A bone health pressed tablet candy enriched with a marine peptide calcium chelate and a method of making the same

Abstract

The application discloses a bone health tablet candy rich in marine peptide calcium chelate and a preparation method thereof, and belongs to the technical field of polypeptides, wherein the calcium chelate peptide provided by the application has an amino acid sequence as shown in SEQ ID No.1, and the specific sequence is GETGPA (F21-1), and the calcium binding capacity of the calcium chelate peptide is 4.72±1.06 μg / mg. The bone health tablet candy provided by the application has good calcium absorption promoting activity. The results of a mouse experiment show that the bone health tablet candy does not cause adverse effects on the growth of mice, can reverse high bone conversion caused by osteoporosis, improves the bone biomechanical properties of osteoporotic mice, and thus achieves the effect of enhancing bone density.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, specifically relating to a bone health compressed candy rich in marine peptide calcium chelates and its preparation method. Background Technology

[0002] Osteoporosis is a metabolic bone disease primarily caused by a disruption of the metabolic balance between bone formation and bone resorption, leading to bone loss. To inhibit bone loss caused by osteoporosis and reduce fracture risk, drugs such as rhanoxifene, alendronate, risedronate, and conjugated estrogen are used to treat postmenopausal osteoporosis. However, drug treatment is limited by factors such as high cost, side effects, and drug resistance. Therefore, developing corresponding bone health products has become a new option. Calcium supplements can help restore the balance between bone resorption and bone formation; therefore, calcium supplementation is considered a safe method for treating osteoporosis. Because calcium chelate peptides can enhance calcium absorption... 2+ Bioavailability provides the possibility for it to exert anti-osteoporosis activity. Studies have found that cod bone calcium chelate peptides and their peptide-calcium chelates have good in vitro and in vivo calcium absorption-promoting activity, and can improve the bone metabolism imbalance in ovarian-induced osteoporosis mice, thus exerting a good anti-osteoporosis effect.

[0003] Chinese patent CN118374565A discloses a method for preparing bovine collagen peptides that increase bone density. A collagen peptide is prepared by enzymatic hydrolysis with bromelain. This collagen peptide can effectively restore serum calcium content and alkaline phosphatase content in osteoporotic rats, thereby increasing bone density.

[0004] Chinese patent CN118415341A discloses a composition for increasing bone density and its preparation method. The composition prepared by combining active ingredients such as hydrolyzed collagen, calcium lactate, D-glucosamine sulfate potassium salt, and casein phosphopeptide can effectively increase the bone density of the lumbar spine in calcium-deficient rats.

[0005] Chinese patent CN118104833A discloses a processing method for a functional food product called Joint Health Nutritional Complex Tablets, which increases bone density. Sea cucumber collagen peptides are used as the main active ingredient, and are combined with high-quality glucosamine substances, minerals and vitamins to form a nutritional tablet that can effectively increase bone density. Summary of the Invention

[0006] The purpose of this invention is to provide a calcium chelating peptide and a peptide-calcium chelate extracted from marine aquatic products, and a calcium supplement preparation made from the said peptides, specifically a bone health compressed candy (IBDF). The IBDF provided by this invention has the functions of promoting calcium absorption and increasing bone density.

[0007] The calcium chelating peptide provided by this invention has the amino acid sequence shown in SEQ ID No. 1, specifically GETGPA(F21-1), and its calcium binding capacity is 4.72±1.06 μg / mg. It can be prepared according to the method disclosed in patent document CN116806917A, specifically by preparing cod bone hydrolysate, and then separating the hydrolysate through steps such as hydroxyapatite affinity chromatography, Sephadex G25 gel filtration chromatography, and C18 reversed-phase high-performance liquid chromatography to obtain the calcium chelating peptide GETGPA(F21-1). Alternatively, it can be prepared through artificial chemical synthesis, such as by a chemical solid-phase synthesis method.

[0008] The calcium chelating peptide provided by this invention has a very strong calcium binding capacity and can be used to prepare peptide-calcium chelates.

[0009] Furthermore, the present invention provides a method for preparing the above-mentioned peptide-calcium chelate as follows: the concentration of calcium chelating peptide is 1.3-1.7 g / L, and Ca... 2+ Chelation was performed at room temperature for 28-32 min at a concentration of 0.4-0.6 mM and pH = 6.8-7.2.

[0010] This invention also provides the application of the above-mentioned calcium chelating peptide or peptide-calcium chelate in the preparation of health food products. Specifically, the health food product is a calcium supplement.

