Preparation method of phosphorylated salmon collagen peptide for promoting bone health

Abstract

The invention discloses a method for preparing a phosphorylated salmon collagen peptide-calcium chelate by taking salmon collagen peptide as a raw material, preparing high-calcium binding active peptide (P-CP) through phosphorylation modification and chelating the high-calcium binding active peptide with calcium ions. The method comprises the following steps: (1) enzymolysis preparation of salmon bone collagen; (2) phosphorylation modification of a peptide fragment mediated by sodium tripolyphosphate; and (3) optimizing a calcium ion chelation process. The chelating rate of the obtained P-CP-Ca calcium reaches 91.21%, and structural characterization proves that a phosphate group (PO4 < 2->) is combined with calcium ions through ionic bonds. Animal experiments show that P-CP-Ca can significantly improve the serum calcium level of a calcium-deficiency mouse (the H group recovers to a normal range), reduce the activity of a bone resorption marker TRAP (Target Related Amplified Protein) (which is 25.4% lower than that of a model group), and improve the bone trabecula microstructure (the bone mineral density is improved by 32.7%). The invention provides a new technology for valuable utilization of fishery by-products and development of functional calcium supplements.

Description

Technical Field

[0001] This invention relates to a method for preparing phosphorylated salmon collagen peptides that promote bone health, belonging to the field of functional foods and bone health. Background Technology

[0002] Osteoporosis is a major global public health challenge, with epidemiological surveys showing a prevalence exceeding 20%. Calcium is a key trace element for maintaining bone density, but traditional calcium supplements (such as calcium carbonate) have low solubility and insufficient absorption in the gastrointestinal tract, and are often accompanied by side effects such as gastrointestinal irritation.

[0003] In recent years, peptides Calcium chelates have become a research hotspot due to their ability to maintain good solubility in the gastrointestinal environment and significantly improve calcium bioavailability. However, the calcium binding sites of natural collagen peptides (CP) mainly originate from carboxyl and amino nitrogen groups, and their calcium chelation capacity is limited by the number of acidic residues.

[0004] Phosphorylation modification can introduce a doubly negatively charged phosphate group (-PO4) onto the peptide chain. 2- Phosphorylated soybean peptides significantly increase the negative charge density of peptides, thereby enhancing their affinity for divalent calcium ions. Previous studies have shown that phosphorylated soybean peptides can form highly efficient calcium carriers and promote osteoblast activity, validating the potential value of phosphorylation in enhancing calcium binding and bone metabolism.

[0005] Salmon bones are a collagen-rich byproduct that makes up about 15%-20% of the fish's body weight. The high collagen content in the bones makes them easily digestible into low-molecular-weight collagen peptides, which are essential for peptide preparation. An ideal raw material for calcium chelates.

[0006] Existing technologies disclose methods for preparing collagen peptide calcium chelates, but do not involve phosphorylation modification; the preparation of phosphorylated soybean peptides has been reported, but their regulatory effect on bone resorption is unknown. Therefore, there is an urgent need for an innovative process that uses salmon bone collagen peptides as a base and enhances the number of calcium binding sites and negative charge density through phosphorylation modification to overcome the dual defects of low absorption of traditional calcium supplements and limited binding sites of natural collagen peptides, thereby meeting the practical needs of osteoporosis prevention and treatment. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a method for preparing phosphorylated salmon collagen peptides that promote bone health, aiming to solve the technical problems of low absorption rate and gastrointestinal irritation associated with traditional calcium supplements (such as calcium carbonate) in the prior art.

[0008] The first technical solution provided by this invention is a method for preparing phosphorylated salmon collagen peptide-calcium chelate (P-CP-Ca) that promotes bone health, comprising the following steps: S1: Salmon bone pretreatment to obtain fish bone meal; S2: Enzymatic hydrolysis to prepare collagen peptides (CP): The fish bone powder of S1 is added to water and hydrolyzed with alkaline protease. After enzyme inactivation, the supernatant is collected by centrifugation and freeze-dried to obtain CP. S3: Phosphorylation modification (P-CP): The CP of S2 is mixed with sodium tripolyphosphate (STP) for phosphorylation modification, and then lyophilized to obtain phosphorylated collagen peptides (P-CP). S4: Calcium chelation: P-CP is mixed with calcium chloride and subjected to a chelation reaction, then freeze-dried to obtain P-CP-Ca.

