Bone health tablet candy rich in marine peptide calcium chelate and preparation method thereof

Abstract

The invention discloses a bone health tablet candy rich in marine peptide calcium chelate and a preparation method thereof, and belongs to the technical field of polypeptide biologication.The calcium chelating peptide provided by the invention has an amino acid sequence as shown in SEQ ID No.1, the specific sequence is GETGPA (F21-1), and the calcium binding capacity is 4.72 + / -1.06 mu g / mg. The bone health tablet candy provided by the invention has relatively good calcium absorption promoting activity. A mouse experiment result shows that no adverse effect is caused to mouse growth, high bone transition caused by osteoporosis can be reversed, the bone biomechanical property of the osteoporosis mouse is improved, and then the effect of enhancing the bone mineral density is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptides, and in particular relates to a bone health compressed candy rich in marine peptide calcium chelate and a preparation method thereof. Background Art

[0002] Osteoporosis is a bone metabolic disease, the main cause of which is the disruption of the metabolic balance between bone formation and bone resorption, resulting in bone loss. In order to inhibit the decrease in bone mass caused by osteoporosis and reduce the risk of fractures, drugs such as raloxifene, alendronate, risedronate and conjugated estrogens are used to treat postmenopausal osteoporosis. However, drug treatment is limited by factors such as high price, side effects and drug resistance. Therefore, the development of corresponding bone health products has become a new choice for people. Calcium preparations can be used to restore the balance between bone resorption and bone formation. Therefore, calcium supplementation is considered to be a safe method for treating osteoporosis. Since calcium chelating peptides can enhance Ca 2+ Bioavailability, which provides the possibility for it to play an anti-osteoporosis activity. Studies have found that cod bone calcium chelate peptide and its peptide calcium chelate have good in vivo and in vitro calcium absorption activity, and can improve the imbalance of bone metabolism in ovarian-induced osteoporosis mice, playing a good anti-osteoporosis effect.

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

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

[0005] Chinese patent CN118104833A discloses a processing method for a functional food joint treasure nutritional compound tablet with the function of increasing bone density. Sea cucumber collagen peptide is used as the main effective ingredient, which is pressed into a nutritional tablet with a compound formula of high-quality aminoglucosidase substances, minerals and vitamins, and can effectively increase bone density. Summary of the invention

[0006] The object of the present invention is to provide a calcium chelating peptide and a peptide calcium chelate extracted from marine aquatic products, and a calcium supplement preparation prepared using the peptide, specifically bone health tablet candy (IBDF). The IBDF provided by the present invention has good functions of promoting calcium absorption and increasing bone density activity.

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

[0008] The calcium chelating peptide provided by the present invention has a very strong calcium binding ability 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: the concentration of calcium chelate peptide is 1.3-1.7 g / L, Ca 2+ Chelation was carried out at room temperature for 28-32 min at a concentration of 0.4-0.6 mM and pH 6.8-7.2.

[0010] The present invention also provides the use of the calcium chelating peptide or peptide calcium chelate in the preparation of health food. Specifically, the health food is a calcium supplement.

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

[0012] The IBDF provided by the present invention follows the concept of "monarch, minister, assistant and envoy" in traditional Chinese medicine theory, with calcium chelating peptides (PBPs) and peptide calcium chelates (PBPs-Ca) as main effective ingredients; calcium carbonate, vitamin D3, glucosamine and chondroitin sulfate as auxiliary ingredients; and β-cyclodextrin, microcrystalline cellulose, magnesium stearate and 4-6% sucralose as auxiliary materials to improve the taste, color, stability and the like of compressed candies.

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

[0014] The IBDF provided by the present invention is milky white in color, uniform in color, smooth in surface, has a light milky flavor, no fishy smell, no peculiar odor, no peculiar taste, good 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] Beneficial effects of the present invention:

[0016] The IBDF provided by the present invention has good calcium absorption promoting activity. The results of mouse experiments show that it has no adverse effect on the growth of mice, and can reverse the high bone turnover caused by osteoporosis, improve the bone biomechanical properties of osteoporotic mice, and thus achieve the effect of enhancing bone density. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of mass spectrometry identification of F21-1 are shown in Figure 1, where (a) is the extracted ion current diagram, (b) is the primary mass spectrum diagram, (c) is the secondary mass spectrum diagram, and (d) is the structure diagram.

