Calcium peptide chelate based on defatted sheep bone powder homologous recombination and preparation method and application thereof

Abstract

The present application relates to a kind of calcium peptide chelate based on defatted sheep bone powder homologous recombination and its preparation method and application, preparation method, including the following steps, defatted sheep bone powder is mixed with water, flavourzyme is added to carry out enzymolysis, enzyme is inactivated after enzymolysis, and the enzymatic hydrolysate containing bone-derived peptide is obtained;Lactic acid is added in enzymatic hydrolysate and is hydrolyzed, so that calcium ion is dissolved, and the mixed solution containing bone-derived peptide and calcium ion is obtained;Mixed solution is carried out chelation under neutral condition, after reaction, aftertreatment is carried out, and calcium peptide chelate is obtained;Calcium peptide chelate prepared according to the above preparation method is used as salty taste enhancer, salt-reducing auxiliary agent or flavor enhancer, and its application in food.The present application has the advantages of homologous resource recombination, improves chelation rate, raw material utilization rate is high, realizes sheep bone by-product high value, process safety, can add chelate to clear soup base material, so as to reduce sodium, the effect of calcium supplement, salty seasoning.

Description

Technical Field

[0001] This invention relates to the technical field of food additives, and in particular to a calcium peptide chelate based on the homologous recombination of defatted sheep bone powder, its preparation method, and its application. Background Technology

[0002] Sheep bones, an important byproduct of livestock slaughtering, are rich in protein and minerals, possessing high development and utilization value. Collagen, accounting for approximately 90% of total bone protein, is an essential nutrient for the human body; the calcium-to-phosphorus ratio is approximately 2:1, an ideal ratio for human absorption, hence sheep bones are hailed as an "ideal natural calcium source." Currently, the utilization pathways of animal bone byproducts mainly include: directly preparing bone meal as a mineral supplement; chemically extracting bone calcium to prepare inorganic calcium salts or bone calcium products; using enzymatic hydrolysis to prepare bone-derived peptides for use as nutritional or flavor peptides; and preparing mineral supplements through the compounding or chelation of exogenous calcium and exogenous peptides.

[0003] Currently, existing technologies still have significant shortcomings. On the one hand, the separate development of proteins and calcium leads to insufficient resource utilization and fails to achieve synergistic effects between bone-derived components. On the other hand, most commercially available calcium supplements are mainly inorganic calcium such as calcium carbonate and calcium phosphate, whose absorption rate in the gastrointestinal tract is typically only 10-20%, and they are prone to precipitation, resulting in low bioavailability. Furthermore, traditional bone mineral extraction largely relies on chemical processing methods such as strong acids and alkalis, which not only destroy the natural properties of the raw materials but also pose safety risks, making it difficult to meet the market's demand for green and natural products. Regarding calcium peptide chelation products, existing solutions mostly use exogenous peptides (such as soybean peptides and fish collagen peptides) to chelate with exogenous calcium salts. The raw material sources are scattered, homology and safety are insufficient, and the products have limited functions, usually focusing only on calcium supplementation and lacking development of functions for food flavor regulation (such as enhancing saltiness and reducing sodium). Currently, homologous calcium peptide chelation products with both calcium supplementation and sodium reduction effects are absent in both domestic and international markets.

[0004] Therefore, how to achieve high-value homologous recombination of calcium and protein components in sheep bone by-products and prepare calcium peptide chelates with both high bioavailability and salty taste enhancement functions to solve the problems of low resource utilization, poor calcium absorption and single product function in the existing technology has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the first objective of this invention is to provide a method for preparing calcium peptide chelates based on homologous recombination of defatted sheep bone powder. By adjusting the ratio of peptide to calcium and technical parameters such as chelation time, temperature, and pH, this method has the advantages of homologous resource recombination, improved chelation rate, high raw material utilization, high value-added utilization of sheep bone by-products, and good process safety.

[0006] The second objective of this invention is to provide a calcium peptide chelate that has the advantages of both nutritional and functional value, namely reducing sodium and supplementing calcium while increasing saltiness and flavor.

[0007] The third objective of this invention is to provide an application of calcium peptide chelates, which can be added to clear broth base to reduce sodium, supplement calcium, and increase saltiness for flavor.