[0009] In some embodiments, the pretreatment is specifically as follows: salmon bones are boiled in boiling water for 10 minutes to remove residual fish meat, and then treated with n-hexane, 10% citric acid solution and 0.08 mol / L NaOH-4% NaCl solution in sequence to degrease, decalcify and remove non-collagenous proteins, freeze-dry and then pulverize.

[0010] In some embodiments, in S2, fish bone powder is added to deionized water at a material-to-liquid ratio of 1:100, the pH is adjusted to 8.0, alkaline protease (12,000 U / g) is added, and the mixture is enzymatically hydrolyzed at 50°C for 5 hours. After enzyme inactivation, the supernatant is collected by centrifugation and freeze-dried to obtain CP.

[0011] In some embodiments, in S3, CP and sodium tripolyphosphate (STP) are mixed at a mass ratio of 1:1 and reacted at 40°C and pH 7.0 for 60 minutes, followed by freeze-drying to obtain phosphorylated collagen peptides (P-CP).

[0012] In some embodiments, in S4, P-CP is mixed with calcium chloride and reacted at 60°C and pH 7.0 for 60 minutes. Free calcium ions are removed by dialysis with a 100 Da dialysis membrane for 12 hours, and then freeze-dried to obtain salmon collagen peptide-calcium chelate (P-CP-Ca).

[0013] The second technical solution provided by the present invention is a salmon collagen peptide-calcium chelate prepared by the method described in the first technical solution.

[0014] The third technical solution provided by the present invention is a composition containing the salmon collagen peptide-calcium chelate described in the second technical solution.

[0015] The present invention provides a fourth technical solution, namely the use of the salmon collagen peptide-calcium chelate described in the second technical solution or the composition described in the third technical solution, in the preparation of a drug for the prevention or treatment of osteoporosis.

[0016] In some embodiments, the amount of salmon collagen peptide-calcium chelate added to the drug is not less than 1108 mg / kg body weight / day.

[0017] In some implementations, the dosage is 133.34-266.68 mg Ca / kg body weight / day.

[0018] The present invention provides a fifth technical solution, namely the application of the salmon collagen peptide-calcium chelate described in the second technical solution or the composition described in the third technical solution in the preparation of a drug for inhibiting bone resorption.

[0019] The sixth technical solution provided by the present invention is salmon bone collagen peptide, wherein the amino acid sequence of the salmon bone collagen peptide is shown in any one of SEQ ID NO.1~9.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses salmon bone by-products as raw materials to achieve high-value utilization of resources, and the calcium chelation rate is increased to 91.21% after phosphorylation modification; 2. P-CP-Ca showed excellent bone health improvement effects in animal experiments. The high-dose group could restore serum calcium to normal levels in calcium-deficient mice, increase bone mineral density by 10.8%, and increase trabecular thickness by 23.5%. 3. Compared to traditional calcium supplements, P-CP-Ca reduces gastrointestinal irritation and improves bioavailability through peptide-calcium chelation, and has the potential to be developed into a functional food. Attached Figure Description

[0021] Figure 1 Three-dimensional reconstruction of the trabecular structure of the mouse femur and tibia.

[0022] Figure 2 Image of a mouse femur.

[0023] Figure 3 The images show the coronal and transverse sections of the mouse tibia.

[0024] Figure 4 It refers to the length of the femur and tibia.

[0025] Figure 5 This refers to the bone mineral density of the femur and tibia.

[0026] Figure 6 This refers to the thickness of the trabecular bone in the femur and tibia.

