changed once. After continuing treatment at 28℃ for another 2 days, the MTC of the sample on the model zebrafish was measured.

[0053] Secondly, the efficacy of bone mineral density enhancement (calcium supplementation) was evaluated: 3dpf transgenic hard-boned green fluorescent zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples (concentrations shown in Table 2) were administered, with alendronate sodium at a concentration of 5.00 μg / mL as a positive control. A normal control group and a model control group were also set up, with each beaker containing 20 mL. Except for the normal control group, all other experimental groups were given water-soluble dexamethasone to establish a zebrafish osteoporosis model. After treatment at 28℃ for 2 days, the solution was changed once. After another 2 days of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software. The intensity of osteogenic fluorescence in the zebrafish was analyzed, and the statistical analysis results of this index were used to evaluate the bone mineral density enhancement (calcium supplementation) efficacy of the samples. Statistical results are expressed as mean ± SE. The formula for calculating the bone mineral density enhancement (calcium supplementation) efficacy of the samples is as follows:

[0054] Statistical analysis using SPSS software showed that p < 0.05 was statistically significant.

[0055] The above is the content of the zebrafish experiment of this invention. The following is the result analysis of each part of the zebrafish experiment.

[0056] Maximum detectable concentration (MTC) results for bone density enhancement (calcium supplementation): Under the experimental conditions, the bone-promoting MTC of aspartic acid chelated calcium prepared in Example 1 was 125 μg / mL, as shown in Table 2. Subsequent experiments were conducted using this maximum detectable concentration.

[0057] Table 2 Results of the concentration exploration test for bone-promoting efficacy of samples (n=30)

[0058] Evaluation results of bone-promoting efficacy: Under the experimental conditions, the nutritional composition prepared in Example 1 exhibited bone-promoting efficacy, specifically demonstrating an effect of increasing bone density. The results are shown in Table 3 and... Figure 5-6 As shown.

[0059] Table 3 Results of the bone-promoting efficacy evaluation test of the samples (n=10)

[0060] Note: Compared with the model control group, p < 0.05 p < 0.01, p < 0.001.

[0061] Example 2 Example 2 discloses a method for producing nano-aspartic acid chelated calcium. Compared with Example 1, the first-stage homogenization pressure and the second-stage homogenization pressure are the same as in Example 1, the third-stage homogenization pressure is 120 MPa, and all other parameters are the same.

[0062] Example 3 Example 3 discloses a method for producing nano-aspartic acid chelated calcium. Compared with Example 1, the first-stage homogenization pressure is 90 MPa, the second-stage homogenization pressure is 100 MPa, and the third-stage homogenization pressure is 120 MPa, while all other aspects are the same.

[0063] Example 4 Example 2 discloses a method for producing nano-aspartic acid chelated calcium. Compared with Example 1, the first-stage homogenization pressure is 60 MPa, the second-stage homogenization pressure is 100 MPa, and the third-stage homogenization pressure is 120 MPa, while all other aspects are the same.

[0064] Example 5 Example 2 discloses a method for producing nano-aspartic acid chelated calcium. Compared with Example 1, the primary homogenization pressure is 90 MPa, the secondary homogenization pressure is 90 MPa, and all other parameters are the same.

[0065] Comparative Example 1 Ordinary micron-sized aspartic acid chelated calcium, batch number 20241001, average particle size 15μm, calcium content 12.1%.

[0066] Comparative Example 2 Commercially available food-grade calcium carbonate, batch number 20240915, with an average particle size of 22μm and a calcium content of 39.2%.

[0067] Comparative Example 3 Commercially available food-grade calcium citrate, batch number 20241005, with an average particle size of 12μm and a calcium content of 21.0%.

[0068] Comparative Example 4 The method is the same as in Example 1, except that the three-stage homogenization process of the high-pressure fluid nanomilling is omitted. Instead, conventional mechanical stirring is used for homogenization for 30 minutes. The remaining steps are the same as in Example 1 to obtain non-nanosized aspartic acid chelated calcium.

