Preparation method and application of chlorella pyrenoidosa protein peptide-calcium chelate

Abstract

The application provides a preparation method of Chlorella pyrenoidosa protein peptide-calcium chelate, and the preparation method comprises the following steps: S1, Chlorella pyrenoidosa protein is dissolved in deionized water, the pH is adjusted to 2-13, stirring is carried out for 1-5 hours, centrifugation is carried out, the supernatant is taken, and a Chlorella pyrenoidosa protein treatment solution is obtained; S2, calcium chloride is added to the Chlorella pyrenoidosa protein treatment solution, water bath chelation is carried out at 35-38 DEG C for 1.8-2.2 hours, centrifugation is carried out, anhydrous ethanol is added to the supernatant, standing is carried out, centrifugation is carried out again, the precipitate is collected, and freeze-drying is carried out, and the Chlorella pyrenoidosa protein peptide-calcium chelate is obtained. The preparation process is simple, no toxic side reagent is used, energy consumption is low, the prepared product is verified to have a calcium absorption promoting effect through animal experiments, and solid theoretical support is provided for the development and application of the Chlorella pyrenoidosa protein peptide-calcium chelate as a new bone health promoter.

Description

Technical Field

[0001] This invention belongs to the field of nutritional supplement preparation technology, specifically relating to a method for preparing a protein-core Chlorella protein peptide-calcium chelate and its application. Background Technology

[0002] Calcium is an essential nutrient for human growth and development, participating in the regulation of various physiological activities and maintaining normal bodily functions. Approximately 99% of the calcium in the human body is distributed in teeth and bones, providing structural support for the hard tissues of bones and teeth. The remaining calcium exists in a free state in tissues such as blood and extracellular fluid, participating in the regulation of numerous physiological processes. Dietary intake is the main route for the human body to obtain calcium, but its absorption and utilization are affected by many factors, resulting in low bioavailability. On the one hand, components such as phytic acid in plant-based foods can bind with calcium, forming insoluble complexes that inhibit calcium absorption. On the other hand, calcium ions easily precipitate in the intestinal environment, leading to calcium loss.

[0003] Traditional calcium supplements mainly fall into three categories: inorganic, organic, and amino acid-based. Inorganic calcium supplements are widely available, and while their main component, calcium carbonate, is abundant, it is poorly soluble in water and easily forms calcium salt precipitates in the intestinal environment, making it difficult for the body to absorb effectively. Excessive intake can also cause gastrointestinal side effects. Organic calcium supplements contain calcium in cationic form, which is less affected by pH and has better stability and water solubility. However, these supplements have strict concentration requirements; concentrations that are too high or too low can affect their calcium supplementation effect. Amino acid-based calcium supplements have abundant raw materials and can increase amino acid intake while supplementing calcium. In these supplements, amino acids and calcium form chelates through coordination bonds, resulting in a stable structure. However, the activity of the finished product is easily affected by production conditions, and the high affinity of amino acids for calcium ions can limit calcium release to some extent, thus affecting the calcium supplementation effect.

[0004] Currently, calcium supplements in chelated form exhibit unique advantages. Studies have shown that chelated calcium can overcome problems such as low bioavailability and poor absorption efficiency at low concentrations, as well as biotoxicity at high concentrations. It also possesses advantages such as low energy consumption, high transport efficiency, and strong stability, making it a highly promising candidate for calcium supplementation. Currently, the preparation of peptide-calcium chelates mainly relies on protease-mediated enzymatic hydrolysis, which specifically or non-specifically cleaves peptide bonds to generate small peptide fragments with calcium-binding activity, thereby achieving effective chelation between peptides and calcium, such as mineral binding, antioxidant activity, and immunomodulation. Enzymatic hydrolysis, with its mild reaction conditions and high specificity, has attracted much attention in the field of protein hydrolysis and the preparation of related bioactive substances. However, high cost and high energy consumption are significant factors restricting its large-scale application. Summary of the Invention

[0005] In view of this, based on pH shift, the present invention provides a method for preparing a protein-core Chlorella protein peptide-calcium chelate, which has a simple preparation process, low cost and low energy consumption.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a protein-core Chlorella protein peptide-calcium chelate, the method comprising the following steps:

[0008] S1. Dissolve Chlorella protein in deionized water, adjust the pH to 2-13, stir for 1-5 hours, centrifuge, and take the supernatant to obtain Chlorella protein treatment solution.

[0009] S2. Add calcium chloride to the protein treatment solution of Chlorella pyrenoidosa, chelate in a water bath at 35~38℃ for 1.8~2.2h, centrifuge, add anhydrous ethanol to the supernatant, let stand, centrifuge again, collect the precipitate, freeze dry, and the product is obtained.

[0010] In some specific embodiments, preferably, the preparation method includes the following steps:

[0011] S1. Dissolve Chlorella protein in deionized water, adjust pH to 13, stir for 4 hours, centrifuge, and take the supernatant to obtain Chlorella protein treatment solution.

[0012] S2. Add calcium chloride to the protein treatment solution of Chlorella pyrenoidosa, chelate in a water bath at 37°C for 2 hours, centrifuge, add anhydrous ethanol to the supernatant, let stand, centrifuge again, collect the precipitate, freeze dry, and the product is obtained.

[0013] Furthermore, in step S1, the stirring speed is 250~350 rpm;

[0014] Centrifugation conditions: 8000~12000 rpm, time 8~12 min.

[0015] In some specific embodiments, preferably, the stirring speed in step S1 is 300 rpm;

[0016] Centrifugation conditions: 10,000 rpm, 10 min.

[0017] Furthermore, in step S2, the mass ratio of the protein nucleus Chlorella protein to calcium chloride is (8~12):1;

[0018] The centrifugation conditions were the same for both centrifugations: 8000~12000 rpm, time 8~12 min;

[0019] The volume ratio of the supernatant to anhydrous ethanol is (8~10):1;

[0020] Let stand for 1.5 to 2 hours.

[0021] In some specific embodiments, preferably, the mass ratio of Chlorella protein to calcium chloride in step S2 is 10:1;

[0022] The two centrifugation conditions were the same: 10,000 rpm, 10 min.

[0023] The volume ratio of the supernatant to anhydrous ethanol was 9:1;

[0024] Let stand for 1.5 hours.

[0025] The protein-core Chlorella protein peptide-calcium chelate prepared by the above preparation method.

[0026] The application of the protein-core Chlorella protein peptide-calcium chelate prepared by the above method in calcium supplement products.