[0027] Figure 7 The trabecular separation between the femur and tibia.

[0028] Different letters indicate significant differences between groups (P<0.05). Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] Test method: 1. The calcium content in the ethanol precipitate (i.e., calcium stably bound to peptides) and the total calcium content in the unprecipitated sample were determined using an inductively coupled plasma spectrometer. Finally, the calcium chelation rate was calculated using the formula above.

[0031] Calcium chelation rate (%) = (calcium content precipitated by ethanol / total calcium content) × 100%.

[0032] 2. Micro-CT analysis of bone mineral density and microstructure of the femur and tibia: High-resolution 3D scanning of bone samples was performed using a Skyscan 1176 CT system (Bruker, Germany); Image reconstruction: The scanned data is reconstructed into a 3D model using NRecon software; Quantitative analysis of parameters: The reconstructed 3D bone model was analyzed using CTAn software to calculate key bone mineral density and bone microstructure parameters. The key parameters reflecting "bone calcium content" and bone quality are: Bone mineral density (BMD): This is the most direct indicator of bone calcium content. The higher the BMD value, the higher the mineral (mainly calcium) content per unit volume of bone.

[0033] Bone volume fraction (BV / TV): This refers to the percentage of bone tissue (not bone marrow or voids) in a given bone volume. Bones with high bone calcium content typically have a higher BV / TV.

[0034] Trabecular bone thickness (Tb.Th): Reflects the average thickness of the trabecular bone. Bone calcium loss leads to thinning of the trabecular bone.

[0035] Trabecular separation (Tb. Sp): Reflects the average distance between trabeculae. Bone calcium loss leads to increased trabecular spacing and separation.

[0036] 3. Statistical Analysis Data are expressed as mean ± standard deviation. One-way ANOVA and Duncan post-hoc tests were performed using SPSS software.

[0037] Raw materials used in the examples: 1. Salmon bones were purchased from a seafood market in Dalian; sodium tripolyphosphate was purchased from Chongqing Wansheng Chuandong Chemical Co., Ltd.; anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; and anhydrous calcium chloride was purchased from Tianjin Damao Chemical Reagent Factory.

[0038] 2. Alkaline protease was purchased from Nanning Pangbo Biotechnology Co., Ltd. Alkaline protease is a proteolytic enzyme produced by deep fermentation, extraction, and purification of Bacillus licheniformis 2709, bred through protoplast mutagenesis. It is a serine-type endopeptide, and its catalytic activity depends on the serine residues in its active site. Chemically, this enzyme is a protein, with optimal activity conditions of 50-60℃ and pH 8.5-10.5. It specifically catalyzes the breaking of peptide bonds within protein molecules, degrading large proteins into smaller polypeptides or amino acids. Enzyme activity is defined as the amount of enzyme (unit: u / g) required per minute to hydrolyze casein to release trichloroacetic acid-soluble matter at 275 nm wavelength, equivalent to the absorbance of 1 μg of tyrosine, under the conditions of 40±0.2℃ and pH 10.5. This enzyme is susceptible to Fe³⁺. + Cu² + Hg + Pb + It is inhibited or destroyed by heavy metal ions and oxidants, and has a strong ability to decompose proteins.

[0039] 3. Kunming (KM) mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., and were SPF grade.

[0040] Example 1 1. Preparation of salmon bone collagen peptides (CP) S1: Salmon bones are boiled in water for 10 minutes to remove residual meat, and then treated sequentially with n-hexane, 10% citric acid, and 0.08 mol / L sodium hydroxide-4% sodium chloride solution to degrease, decalcify, and remove non-collagenous proteins; S2: Add water to the treated bone meal at a ratio of 1:100, adjust the pH to 8 with 1M hydrochloric acid / sodium hydroxide, add alkaline protease (12000U / g), and enzymatically hydrolyze at 50℃ for 5h; S3: Boil the enzymatic hydrolysate for 10 min to terminate the reaction, centrifuge at 10000 rpm to collect the supernatant, concentrate by rotary evaporation, and freeze dry to obtain CP dry powder.

