A method for preparing a peptide calcium chelate and a method for confirming osteogenic physiological activity thereof

Abstract

This invention discloses a method for preparing peptide-calcium chelates and a method for confirming their osteogenic physiological activity. The prepared peptide-calcium chelates exhibit good thermal stability and osteoblast proliferation-promoting activity. The combined use of ultrasound and microwave treatment significantly enhances the chelation effect between peptides and calcium. The method for confirming the osteogenic physiological activity of peptide-calcium chelates includes steps such as preparing samples of the peptide-calcium chelates to be confirmed, rat animal experiments, sample preparation, comparison of bone turnover markers, comparison of bone microstructure, and histological comparison. This method has a good ability to evaluate the anti-osteoporosis activity of peptide-calcium chelates and can provide a theoretical basis for guiding the future development of bioactive functional foods using collagen peptide-calcium chelates.

Description

Technical fields:

[0001] This invention relates to the field of biomaterials technology, specifically to a method for preparing a peptide-calcium chelate and a method for confirming its osteogenic physiological activity. Background technology:

[0002] Currently, my country's livestock and poultry meat processing industry produces approximately 17 million tons of livestock and poultry bone by-products annually, but the overall processing and utilization rate is less than 10% of the total output. Collagen accounts for over 90% of the organic matrix in livestock and poultry bones, making it an important source of food-grade natural collagen with significant development and utilization value. Natural collagen has a triple helix structure that is poorly soluble in water, making it difficult for the human body to digest, absorb, and utilize. However, some collagen hydrolysates obtained after enzymatic hydrolysis possess unique biological activities, attracting the attention of researchers. Collagen peptides exhibit various physiological activities, including enhancing immunity, inhibiting ACE, improving arthritis, and improving calcium chelation capacity.

[0003] The strong calcium chelating ability of collagen peptides is a current research hotspot. Compared with other calcium supplements, collagen peptide calcium chelates can promote the body's calcium absorption and improve bioavailability, without affecting the gastrointestinal tract. Currently, the study of the osteogenic activity of collagen peptide calcium chelates mainly focuses on cell experiments, lacking comprehensive evaluation studies based on animal experiments, which restricts the confirmation of the osteogenic physiological activity of bone collagen peptide calcium chelates and their development and application. Summary of the Invention:

[0004] The first objective of this invention is to provide a method for preparing a peptide-calcium chelate, which yields a peptide-calcium chelate with good chelation effect.

[0005] A second objective of this invention is to provide a peptide-calcium chelate.

[0006] The third objective of this invention is to provide a method for confirming the osteogenic physiological activity of peptide-calcium chelates, which can be used to evaluate the anti-osteoporosis activity of collagen peptide-calcium chelates.

[0007] The first objective of this invention is achieved by the following technical solution:

[0008] A method for preparing a peptide-calcium chelate includes the following steps: S1, preparing collagen protein; S2, preparing collagen peptides; S3, preparing collagen peptide-calcium chelate, wherein step S3 includes the following steps:

[0009] S31. Preparation of mixed solution: Dissolve the collagen peptides obtained in step S2 in deionized water to form a collagen peptide solution. Add calcium chloride to the collagen peptide solution and stir at 60°C for 60 min to obtain a mixed solution.

[0010] S32. Preparation of peptide-calcium chelate solution: The mixed solution is subjected to microwave and ultrasonic treatment to obtain a peptide-calcium chelate solution. The special effects of ultrasonic technology (cavitation effect, mechanical effect, thermal effect, chemical effect) have advantages such as assisting extraction, accelerating reaction, and changing the molecular properties of proteins, promoting the interaction between peptides and calcium ions. Microwaves are electromagnetic waves that can cause changes in the polarity orientation of water under high-frequency conditions with an external electric field, thereby further inducing molecular motion and mutual friction, which has a certain impact on the internal structure of substances. Both ultrasound and microwaves affect the folding dynamics of peptides, causing changes in the natural folding structure, accelerating the chelation of peptide molecules with calcium ions, thereby increasing the calcium content.

[0011] S33. Preparation of peptide-calcium chelate: After adding ethanol to the peptide-calcium chelate solution, centrifuge, freeze-dry the collected precipitate, and obtain the peptide-calcium chelate.

[0012] Further, in step S31, the ratio of collagen peptides to deionized water is 1:15 to 1:25; the weight ratio of calcium chloride to the collagen peptide solution is 1:4 to 1:5.