[0069] Comparative Example 5 L-aspartic acid and calcium chloride were physically mixed at a molar ratio of 2:1 and stirred until homogeneous to obtain a physical mixture.

[0070] The average particle size, pH, solubility, dissolution rate, bone density enhancement efficacy, and incidence of diarrhea / bloating in Examples 1-5 and Comparative Examples 1-5 were tested, and the specific results are shown in Table 4 below: Table 4 Comparison of Product Items between Examples and Comparative Examples

[0071] As shown in Table 4 above, the product of this invention has better average particle size, pH, solubility, and bone density increase than traditional organic and inorganic calcium, achieving the technical effects of high absorption, low irritation, and high efficiency in increasing bone density.

[0072] Furthermore, the nano-aspartic acid chelated calcium obtained in Example 1 of this invention was compared with commercially available micron-sized aspartic acid calcium, commercially available food-grade calcium carbonate, and commercially available food-grade calcium citrate in terms of their relief of bone and joint pain and osteoporosis through human trials, as detailed below: Eighty participants were selected for a trial, excluding individuals with fractures, osteonecrosis, paralysis, or those taking other medications to improve bone density. Individuals experiencing joint pain and leg cramps requiring crutches or support were also selected to observe whether the trial improved their pain and cramping symptoms. Four groups of 40 participants were chosen: 20 men and 20 women aged 55-65, and 20 men and 20 women aged 66-75. Each group consisted of 20 participants: 5 men and 5 women aged 55-65, and 5 men and 5 women aged 66-75. The trial lasted 90 days, with a dosage of 2.0g twice daily. Improvements and ineffectiveness in joint pain and leg cramping were recorded, and the effectiveness rate was calculated. The effectiveness rate was calculated as: (Number of participants whose joint pain or leg cramping improved after the trial / Total number of participants). 100%, the results are shown in Table 5: Table 5. Effects on relieving joint pain and leg cramps

[0073] As shown in Table 5, the nano-aspartic acid chelated calcium of the present invention is superior to commercially available calcium products in relieving joint pain and leg cramps, effectively verifying the beneficial effects of the nano-aspartic acid chelated calcium of the present invention on joints.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing nano-aspartic acid chelated calcium to increase bone density, characterized in that, The method includes: S1 chelation reaction: Using L-aspartic acid and calcium source as raw materials, the chelation reaction is carried out in water with a weight of 2-5 times that of the raw materials. The reaction conditions are: temperature 40-65℃, pH value 6.2-7.5, and molar ratio of L-aspartic acid to calcium source of 2.6-3.5:0.8-1.

2. S2 post-processing: After the reaction is completed, multi-stage filtration and multi-stage homogenization are alternately used for processing, followed by repeated processing with high-pressure fluid nanomilling, and finally spray drying to obtain nano-aspartic acid chelated calcium; the conditions for high-pressure fluid nanomilling are: pressure 80-120MPa, time 60-90min.

2. The preparation method according to claim 1, characterized in that, The calcium source is selected from calcium hydroxide, calcium carbonate, calcium oxide, calcium chloride, calcium citrate, or calcium lactate.

3. The preparation method according to claim 1, characterized in that, The calcium source is selected from calcium hydroxide, and the molar ratio of L-aspartic acid to calcium hydroxide is 3.5:

1.

4. The preparation method according to claim 1, characterized in that, Step S2 specifically includes: after the reaction is completed, the product undergoes primary filtration, primary homogenization, secondary filtration, secondary homogenization, tertiary filtration, high-pressure fluid nanomilling, settling, supernatant filtration, and spray drying to obtain nano-aspartic acid chelated calcium. The conditions for primary filtration are: 100-300 mesh filter opening; secondary filtration is also 100-300 mesh filter opening; tertiary filtration is 100-300 mesh filter opening; supernatant filtration is 300-500 mesh filter opening; primary homogenization is 60-90 MPa pressure and 60-90 min homogenization time; secondary homogenization is 80-120 MPa pressure and 60-90 min homogenization time; settling time is 18-24 h; and spray drying is 180-240℃ inlet air temperature and 115-135℃ outlet air temperature.