[0041] 2. Phosphorylation modification (preparation of P-CP) S4: Dissolve CP dry powder to a 20 mg / mL solution, add STP (1 mg / mL), and react with magnetic stirring at 40°C for 60 min; S5: The reaction solution was dialyzed through a 100 Da dialysis membrane for 12 hours and then freeze-dried to obtain P-CP.

[0042] 3. Preparation of P-CP-Ca complex S6: P-CP is dissolved to a concentration of 15 mg / mL, and an equal mass of calcium chloride is added. The mixture is then reacted at 60°C for 60 min. S7: The product was dialyzed to remove free calcium, and then freeze-dried to obtain the P-CP-Ca complex with a calcium chelation rate of 91.21%.

[0043] Example 2 1. Animal grouping and feeding Animal species: Male Kunming (KM) mice, 3 weeks old.

[0044] Keeping environment: Temperature around 25℃, free access to drinking water.

[0045] Grouping: Control group (Con, 10 animals): fed standard feed (containing 5000 ppm calcium).

[0046] Model group (60 animals): Feed them a low-calcium diet (containing 5 ppm calcium) for 3 weeks to establish a calcium deficiency model.

[0047] Intervention group: Sixty mice that successfully developed the model were randomly divided into 6 groups (n=10 per group) and received intervention on a continuous low-calcium diet. Mod group: Continue with low-calcium diet; Calcium carbonate group (Ca): low calcium diet + calcium carbonate (calculated as calcium ions: 133.34 mg / kg body weight). CP-Ca group (CP): low-calcium feed + ordinary peptide calcium chelate (CP dry powder in Example 1, calculated as calcium ions: 133.34 mg / kg body weight).

[0048] P-CP-Ca-L group (L): low-calcium diet + phosphorylated peptide calcium chelate (P-CP-Ca complex in Example 1, low dose, 66.67 mg / kg body weight as calcium ions).

[0049] P-CP-Ca-M group (M): low-calcium diet + phosphorylated peptide calcium chelate (P-CP-Ca complex in Example 1, medium dose, 133.64 mg / kg body weight as calcium ions).

[0050] P-CP-Ca-H group (H): low-calcium diet + phosphorylated peptide calcium chelate (P-CP-Ca complex in Example 1, high dose, as calcium ions: 266.68 mg / kg body weight).

[0051] 2. Intervention methods Administration route: once daily via oral gavage.

[0052] Intervention duration: 5 weeks.

[0053] Fasting: After the 5-week gavage procedure, fast for 12 hours (water is not prohibited).

[0054] 3. Sample Collection Method of execution: Death by disembowelment after anesthesia.

[0055] Sample collection: Serum, femur and tibia were collected.

[0056] 4. Testing Indicators and Procedures Serum biochemical indicators: Detect the levels of calcium, phosphorus, alkaline phosphatase (AKP), tartrate-resistant acid phosphatase (TRAP), and osteocalcin (OCN).

[0057] Organ index: Remove the heart, liver, spleen, and kidneys, weigh them, and calculate their ratio to body weight (organ coefficient).

[0058] Bone parameter measurement: Bone length and width: The length and width of the left femur and tibia were measured using electronic calipers.

[0059] Bone weight and bone coefficient: After removing muscle and fat, weigh the body and calculate the ratio of bone weight to body weight.

[0060] Maximum load (bone strength): The maximum force that the bone can withstand when it breaks is measured using a three-point bending test with a texture analyzer (TA.XT. plus).

[0061] Micro-CT scan: The femur and tibia were scanned using the Skyscan 1176 CT system, and parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analyzed using the accompanying software (NRecon, DataViewer, CTAn).

[0062] Histological staining: H&E staining: Staining colon and femoral tissues to observe intestinal inflammation and trabecular bone structure.

[0063] TRAP staining: Staining the cross-section of the femoral epiphysis to quantitatively assess osteoclast activity.