[0011] Furthermore, the present invention provides a calcium supplement, specifically a bone health compressed candy (IBDF), comprising the above-mentioned calcium chelating peptide and peptide-calcium chelate.

[0012] The IBDF provided by this invention follows the concept of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine theory, with calcium chelate peptides (PBPs) and peptide-calcium chelates (PBPs-Ca) as the main active ingredients; calcium carbonate, vitamin D3, glucosamine, and chondroitin sulfate as auxiliary ingredients; and β-cyclodextrin, microcrystalline cellulose, magnesium stearate, and 4-6% sucralose as excipients to improve the taste, color, and stability of the compressed candy.

[0013] Its specific formula (by weight percentage) is as follows: PBPs 18-22%, PBPs-Ca 8-12%, calcium carbonate 13-17%, vitamin D3 5500-6500 IU, glucosamine 15-17%, chondroitin sulfate 15-17%, β-cyclodextrin 5-7%, microcrystalline cellulose 8-12%, magnesium stearate 1-3%, and sucralose 4-6%.

[0014] The IBDF provided by this invention is milky white in color, uniform in color, smooth in surface, has a light milky aroma, no fishy smell, no off-odor, no odor, has excellent sensory quality, delicate taste, long aftertaste, and is in the form of small round pieces with a relatively smooth surface and no obvious particles.

[0015] The beneficial effects of this invention are:

[0016] The IBDF provided by this invention has good calcium absorption-promoting activity. Mouse experiments showed that it did not adversely affect mouse growth, and could reverse the high bone turnover caused by osteoporosis, improve bone biomechanical properties in osteoporotic mice, thereby increasing bone density. Attached Figure Description

[0017] Figure 1 The images show the mass spectrometry identification results of F21-1, where (a) is the extracted ion chromatogram, (b) is the first-order mass spectrum, (c) is the second-order mass spectrum, and (d) is the structural diagram.

[0018] Figure 2 This is a picture of an IBDF.

[0019] Figure 3 The effect of IBDF intervention on calcium bioavailability in osteoporotic mice is shown in the graphs: (a) shows the calcium absorption rate of mice and (b) shows the calcium retention rate of mice.

[0020] Figure 4 To show the effect of IBDF intervention on the bone biomechanical properties of osteoporotic mice, (a) maximum load on the mouse tibia, and (b) structural strength diagram of the mouse tibia.

[0021] Figure 5 To illustrate the effects of IBDF intervention on bone metabolism-related indicators in osteoporotic mice, (a) shows the serum ALP content of mice, (b) shows the serum OCN content of mice, (c) shows the serum Cath-K content of mice, (d) shows the serum CTX-1 content of mice, and (e) shows the serum TRACP-5b content of mice. Detailed Implementation

[0022] This invention extracts and identifies a novel marine calcium chelating peptide, GETGPA, using hydroxyapatite affinity chromatography, Sephadex G25 gel filtration chromatography, and C18 reversed-phase high-performance liquid chromatography combined with Q-Orbitrap. The specific amino acid sequence of the peptide is GETGPA, and its calcium chelating activity reaches 4.72±1.06 μg / mg.

[0023] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] In the following embodiments, the methods for determining calcium chelation activity are as follows:

[0025] Dissolve 3 mg of sample in 2 mL of 5 mmol / L CaCl2 and incubate at 37 °C for 20 min. Add 4 mL of 20 mmol / L sodium phosphate buffer (pH 7.5) to the reaction system and incubate at 37 °C for another 30 min. Centrifuge at 8000 rpm for 20 min to remove calcium phosphate precipitate. Filter the supernatant through a 0.22 μm filter, take 2 mL, add 10 mL of nitric acid for digestion, and determine the calcium content according to GB 5009.92-2016 (atomic absorption spectrometry). A blank control group without peptides was included. The formula for calculating calcium chelation activity is as follows:

[0026]

[0027] Wherein, Cs is the calcium content in the supernatant of each sample group, in μg; Cc is the calcium content in the supernatant of the control group, in μg; and Ms is the sample mass, in mg.

[0028] In the following embodiments, the methods for establishing osteoporosis mouse models are as follows:

[0029] (1) Fast for 12 hours the day before surgery, and administer an intraperitoneal injection of anesthetic at a dose of 1% of body weight (3.5%, m / v).