[0013] Furthermore, in step S32, the microwave power is 600-800W and the time is 10-20min, while the ultrasonic power is 140-180W and the time is 10-20min.

[0014] Furthermore, in step S33, the freeze-drying temperature is -38°C and the time is 48 hours.

[0015] Further, step S1 includes: crushing, defatting, and decalcifying animal bones to obtain collagen.

[0016] Further, step S2 includes: adding alkaline protease to the collagen protein prepared in step S1 for enzymatic hydrolysis, and obtaining collagen peptides after enzymatic hydrolysis.

[0017] The second objective of this invention is achieved by the following technical solution:

[0018] The peptide calcium chelate prepared by the above-mentioned method.

[0019] The third objective of this invention is achieved by the following technical solution:

[0020] A method for confirming the osteogenic physiological activity of peptide-calcium chelates, comprising the following steps:

[0021] P1. Prepare the peptide-calcium chelate sample to be confirmed: Use the above-mentioned peptide-calcium chelate as the peptide-calcium chelate sample to be confirmed.

[0022] P2. Rat animal experiment: Osteoporotic rats were randomly divided into collagen peptide calcium chelate group, collagen peptide group, osteoporosis group and N (N≥1) commercial calcium supplement groups, and a sham surgery group was set up, for a total of 4+N experimental groups; the specific diet of each experimental group is as follows: (1) Sham surgery group: oral administration of physiological saline; (2) Collagen peptide group: oral administration of collagen peptide; (3) Collagen peptide calcium chelate group: oral administration of the peptide calcium chelate sample to be confirmed in step P1; (4) Osteoporosis group: oral administration of physiological saline; (5) Commercial calcium supplement group: oral administration of commercial calcium supplement; the dietary intervention lasted for 12 weeks;

[0023] P3. Sample preparation: After 12 weeks of dietary intervention, rats in each experimental group were sacrificed, and serum was obtained by centrifuging the collected blood. The right femur, heart, liver, spleen, lung, kidney, and ileum of rats in each experimental group were stored in 4% paraformaldehyde.

[0024] P4. Comparison of bone turnover markers: The levels of bone turnover markers in the serum were measured using an ELISA kit, and the levels of bone turnover markers in each experimental group were compared.

[0025] P5. Comparison of bone microstructure: Micro-CT imaging was performed on the right femur, and the bone morphology parameters were calculated. The right femur images and bone morphology parameters of each experimental group were compared.

[0026] P6. Histological comparison: The heart, liver, spleen, lung, kidney and ileum of rats in each experimental group were sectioned, stained and observed under a microscope. The area of ​​the ileal villi was calculated and the tissue images and the area of ​​the ileal villi of each experimental group were compared.

[0027] Furthermore, the bone turnover markers include alkaline phosphatase, osteocalcin, type I procollagen N-terminal propeptide, tartrate-resistant acid phosphatase, type I collagen C-terminal peptide, and deoxypyridine.

[0028] Furthermore, the skeletal morphology parameters include bone density, bone volume / total volume ratio, cortical width, trabecular thickness, trabecular spacing, and number of trabeculae.

[0029] Beneficial effects:

[0030] This invention provides a method for preparing peptide-calcium chelates. The prepared peptide-calcium chelates have good thermal stability and osteoblast proliferation-promoting activity. The combined use of ultrasound and microwave treatment methods can significantly improve the chelation effect between peptides and calcium.

[0031] This invention provides a method for confirming the osteogenic physiological activity of peptide-calcium chelates. Through the establishment of an osteoporotic rat model, the method utilizes physiological and biochemical indicators such as bone microstructure, histopathology, and serum bone turnover markers to evaluate the anti-osteoporosis activity of peptide-calcium chelates. This method can provide a theoretical basis for guiding the future development of bioactive functional foods using bone collagen peptide-calcium chelates. Attached image description:

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The effect of microwave treatment with different power on the calcium content in the peptide calcium chelate in Example 2;

[0034] Figure 2 The effect of different power ultrasonic treatments on the calcium content in the peptide calcium chelate in Example 3;

[0035] Figure 3 The thermal stability results of the sample prepared by the method of the present invention in Example 4 and the sample prepared by the hydrothermal method are shown.