[0008] To achieve the first objective mentioned above, the present invention provides the following technical solution: A method for preparing calcium peptide chelates based on homologous recombination of defatted sheep bone powder includes the following steps: S1. Defatted sheep bone powder is mixed with water, flavored protease is added for enzymatic hydrolysis, and the enzyme is inactivated after hydrolysis to obtain an enzymatic hydrolysate containing bone-derived peptides. S2 adds lactic acid to the enzymatic hydrolysate obtained in S1 to hydrolyze calcium ions, thereby dissolving calcium ions and obtaining a mixture containing bone-derived peptides and calcium ions. S3 involves chelating the mixture obtained in S2 under neutral conditions, followed by post-processing to obtain calcium peptide chelates.

[0009] Further, in step S1, fresh sheep bones are pre-washed, chopped, and pulverized to below 100 mesh using a high-speed pulverizer. The residual fat in the sheep bone powder is removed with 4 to 6 times its volume of anhydrous ethanol, and then dried in an oven at 40 to 60°C to constant weight to obtain defatted sheep bone powder.

[0010] Furthermore, in S1, the amount of flavor protease added is controlled to be 6000~8000 U / g defatted sheep bone powder, the enzymatic hydrolysis temperature is 40~60℃, the enzymatic hydrolysis time is 2~4h, and the enzymatic hydrolysis pH is 6~8.

[0011] Further, in step S1, after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90-110°C for 5-15 minutes to obtain an enzymatic hydrolysate containing bone-derived peptides.

[0012] Furthermore, in S2, the concentration of lactic acid is controlled to be 1.8~2.0 mol / L, the ratio of enzymatic hydrolysate to lactic acid is 1:6~12, the hydrolysis temperature is 40~60℃, and the hydrolysis time is 40~60 min.

[0013] Furthermore, in S3, the pH of the chelation reaction is controlled to be 6.5-7.5, the reaction temperature to be 55-65°C, and the reaction time to be 55-65 min. Further, in step S3, after the reaction is complete, 4 to 6 times the volume of anhydrous ethanol is added to the reaction solution for precipitation, and the precipitate is washed multiple times with anhydrous ethanol. After centrifugation, the precipitate is freeze-dried to obtain the calcium peptide chelate.

[0014] To achieve the second objective mentioned above, the present invention provides the following technical solution: A calcium peptide chelate based on homologous recombination of defatted sheep bone powder was prepared according to the above preparation method.

[0015] To achieve the third objective mentioned above, the present invention provides the following technical solution: An application of a calcium peptide chelate based on the homologous recombination of defatted sheep bone powder, wherein the calcium peptide chelate prepared according to the above preparation method is used in food as a saltiness enhancer, salt reduction aid or flavor enhancer.

[0016] Furthermore, the food products include meat products, dairy products, noodle soup bases, seasonings, and pre-prepared foods.

[0017] In summary, the beneficial technical effects of the present invention are as follows: 1. Since this invention uses defatted sheep bone powder as the only homologous raw material, it obtains bone peptides and bone calcium from the same raw material through enzymatic hydrolysis and lactic acid hydrolysis, respectively. Then, it carries out homologous recombination and chelation to achieve integrated synergistic utilization of calcium and peptides. Therefore, it achieves the effects of high value of sheep bone by-products, high raw material utilization rate, and good product homology and safety, avoiding the source dispersion and safety hazards caused by exogenous calcium or peptide sources. 2. In this invention, flavor protease and food-grade lactic acid are preferentially selected for mild treatment, and the chelation conditions are optimized to promote the formation of a stable coordination structure between calcium ions and peptides. Because this structure can protect calcium ions and enable them to be absorbed as a whole through small intestinal oligopeptide transporters, it significantly improves the solubility and bioavailability of calcium. At the same time, the spatial conformational change after the peptides chelate with calcium ions can specifically block bitter taste receptors and activate salty taste receptors. Therefore, calcium bioavailability is far superior to that of traditional inorganic calcium (effectively avoiding gastrointestinal precipitation), and it also has the synergistic effect of increasing saltiness and reducing sodium. 3. The method of the present invention involves sequentially performing flavor protease-targeted enzymatic hydrolysis (releasing salty-enhancing peptides rich in carboxyl and amino groups), mild lactic acid hydrolysis (breaking the hydroxyapatite lattice to release active calcium ions), and an "in-situ dissociation-targeted replacement" chelation reaction under neutral conditions on the same defatted sheep bone powder. This allows calcium ions to be encapsulated by homologous polypeptides to form stable cyclic coordination compounds. No strong acids, strong bases, or exogenous additives are required, making the process green and safe. Therefore, the resulting chelates can be directly used as salty-enhancing agents, salt-reducing aids, and calcium-supplementing ingredients in low-sodium foods (such as soup bases, seasonings, and meat products), achieving a comprehensive effect of integrated nutritional fortification and flavor regulation. Attached Figure Description