[0064] The results are as follows Figure 1As shown, this is a 3D reconstruction image of the trabecular structure of the femur and tibia. This image visually displays the differences in the internal microstructure of the bones of mice in each group. Control group (Con): The bone structure is dense and intact, with a tightly connected trabecular network. Model group (Mod): The bone structure is severely damaged, characterized by sparse and broken trabeculae and numerous cavities, a typical morphology of osteoporosis. Calcium carbonate group (Ca) and unphosphorylated peptide calcium group (CP): There is some improvement compared to the model group, but the trabeculae are still relatively thin and sparse, with limited recovery. P-CP-Ca groups (L, M, H): The trabecular structure is significantly repaired, becoming thicker and more connected. Among them, the structures of the medium-dose (M) and high-dose (H) groups are very close to the control group, showing the best recovery effect.

[0065] Figures 2-3 These are direct images and coronal / transverse views of the femur and tibia, and these images are... Figure 1 The images corroborate each other, showcasing the overall morphology and density of the bones from different perspectives. The density of the white spots represents the bone mineral density. The white spots in the control group are dense; those in the model group appear sparse and pale. The P-CP-Ca treatment groups, especially the H group, have bone images with brightness and density closest to the control group.

[0066] Figure 4 The bone lengths are those of the femur and tibia. The bone length in the calcium deficiency model group (Mod) was significantly lower than that in the control group (Con), indicating that calcium deficiency directly inhibits longitudinal bone growth. All calcium supplementation groups showed some recovery, but the P-CP-Ca group (L, M) showed the best recovery, almost indistinguishable from the control group, while the calcium carbonate (Ca) and unphosphorylated peptide calcium (CP) groups showed weaker effects.

[0067] Figure 5 Bone mineral density is a core quantitative indicator for measuring bone calcium content. The model group (Mod) had the lowest bone mineral density values ​​(femur 1.20, tibia 1.14). All calcium supplementation groups showed an increase in bone mineral density, but the P-CP-Ca group was significantly more effective than the others. In particular, the M group (highest femur bone mineral density, 1.33) and the H group (highest tibia bone mineral density, 1.33) had bone mineral density values ​​closest to the control group, demonstrating the remarkable efficacy of P-CP-Ca in promoting calcium deposition and increasing bone mineral density.

[0068] Figures 6-7 These are trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Figure 6Trabecular bone thickness analysis showed that the model group had the greatest trabecular separation (0.11 mm for femur and 0.10 mm for tibia). The P-CP-Ca groups (especially the M group with 0.15 mm for femur and the H group with 0.15 mm for tibia) significantly increased trabecular bone thickness, with the Con group (0.20 mm for femur and 0.17 mm for tibia) showing the closest recovery to normal levels. Thicker trabecular bone implies greater skeletal mechanical strength. Figure 7 Trabecular separation was highest in the model group, indicating large gaps and a sparse structure between trabeculae. P-CP-Ca treatment significantly reduced separation, making the trabecular network more compact, which directly explains its anti-osteoporosis mechanism.

[0069] Example 3: Phosphorylated peptides significantly enrich calcium signaling pathways 1. Sample preparation: Reagent kit: High-Select TM Fe-NTA phosphorylated peptide enrichment kit; Washing buffer: 0.1% TFA / H2O; Elution buffer: a mixture of 50% ACN and 5% NH4OH; Subsequent processing: Vacuum drying after elution.

[0070] Phosphorylated collagen peptide (P-CP) samples were specifically enriched using the High-Select™ Fe-NTA Phosphorylated Peptide Enrichment Kit. Phosphorylated peptides were isolated from complex mixtures of peptides.

[0071] 2. Amino acid sequence identification and data analysis Instrument platform: HPLC-MS / MS; Mass spectrometer: Orbitrap Exploris 480; Liquid chromatography system: Dionex Ultimate 3000 RSLCnano nano liquid chromatography system; Liquid chromatography-tandem mass spectrometry analysis: The enriched phosphorylated peptides were analyzed using an HPLC-MS / MS system. Specifically, an Orbitrap Exploris 480 mass spectrometer coupled with a Dionex Ultimate 3000 RSLCnano nanofluid chromatograph was used.