5. The preparation method according to claim 4, characterized in that, The primary filtration and supernatant filtration both use pipeline filters, while the secondary and tertiary filtration both use circular sieve filters.

6. The preparation method according to claim 1, characterized in that, The method includes: (1) Chelation reaction: L-aspartic acid and calcium hydroxide are used as raw materials. The chelation reaction is carried out in water with a weight of 2-5 times that of the raw materials. The reaction conditions are: temperature 40-65℃, pH value 6.2-7.5, reaction time 30-60min, and molar ratio of L-aspartic acid to calcium hydroxide 2.6-3.5:0.8-1.

2. After the reaction is completed, the reaction solution is sent to step (2). (2) First-stage homogenization: First-stage filtration is performed using a 100-300 mesh pipeline filter. The filtrate is then homogenized to obtain homogenate liquid one. The homogenization pressure is 60-90 MPa and the homogenization time is 60-90 min. Homogenate liquid one is then sent to step (3). (3) Secondary homogenization: The filtrate is filtered in two stages using a 100-300 mesh round sieve filter. The filtrate is then homogenized in two stages to obtain homogenized liquid II. The homogenization pressure is 80-120 MPa and the homogenization time is 60-90 min. Homogenized liquid II is then sent to step (4). (4) High-pressure fluid nanomilling treatment: Three-stage filtration is carried out using a 100-300 mesh round sieve filter. The filtrate is sent to the high-pressure fluid nanomill for repeated treatment to obtain homogenized liquid three. The treatment pressure is 80-120MPa and the treatment time is 60-90min. Homogenized liquid three is sent to step (5). (5) Filtration: Let the liquid stand in the settling tank for 18-24 hours, and filter the supernatant using a 300-500 mesh pipeline filter; send the filtrate to step (6); (6) Spray drying: The filtrate is spray dried. The conditions for spray drying are: inlet air temperature of 180-240℃ and outlet air temperature of 115-135℃.

7. The preparation method according to claim 1, characterized in that, The production is carried out using the following preparation system, which includes: a packaging device, a batching device, and a reaction vessel, a primary pipeline filter, a primary homogenizer, a primary transfer tank, a secondary circular sieve filter, a secondary homogenizer, a secondary transfer tank, a tertiary circular sieve filter, a high-pressure fluid nanomill, a settling tank, a supernatant pipeline filter, and a spray drying device connected in sequence by pipelines. The batching device is connected to the reaction vessel, and the spray drying device and the packaging device are connected in sequence.

8. A nano-aspartic acid chelated calcium for increasing bone density, characterized in that, The aspartic acid chelated calcium nanoparticles were prepared using L-aspartic acid and a calcium source. After the reaction, the nanoparticles were subjected to alternating multi-stage filtration and multi-stage homogenization, followed by repeated processing using a high-pressure fluid nanomill. The parameters of the nano-aspartic acid chelated calcium were: bulk density 0.14-0.20 g / mL, solubility ≥8.0 g / 100 mL at 25 °C, and hydrodynamic particle size D. 50 It is 150-220nm.

9. The nano-aspartic acid chelated calcium according to claim 8, characterized in that, The nano-aspartic acid chelated calcium has a stable double five-membered ring chelate structure. The calcium ion is connected to the nitrogen atoms in the two L-aspartic acid molecules by coordination bonds and to the carboxyl oxygen atoms by covalent bonds.

10. The nano-aspartic acid chelated calcium according to claim 8, characterized in that, The nano-aspartic acid chelated calcium is obtained by sequentially performing the following steps: chelation reaction, primary filtration, primary homogenization, secondary filtration, secondary homogenization, tertiary filtration, high-pressure fluid nanomilling treatment, settling, supernatant filtration, and spray drying; the conditions for the high-pressure fluid nanomilling treatment are: pressure of 80-120 MPa and time of 60-90 min.

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