[0027] Furthermore, the calcium supplement products include liquid formulations and solid formulations.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention provides a simple and low-cost (requiring only basic operations such as dissolving, stirring, and centrifugation) pH-shifting method for preparing Chlorella protein peptide-calcium chelates, achieving a calcium chelation rate as high as 44.04%. Furthermore, the prepared product exhibits excellent bioavailability. Rat experiments demonstrate that the chelate prepared in this application is more easily absorbed and utilized by the body, more effectively alleviating calcium deficiency-induced imbalances and promoting bone growth and development. Measurements of duodenal and skeletal parameters also reflect good absorption. Attached Figure Description

[0030] Figure 1 This is a diagram showing the effect of different pH levels on protein conformation and activity in Example 2 of the present invention; where A represents the chelation rate under different pH conditions, B represents the change in chelation rate with pH treatment time, C represents the effect of different pH treatments on free sulfhydryl content, and D represents the dynamic effect of pH treatment time on free sulfhydryl content.

[0031] Figure 2 The results are the physicochemical properties of the product under the optimal pH offset conditions in Example 2 of this invention; where A is the zeta potential of pH-pro and pH-Ca, B is the UV-Vis absorption spectrum of pH-Ca, C is the Fourier transform infrared spectrum, and D is the X-ray diffraction result.

[0032] Figure 3The results of the effects of different calcium sources on serum calcium and phosphorus metabolism, bone metabolism and inflammatory factors in calcium-deficient rats in Example 3 of this invention are shown. Among them, A is the serum calcium level, B is the serum phosphorus level, C is the serum ALP level, D is the PTH level, E~F are bone formation markers, G~H are bone resorption markers, and I~K are inflammatory factor detection.

[0033] Figure 4 The results of duodenal parameter measurements in Example 2 of this invention are shown; where A is the microstructure of the duodenum, B is the goblet cell count, C is the villus height, D is the villus area, and E is the crypt depth.

[0034] Figure 5 The effect of different calcium supplements on the morphological development of long bones in rats is shown in Example 2 of this invention; where A is bone length, B is bone diameter, C is dry matter weight index, D is bone calcium content, E is bone fracture strength, F is bone fracture work, and G is bone stiffness.

[0035] Figure 6 The results of the femoral micro-CT analysis in Example 2 of the present invention are shown below; where A represents the morphological characteristics of bone tissue, B represents the BV / TV value, and C~I represent Tb.Th (trabecular thickness), Tb.Sp (trabecular separation), Tb.N (number of trabeculae), Tb.Pf (trabecular pattern factor), and BMD trabecular index, respectively. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0037] Key experimental material sources and physicochemical parameters:

[0038] The Chlorella protein powder was provided by Shaanxi Baichuan Biotechnology Co., Ltd.

[0039] Anhydrous calcium chloride, sodium hydroxide, hydrochloric acid, and anhydrous ethanol were all sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0040] The protein free thiol content detection kit is from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

[0041] The serum calcium, serum phosphorus, and alkaline phosphatase kits were obtained from the Nanjing Jiancheng Biotechnology Institute in China.

[0042] Osteocalcin, type I procollagen N-terminal peptide (PⅠNP), type I procollagen C-terminal peptide (CTX-Ⅰ), antitartrate phosphatase 5b (TRAP-5b), tumor necrosis factor α (TNF-α), interleukin-17 (IL-17), and interleukin-6 (IL-6) are all sourced from Jiangsu Enzyme Immunosorbent Assay Co., Ltd.

[0043] All reagents used in the experiment were of analytical grade.

[0044] Example 1

[0045] This embodiment provides a method for preparing a protein-core Chlorella protein peptide-calcium chelate, comprising the following steps:

[0046] S1. Dissolve Chlorella proteoglycans protein in deionized water (100 mg / mL), adjust the pH to 13 with 6M sodium hydroxide, and after the pH of the solution stabilizes, stir at 300 rpm for 4 hours on a magnetic stirrer. After the reaction is complete, centrifuge at 10,000 rpm for 10 minutes, collect the supernatant, and adjust the pH to 7 to obtain the Chlorella proteoglycans protein treatment solution (pH-pro).

[0047] S2. Take pH-pro and, based on the mass of Chlorella protein, add anhydrous calcium chloride to pH-pro at a mass ratio of 10:1. Incubate in a 37℃ constant temperature water bath for 2 hours to form a calcium chelate. After incubation, centrifuge at 10000 rpm for 10 minutes, and take 0.1 mL of the supernatant as the stock solution for total calcium determination. Add 9 volumes of anhydrous ethanol to the remaining supernatant to remove unchelated free calcium. After standing for 1.5 hours, centrifuge at 10000 rpm for 10 minutes, collect the precipitate, and freeze-dry it to obtain the Chlorella protein peptide-calcium chelate (pH-Ca).

[0048] Furthermore, to investigate the effects of different pH values ​​and stirring times on the product, experiments were conducted sequentially for pH and stirring time. The pH values ​​were set as follows: 2, 4, 6, 7, and 9; the stirring times were set as follows: 1, 2, 3, and 5 hours.

[0049] Example 2

[0050] To understand the performance of the products prepared under different conditions in Example 1, the following characterization or tests were performed in this example:

[0051] 2.1 Calculation of calcium chelation rate

[0052] A standard curve was plotted using a standard calcium solution, and the chelated calcium content and total calcium content were determined using an atomic absorption spectrometer (AA-6300C, Shimadzu, Japan) (results are shown in [see table]). Figure 1(AB). The formula for calculating the calcium chelation rate is as follows:

[0053]

[0054] In the above formula: C 1 represents the calcium ion concentration (µg / mL) in the chelate. V 1 represents the volume (mL) of the chelate solution. X 1 represents the dilution factor of the chelate solution; C 2 represents the total calcium ion concentration (µg / mL) in the original solution. V 2 represents the volume of the solution reacting (mL). X 2 represents the dilution factor of the original solution.

[0055] 2.2 Determination of free thiol content

[0056] The free sulfhydryl content was determined using a protein free sulfhydryl content assay kit with reduced glutathione as the standard under different pH shifts and treatment times (results are shown in the figure). Figure 1 CD).

[0057] 2.3 Determination of hydrolyzed amino acid content

[0058] Amino acid composition analysis was performed using a fully automated amino acid analyzer (Biochrom 30+, Biochrom, UK) according to the People's Republic of China National Standard (GB 5009.124-2016, Determination of Amino Acids in Food). 50 mg of sample was weighed and transferred to a hydrolysis tube, mixed with 10 mL of 6 M HCl at a 1:1 (v / v)d concentration, and the tube was purged with nitrogen for 30 seconds and sealed. The tube was then placed in an oil bath at 110°C for 22-24 hours for hydrolysis. After hydrolysis, the sample was cooled to room temperature, filtered through a 0.45 μm membrane into a 50 mL volumetric flask, and diluted to volume. 2 mL of the diluted sample was then transferred to… The plate inside the weighing bottle was heated at 85°C twice to remove acidity until a small amount of solid or residue remained at the bottom of the bottle. 1 mL of sodium citrate buffer was added to dissolve the solid completely. The solution was then filtered through a 0.22 µm filter and amino acid analysis was performed (results are shown in Table 1).