[0072] Database search and identification: The raw data acquired by mass spectrometry was searched in the UniProt database against Atlantic salmon (Salmo salar) using ProteomeDiscoverer 2.4 software to identify the specific peptide amino acid sequences and their corresponding phosphorylation modification sites (as shown in Tables 1-3).

[0073] Table 1. Phosphorylated peptides with only one phosphorylation site

[0074] Table 2. Phosphorylated peptides with two phosphorylation sites

[0075] Table 3 Phosphorylated peptides with three phosphorylation sites

[0076] As shown in Tables 1-3, serine is the most prevalent phosphorylation site. Among phosphorylated peptides, serine (Ser) has the highest probability of phosphorylation and is the main modification site. Threonine (Thr) is the next most frequent phosphorylation site. Tyrosine (Tyr) has the lowest phosphorylation rate (13.0%). This is consistent with the common serine / threonine phosphorylation trend in organisms.

[0077] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a phosphorylated salmon collagen peptide-calcium chelate that promotes bone health, characterized in that, Includes the following steps: S1, salmon bone pretreatment to obtain fish bone powder; S2, enzymatic hydrolysis to prepare collagen peptides: the fish bone powder from S1 was added to water and hydrolyzed with alkaline protease, the enzyme was inactivated, the supernatant was collected by centrifugation, and lyophilized to obtain CP; S3, phosphorylation modification: the CP from S2 was mixed with sodium tripolyphosphate for phosphorylation modification, and lyophilized to obtain phosphorylated collagen peptides; S4, calcium chelation: the phosphorylated collagen peptides were mixed with calcium chloride for chelation reaction, and then lyophilized to obtain salmon collagen peptide-calcium chelate.

2. The method according to claim 1, characterized in that, The pretreatment is as follows: salmon bones are boiled in boiling water for 10 minutes to remove residual fish meat, and then treated with n-hexane, 10% citric acid solution and 0.08 mol / L NaOH-4% NaCl solution in sequence to degrease, decalcify and remove non-collagenous proteins, freeze-dry and then pulverize.

3. The method according to claim 1, characterized in that, In S2, fish bone powder was added to deionized water at a ratio of 1:100, the pH was adjusted to 8.0, 12,000 U / g alkaline protease was added, and the mixture was enzymatically hydrolyzed at 50°C for 5 hours. After enzyme inactivation, the supernatant was collected by centrifugation and freeze-dried to obtain CP.

4. The method according to claim 1, characterized in that, In S3, CP and sodium tripolyphosphate are mixed at a mass ratio of 1:1 and reacted at 40°C and pH 7.0 for 60 minutes, followed by freeze-drying to obtain phosphorylated collagen peptides.

5. The method according to claim 1, characterized in that, In S4, P-CP was mixed with calcium chloride and reacted at 60°C and pH 7.0 for 60 minutes. Free calcium ions were removed by dialysis with a 100 Da dialysis membrane for 12 hours, and the mixture was freeze-dried to obtain salmon collagen peptide-calcium chelate.

6. Salmon collagen peptide-calcium chelate prepared by the method according to any one of claims 1 to 5.

7. A composition containing the salmon collagen peptide-calcium chelate of claim 6.

8. The use of the salmon collagen peptide-calcium chelate of claim 6 or the composition of claim 7 in the preparation of a medicament for the prevention or treatment of osteoporosis.

9. The use of the salmon collagen peptide-calcium chelate of claim 6 or the composition of claim 7 in the preparation of a medicament for inhibiting bone resorption.

10. Salmon bone collagen peptides, characterized in that, The amino acid sequence of the salmon bone collagen peptide is shown in any one of SEQ ID NO. 1 to 9.

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