[0030] (2) Fix the anesthetized mouse on the surgical board, use a hair removal machine to remove the mouse hair on the abdomen, and use a blade to remove all the downy hair to expose the smooth skin.

[0031] (3) After cleaning the surgical site with iodine, cut open the skin and muscle layers of the mouse's abdomen with a scalpel.

[0032] (4) After exposing the adipose tissue, use tweezers to hold the fat and gently move it to the side. Slowly find the mouse uterus, which is shaped like a "Y", along the fat and find the ovary at the end of the fallopian tube. Avoid the capillaries on the fallopian tube wall and cut off the two ovaries with scissors. In the sham surgery group, only a small amount of fat next to the ovary is cut off.

[0033] (5) The mouse's internal organs were restored to their original positions. 50,000 units of penicillin were dripped into the abdominal cavity. The mouse's muscle layer was sutured, and then the outer skin was sutured. After suturing, 50,000 units of penicillin were dripped into the wound to prevent infection.

[0034] (6) After surgery, each mouse is placed in a cage and its condition is observed at all times. Once the mouse wakes up, it can be given an appropriate amount of water.

[0035] (7) Mice were injected with 50,000 units of penicillin daily for 3 days after surgery. Mice with open wounds needed to be sutured again. The formal experiment could be carried out after one week when the wounds had healed. The experimental groups and gavage dosages are shown in Table 1.

[0036] Table 1. Animal experimental groupings and gavage dosages

[0037] Group serial number Mouse number Gavage sample Sham surgery group Sham n=10 physiological saline Model group Model n=10 physiological saline Positive control group Aln n=10 1 mg / kg body weight / day alendronate sodium by gavage Calcium carbonate control group <![CDATA[CaCO3]]> n=10 <![CDATA[Ca 2+ The dosage is 104 mg / kg body weight / day. Cod bone peptide calcium chelate group PBPs-Ca n=10 <![CDATA[Gavage with PBPs-Ca (Ca 2+ dose is 104 mg / kg body weight / d)]]> Bone health compressed candy rich in marine peptide calcium chelates IBDF n=10 <![CDATA[Gavage with bone health chewable candies rich in marine peptide calcium chelate (Ca 2+ dose is 104 mg / kg body weight / d)]]>

[0038] In the following embodiments, the methods for determining calcium bioavailability are as follows:

[0039] After 12 weeks of continuous gavage, mice were placed in metabolic cages for calcium metabolism experiments. The food intake of mice in each cage was recorded for 12 hours, and urine and feces were collected and weighed. The calcium content in feed, urine and feces was measured, and calcium metabolism-related indicators were calculated according to Table 2.

[0040] Table 2 Calculation formulas for calcium metabolism-related indicators

[0041] Relevant indicators formula Calcium intake Calcium intake (mg / d) = Calcium in feed + Calcium administered orally Calcium absorption Calcium absorption (mg / d) = Ingested calcium - Fecal calcium Calcium absorption rate Calcium absorption rate (%) = (Absorbed calcium / Ingested calcium) × 100% Calcium retention Calcium retention (mg / d) = Calcium intake - Fecal calcium - Urinary calcium Calcium retention rate Calcium retention rate (%) = (Retained calcium / Ingested calcium) × 100%

[0042] In the following embodiments, the methods for measuring bone biomechanical properties are as follows:

[0043] The left and right tibias were preserved in 10% formaldehyde fixative for the determination of maximum load and structural strength. The same part of the tibia was selected as the stress point. For specific operating procedures, please refer to the instruction manual of YLS-16A Small Animal Bone Strength Tester.

[0044] In the following embodiments, the methods for measuring bone metabolism-related biochemical indicators are as follows:

[0045] After 12 weeks of gavage, each mouse was fasted for 12 hours but allowed free access to water. Before collection, all mice were weighed, and blood was collected by enucleation. Following blood collection, the mice were euthanized by cervical dislocation. Serum was centrifuged at 4 °C, 8000 r / min for 40 min, and immediately aliquoted into centrifuge tubes and labeled. The levels of alkaline phosphatase (ALP), osteocalcin (OCN), cathepsin K (Cath-K), type I collagen C-terminal peptide (CTX-1), and tartrate-resistant acid phosphatase isoform 5b (TRAP-5b) in mouse serum were determined according to the instructions of the respective ELISA kits.