[0059] Table 1. Amino acid composition characteristics of protein hydrolysates after treatment under different pH conditions.

[0060]

[0061] 2.4 Zeta potential test

[0062] Using a nanoparticle size potentiometer (NanoZS, Malvern, UK), pH-pro and pH-Ca were tested under optimal pH offset conditions. Solutions with a concentration of 1 mg / mL were prepared for measurement (results are shown in [link to results]). Figure 2 A).

[0063] 2.5 Ultraviolet-Visible Absorption Spectroscopy Test

[0064] Solutions of pH-pro and pH-Ca, which exhibited the highest calcium chelation rates, were prepared at 0.5 mg / mL and loaded into quartz cuvettes. The samples were then scanned at wavelengths of 200-700 nm using a UV-1100 Rayleigh Analytical Instrument Co., Ltd., China, with a scan speed of 60 nm / min and a bandwidth of 2.0 nm. Each sample was scanned three times, and the average value was used to plot the corresponding UV spectrum (see results below). Figure 2 B).

[0065] 2.6 FTIR Test

[0066] Fourier transform infrared spectroscopy (FIR) was used to compare and analyze the changes in peptide bonds and functional groups before and after chelation. pH-pro and pH-Ca, which had the highest chelation rates, were mixed with dry potassium bromide at a ratio of 1:100, thoroughly ground in a mortar, and then compressed into thin tablets using a tablet press. The tablets were then examined at 400-4000 cm⁻¹. -1 A full-wavelength scan was performed within the specified range, with 32 scans conducted, and the potassium bromide background was removed. Each sample was tested three times to obtain the average value (results are shown in [link to results]). Figure 2 C).

[0067] 2.7 X-ray testing

[0068] The changes in crystallinity of pH-pro and pH-Ca were determined using an X-ray diffractometer (Bruker D8 X-ray, Brabender, Germany). A suitable amount of lyophilized sample was thoroughly ground, and the sample was scanned at a voltage of 40 kV, a current of 40 mA, and a scanning speed of 4° / min within the range of 5° to 75°. The diffraction intensity of the sample was measured (see results). Figure 2 D).

[0069] Based on the above series of physicochemical characterizations and measurements, the following conclusions can be drawn:

[0070] Different acid-base levels can affect the conformation and activity of proteins, thereby regulating the chelation efficiency of Chlorella protein peptides with calcium. Figure 1A shows the calcium chelation rate of Chlorella protein peptide-calcium chelates under different pH conditions. Between pH 2 and 4, the calcium chelation rate increases. In this acidic environment, the increased hydrogen ion concentration promotes protonation of the acidic groups of the protein peptide, inducing the gradual unfolding of the protein structure and exposing previously buried calcium binding sites, creating favorable conditions for the calcium chelation reaction. Between pH 4 and 6, the chelation rate decreases. Under a slightly acidic environment, the conformational changes of the protein peptide tend to be gradual, and some binding sites may recfold and remain hidden. Between pH 6 and 13, the chelation rate increases significantly. The strongly alkaline environment not only accelerates the complete unfolding of the protein structure but also induces deprotonation of amino groups. At this point, a large number of coordination reactions occur between calcium ions and carboxyl and amino groups.

[0071] The calcium chelation rate reached its maximum at pH 13, at 44.04 ± 1.34%, and was significantly different from the other five pH levels (P < 0.05). The change in chelation rate with pH treatment time is shown below. Figure 1 As shown in B, the protein solution rapidly adjusted to 13% served as a control (31.83±0.97%). With increasing treatment time, the overall chelation rate showed a slow upward trend. At 1 hour, the chelation rate was 34.03±0.44%, indicating that the protein conformation was not fully unfolded and the binding sites were only partially exposed. The chelation rate reached its peak at 4 hours, at 40.23±0.36%, indicating that the protein molecules were fully unfolded at this time.

[0072] The amino acid composition of protein hydrolysates under different pH treatments is shown in Table 1. Amino acid profile analysis can reflect changes in anionic ligand characteristics, protein unfolding, and the degree of ionization and accessibility of binding sites during hydrolysis. These factors collectively determine the effective coordination probability of a single peptide chain, thus affecting the calcium chelation rate. Therefore, analyzing the amino acid composition under different pH conditions is of great significance for elucidating the calcium chelation mechanism.

[0073] The results showed that the contents of aspartic acid (Asp) and glutamate (Glu) increased in the acidic to near-neutral range; however, at pH=13, the contents of Asp and Glu decreased, but the calcium chelation rate reached its maximum. This phenomenon can be explained by the following mechanism: under high pH conditions, protein peptide molecules undergo extensive deprotonation, which enhances the negative electrostatic potential around the oxygen donor and significantly increases the affinity of the binding site. Previous studies have confirmed that peptides with stronger negative electrostatic potentials can coordinate with calcium ions more efficiently and are beneficial to calcium absorption and utilization. In addition, strong alkaline treatment can shorten the peptide chain length and increase the density of terminal carboxylic acid groups. Even if the contents of Asp and Glu on the side chains decrease, the terminal carboxylic acid groups can still cooperate with the carbonyl groups of the adjacent main chain to participate in calcium coordination, indicating that the actual number of functional oxygen donors cannot be accurately assessed based solely on amino acid composition data.

[0074] Meanwhile, a strongly alkaline environment promotes the deamidation reaction of proteins and the formation of transient succinimide. This process interferes with the accuracy of traditional amino acid analysis results and may lead to an underestimation of the calcium chelating capacity of Asp and Glu after hydrolysis. Therefore, the decrease in Asp and Glu content measured at pH=13 is partly due to errors in the analytical process, rather than a reduction in the actual number of oxygen donors. In summary, the calcium chelation rate reaches its maximum at pH=13, mainly attributed to improved binding site efficiency and the participation of terminal oxygen donors in coordination.

[0075] The zeta potentials of pH-pro and pH-Ca are as follows: Figure 2 As shown in Figure A, the zeta potential of all samples was negative. When pH-pro was mixed with calcium ions to form pH-Ca, the absolute value of the zeta potential significantly decreased from -8.65 ± 0.58 mV to -6.65 ± 0.17 mV (P < 0.05). This decrease in zeta potential is a typical characteristic of peptide-calcium systems, indicating that calcium ions can promote the formation of more stable peptide-calcium complexes through charge shielding and bridging. Recent studies on peptide-calcium chelates have generally found that a decrease in zeta potential is often accompanied by an increase in particle size; this pattern further confirms the good stability of the calcium-peptide complex formed in this study.