[0036] Figure 4 The results of promoting osteoblast proliferation of the samples prepared by the method of the present invention and the samples prepared by the hydrothermal method in Example 5;

[0037] Figure 5 The changes in body weight of rats in each experimental group during the dietary intervention period in Example 7;

[0038] Figure 6 For the histopathological comparison of the viscera and ileum of rats in different experimental groups in Example 7: (A) Histopathological comparison of visceral tissues in different groups; (B) Histological comparison of ileal villi in different groups; (C) Relative area of ​​ileal villi in different groups;

[0039] Figure 7 The levels of bone turnover markers in different experimental groups of rats in Example 7 are as follows: (A) serum calcium; (B) serum phosphorus; (C) bone alkaline phosphatase; (D) osteocalcin; (E) type I procollagen N-peptide; (F) serum type I collagen C-terminal peptide; (G) deoxypyridine; (H) tartrate-resistant acid phosphatase;

[0040] Figure 8Comparison of Micro-CT images and skeletal morphology parameters of the right femur of rats in different experimental groups in Example 7: (A) Representative Micro-CT image of the trabecular microstructure of the right femur; (B) Bone mineral density; (C) Bone volume / total volume ratio; (D) Cortical width; (E) Trabecular thickness; (F) Trabecular spacing; (G) Number of trabeculae. Detailed implementation method:

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] In the following examples and comparative examples, all reagents were commercially available.

[0043] Example 1: A method for preparing a peptide-calcium chelate

[0044] A method for preparing a peptide-calcium chelate includes S1 preparing bovine bone collagen, S2 preparing collagen peptides, and S3 preparing a collagen peptide-calcium chelate.

[0045] S1, the preparation of bovine bone collagen includes the following steps S11-S13:

[0046] S11. Degreasing of raw materials: After crushing the beef bones, add 0.1M sodium hydroxide solution and stir at 4℃ for 6 hours, wherein the volume ratio of beef bones to sodium hydroxide solution is 1:4 to 1:5; then pour out the liquid to obtain beef bone residue, wash the beef bone residue with distilled water until pH 6.5 to 7.0, and then drain the water; add the neutral beef bone residue to 10% n-hexane solution and stir at 4℃ for 12 to 16 hours, wherein the material-to-liquid ratio (w / v) of beef bone residue to n-hexane solution is 1:4 to 1:5; then pour out the liquid, wash the beef bone residue with distilled water until neutral, and then drain the water to obtain defatted beef bones;

[0047] S12, Raw material decalcification: Defatted beef bones are added to a 0.25M disodium EDTA solution and stirred at 4°C for 12 hours. The ratio of defatted beef bones to disodium EDTA solution (w / v) is 1:4 to 1:5. The liquid is then poured off, and the defatted beef bone residue is rinsed with distilled water until neutral. The water is then drained to obtain decalcified beef bones. Steps S11 and S12 remove fat and calcium salts from the beef bones and remove non-collagenous components, thereby improving collagen purity.

[0048] S13. Preparation of bovine bone collagen: Decalcified bovine bone was added to 0.5M acetic acid solution and pepsin, and stirred at 4℃ for 20-24h. The mixture was then filtered through double-layer gauze to obtain crude collagen filtrate. The volume ratio of decalcified bovine bone to acetic acid solution was 1:4-1:5, and the mass ratio of decalcified bovine bone to pepsin was 1:0.010-1:0.016. Sodium chloride was added to the filtrate to a final concentration of 25%, and the mixture was stirred to precipitate flocculent precipitate. The precipitate was collected by centrifugation at 10000×g for 20min. The precipitate was freeze-dried at -38℃ for 48h to obtain bovine bone collagen with a purity of over 90%.

[0049] S2. The steps for preparing collagen peptides are as follows: Bovine bone collagen is added to alkaline protease for enzymatic hydrolysis at a temperature of 50℃ for 5 hours. The hydrolyzed solution is then freeze-dried at -38℃ for 48 hours to obtain powdered collagen peptides. Analysis showed that approximately 90% of the obtained collagen peptides had a relative molecular weight of less than 1000 Daltons.

[0050] S3. The step of preparing collagen peptide calcium chelate includes the following steps S31-S33:

[0051] S31. Preparation of mixed solution: Dissolve collagen peptides in deionized water (the ratio of collagen peptides to deionized water is 1:15 to 1:25) to form a collagen peptide solution, then add calcium chloride (the weight ratio of calcium chloride to collagen peptide solution is 1:4 to 1:5), stir at 60°C for 60 min to obtain a mixed solution.