[0046] Example 1: Preparation of a novel marine calcium chelating peptide

[0047] According to the inventor's previously disclosed patent CN116806917A, a fish bone gelatin calcium chelate peptide and its preparation method, the specific steps are as follows: fish bones are softened at 121 °C for 50 min, then wet-milled using an ultra-micro grinder, and distilled water is added at a ratio of 1:4 (w / v). The mixture is fully extracted at 80 °C for 4 h to obtain cod bone gelatin. The concentration of cod bone gelatin is 100 g / L. Papain with a substrate protein content of 1% is added, and the mixture is hydrolyzed at pH=6.5 and 55 °C for 4 h. The enzyme is inactivated by boiling for 15 min. After centrifugation at 8000 rpm / min for 15 min, the supernatant is collected, concentrated, and freeze-dried to obtain the enzymatic hydrolysate.

[0048] Example 2: Identification of marine calcium chelate peptide sequences

[0049] The amino acid sequence of marine calcium chelate peptides was identified using UHPLC-Q-Orbitrap. The column used was an Agilent Advance Bio Peptide Map C18 column. UHPLC parameters: Mobile phase A: 0.1% formic acid-water; Mobile phase B: 0.1% formic acid-acetonitrile; Flow rate: 0.15 mL / min; Injection volume: 10 μL; Column temperature: 40 °C; Gradient elution program: 0–20 min, 2%–25% B; 20–30 min, 25–45% B; 30–35 min, 45%–85% B; 35–40 min, 85% B; 40–42 min, 85–2% B; 42–52 min, 2% B; Scan range: 100–1200 m / z; Electrospray ionization mode: positive ion electrospray; Electrospray voltage: 3600 V. The identification results of the peptides in the liquid phase fraction are as follows: Figure 1 As shown, the polypeptide sequence was identified as GETGPA.

[0050] Example 3 Synthesis of marine calcium chelate peptide GETGPA

[0051] The identified marine calcium chelating peptide GETGPA was produced using a solid-phase peptide synthesis method. The peptide underwent desalting treatment and achieved a purity >95%. The calcium binding capacity of the cod bone peptide GETGPA prepared by the solid-phase synthesis method was verified, and its calcium chelation amount was 4.72 ± 1.06 mg / g.

[0052] Example 4: Preparation of marine peptide calcium chelates

[0053] When the CaCl2 concentration was adjusted to 0.5 mM, the marine calcium chelate peptide concentration was adjusted to 1.5 g / L, and the solution pH was adjusted to 7.0, the chelate was chelated at room temperature for 0.5 h, precipitated with 8 times the volume of ethanol, and then freeze-dried to obtain the marine peptide calcium chelate.

[0054] Example 5: Effect of IBDF intervention on calcium bioavailability in osteoporotic mice

[0055] The results of calcium bioavailability of each sample in mice are as follows: Figure 3 As shown, the calcium absorption rate and calcium retention rate in the Model group were 35.05% and 32.80%, respectively, significantly lower than those in the Sham group (p<0.05). The calcium absorption rate and calcium retention rate in the Aln and CaCO3 groups showed no significant changes compared to the Model group (p>0.05). The calcium absorption rate and calcium retention rate in the PBPs-Ca group increased by 59.40% and 50.47%, respectively, compared to the Model group; the calcium absorption rate and calcium retention rate in the IBDF group increased by 62.41% and 73.55%, respectively, compared to the Model group (p<0.05). Further supplementation with the same amount of Ca... 2+ Under certain conditions, PBPs-Ca and IBDF interventions were more effective than CaCO3. IBDF can competitively bind to CaCO3 with phosphates, oxalic acid, phytic acid, tannic acid, and other substances in the digestive tract. 2+ This keeps it in a dissolved state, making it easier for the small intestine to absorb. The results of this study indicate that IBDF has a good effect on promoting calcium absorption.

[0056] Example 6: Effects of IBDF intervention on bone biomechanical properties in osteoporotic mice

[0057] Bone biomechanical parameters directly reflect changes in bone strength and are of great significance for diagnosing osteoporosis, assessing fracture risk, and monitoring treatment effectiveness. For example... Figure 4 As shown, compared with the Sham group, the maximum load in the Model group decreased from 1.16±0.18 N to 0.93±0.07 N, and the structural strength decreased from 2.26±0.28 N to 1.61±0.34 N (p<0.05). This result indicates that osteoporosis modeling was successful in the Model group, and the biomechanical properties of mouse bones decreased. Compared with the Model group, the maximum load in the Aln and CaCO3 intervention groups significantly increased to 1.12±0.13 N and 1.09±0.05 N, respectively (p<0.05), and the structural strength increased to 1.96±0.47 N and 1.87±0.46 N, respectively. The maximum load and structural strength in the PBPs-Ca intervention group were significantly higher than those in the Model group, reaching 1.15±0.15 N and 2.43±0.31 N, respectively (p<0.05). The maximum load and structural strength of the IBDF intervention group were significantly higher than those of the Model group, reaching 1.17±0.05 N and 2.49±0.30 N, respectively (p<0.05). These results indicate that IBDF improves the bone biomechanical properties of osteoporotic mice to some extent and reduces the risk of fracture.