[0076] like Figure 2 As shown in Figure B, the dynamic changes in the ultraviolet-visible absorption spectrum (UV-Vis) provide direct molecular-level evidence for the chelation reaction. The results show that the absorption peak near 210 nm mainly corresponds to the characteristic absorption of intramolecular carbonyl groups (C=O), carboxyl groups (-COOH), and amide bonds (amide I band). Compared to pH-pro, the absorption intensity and the position of the maximum absorption peak of pH-Ca both change significantly: the maximum absorption wavelength (λ...) max The blue shift occurred from 207 nm to 204 nm (Δλ = -3 nm). This blue shift is attributed to the interaction between calcium ions and the carboxyl oxygen (O2) in aspartic acid / glutamate residues. - The preferential formation of a five-membered chelate structure by the amide nitrogen (N) and the coordinating atoms leads to a redistribution of electron density around the coordinating atoms, which in turn changes the electronic transition characteristics of the carbonyl and carboxyl groups, confirming the formation of a new peptide-calcium complex.

[0077] Changes in absorption peaks in Fourier transform infrared (FTIR) spectroscopy provide crucial evidence for the binding modes of metal ions and organic ligands. For example... Figure 2 As shown in C, compared to pH-pro, pH-Ca at 3390 cm⁻¹ -1 The absorption peak at that point shifts towards higher wavenumbers to 3412 cm⁻¹. -1The absorption peak corresponds to the stretching vibration of the NH and OH bonds, indicating a significant enhancement of hydrogen bonding in the complex after the chelation reaction. Simultaneously, the inductive effect or enhanced dipole field induced by the chelation reaction increases the electron cloud density, suggesting that the amino group may participate in the calcium coordination process. Furthermore, at 1647 cm⁻¹... -1 The amide I band (mainly corresponding to the C=O stretching vibration) extends to 1649 cm⁻¹ -1 The shift confirms that the carbonyl group in the protein backbone participates in calcium ion coordination.

[0078] XRD technology was used to analyze the changes in crystal structure during calcium chelation. The results are as follows: Figure 2 As shown in Figure D, a broad diffraction peak was observed at 2θ = 20.5 in the pH-pro sample, indicating an amorphous structure without a distinct crystalline phase. After calcium ion chelation, the diffraction peak shifted towards 2θ = 21.0° ± 0.2° (Δ2θ = +0.5°) and the peak width increased. This shift and broadening phenomenon indicates the formation of a novel amorphous pH-Ca complex with a short-range ordered structure. Specifically, when the coordination bond between the carboxyl and amino groups of the protein nucleus chlorella peptide chelates with Ca... 2+ When chelation occurs, the original intermolecular interactions (such as hydrogen bonds and electrostatic interactions) are disrupted, leading to the reconstruction of the amorphous network structure. These structural changes are in high agreement with the FTIR detection results, further confirming the success of the chelation reaction.

[0079] Example 3

[0080] Furthermore, to understand the biological properties of the prepared product (using the optimal process paper cup with pH=13 and stirring time of 4h as the research object), the following animal experiments were also conducted:

[0081] 3.1 Animal husbandry

[0082] All feeds were adjusted according to AIN-93G standards and produced by Anhui Kuibu Shuyu Biotechnology Co., Ltd. The standard feed contains 0.5% calcium, and the low-calcium feed contains 0.1% calcium. Specific ingredients are shown in Table 2 below.

[0083] Table 2 AIN-93G Standard Feed Details

[0084]

[0085] 3.2 Animal feeding experiment

[0086] Twenty-five 3-week-old SPF-grade SD rats, weighing 95±10 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and raised in the Animal Experiment Center of Huazhong Agricultural University, with the license number SYXK(E)2020-0084 and the ethical number HZAURA-2025-0029. Under the conditions of an SPF-grade rat room (temperature 22±2°C, humidity 55±15%, light cycle 12 h), the rats were adapted to the corresponding feed for one week and then randomly divided into 5 groups according to body weight (normal diet group NC, low-calcium model group Model, calcium carbonate (CaCO3), untreated protein-calcium chelate group Native-pro-Ca (i.e., omitting the pH adjustment process and keeping the rest unchanged), and protein peptide-calcium chelate pH-Ca after pH offset treatment), with 10 rats in each group. During the feeding period, the rats could freely eat and drink. The normal diet group was fed with the standard AIN-93G feed, the low-calcium model group was fed with the low-calcium feed, and while the other 3 groups were fed with the low-calcium feed, calcium carbonate, Native-pro-Ca, and pH-Ca were respectively gavaged in the form of calcium solution once a day for 4 weeks. The gavage dose was set at 5 times the daily requirement of 800 mg calcium for an adult weighing 60 kg, i.e., 66.67 mg / kg.

[0087] 3.3 Animal growth experiment

[0088] After grouping, the initial body weights of the SD rats in each group were recorded. During the gavage period, the body weights were measured every 2 days. After 4 weeks of feeding, the body weight changes of the rats in each group were calculated and the differences between the groups were analyzed. And the relevant indexes of the rats were tested as follows:

[0089] 3.3.1 Collection of animal tissues

[0090] After 4 weeks of feeding, the rats were fasted for 12 h, their whiskers were cut off, and blood was taken from the eyeballs into 15 mL centrifuge tubes. The tubes were left to stand at 4°C for 2-3 h. After the blood coagulated, it was centrifuged at 4000 rpm / min for 10 minutes, and the serum was quickly separated and stored in a -80°C refrigerator. After the rats were sacrificed by cervical dislocation, the abdominal cavity of the rats was opened along the midline of the abdomen, and the liver, spleen, and kidneys were collected; the duodenum was taken, one part was fixed with 4% paraformaldehyde fixative, and the other part was fixed with RNA fixative for subsequent experiments; the femurs and tibias of both sides of the rats were taken, and after removing the muscle and fascia tissues, one side of the femur and tibia was placed in 4% paraformaldehyde fixative, and the other side was wrapped with gauze moistened with physiological saline and stored at -20°C.

[0091] 3.3.2 Determination of organ index

[0092] The collected liver, spleen, and kidneys were defatted, the surface was washed with physiological saline, and after drying with filter paper, the weights were recorded and the differences between the groups were analyzed and compared (the results are shown in Table 3).

[0093] Table 3. Details of body weight gain and related organ weight in rats from different treatment groups.

[0094]

[0095] Note: Data are expressed as mean ± standard deviation, sample size n=5. The same superscript letter in the same column indicates no significant difference (P>0.05).