[0052] S32. Preparation of peptide-calcium chelate solution: The mixed solution is subjected to microwave treatment and ultrasonic treatment at room temperature to obtain peptide-calcium chelate solution, wherein the microwave power is 600W and the duration is 10-20min, and the ultrasonic power is 140W and the duration is 10-20min.

[0053] S33. Preparation of peptide-calcium chelate: After adding ethanol to the peptide-calcium chelate solution, centrifuge at 8000×g for 15min, and freeze-dry the collected precipitate at -38℃ for 48 hours to obtain the peptide-calcium chelate; the volume ratio of peptide-calcium chelate solution to ethanol is 1:8.

[0054] The calcium content of the peptide-calcium chelate prepared by the above method was 76.31 mg / g, indicating that the combined ultrasonic and microwave treatment promoted the chelation of collagen peptides and calcium ions.

[0055] Example 2: A method for preparing a peptide-calcium chelate

[0056] A method for preparing a peptide-calcium chelate is the same as that in Example 1, except that the microwave power is 0-800W and the ultrasonic power is 120W.

[0057] like Figure 1 As shown, under ultrasonic treatment at 120W, within the microwave power range of 0–800W, the calcium content initially increased and then stabilized with increasing microwave power. When the microwave power was 600W, the calcium content reached 68.40 mg / g, which was 28.0% higher than the 53.43 mg / g without microwave treatment.

[0058] Example 3: A method for preparing a peptide-calcium chelate

[0059] A method for preparing a peptide-calcium chelate is the same as that in Example 1, except that the microwave power is 400W and the ultrasonic power is 100-180W.

[0060] like Figure 2 As shown, under microwave treatment at 400W, within the ultrasonic power range of 100–180W, the calcium content initially increased and then stabilized with increasing ultrasonic power. When the ultrasonic power was between 140–180W, the peptide calcium chelation rate reached a stable level with no significant difference. At an ultrasonic power of 140W, the calcium content was 71.80 mg / g, which was 23.7% higher than the 58.03 mg / g obtained with ultrasonic treatment at 100W.

[0061] Example 4: Thermal stability test of peptide-calcium chelate

[0062] Experimental group ①: CP-Ca (hydrothermal). The preparation method of the sample in this experimental group is the same as that in Example 1. The difference is that after the mixed solution is prepared in step S31, it is not passed through S32. The mixed solution is directly used as the peptide-calcium chelation solution to prepare peptide-calcium chelates. Experimental group ②: CP-Ca (microwave-ultrasound). The preparation method of the sample in this experimental group is the same as that in Example 1.

[0063] The sample from the experimental group was dissolved in ultrapure water (10 mg / mL), heated to 50–80 °C and incubated for 1 h. After incubation, ethanol was added, and the mixture was centrifuged at 8000 × g for 15 min to obtain the peptide-calcium chelate. A control group, Contrl, was set up, which was an aqueous solution of the sample (25 °C). The calcium content in the chelate was determined by flame atomic absorption spectrometry according to GB5009.92—2016, and the calcium retention rate was calculated according to formula (1).

[0064] Calcium retention rate = Calcium content of experimental group / Calcium content of control group × 100% Equation (1)

[0065] The results are as follows Figure 3As shown, the peptide-calcium chelate of experimental group ② showed no significant difference in temperature between 50 and 80℃, exhibiting high thermal stability. At 80℃, the calcium retention rate of the peptide-calcium chelate of experimental group ② was 96%, significantly higher than that of the peptide-calcium chelate prepared in experimental group ①. This indicates that the combined microwave and ultrasound treatment method can effectively promote the calcium chelation effect of collagen peptides.

[0066] Example 5: Cell proliferation rate test of peptide-calcium chelate

[0067] MC3T3-E1 cells were cultured in α-MEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator. Cells were seeded in 96-well plates and incubated for 24 hours. Then, MC3T3-E1 cells were incubated for 72 hours with the sample from experimental group ① (concentration 50 μg / mL) and different concentrations of sample from experimental group ② (50 μg / mL, 75 μg / mL, 100 μg / mL, 200 μg / mL, and 300 μg / mL). Cells treated with culture medium (without sample) served as a control group (concentration 50 μg / mL). Cell proliferation was detected using the MTT assay kit according to the manufacturer's instructions.