[0018] Figure 1This is a scanning electron microscope image of the bone-derived peptide and calcium peptide chelate of Example 1 of the present invention; wherein, the top left SBPHs1000×, the middle left SBPHs10000×, the bottom left SBPHs20000×, the top right SBPHs-Ca1000×, the middle right SBPHs-Ca10000×, and the bottom right SBPHs-Ca20000×.

[0019] Figure 2 This is a Zeta potential diagram of the bone-derived peptide and calcium peptide chelate of Example 1 of the present invention.

[0020] Figure 3 This is the Fourier transform infrared spectrum of the bone-derived peptide and calcium peptide chelate of Example 1 of the present invention.

[0021] Figure 4 This is the ultraviolet spectrum of the bone-derived peptide and calcium peptide chelate of Example 1 of the present invention.

[0022] Figure 5 This is an X-ray diffraction pattern of the bone-derived peptide and calcium peptide chelate of Example 1 of the present invention.

[0023] Figure 6 This is a graph showing the calcium retention rate of the calcium peptide chelate in simulated gastrointestinal fluid digestion during Example 1 of the present invention. Detailed Implementation

[0024] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1: A method for preparing calcium peptide chelates based on homologous recombination of defatted sheep bone powder, as disclosed in this invention, includes the following steps: S1. Fresh sheep bones are first washed, chopped, and pulverized to below 100 mesh using a high-speed grinder. Residual fat in the sheep bone powder is removed with 4-6 times its volume of anhydrous ethanol. The powder is then dried in an oven at 50°C to constant weight to obtain defatted sheep bone powder. 100g of defatted sheep bone powder is mixed with 1150mL of water and treated in a constant-temperature water bath with shaking for 10 minutes until completely dissolved. Then, 35g of flavor protease is added for enzymatic hydrolysis. During hydrolysis, the pH is adjusted to 7 using 0.5mol / L NaOH and 0.5mol / L HCl solutions. The mixture is then placed in a 50°C constant-temperature shaking incubator for 2 hours with shaking. After hydrolysis, the enzyme is inactivated in a 100°C water bath for 15 minutes. After cooling to room temperature, an enzymatic hydrolysate containing bone-derived peptides is obtained. S2: Add 8 times the volume of 1.8 mol / L lactic acid to the enzymatic hydrolysate obtained in S1, and treat it under constant temperature water bath shaking for 10 min to completely dissolve it. Adjust the pH to be constant with 0.5 mol / L NaOH and 0.5 mol / L HCl solutions, and place it in a 50℃ constant temperature shaking incubator water bath for 50 min to dissolve calcium ions. Cool to room temperature to obtain a mixed solution containing bone peptides and calcium ions. S3 The pH of the mixture obtained in S2 was adjusted to 7 using 0.5 mol / L NaOH and 0.5 mol / L HCl solutions. The chelation reaction was carried out in a 60℃ water bath for 60 min. After the reaction was completed, 5 times the volume of anhydrous ethanol was added to the reaction solution to precipitate the mixture. The precipitate was washed several times with anhydrous ethanol, centrifuged, and then freeze-dried to obtain the calcium peptide chelate.

[0026] Examples 2-13: These are calcium peptide chelates based on homologous recombination of defatted sheep bone powder disclosed in this invention. They were prepared according to the preparation method of Example 1, but differ from Example 1 in that, in S1, this example uses the degree of hydrolysis and salty sensory properties as evaluation indicators to study the effects of different enzyme dosages (3000 U / g, 4000 U / g, 5000 U / g, 6000 U / g, 7000 U / g, 8000 U / g) and enzymatic hydrolysis times (1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h) on bone-derived peptides. The results are shown in Table 1.