[0096] 3.3.3 Determination of serum biochemical indicators

[0097] Serum calcium, serum phosphorus, and alkaline phosphatase were measured according to the instructions provided by Nanjing Jiancheng Biotechnology Institute, China. Osteocalcin, type I procollagen N-terminal peptide (PⅠNP), type I procollagen C-terminal peptide (CTX-Ⅰ), antitartrate phosphatase 5b (TRAP-5b), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), and interleukin-6 (IL-6) were measured according to the instructions provided by Jiangsu Meimian Industrial Co., Ltd.

[0098] 3.3.4 Duodenal parameters

[0099] Approximately 1 cm of tissue was harvested from the same location of the duodenum, fixed in 4% paraformaldehyde, routinely embedded in paraffin, and then serially sectioned (4 µm). After HE staining and mounting on neutral resin film, villus height, area, goblet cell count, and crypt depth were analyzed using Case Viewer 2.4 scanning software.

[0100] 3.3.5 Bone length and diameter

[0101] The left femur and tibia of rats, stored at -20°C, were thawed at room temperature. The length and diameter of the rat femur and tibia were measured using Exploit digital calipers (Exploit Tools Co., LTD).

[0102] 3.3.6 Determination of bone dry weight and bone calcium content

[0103] The fractured femur and tibia were dried in a 110°C oven until constant weight, weighed on an analytical balance, and the dry weight was recorded. The dry weight index was calculated using the following formula:

[0104] DW(10) -3 = (Bone weight / Rat body weight) × 1000

[0105] The femur, dried to constant weight, was transferred to a digestion tube, and 10 mL of mixed acid (nitric acid: perchloric acid = 4:1, v / v) was added. The mixture was then digested on an electric furnace at a programmed temperature until it became clear and transparent. After making up to a suitable volume and diluting to a suitable concentration, the bone calcium content (mg / g) was determined using an atomic absorption spectrophotometer.

[0106] 3.3.7 Determination of Bone Tissue Biomechanics

[0107] The thawed femur and tibia were placed on a stage, and the experimental parameters were as follows: span 10 mm, speed 1 mm / s, displacement 5 mm. The "three-point fracture method" was used to measure and record the fracture force, fracture work and stiffness of the femur and tibia.

[0108] 3.3.8 Micro-CT Analysis of the Femur

[0109] The femur was removed from the paraformaldehyde fixation solution, and excess liquid was wiped off with gauze. A Micro-CT scan (Ping Sheng Medical Technology, NMC-200) was performed with the following parameters: scan resolution 35 μm, scan voltage 80 kV, and scan current 0.06 mA. Recon software was used to perform 3D reconstruction of the original images. Finally, Avatar data analysis software was used to analyze the target region ROI. All samples were analyzed within the same region, and the following parameters were calculated: bone volume fraction (BV / TV, %), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), and trabecular number (Tb.N, mm). -1 ), trabecular pattern factor (Tb.Pf, mm) -1 Bone mineral density (BMD, mg / cm³) 3 Cortical bone thickness (Ct.Th, mm) and cortical bone area (Ct.ar, mm) 2 ).

[0110] 3.3.9 Osteocalcin immunohistochemical staining of bone tissue and number of osteoblasts

[0111] Femoral tissue was extracted from each rat and fixed in 4% (v / v) paraformaldehyde (PBS). After 48 hours, all femoral samples were decalcified in 10% EDTA (pH 7.4) for 3 weeks. After decalcification, the samples underwent gradient dehydration, clearing, paraffin embedding, and sectioning. The sections were stained with hematoxylin and observed morphologically under an optical microscope.

[0112] 3.3.10 TRAP staining and osteoclast count in bone tissue

[0113] Bone tissue sections were stained, and the number of osteoclasts was analyzed using ImagePro Plus software.

[0114] Finally, the obtained data were repeated three times and the average was taken. Data processing was performed using Origin 2025 and GraphpadPrism 10. Differences with P < 0.05 were statistically analyzed using one-way ANOVA, with different letters indicating significant differences.

[0115] The following conclusions can be drawn from the above series of biological tests:

[0116] Table 3 shows the weight gain and related organ weights of rats in different treatment groups. The results showed no significant difference in weight gain among the groups (P>0.05), indicating that different calcium supplements had no significant effect on the overall growth rate of rats. However, the liver weight of the model group (11.13±0.66g) was significantly higher than that of the control group (9.86±0.74g), which is speculated to be due to a compensatory response triggered by calcium deficiency, leading to enhanced liver metabolic activity. There were no significant differences in spleen and kidney weight among the groups, indicating that the calcium supplement dosage used in this study did not cause significant organ toxicity and had good safety.

[0117] Figure 3 This study systematically demonstrated the regulatory effects of different calcium sources on serum calcium and phosphorus metabolism, bone metabolism, and inflammatory factors in calcium-deficient rats. Serum calcium and serum phosphorus play important roles in maintaining normal physiological functions. Figure 3 As shown in Figure A, there was no significant difference between the model group (2.30±0.14mmol / L) and the normal group (2.35±0.18mmol / L) (P>0.05), which is presumably because the body maintains blood calcium homeostasis through bone calcium mobilization and renal reabsorption. However, the group supplemented with calcium carbonate (2.01±0.18mmol / L) showed a significant difference from the normal group (P<0.05), possibly because the alkaline environment in the intestine led to calcium precipitation and reduced bioavailability. The blood calcium levels of Native-pro-Ca (2.11±0.07mmol / L) and pH-Ca (2.21±0.02mmol / L) were similar to those of the normal group, indicating that the sample can be well absorbed in rats.

[0118] Serum phosphorus levels at Figure 3 B indicates that only the model group (2.13±0.02mmol / L) and the pH-Ca group (2.45±0.06mmol / L) showed significant differences, while no significant differences were observed overall. ALP is closely related to osteoblast activity and can reflect the physiological activity of bone.

[0119] Depend on Figure 3As shown in Figure C, the serum ALP level in the model group rats (235.51±101.89 U / 100 mL) was significantly higher than that in other groups, which is consistent with the findings of Chen et al. After gavage with CaCO3, Native-pro-Ca, and pH-Ca, the ALP levels decreased to 159.37±56.84 U / 100 mL, 152.69±42.39 U / 100 mL, and 66.48±13.62 U / 100 mL, respectively. However, there was no significant difference in ALP levels between the pH-Ca group and the NC group (P>0.05), indicating that pH-Ca can effectively inhibit bone metabolism and enhance bone stability.