[0068] The results are as follows Figure 4 As shown, the peptide-calcium chelate prepared in experimental group ② exhibited higher osteoblast proliferation activity, with a significantly increased osteoblast proliferation rate of 8% compared to the 112% cell proliferation rate of the peptide-calcium chelate prepared by the hydrothermal method in experimental group ①. When the concentration of the peptide-calcium chelate sample prepared in experimental group ② was 100 mg / mL for cell incubation, its cell proliferation rate reached 135%. The results indicate that the combined microwave and ultrasound treatment method can improve the osteoblast proliferation activity of the peptide-calcium chelate.

[0069] Example 6: Method for confirming the osteogenic physiological activity of peptide-calcium chelates

[0070] The osteogenic physiological activity of the peptide calcium chelate was confirmed by the following steps P1-P6.

[0071] P1. Prepare the peptide-calcium chelate sample to be confirmed: Use the peptide-calcium chelate obtained in Example 1 as the peptide-calcium chelate sample to be confirmed.

[0072] P2. Rat animal experiments:

[0073] Eight-month-old female SD rats (weighing 392.02±34.01 g) were housed in a suitable environment with a temperature of 25±2℃ and a relative humidity of 55±5%. After one week of acclimatization, a rat model (osteoporotic rats) was established by ovariectomy. Two weeks after the operation, the osteoporotic rats were randomly divided into four groups, and a sham-operated group was set up, for a total of five experimental groups (n=6 in each group). The specific diets of each group were as follows: (1) Sham-operated group: oral administration of physiological saline (1 mL / 100 g / day); (2) Collagen peptide group: oral administration of collagen peptide 525 mg / day. kg / day (collagen peptide content is the same as CP-Ca group); (3) Collagen peptide calcium chelate group (hereinafter referred to as CP-Ca group): peptide calcium chelate prepared by gavage step P1 (500mg / kg / day); (4) Calcium lactate group: calcium lactate 41.9mg / kg / day by gavage (calcium content is the same as CP-Ca group); (5) Osteoporosis group: physiological saline by gavage (1mL / 100g / day); All rats can freely ingest AIN-93 pellet feed (calcium content 1.0~1.8%) and deionized water, and the dietary intervention lasts for 12 weeks.

[0074] P3. Sample Preparation:

[0075] After 12 weeks of dietary intervention, rats in each group were sacrificed, and the collected blood was centrifuged at 2000×g for 15 min to obtain serum. The right femur and internal organs (heart, liver, spleen, lung, kidney, and ileum) of the rats were stored in 4% paraformaldehyde.

[0076] P4, Histological Comparison:

[0077] After storing the ileum in 4% paraformaldehyde for 24 hours, it was embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) and observed under a microscope (Olympus, Tokyo, Japan). The area of ​​the ileal villi was calculated using ImageJ software (RawakSoftware Inc., Stuttgart, Germany). The intervillous spacing and area of ​​the ileal villi were compared among the groups.

[0078] P5, Comparison of bone turnover markers:

[0079] The levels of bone turnover markers in serum were measured using an ELISA kit, including alkaline phosphatase (BAP), osteocalcin (OCN), type I procollagen N-terminal propeptide (PINP), tartrate-resistant acid phosphatase (TRAP), type I collagen C-terminal peptide (S-CTX), and deoxypyridine (DPD). The levels of bone turnover markers in each group were compared.

[0080] P6. Comparison of bone microstructure:

[0081] Micro-CT (micro-computed tomography) imaging was performed using an Inveon Micro-PET / CT scanner (Siemens, Germany); X-ray images were reconstructed using CTAN software (version 1.17.7.2) and CTvox software (version 3.30). Skeletal morphological parameters were calculated using existing techniques, including bone mineral density (BMD), bone volume to total volume ratio (BV / TV), cortical width (Cw.T), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N). Images and skeletal morphological parameters were compared among the groups.

[0082] The order of P4, P5, and P6 can be interchanged. This method can also be used to confirm the osteogenic physiological activity of peptide-calcium chelates prepared by other methods.

[0083] Example 7: Confirmation Results of Osteogenic Physiological Activity of Peptide-Calcium Chelate

[0084] The peptide-calcium chelate prepared in Example 1 was confirmed according to the confirmation method of Example 6, and the results are as follows.