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

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

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

[0021] Figure 5 The effects of IBDF intervention on bone metabolism-related indicators in osteoporotic mice, (a) is the graph of mouse serum ALP content, (b) is the graph of mouse serum OCN content, (c) is the graph of mouse serum Cath-K content, (d) is the graph of mouse serum CTX-1 content, and (e) is the graph of mouse serum TRACP-5b content. DETAILED DESCRIPTION

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

[0023] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0024] In the following examples, the method for determining calcium chelating activity is as follows:

[0025] 3 mg of sample was dissolved in 2 mL of 5 mmol / L CaCl2 and incubated at 37 °C for 20 min. 4 mL of 20 mmol / L sodium phosphate buffer (pH 7.5) was added to the reaction system and incubated at 37 °C for 30 min. The mixture was centrifuged at 8000 rpm / min for 20 min to remove calcium phosphate precipitate. After the supernatant was filtered through a 0.22 μm filter, 2 mL was taken and digested with 10 mL of nitric acid. The calcium content was determined according to GB 5009.92-2016 (atomic absorption method). A blank control group without polypeptide was set up. The calculation formula for calcium chelation activity is as follows:

[0026]

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

[0028] In the following examples, the method for establishing the osteoporosis mouse model is as follows:

[0029] (1) The day before surgery, the patients were fasted for 12 h and anesthetic was injected intraperitoneally at a dose of 1% of body weight (3.5%, m / v).

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

[0031] (3) After wiping the surgical site clean with iodine, use a scalpel to cut open the mouse's abdominal skin and muscle layers.

[0032] (4) After exposing the adipose tissue, grasp the fat with forceps and gently move it aside. Slowly find the "Y"-shaped mouse uterus along the fat, and find the ovaries at the end of the fallopian tube. Avoid the capillaries on the wall of the fallopian tube and use scissors to cut off the ovaries on both sides. In the sham operation group, only a small amount of fat next to the ovaries was 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 muscle layer of the mouse was sutured, and then the outer skin layer was sutured. After the suturing was completed, 50,000 units of penicillin were dripped into the wound to prevent wound infection.

[0034] (6) After surgery, place each mouse individually in a cage and observe its condition at all times. After the mouse wakes up, give it an appropriate amount of water.

[0035] (7) Within 3 days after surgery, 50,000 units of penicillin were injected daily. If the wound of the mouse was open, it was necessary to suture it again. After one week, the formal experiment could be carried out after the wound was healed. The experimental groups and oral gavage doses are shown in Table 1.

[0036] Table 1 Animal experimental groups and gavage doses

[0037] Group serial number Number of mice Gavage samples Sham operation group Sham n=10 Normal saline Model Group Model n=10 Normal saline Positive control group Aln n=10 1 mg / kg body weight / d oral administration of alendronate Calcium carbonate control group <![CDATA[CaCO3]]> n=10 <![CDATA[Ca 2+ The dose 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 with marine peptide calcium chelate 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 examples, the method for determining calcium bioavailability is as follows:

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

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

[0041] Related indicators formula Calcium intake Calcium intake (mg / d) = calcium in feed + calcium ingested by oral administration Calcium absorption Calcium absorption (mg / d) = calcium intake - fecal calcium Calcium absorption rate Calcium absorption rate (%) = (calcium absorption / calcium intake) × 100% Calcium retention Calcium retention (mg / d) = calcium intake - fecal calcium - urine calcium Calcium retention Calcium retention rate (%) = (calcium retention / calcium intake) × 100%

[0042] In the following embodiments, the method for determining the biomechanical properties of bones is as follows:

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

[0044] In the following embodiments, the determination method of the biochemical indexes related to bone metabolism is as follows:

[0045] After 12 weeks of intragastric administration, each mouse was fasted for 12 h but not water. The mice were weighed before sampling, and their eyeballs were removed for blood collection. After blood collection, the mice were killed by dislocation of the neck. The serum was centrifuged at 4 °C, 8000 r / min, for 40 min, and immediately dispensed 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 isomer 5b (TRAP-5b) in mouse serum were determined according to the instructions of the corresponding 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 a preparation method thereof, the specific steps are: after the fish bone is softened at 121 ° C for 50 min, it is wet-ultrafinely ground with an ultrafine grinder, distilled water is added at a ratio of 1:4 (w / v), and 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, hydrolyzed at pH = 6.5, 55 ° C for 4 h, boiled for 15 min to inactivate the enzyme, and after centrifugation at 8000 rpm / min for 15 min, the supernatant is concentrated and freeze-dried to obtain an enzymolysate.

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

[0049] UHPLC-Q-Orbitrap was used to identify the amino acid sequence of marine calcium chelating peptides, and the chromatographic column used was 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. Scanning range: 100-1200 m / z, electrospray mode: electrospray positive ion, electrospray voltage 3600 V. The peptides in the liquid phase were identified as follows. Figure 1 As shown, the polypeptide sequence was identified as GETGPA.