[0120] When the body experiences significant calcium deficiency, CaSR in the parathyroid glands promotes rapid secretion of PTH, thereby regulating the dynamic balance between blood calcium and bone calcium, and the PTH level. Figure 3 D) Further verification of the model's success (model group 50.81±3.98ng / L, normal group 7.35±2.72ng / L, P<0.05), while the pH-Ca group (25.76±1.94pg / mL) was significantly lower than the Native-pro-Ca group (38.53±5.42pg / mL, P<0.05), indicating that the chelate after pH shift treatment is more easily absorbed and utilized by the body, and can more effectively alleviate the body's imbalance caused by calcium deficiency.

[0121] Figure 3 E~F are bone formation markers. Bone formation markers show that the model group's BGP (… Figure 3 E, 0.92±0.25ng / mL) and PINP ( Figure 3 The levels of PINP (1.52±0.30 ng / mL) were significantly lower than those in the normal group (2.41±0.58 and 2.34±0.18 ng / mL, P<0.05). After pH-Ca intervention, the levels recovered to 2.34±0.16 and 3.44±0.10 ng / mL, respectively. The PINP level was significantly higher than that in the NC group (P<0.05), confirming that this substance has a promoting effect on bone growth and development.

[0122] Figure 3 G~H are markers of bone resorption. Figure 3 In group G, the levels of TRACP-5b varied among the groups. The level in the Model group (207.26±40.60 pg / mL) was statistically significantly higher than that in the NC group (33.45±7.27 pg / mL), indicating that a low-calcium diet leads to increased osteoclast activity. After gavage with CaCO3 and Native-pro-Ca, there was a slight decreasing trend, but no significant difference (P>0.05). After gavage with pH-Ca, the level decreased significantly and returned to normal levels (84.94±8.04 pg / mL). Figure 3 H represents the CTX-1 content. CTX-1 is a marker of bone turnover; higher levels indicate greater bone calcium release and bone resorption. Comparing the Model group (3.00±0.41 ng / mL) and the NC group (1.71±0.06 ng / mL), the Model group showed a statistically significant difference in rapidly initiating bone mobilization. Supplementation with CaCO3, Native-pro-Ca, and pH-Ca decreased CTX-1 levels, but CaCO3 and Native-pro-Ca showed no significant difference compared to the Model group. However, CTX-1 activity in pH-Ca returned to normal levels (2.20±0.11 ng / mL), indicating that pH-Ca successfully inhibited bone resorption. Increased bone resorption has been shown to be closely related to the body's immune system, and IL-6, IL-17, and TNF-α promote osteoclast formation. Therefore, the levels of pro-inflammatory cytokines are valuable for assessing the body's skeletal status.

[0123] Inflammatory factor detection ( Figure 3 IK assays revealed that IL-6 (20.94±1.36 pg / mL), IL-17 (14.41±1.50 pg / mL), and TNF-α (108.00±9.68 pg / mL) in the model group were significantly higher than those in the normal group (10.61±1.39, 6.66±0.64, and 32.57±4.19 pg / mL, P<0.05). In the pH-Ca group, IL-6 (9.67±1.19 pg / mL) and TNF-α (41.67±0.87 pg / mL) returned to normal levels (P>0.05), while CaCO3 and Native-pro-Ca levels remained significantly higher than those in the normal group (P<0.05). This suggests that pH-Ca indirectly improves bone health by regulating the immune microenvironment. In summary, pH-shifted chelated calcium effectively reverses calcium deficiency-induced bone metabolism disorders through a synergistic effect of optimizing calcium absorption, inhibiting bone resorption, promoting bone formation, and anti-inflammation.

[0124] The microstructure of the duodenum in each experimental group was observed using HE staining technique. Figure 4A). In the normal control group (NC group), the duodenal villi showed dense, regular, finger-like projections with uniform villus height and clear crypt structures. The mucosal layer was intact without breakage, reflecting the normal proliferation and differentiation state of intestinal epithelial cells. In contrast, the calcium-deficient model group (Model group) showed significant pathological changes, with significantly shorter villi and larger gaps. After supplementation with various substances, the intestinal damage in calcium-deficient rats was improved, and the pH-Ca group showed better results than CaCO3 and Native-pro-Ca, indicating that this preparation can effectively rebuild the intact structure of the intestinal epithelium. The core function of goblet cells is to secrete mucin, forming an intestinal mucus barrier to resist pathogen invasion and inflammatory damage, further revealing the differences in intestinal barrier function among the groups. The number of goblet cells in the NC group remained stable, reflecting normal intestinal barrier function. In the Model group, due to intestinal microenvironment imbalance triggering a compensatory mechanism, the number of goblet cells increased to (41.92±8.24). Although the CaCO3 and Native-pro-Ca groups showed improvement, the number of goblet cells did not return to normal. The number of goblet cells in the pH-Ca group was closer to that in the NC group, suggesting that it is more conducive to calcium absorption and can effectively improve the intestinal compensatory response caused by calcium deficiency, maintaining mucus barrier function. Although there were no significant differences in villus height and villus area among the groups (P>0.05), the pH-Ca group showed significant advantages in both mucosal repair quality and barrier function recovery. In summary, all three calcium supplements can improve intestinal damage induced by a low-calcium diet through different mechanisms. Among them, the pH-Ca group, with its targeted release characteristics, achieved dual regulation of structural repair and functional reconstruction, providing experimental evidence for the development of highly efficient calcium absorption enhancers.

[0125] pass Figure 5 The AB system was used to evaluate the effects of different calcium supplements on the morphological development of long bones in rats, with a focus on analyzing the longitudinal growth characteristics of the femur and tibia. Regarding longitudinal growth ( Figure 5 A) In the calcium deficiency model group (Model group), the femur length was reduced by 1.00 mm and the tibia shortened by 1.28 mm compared to the normal control group (NC group), confirming that a low-calcium diet significantly inhibits bone growth. However, after intervention with pH-shifted Chlorella protein peptide-calcium chelate (pH-Ca group), the femur length was 0.81 mm longer and the tibia length was 0.98 mm longer than the NC group. This improvement was significantly better than that of the calcium carbonate group (CaCO3 group, femur / tibia still 0.40 / 0.04 mm shorter) and the natural protein calcium group (Native-pro-Ca group, 0.86 / 0.81 mm shorter), indicating that this preparation can effectively reverse low-calcium-induced bone growth retardation. Lateral growth indices ( Figure 5B) Shows differentiated responses to bone mass accumulation. In femoral diameter measurement, the NC group measured 3.36±0.14 mm, while the Model, CaCO3, and Native-pro-Ca groups maintained a range of 3.18–3.32 mm. The pH-Ca group, however, showed a significant increase in femoral diameter to 3.61±0.08 mm, 0.43 mm thicker than the Model group and even exceeding the NC group by 0.25 mm, suggesting a unique effect in promoting cortical bone thickening. In tibial diameter analysis, the NC group (3.31±0.04 mm) was significantly higher than the other groups. The CaCO3 group (2.46±0.11 mm), Native-pro-Ca group (2.44±0.09 mm), and Model group (2.43±0.07 mm) were close. The pH-Ca group, however, reached 2.72±0.05 mm, 0.26 mm, 0.28 mm, and 0.29 mm thicker than the other three groups, respectively. Although it did not return to the NC level, it still demonstrated a significant bone accumulation effect. Chlorella protein peptide-calcium chelate prepared via pH shifting technology exhibits significant advantages in promoting bone development: it not only effectively restores longitudinal growth inhibited by a low-calcium diet, but also substantially enhances bone mass accumulation by increasing cortical bone thickness, with particularly pronounced effects on the femur. This discovery provides a new theoretical basis for developing calcium fortifiers targeting bone health.