[0085] like Figure 5 As shown, the body weight of rats in each group gradually increased with the increase in the number of weeks of dietary intervention; among them, the osteoporosis group had the highest body weight, while the sham-operated group had the lowest body weight. In the osteoporosis group, the weight gain was mainly due to the decrease in estrogen levels in osteoporotic rats. Compared with the osteoporosis group, the body weight of rats in the collagen peptide group and the CP-Ca group decreased from week 3 to week 11, mainly because collagen peptides can inhibit the weight gain caused by the decrease in estrogen.

[0086] like Figure 6 As shown in Figure A, histopathological examination of the heart, liver, spleen, lungs, and kidneys of rats in the sham-operated group, CP-Ca group, and osteoporosis group showed no significant differences among the groups. This indicates that ovariectomy did not cause tissue damage, and that the peptide-calcium chelate prepared in Example 1 is a healthy and reliable calcium supplement.

[0087] To assess the effects of peptide-calcium chelates on the small intestine, the histological structure of the ileal villi was observed using H&E staining. Figure 6 As shown in Figure B, the distance between adjacent ileal villi in the osteoporosis group was relatively larger than that in the sham-operated group. The villi spacing in the calcium lactate group was also large, while the ileal villi in the collagen peptide and CP-Ca groups showed a compact structure. This is likely because calcium lactate caused some damage to the regularity and fullness of the ileal villi, a drawback of traditional inorganic calcium supplements. Data processing of histological images was performed to quantify the relative villi area, such as... Figure 6As shown in Figure C, the relative villous area of ​​the collagen peptide group and the CP-Ca group was significantly larger than that of the calcium lactate group and the osteoporosis group. These results indicate that the peptide-calcium chelate prepared in Example 1 is a calcium supplement that can protect the small intestinal mucosa and has no side effects.

[0088] Bone turnover markers are important indicators for assessing bone formation and bone resorption. Serum calcium, serum phosphorus, alkaline phosphatase (BAP), osteocalcin (OCN), and type I procollagen N-peptide (PINP) are markers of bone formation, while serum type I collagen C-terminal peptide (S-CTX), deoxypyridine diphosphate (DPD), and tartrate-resistant acid phosphatase (TRAP) are markers of bone resorption. Figure 7 As shown in Figure A, the serum calcium level in the osteoporosis group was significantly lower than that in the sham surgery group, collagen peptide group, and CP-Ca group. The serum calcium level in the CP-Ca group was higher than that in the osteoporosis group and the calcium lactate group. Figure 7 As shown in Figure B, the serum phosphorus levels were higher in the sham surgery group and the collagen peptide group, followed by the osteoporosis group, the CP-Ca group, and the calcium lactate group. Meanwhile, as... Figure 7 As shown in the CH diagram, the levels of BAP, OCN, and PINP in the osteoporosis group were significantly lower than those in the sham-operated group, while the levels of S-CTX, DPD, and TRAP were significantly increased. Compared with osteoporotic rats, the CP-Ca group showed increased serum levels of bone formation markers BAP, OCN, and PINP, and decreased levels of bone resorption markers S-CTX, DPD, and TRAP. These results indicate that dietary interventions in the collagen peptide group, CP-Ca group, and calcium lactate group alleviated osteoporosis symptoms, with the CP-Ca group showing the most significant effect, effectively promoting bone formation and inhibiting bone resorption.

[0089] like Figure 8 As shown in Figure A, compared with the sham surgery group, the osteoporosis group had sparser bone trabeculae with larger gaps. After intervention with collagen peptides, calcium lactate, and CP-Ca, bone structure was significantly improved to varying degrees. In particular, after dietary intervention in the CP-Ca group, bone microstructure and distal trabecular connectivity were significantly improved, approaching the level of the sham surgery group and superior to the collagen peptide and calcium lactate groups. Further quantitative analysis of bone microstructure included bone mineral density (BMD), bone volume / total volume ratio (BV / TV), cortical width (Cw.T), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). Among these, bone mineral density is an important indicator for diagnosing osteoporosis. Figure 8 As shown in BG, compared with the osteoporosis group, the BMD, BV / TV and Tb.N parameters of the CP-Ca group were significantly increased (P<0.05), indicating strong anti-osteoporosis activity.

[0090] In summary, this invention has confirmed the osteogenic activity of collagen peptide calcium chelates in animals through animal experiments, thereby clarifying their anti-osteoporosis activity. The method of this invention has a good ability to evaluate the anti-osteoporosis activity of collagen peptide calcium chelates and can provide a theoretical basis for guiding the future development of bioactive functional foods using collagen peptide calcium chelates.