[0027] Determination of Enzymatic Hydrolysis Degree (DH): Amino peptide nitrogen (AN) was determined by adding 5 mL of the enzymatic hydrolysis solution and 25 mL of distilled water to a 150 mL Erlenmeyer flask. Titration with 0.5 mol / L NaOH standard solution was carried out while stirring until the pH reached 8.2. Immediately afterwards, 5 mL of formaldehyde solution was added, and titration with 0.5 mol / L NaOH solution was continued while stirring until the pH reached 9.2. The volume of NaOH solution consumed in raising the pH from 8.2 to 9.2 after adding formaldehyde was recorded as V1. Simultaneously, 5.0 mL of distilled water was used for a blank test, recorded as V0. Three parallel experiments were performed, and AN was calculated. DH (%) = AN / TPN * 100%; TPN (measured by Kjeldahl method) is the protein content of the sample.

[0028] Sensory evaluation of saltiness: The sensory evaluation team consisted of 20 members (aged 22-30) all of whom had received sensory training. All samples in the experimental group were uniformly placed in the same beverage cup. Enzymatically hydrolyzed lyophilized powders prepared under different conditions were prepared to a concentration of 10 g / L. Samples were evaluated using a scoring system of 0-10 (1-3 points: slightly salty or unpleasant; 4-7 points: moderately salty; 8-10 points: distinctly salty and rich in flavor). 3.5 g / L NaCl was used as the standard for saltiness evaluation, with a standard score of 5 points. Beverage cups were randomly numbered with three digits. Before tasting, the sensory evaluators rinsed their mouths with purified water, placed 2-3 mL of sample in their mouths for 10 seconds to allow the sample solution to fully disperse in the oral cavity, and rinsed their mouths with purified water after tasting.

[0029] Table 1 Example Enzyme addition amount / U / g Enzymatic hydrolysis time / h Degree of enzymatic hydrolysis / % Saltiness rating 2 3000 2.0 12.1 2.8 3 4000 2.0 13.2 3.95 4 5000 2.0 17.1 3.8 5 6000 2.0 16.4 3.7 6 7000 2.0 18.2 6.4 7 8000 2.0 18.3 6.05 8 7000 1.5 18.6 5.73 9 7000 2.0 19.6 6.64 10 7000 2.5 19.5 6.51 11 7000 3.0 19.4 6.41 12 7000 3.5 19.3 6.2 13 7000 4.0 19.5 6.3 Examples 14-35: These are calcium peptide chelates based on homologous recombination of defatted sheep bone powder disclosed in this invention. They were prepared according to the preparation method of Example 1, but differ from Example 1 in that, in S2, this example uses calcium dissolution rate as the evaluation index to study the effects of different concentrations (0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mol / L), material-liquid ratios (6∶1, 8∶1, 10∶1, 12∶1, 14∶1 and 16∶1), extraction temperatures (40, 50, 60, 70, 80 and 90℃), and extraction times (40, 50, 60, 70, 80 and 90 min) on calcium dissolution. The results are shown in Table 2.

[0030] Calcium content determination: 0.1 mL of CaCl2 solution at different concentrations (0, 2, 4, 6, 8, 10 mg / mL) was added to six 100 mL volumetric flasks, respectively. Then, 10 mL of 0.002 mol / L Acid Chrome Blue K and 5 mL of pH 11.5 sodium tetraborate buffer were added to each flask. Finally, the volume was adjusted to 100 mL with deionized water, and the absorbance (A) at 450 nm was measured. 450nm A standard curve was plotted with CaCl2 concentration on the x-axis and absorbance on the y-axis, and the calcium content was calculated based on the standard curve.