[0126] Figure 5The C-system analysis investigated the effects of different calcium supplements on the dry weight index (DW) of rats. This index, a core evaluation parameter for bone calcium deposition, directly reflects the degree of bone mineralization and biomechanical properties. The results showed that the dry weight indices of the femur and tibia in the normal control group (NC group) were 1.76±0.17 and 1.64±0.13, respectively, while those in the model group significantly decreased to 1.07±0.06 and 0.96±0.16 (P<0.05 vs NC), confirming that a low-calcium diet led to severe insufficient bone mineralization and significantly deteriorated bone growth. Although calcium supplementation with CaCO3 and Native-pro-Ca increased the dry weight index (femur: 1.25±0.06, 1.21±0.05; tibia: 1.15±0.17, 1.12±0.08), the increase was still close to that in the model group, and the effect was not significant (P>0.05). In contrast, the pH-shifted Chlorella protein peptide-calcium chelate (pH-Ca group) exhibited a significant bone mineralization-promoting effect: its femoral dry weight index reached 1.56±0.05, and its tibial dry weight index reached 1.51±0.05, which were not only significantly higher than the Model group, CaCO3 group, and Native-pro-Ca group (P<0.05), but also showed no statistically significant difference compared with the NC group (P>0.05), suggesting that this preparation can effectively restore the bone mineralization level inhibited by a low-calcium diet. This is highly consistent with the report by Hua et al., further demonstrating that Chlorella protein calcium carriers can enhance bone strength and prevent the occurrence of osteoporosis.

[0127] Figure 5 D reflects bone calcium content. Biomechanical parameters of bone tissue reflect bone strength, toughness, and stability. This experiment verified the bone-strengthening effect of pH-shifted Chlorella protein peptide-calcium chelate (pH-Ca) from three dimensions: bone strength, toughness, and resistance to deformation. Figure 5 (E~G). The fracture forces of the femur and tibia in the calcium deficiency model group were significantly lower than those in the NC group (P<0.05); this indicates that calcium deficiency directly weakens the load-bearing capacity of bones. After calcium supplementation (CaCO3 and Native-pro-Ca), although the maximum fracture forces of the femur and tibia showed an increasing trend, the tibia did not reach a level significantly different from the Model group (P>0.05). However, when intervention was performed using pH-shifted Chlorella protein peptide-calcium chelate (pH-Ca), the maximum fracture forces of both the femur and tibia were significantly increased (P<0.05), showing a good bone strengthening effect. Figure 5F represents the fracture work. In the NC group, the fracture work of the femur and tibia was at a relatively high level, while it was significantly lower in the Model group (femoral fracture work was 3671 J, tibial fracture work was 2228 J) (P < 0.05), suggesting that calcium deficiency reduces bone toughness. After CaCO3 and Native-pro-Ca supplementation, bone toughness improved to some extent, but there was still no significant difference compared with the Model group. Conversely, after pH-Ca intervention, the femoral fracture work increased to approximately 7254 J, and the tibial fracture work increased to approximately 5521 J, both of which showed a significant increase in fracture work compared with the Model group (P < 0.05). Figure 5 G represents stiffness, reflecting the bone's resistance to deformation. In the NC group, the stiffness of the femur and tibia was at a high level, with both exceeding 7000 N / mm. In contrast, the stiffness of the femur in the calcium-deficient model group (Model) dropped significantly to approximately 1836 N / mm, and the stiffness of the tibia also decreased significantly to approximately 2866 N / mm. This indicates that long-term calcium deficiency weakens the bone's resistance to deformation. After CaCO3 and Native-pro-Ca supplementation, the stiffness of the femur and tibia improved to varying degrees, but there was no significant difference compared to the Model group (P > 0.05). When pH-Ca was used for intervention, the stiffness of the femur increased to approximately 4551 N / mm, and the stiffness of the tibia increased to approximately 6411 N / mm, both significantly improved compared to the Model group. These experimental results demonstrate that pH-Ca can effectively enhance the bone's resistance to deformation and promote bone development.

[0128] Micro-CT technology uses X-rays to scan bone structures and performs quantitative analysis of microstructures through three-dimensional reconstruction. Due to its high efficiency and high resolution, this technology is widely used in many research fields such as bone tissue engineering and osteoporosis. The rat femur consists of cancellous bone, cortical bone, and medullary cavity. Cortical bone, also known as compact bone due to its dense structure, is mainly distributed on the surface of the bone body and in the outer layer of the bone shaft, serving as a key structure supporting the body due to its excellent compressive strength. Cancellous bone can be considered a structural extension of cortical bone, concentrated in the bone cavities at both ends of the femur, and is composed of numerous needle-like or sheet-like trabeculae interwoven to form a sponge-like morphology. In calcium-deficient rat model studies, the morphological characteristics of the cancellous and cortical bone of the femur are often used as key indicators for assessing bone growth and development. The coronal plane, trabeculae, and cortical bone of the femur in each experimental group are shown below. Figure 6 As shown in Figure A, the white, dotted or sheet-like tissue in the coronal plane of the femur is the trabecular bone. The thickness on both sides of the bone can visually reflect the growth status of the cortical bone. Through comparative analysis... Figure 6The morphological characteristics of bone tissue presented in group A lead to the following key conclusions: In the Model group, the bone cavity exhibited a highly pronounced internal cavitation feature, and the arrangement of trabeculae was disordered and extremely sparse, with a significant reduction in their number compared to normal levels. These intertwined and synergistic changes resulted in a devastating disruption of the integrity of the bone microstructure, greatly weakening the load-bearing and damage-resistant properties of the bone from a biomechanical perspective, reflecting significant degenerative changes in bone tissue under calcium deficiency. Simultaneously, the thickness of cortical bone in this group was significantly thinner than that in the normal control group (NC group). This microstructural change clearly reflects the imbalance in bone metabolism. As a dynamic organ, the disruption of metabolic balance in the skeleton signifies a disorder in the coupling relationship between bone formation and bone resorption. Further observation of the effects of different interventions on bone tissue revealed differential results. After supplementation with CaCO3 and Native-pro-Ca, the number of trabeculae showed a certain degree of increase, and the proportion of spongy bone tissue improved.