[0091] 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 a peptide-calcium chelate, comprising the steps of: S1, preparing collagen protein; S2, preparing collagen peptides, characterized in that, It also includes step S3, preparing collagen peptide calcium chelates, wherein step S3 includes the following steps: S31. Preparation of mixed solution: Dissolve the collagen peptides obtained in step S2 in deionized water to form a collagen peptide solution. Add calcium chloride to the collagen peptide solution and stir at 60°C for 60 min to obtain a mixed solution. S32. Preparation of peptide-calcium chelate solution: The mixed solution is subjected to microwave treatment and ultrasonic treatment to obtain peptide-calcium chelate solution; S33. Preparation of peptide-calcium chelate: After adding ethanol to the peptide-calcium chelate solution, centrifuge, freeze-dry the collected precipitate, and obtain the peptide-calcium chelate.

2. The method for preparing a peptide-calcium chelate according to claim 1, characterized in that, In step S31, the ratio of collagen peptides to deionized water is 1:15 to 1:25; the weight ratio of calcium chloride to collagen peptide solution is 1:4 to 1:

5.

3. The method for preparing a peptide-calcium chelate according to claim 1, characterized in that, In step S32, the microwave power is 600-800W and the time is 10-20min, while the ultrasonic power is 140-180W and the time is 10-20min.

4. The method for preparing a peptide-calcium chelate according to claim 1, characterized in that, In step S33, the freeze-drying temperature is -38℃ and the time is 48h.

5. The method for preparing a peptide-calcium chelate according to claim 1, characterized in that, Step S1 includes: crushing, defatting, and decalcifying animal bones to obtain collagen.

6. The method for preparing a peptide-calcium chelate according to claim 5, characterized in that, Step S2 includes: adding alkaline protease to the collagen protein prepared in step S1 for enzymatic hydrolysis, and obtaining collagen peptides after enzymatic hydrolysis.

7. The peptide calcium chelate prepared by the method for preparing a peptide calcium chelate according to claim 1.

8. The method for confirming the osteogenic physiological activity of the peptide-calcium chelate according to claim 7, characterized in that, It includes the following steps: P1. Prepare the peptide-calcium chelate sample to be confirmed: Use the peptide-calcium chelate of claim 7 as the peptide-calcium chelate sample to be confirmed. P2. Rat animal experiment: Osteoporotic rats were randomly divided into collagen peptide calcium chelate group, collagen peptide group, osteoporosis group and N commercially available calcium supplement groups, and a sham surgery group was set up, for a total of 4+N experimental groups; the specific diet of each experimental group is as follows: (1) Sham surgery group: oral administration of physiological saline; (2) Collagen peptide group: oral administration of collagen peptide; (3) Collagen peptide calcium chelate group: oral administration of the peptide calcium chelate sample to be confirmed in step P1; (4) Osteoporosis group: oral administration of physiological saline; (5) Commercially available calcium supplement group: oral administration of commercially available calcium supplement; the dietary intervention lasted for 12 weeks; P3. Sample preparation: After 12 weeks of dietary intervention, rats in each experimental group were sacrificed, and serum was obtained by centrifuging the collected blood. The right femur, heart, liver, spleen, lung, kidney, and ileum of rats in each experimental group were stored in 4% paraformaldehyde. P4. Comparison of bone turnover markers: The levels of bone turnover markers in the serum were measured using an ELISA kit, and the levels of bone turnover markers in each experimental group were compared. P5. Comparison of bone microstructure: Micro-CT imaging was performed on the right femur, and the bone morphology parameters were calculated. The right femur images and bone morphology parameters of each experimental group were compared. P6. Histological comparison: The heart, liver, spleen, lung, kidney and ileum of rats in each experimental group were sectioned, stained and observed under a microscope. The area of ​​the ileal villi was calculated and the tissue images and the area of ​​the ileal villi of each experimental group were compared.

9. The method for confirming the osteogenic physiological activity of the peptide-calcium chelate according to claim 8, characterized in that, The bone turnover markers include alkaline phosphatase, osteocalcin, type I procollagen N-terminal propeptide, tartrate-resistant acid phosphatase, type I collagen C-terminal peptide, and deoxypyridine.

10. The method for confirming the osteogenic physiological activity of the peptide-calcium chelate according to claim 8, characterized in that, The skeletal morphology parameters include bone density, bone volume / total volume ratio, cortical width, trabecular thickness, trabecular spacing, and number of trabeculae.

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