[0031] Table 2 Example lactic acid concentration / mol / L Material-to-liquid ratio / g / mL Hydrolysis temperature / ℃ Hydrolysis time / min Calcium content / mg / mL 14 0.6 1：10 50 60 0.9 15 0.9 1：10 50 60 2.8 16 1.2 1：10 50 60 4.6 17 1.5 1：10 50 60 7.8 18 1.8 1：10 50 60 17.2 19 2.0 1：10 50 60 11.2 20 1.8 1：6 50 60 9.8 21 1.8 1：8 50 60 10.9 22 1.8 1：10 50 60 9.8 23 1.8 1：12 50 60 9.6 24 1.8 1：14 50 60 9.0 25 1.8 1：16 50 60 4.1 26 1.8 1：8 40 60 14.6 27 1.8 1：8 50 60 17.2 28 1.8 1：8 60 60 15.9 29 1.8 1：8 70 60 11.1 30 1.8 1：8 80 60 7.8 31 1.8 1：8 50 40 18.2 32 1.8 1：8 50 50 22.13 33 1.8 1：8 50 60 21.2 34 1.8 1：8 50 70 20.3 35 1.8 1：8 50 80 21.1 Examples 36-50: These are calcium peptide chelates based on homologous recombination of defatted sheep bone powder disclosed in this invention. They were prepared according to the preparation method of Example 1, but differ from Example 1 in that, in S3, this example uses chelation rate and salty sensory properties as evaluation indicators to study the effects of different pH (4, 5, 6, 7, 8, 9), chelation time (30, 40, 50, 60, 70, 80 min), and chelation temperature (30, 40, 50, 60, 70 °C) on calcium chelation of bone peptides. The results are shown in Table 3.

[0032] Measurement indicators and methods Chelation rate determination: 0.1 mL of CaCl2 solutions of different concentrations (0, 2, 4, 6, 8, 10 mg / mL) were added to six 100 mL volumetric flasks, respectively. Then, 10 mL of 0.002 mol / L Acid Chrome Blue K and 5 mL of pH 11.5 sodium tetraborate buffer were added to each flask. Finally, the volume was adjusted to 100 mL with deionized water. The absorbance at 450 nm was measured. A standard curve was plotted with CaCl2 concentration on the x-axis and absorbance on the y-axis. The calcium content and chelation rate were calculated based on the standard curve.

[0033] Sensory evaluation of saltiness and bitterness: The sensory evaluation team consisted of 20 members (aged 22-30) all of whom had received sensory training. All samples in the experimental group were uniformly placed in the same beverage cup. Enzymatically hydrolyzed lyophilized powders prepared under different conditions were prepared to a concentration of 10 g / L. Samples were evaluated using a scoring system of 0-10 (1-3 points: weak or unpleasant taste; 4-7 points: moderate taste; 8-10 points: distinct and rich taste). 3.5 g / L NaCl and 0.8 g / L quinine were used as evaluation standards for saltiness and bitterness, with a standard score of 5. Beverage cups were randomly numbered with three digits. Before tasting, the sensory evaluators rinsed their mouths with purified water, placed 2-3 mL of sample in their mouths for 10 seconds to allow the sample solution to fully disperse in the oral cavity, and rinsed their mouths with purified water after tasting.

[0034] Table 3 Example Reaction time / min Reaction temperature / ℃ reaction pH Chelation rate / % Saltiness rating Bitterness rating 36 30 50 7 81.5 2.8 2.5 37 40 50 7 85.6 2.4 2.4 38 50 50 7 85.1 2.7 2.6 39 60 50 7 88.7 3.6 2.1 40 70 50 7 82.4 2.3 2.8 41 60 30 7 75.9 2.8 2.9 42 60 40 7 83.6 3 3.2 43 60 50 7 84.1 3.1 3.3 44 60 60 7 88.9 4.3 3.1 45 60 70 7 80.7 3.4 3.2 46 60 60 5 86 2.2 3.1 47 60 60 6 89.1 3.9 3.1 48 60 60 7 89.3 5 2.4 49 60 60 8 84.6 5 3.2 50 60 60 9 82 3.5 3.3 Example 51: This invention discloses an application of a calcium peptide chelate based on homologous recombination of defatted sheep bone powder. The calcium peptide chelate prepared according to the preparation method of Example 1 is used as a saltiness enhancer, salt reduction aid, or flavor enhancer in food. The food includes, but is not limited to, meat products, dairy products, noodle soup bases, seasonings, and pre-prepared prepared foods.