[0058] Example 7: Effects of IBDF intervention on bone metabolism-related biochemical indicators in osteoporotic mice

[0059] ALP, OCN, Cath-K, CTX-I, and TRAP-5b are important biomarkers in bone resorption and bone formation, playing different roles in the pathophysiology of osteoporosis and can be used to assess the bone metabolic status of osteoporosis patients. Changes in bone metabolism biomarkers in different groups of mice are shown below. Figure 5 As shown, compared with the Sham group, the levels of all biomarkers in Model mice were significantly increased (p<0.05). This result indicates that bone resorption and bone formation in Model mice are abnormally active, exhibiting an abnormal state of high bone turnover, which is consistent with the metabolic disorders in ovariectomized mice. Compared with the Model group, the serum ALP, OCN, Cath-K, CTX-I, and TRAP-5b levels in the Aln group were significantly reduced by 7.47%, 5.07%, 12.83%, 22.44%, and 8.64%, respectively (p<0.05). This result indicates that Aln improves osteoporosis by restoring the balance between bone resorption and bone formation and regulating bone remodeling. There were no significant changes in bone metabolism biomarkers in the CaCO3 intervention group compared with the Model group. The serum ALP, OCN, Cath-K, CTX-I, and TRAP-5b levels in the PBPs-Ca intervention group were significantly lower than those in the Model group by 5.23%, 17.63%, 17.60%, 10.89%, and 13.05%, respectively (p<0.05). The serum ALP, OCN, Cath-K, CTX-I, and TRAP-5b levels in the IBDF group were significantly lower than those in the Model group by 7.39%, 17.68%, 20.53%, 9.75%, and 17.44%, respectively (p<0.05), showing superior efficacy compared to the PBPs-Ca intervention group. These results indicate that both PBPs-Ca and IBDF can improve osteoporosis by regulating bone metabolism balance and reducing high bone turnover in osteoporotic mice, and that the various active ingredients in IBDF exhibit a good synergistic effect.

[0060] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention and are not intended to limit the scope of the invention in any way. Various changes and modifications can be made to the invention without departing from its scope, and all such changes and modifications fall within the scope of the claims.

Claims

1. A calcium chelating peptide, characterized in that, Its amino acid sequence is shown in SEQ ID No. 1, specifically GETGPA.

2. The application of the calcium chelating peptide according to claim 1 in the preparation of peptide-calcium chelates.

3. A peptide-calcium chelate, characterized in that, It contains the calcium chelating peptide as described in claim 1.

4. The method for preparing the peptide-calcium chelate according to claim 3 is characterized in that, The preparation method is as follows: the concentration of calcium chelating peptide is 1.3-1.7 g / L, Ca... 2+ Chelation was performed at room temperature for 28-32 min at a concentration of 0.4-0.6 mM and pH = 6.8-7.

2.

5. The application of the calcium chelate peptide of claim 1 or the peptide-calcium chelate of claim 3 in the preparation of health food products.

6. A health food product, characterized in that, It contains the calcium chelating peptide of claim 1 or the peptide-calcium chelate of claim 3.

7. The health food according to claim 6, characterized in that, The health food product in question is a calcium supplement.

8. The health food according to claim 7, characterized in that, The calcium supplement mentioned is a bone health compressed candy.

9. The bone health compressed candy according to claim 8, characterized in that, It contains the calcium chelating peptide of claim 1 and the peptide-calcium chelate of claim 3.

10. The bone health compressed candy of claim 8, characterized in that, The weight percentages of each component are as follows: calcium chelate peptide 18%-22%, peptide-calcium chelate 8%-12%, calcium carbonate 13%-17%, vitamin D3 5500-6500 IU, glucosamine 15%-17%, chondroitin sulfate 15%-17%, β-cyclodextrin 5%-7%, microcrystalline cellulose 8%-12%, magnesium stearate 1%-3%, and sucralose 4%-6%.

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