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

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

[0052] Example 4 Preparation of marine peptide calcium chelate

[0053] The concentration of CaCl2 was adjusted to 0.5 mM, the concentration of marine calcium chelate peptide was adjusted to 1.5 g / L, and the pH of the solution was adjusted to 7.0. The chelate was carried out at room temperature for 0.5 h, and the chelate was precipitated with 8 volumes of ethanol. The marine peptide calcium chelate was obtained after freeze-drying.

[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 in the results, the calcium absorption rate and calcium retention rate of the Model group were 35.05% and 32.80%, respectively, which were significantly lower than those of the Sham group (p<0.05). The calcium absorption rate and calcium retention rate of the Aln and CaCO3 groups were not significantly different from those of the Model group (p>0.05). The calcium absorption rate and calcium retention rate of the PBPs-Ca group were 59.40% and 50.47% higher than those of the Model group, respectively; the calcium absorption rate and calcium retention rate of the IBDF group were 62.41% and 73.55% higher than those of the Model group, respectively (p<0.05). 2+ Under these conditions, the intervention effects of PBPs-Ca and IBDF were better than those of CaCO3. IBDF can competitively bind to Ca with phosphate, oxalic acid, phytic acid, tannic acid, etc. in the digestive tract. 2+ It keeps the solution in a dissolved state and is easier to be absorbed by the small intestine. The results of this study show that IBDF has a good effect in promoting calcium absorption.

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

[0057] Bone biomechanical indices can directly reflect changes in bone strength and are of great significance for diagnosing osteoporosis, assessing fracture risk, and monitoring treatment effects. Figure 4 As shown in the figure, compared with the Sham group, the maximum load of 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 showed that the osteoporosis modeling in the Model group was successful and the biomechanical properties of the mouse bones decreased. Compared with the Model group, the maximum load of the Aln and CaCO3 intervention groups increased significantly 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 of the PBPs-Ca intervention group were significantly higher than those of 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). The results showed that IBDF improved the bone biomechanical properties of osteoporotic mice to a certain extent and reduced the risk of fracture.

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

[0059] ALP, OCN, Cath-K, CTX-I and TRAP-5b are important biomarkers in the process of bone resorption and bone formation. They play different roles in the pathophysiological process of osteoporosis and can be used to evaluate the bone metabolism status of patients with osteoporosis. Figure 5 As shown in the figure, compared with the Sham group, the levels of various biomarkers in the Model mice were significantly increased (p<0.05), indicating that the bone resorption and bone formation in the Model mice were abnormally active, presenting an abnormal state of high bone turnover, which was consistent with the metabolic disorder of mice after ovariectomy. 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), indicating that Aln regulates bone remodeling by restoring the balance between bone resorption and bone formation, thereby improving osteoporosis. There was no significant change 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 decreased by 5.23%, 17.63%, 17.60%, 10.89% and 13.05% (p<0.05) compared with those in the Model group, respectively; the serum ALP, OCN, Cath-K, CTX-I and TRAP-5b levels in the IBDF group were significantly decreased by 7.39%, 17.68%, 20.53%, 9.75% and 17.44% (p<0.05) compared with those in the Model group, respectively, and the effect was better than that in the PBPs-Ca intervention group. These results indicate that both PBPs-Ca and IBDF can reduce the high bone turnover in osteoporotic mice by regulating the balance of bone metabolism, thereby improving osteoporosis, and the effective components in IBDF have a good synergistic effect.

[0060] The above embodiments show and describe the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for illustrating the principles of the present invention, and are not intended to limit the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the protection claimed.

Claims

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

2. Use of the calcium chelating peptide according to claim 1 in the preparation of peptide calcium chelates.

3. A peptide calcium chelate, characterized in that: Contains the calcium chelating peptide according to 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 carried out at room temperature for 28-32 min at a concentration of 0.4-0.6 mM and pH 6.8-7.

2.

5. Use of the calcium chelating peptide according to claim 1 or the peptide calcium chelate according to claim 3 in the preparation of health food.

6. A health food, characterized in that: Contains the calcium chelating peptide according to claim 1 or the peptide calcium chelate according to claim 3.

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

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

9. The bone health compressed candy according to claim 8, characterized in that: Contains the calcium chelating peptide according to claim 1 and the peptide calcium chelate according to claim 3.

10. The bone health compressed candy according to claim 8, characterized in that: The weight percentages of the various components are: 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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