[0035] (1) Sensory experiment to verify the synergistic saltiness enhancement of peptide-calcium chelates using the 3-AFC forced selection method All 40 evaluators selected the peptide-calcium chelate (Sample A: calcium-peptide chelate solution prepared in Example 1) as the saltiest sample (100%), while the free calcium solution (Sample B, free calcium ion solution) and the pure peptide solution (Sample C, pure peptide solution) were not selected (0%). The 95% confidence interval calculated using the Clopper-Pearson method shows that even under the most conservative estimate, the proportion of the real population selecting Sample A as the "saltiest" is still as high as 91.2% [91.2%, 100%], while the proportion selecting Sample B or C is no more than 8.8% [0%, 8.8%]. This confidence interval completely excludes the random probability of 33.3%, further supporting the conclusion that the peptide-calcium chelate significantly enhances the perception of saltiness.

[0036] (2) Stability of Peptide Calcium in Gastrointestinal Fluid 1) Simulated gastric juice digestion: The preparation method of simulated gastric juice SGF is as follows: Take 0.384 mL of concentrated hydrochloric acid and add it to 80 mL of deionized water. After mixing, add 1 g of pepsin and dissolve it completely. Then add water to make up to 100 mL to obtain simulated human gastrointestinal juice. At this time, the pH value of simulated gastric juice is about 2.0. Prepare 10 mg / mL peptide calcium chelate. Take 20 mL of the above solution into 100 mL conical flasks and adjust the pH to 2.0 with 1 mol / L HCl solution. Add 20 mL of simulated gastric juice SGF and shake in a 37℃ water bath (0, 15, 30, 60, 120 min). Then remove and place in boiling water for 5 min to inactivate the enzyme. After the sample is cooled to room temperature, centrifuge at 8000 rpm for 10 min using a refrigerated centrifuge. Take the supernatant and place it in a digestion tube. Then determine the free calcium content by flame atomic absorption spectrometry.

[0037] 2) Simulated intestinal fluid digestion: Preparation of simulated intestinal fluid SIF: Dissolve 0.68g of KH2PO4 in 50mL of water and adjust the pH to 6.8 with NaOH; dissolve 1g of trypsin in a small amount of water, mix the two solutions and add water to 100mL; after the above simulated gastric fluid digestion for 120min, inactivate the enzyme in a boiling water bath for 5min, adjust the pH to 7.6 with 0.5mol / L NaOH solution, add 5mL of intestinal fluid SIF, shake in a 37℃ water bath for 0, 15, 30, 60 and 120min respectively, then inactivate the enzyme in a 100℃ water bath for 5min. After the sample is cooled to room temperature, centrifuge at 8000rpm for 10min using a refrigerated centrifuge, determine the free calcium content by flame atomic absorption spectrometry, and calculate the calcium retention rate of the sample.

[0038] (3) Structural characterization experiments: 1) Scanning Electron Microscopy (SEM): Freeze-dried bone peptides (SBPHs) and calcium peptide chelates (SBPHs-Ca) powder samples were uniformly coated onto the SEM sample column and sputter-coated with gold. The scanning conditions were: accelerating voltage 15.0 kV, beam current 6.9 × 10⁻⁶. −2 mA, working distance 6.7mm. The image was clarified by adjusting the focus. Morphology was observed at magnifications of 1000× and 20000×, and the results are as follows. Figure 1 As shown.

[0039] from Figure 1 It can be seen that the surface of SBPHs exhibits a compact, smooth, sheet-like structure with an irregular shape, while the surface of SBPHs-Ca appears loose, with many pores of varying sizes, exhibiting a flocculent structure, which is significantly different from SBPHs. This difference may be due to the different properties of SBPHs and Ca... 2+ The coordination bonds formed by these bonds lead to the folding and aggregation of the internal structure, disrupting the original dense structure of SBPHs. Furthermore, the bridging effect formed by the binding of amino and carboxyl groups in the peptides to calcium also alters the surface properties.

[0040] 2) Zeta potential: The zeta potential of SBPHs and SBPHs-Ca was analyzed using a Zetasizer Nano ZS90 particle size analyzer. The sample (1 mg / mL) was added to a U-shaped cell and equilibrated at 25°C for 60 s. All measurements were performed at 25°C and repeated 12 times. The results are as follows: Figure 2 As shown.