[0129] However, a significant gap remained in the intervention group, indicating that bone remodeling had not fully recovered to normal levels, suggesting potential limitations in the effectiveness of these two interventions. In stark contrast, the experimental group supplemented with pH-regulated chelated calcium (pH-Ca) exhibited a more significant bone tissue repair effect. Trabecular bone structure was denser, its network integrity was significantly improved, and the connections between trabeculae were tighter, forming a stronger support network. Cortical bone thickness was significantly increased compared to the Model group and the aforementioned intervention groups. These optimizations in morphological parameters suggest that pH-Ca intervention can precisely regulate bone metabolism and effectively promote the restoration of bone metabolic balance. Its mechanism of action may involve the regulation of key signaling pathways in bone formation and resorption, coordinating interactions between cells and molecules to restore bone tissue to a healthy physiological state.

[0130] In summary, pH-Ca demonstrates significant potential value as a functional food additive in improving bone health, providing an important theoretical basis for the development of novel bone metabolism regulators.

[0131] Indicators describing the skeleton, such as Figure 6 As shown in Figures B to I, BV / TV is a key indicator for measuring changes in bone mass and has significant reference value. Figure 6As shown in Figure B, the bone volume fraction in the NC group was significantly higher than that in other groups. This result strongly indicates that the normal control group rats have abundant bone mass and maintain good bone structure integrity. The bone volume fraction in the Model group was significantly lower than that in the NC group, which clearly indicates successful modeling. The model group rats experienced significant bone loss, and bone metabolic homeostasis was disrupted. Further analysis revealed that the bone volume fraction in the CaCO3 group and the Native-pro-Ca group was not significantly different from that in the Model group (P > 0.05). This suggests that simply supplementing with CaCO3 or Native-pro-Ca has a weak effect on improving bone loss in this model rat, and is unlikely to effectively stimulate bone formation or inhibit bone resorption. In contrast, the bone volume fraction of rats in the pH-Ca group was not only significantly higher than that in the Model group, CaCO3 group, and Native-pro-Ca group, but also nearly reached the level of the NC group. This indicates that the calcium preparation treated with pH has a significant advantage in increasing the bone volume fraction of model rats. It is speculated that this may be achieved by optimizing the calcium absorption and utilization mechanism or by precisely regulating bone metabolism-related signaling pathways, thereby effectively promoting bone formation or inhibiting bone resorption, and ultimately achieving effective improvement in bone loss.

[0132] Bone growth can also be reflected by the bone's geometry and the microstructure of the trabecular bone structure. Tb.Th (trabecular thickness), Tb.Sp (trabecular separation), Tb.N (number of trabeculae), Tb.Pf (trabecular pattern factor), and BMD are all key parameters describing trabecular bone. Figure 6(BG). Among the first four indicators, the trabecular bone structure of rats in the NC group was characterized by density and good connectivity, forming a fine network of healthy trabecular bone. In stark contrast, rats in the Model group showed obvious bone degeneration, specifically sparse and broken trabecular bone. Although the CaCO3 group and the Native-pro-Ca group showed some improvement compared to the Model group, they still lagged far behind the NC group, indicating that these two calcium preparations had limited effects on trabecular bone repair. The pH-Ca group was closer to the NC group in terms of Tb.Th and Tb.N, and its Tb.Sp and Tb.Pf were significantly better than the other three groups, showing its advantage in improving the microstructure of trabecular bone. Bone mineral density is closely related to bone strength and the stability of the internal bone structure, and is an important indicator for evaluating low-calcium osteoporosis models. Compared with NC, Mddel's BMD decreased significantly, which confirmed that a large amount of bone mineral was lost and bone metabolism was disordered after successful modeling. While the bone mineral density of the CaCO3 and Native-pro-Ca groups showed some increase compared to the Model group, it was still significantly lower than that of the NC group (P < 0.05). This indicates that these two calcium preparations had limited effect in promoting bone mineral deposition and failed to restore bone mineral density to normal levels. In contrast, the bone mineral density of the pH-Ca group was not only significantly higher than that of the Model, CaCO3, and Native-pro-Ca groups, but also close to that of the NC group. This result clearly demonstrates that the pH-shifted calcium preparation (pH-Ca) can more effectively promote bone mineral deposition by optimizing calcium absorption and utilization efficiency, significantly increasing bone mineral density, thereby enhancing bone strength and improving bone health.

[0133] Ct.Th and Ct.ar ( Figure 6 H and I are parameters of cortical bone. The NC group showed significantly better cortical bone thickness and area, while the Model group showed a significant decrease (P>0.05), indicating cortical bone morphological degeneration. Although the CaCO3 and Native-pro-Ca groups showed improvement, they were still inferior to the NC group. The pH-Ca group was significantly better than the Model group and the first two calcium preparation groups, and was more conducive to cortical bone morphological repair.

[0134] In summary, this application successfully prepared Chlorella protein peptide-calcium chelate using pH shift. The preparation process is simple, does not involve hazardous reagents, and does not consume a large amount of energy. Furthermore, the prepared product has good bioavailability, providing solid theoretical support for the development and application of Chlorella protein peptide-calcium chelate as a novel bone health promoter.

[0135] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0136] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a protein peptide-calcium chelate of Chlorella pyrenoidosa protein, characterized by, The preparation method comprises the following steps: S1, taking the Chlorella pyrenoidosa protein to be dissolved in deionized water, adjusting the pH to 13, stirring for 4h, centrifuging, taking the supernatant, adjusting the pH to 7, and obtaining a Chlorella pyrenoidosa protein treatment liquid; S2, adding calcium chloride to the Chlorella pyrenoidosa protein treatment liquid, chelating in a 37 DEG C water bath for 2h, centrifuging, adding anhydrous ethanol to the supernatant, standing, centrifuging again, collecting the precipitate, and freeze-drying, thereby obtaining the Chlorella pyrenoidosa protein peptide-calcium chelate; The stirring speed in step S1 is 300 rpm; The centrifugation condition is 10000 rpm for 10 min; The mass ratio of Chlorella pyrenoidosa protein to calcium chloride in step S2 is 10:1; The centrifugation condition is 10000 rpm for 10 min; The volume ratio of supernatant to anhydrous ethanol is 9:1; The standing time is 1.5h.

2. The Chlorella pyrenoidosa protein peptide-calcium chelate prepared by the preparation method in claim 1.

Images