[0041] from Figure 2 As can be seen, the Zeta potential is a physicochemical index that reflects the surface charge state of a material. The figure shows that, compared to SBPHs, the Zeta potential of SBPHs-Ca decreases significantly from -11.7 mV to -32.4 mV.

[0042] 3) Fourier transform infrared spectroscopy analysis: Take 3 mg each of lyophilized SBPHs and SBPHs-Ca, place them in an agate mortar, add 100 mg of dried, spectroscopically pure KBr, mix and grind evenly until it adheres to the wall, place in the measuring chamber, and use a Fourier transform infrared spectrometer at 400~4000 cm⁻¹. -1 The infrared spectrum obtained by scanning was processed using OMNIC 8.2 data processing software. The spectrum was obtained through 64 scans with a resolution of 4 cm⁻¹. -1 / 2cm -1 The result is as follows Figure 3 As shown.

[0043] from Figure 3 It can be seen that 3500~3100cm -1The absorption peak is due to the stretching vibration of the NH and OH bonds. At 3242.73 cm⁻¹ -1 The amide A band of SBPHs was observed, which is associated with NH stretching. Nitrogen atoms can form coordination bonds with calcium ions by donating electron pairs; when peptide-calcium chelates are formed, this band shifts to 3245.328 cm⁻¹. -1 This indicates that the -N-Ca bond has replaced the NH group. Important vibrational modes of the amide are located at 1700 and 1600 cm⁻¹. -1 The amide I band between these wavelengths primarily originates from the C=O stretching vibration of peptide bonds. In SBPHs-Ca, it is located at 1636.61 cm⁻¹. -1 The SBPHs band shifted to 1648.33 cm. -1 This indicates that C=O plays a crucial role in covalent bonding reactions. 531.99cm -1 The wavenumber changes at the ligase are caused by the stretching and deformation vibrations of single bonds. These results indicate that the carboxyl oxygen and amino nitrogen atoms play a major role in the chelation of peptides with calcium. It can be seen that the infrared spectrum of the peptides used in this study changed significantly after chelation with calcium, confirming that chelation occurred between the peptides and calcium, forming new compounds.

[0044] 4) Ultraviolet spectroscopy analysis: SBPHs and SBPHs-Ca were dissolved to a concentration of 1 mg / mL. The absorbance was recorded using a TV-1810 spectrophotometer in the wavelength range of 250-400 nm. Deionized water was used as a blank for calibration before measurement. The results are as follows: Figure 4 As shown.

[0045] from Figure 4 As can be seen, a distinct absorption peak is present at approximately 300 nm, with the absorbance decreasing from 0.64 to 0.60, and the wavelength of the absorption peak also changing. This indicates that the peptide and Ca²⁺ did indeed undergo a chelation reaction, accompanied by the formation of a new substance. This may be due to the transformation of n→π* electrons in the ligand (C=O). After the peptide calcium chelation, the electron cloud of the amide bond and the absorption characteristics of the ligand change. Studies have shown that the chirality of chromophores (C=O and -COOH) and auxochromes (-OH and -NH₂) can change due to the binding of ligands to calcium ions. The above results can preliminarily prove the existence of binding behavior between SBPHs and Ca²⁺.

[0046] 4) X-ray diffraction: Following the method of Zhang et al., SBPHs and SBPHs-Ca were uniformly ground, loaded into a sample plate, and placed in a support. Using Cu as the target anode material, the crystal structures of SBPHs and SBPHs-Ca were studied using X-ray diffraction. The scanning angle was 2θ, 10-80°, 40kV, 40mA, and the scanning speed was 5° / min, employing continuous scanning. The results are shown below. Figure 5 As shown.

[0047] from Figure 5 As can be seen, SBPHs exhibit multiple sharp diffraction peaks, proving that they possess a long-range ordered crystal structure. However, after chelation with calcium ions, all characteristic diffraction peaks disappear, leaving only a diffuse background, indicating that SBPHs-Ca is amorphous. The disappearance of the crystal structure is attributed to the coordination of Ca²⁺ with the peptide chain, disrupting the original hydrogen bond network and molecular stacking mode. This change is consistent with the typical structural characteristics of peptide-calcium chelates and is beneficial for improving calcium solubility and bioavailability.

[0048] 5) Dietary nutrients are typically digested in the stomach and then absorbed in the intestines: the stability of calcium supplements in the gastrointestinal tract directly affects the body's absorption and utilization of calcium. In the gastrointestinal environment, H... + Factors such as proteases can induce calcium release, producing insoluble precipitates and Ca(OH)2, leading to reduced calcium bioavailability. Therefore, evaluating the stability of SBPHs-Ca in a simulated gastrointestinal environment is of great significance.

[0049] like Figure 6 Simulated gastrointestinal digestion revealed that during the gastric digestion stage, the calcium retention rate was 53%, with a significant release of calcium ions in the stomach. This indicates that SBPHs-Ca underwent partial dissociation under the action of pepsin. It also suggests that SBPHs-Ca is significantly affected by pH; under acidic conditions, H+... + Peptides readily compete with calcium ions for electron-donating groups. Acidic conditions can also lead to certain structural changes in peptides, affecting their ability to bind calcium ions. Simultaneously, pepsin induces the degradation of chelates; peptides are hydrolyzed by pepsin into small peptides or amino acids. Some small peptides are further degraded into amino acids by brush border enzymes and absorbed, while others are absorbed intact. Calcium retention increases in the intestine, resulting in good stability. Studies have shown that SBPHs-Ca exhibits some tolerance to trypsin, and neutral conditions are beneficial for maintaining the stability of peptide-calcium chelates. It has also been found that calcium ions dissociate from the peptide-calcium complex during gastric digestion, but spontaneously bind to peptides at appropriate pH during intestinal digestion. Therefore, SBPHs-Ca possesses good stability and high calcium bioavailability, making it suitable for use as a food ingredient.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing calcium peptide chelates based on homologous recombination of defatted sheep bone powder, characterized in that: Includes the following steps, S1 mix defatted sheep bone powder with water, add flavor protease for enzymatic hydrolysis, and inactivate the enzyme after hydrolysis to obtain an enzymatic hydrolysate containing bone peptides. S2 adds lactic acid to the enzymatic hydrolysate obtained in S1 to hydrolyze calcium ions, thereby dissolving calcium ions and obtaining a mixture containing bone-derived peptides and calcium ions. S3 involves chelating the mixture obtained in S2 under neutral conditions, followed by post-processing to obtain calcium peptide chelates.

2. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In step S1, fresh sheep bones are pre-washed, chopped, and pulverized to below 100 mesh using a high-speed pulverizer. The residual fat in the sheep bone powder is removed with 4 to 6 times its volume of anhydrous ethanol, and then dried in an oven at 40 to 60°C to constant weight to obtain defatted sheep bone powder.

3. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In S1, the amount of flavor protease added is controlled to be 6000~8000 U / g defatted sheep bone powder, the enzymatic hydrolysis temperature is 40~60℃, the enzymatic hydrolysis time is 2~4h, and the enzymatic hydrolysis pH is 6~8.

4. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In step S1, after enzymatic hydrolysis, the enzyme is inactivated at 90-110°C for 5-15 minutes to obtain an enzymatic hydrolysate containing bone-derived peptides.

5. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In S2, the concentration of lactic acid is controlled at 1.8~2.0 mol / L, the ratio of enzymatic hydrolysate to lactic acid is 1:6~12, the hydrolysis temperature is 40~60℃, and the hydrolysis time is 40~60 min.

6. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In S3, the pH of the chelation reaction is controlled at 6.5~7.5, the reaction temperature at 55~65℃, and the reaction time at 55~65min.

7. The method for preparing a calcium peptide chelate based on homologous recombination of defatted sheep bone powder according to claim 1, characterized in that: In step S3, after the reaction is complete, 4 to 6 times the volume of anhydrous ethanol is added to the reaction solution for precipitation. The precipitate is then washed multiple times with anhydrous ethanol, centrifuged, and freeze-dried to obtain the calcium peptide chelate.

8. A calcium peptide chelate, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. An application of a calcium peptide chelate, characterized in that: The application of calcium peptide chelates prepared by any one of the preparation methods according to claims 1 to 7 in food as saltiness enhancers, salt reduction aids or flavor enhancers.

10. The application of the calcium peptide chelate according to claim 9, characterized in that: The food products include meat products, dairy products, noodle soup bases, seasonings, and pre-prepared foods.

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