Calcium peptide chelate based on bovine bone collagen, preparation method and application

Abstract

The invention belongs to the technical field of calcium peptide chelates, and particularly relates to a calcium peptide chelate based on bovine bone collagen, a preparation method and application. According to the calcium peptide chelate and the preparation method thereof, bovine bone collagen serves as chelating peptide and reacts with a calcium agent in a deep eutectic solvent medium, the calcium peptide chelate is provided, it is verified that the chelating peptide has good solubility in a deep eutectic solvent, the calcium chelation rate is effectively increased, and the prepared calcium peptide chelate has good gastrointestinal stability and can be used for preparing the calcium peptide chelate. When the compound is applied to the calcium supplement, adverse stimulation to the stomach can be effectively avoided, and the bioavailability is improved.

Description

Technical Field

[0001] This invention belongs to the field of calcium peptide chelate technology, specifically relating to a calcium peptide chelate based on bovine bone collagen, its preparation method, and the application of the calcium peptide chelate in the preparation of calcium supplements. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Calcium peptide chelates are composed of the carboxyl and amino groups of a peptide chain and calcium. 2+ The water-soluble complex formed by stable coordination can be directly absorbed by the intestinal mucosa without the need for gastric acid mediation, increasing absorption efficiency to over 60%, making it a more active calcium supplement. Conventional calcium supplements (such as calcium carbonate and calcium phosphate) easily form insoluble precipitates in the gastrointestinal tract, relying on gastric acid for dissolution, resulting in low absorption efficiency. The dissolution of calcium supplements can also lead to an increase in local pH, irritating the gastrointestinal mucosa, and unabsorbed calcium can easily deposit in the intestines, causing constipation. In contrast, the peptide chains in calcium peptide chelates act as carriers, avoiding the formation of calcium precipitates. 2+ It forms precipitates with oxalic acid and phytic acid, while the peptide chains promote intestinal peristalsis and alleviate calcium deposition problems. Furthermore, the biocompatibility of chelated calcium comes from natural collagen peptides, with no chemical additive residues, making it safer for long-term use.

[0004] In view of the current state of research, this invention suggests that using peptide chains with antioxidant activity as chelating peptides in the preparation process may have a synergistic effect with calcium, thereby achieving synergistic supplementation of calcium and collagen and jointly maintaining bone and joint function. Summary of the Invention

[0005] To achieve the above-mentioned technical objectives, in a first aspect, the present invention provides a calcium peptide chelate based on bovine bone collagen, wherein the chelate uses bovine bone collagen with a molecular weight of 500-3000 Da as a chelating peptide to bind with calcium salt.

[0006] Existing research indicates that eutectic solvents (DES) can enhance the chelation efficiency of metal ions and biomolecules (such as peptides and polysaccharides) compared to traditional aqueous solutions, and the resulting products exhibit good stability. This invention investigates chelated peptides and chelation processes. Results show that chelated peptides derived from bovine bone show significantly improved solubility in the DES system, and the prepared chelates exhibit good calcium binding rates and stability.

[0007] Therefore, in a second aspect, the present invention provides a method for preparing the calcium peptide chelate described in the first aspect, comprising the following steps: dissolving a chelate peptide with a molecular weight of 500-3000 Da in a eutectic solvent, adding a calcium salt, and reacting at 40-60°C for 1-3 hours; wherein the eutectic solvent comprises at least choline chloride.

[0008] The aforementioned chelated peptides are derived from enzymatic hydrolysates of bovine bone, hide, fish bone, or fish skin. In some embodiments with better results, the chelated peptides are enzymatic hydrolysates of bovine bone, prepared as follows: decalcified bone powder is added to a compound enzyme and enzymatically hydrolyzed at 40-50°C for 20-40 minutes. After the enzymatic hydrolysis reaction, the fraction with a molecular weight of 500-3000 Da is selected by ultrafiltration. Further, the compound enzyme is obtained by mixing trypsin, alkaline protease, and collagenase in a mass ratio of 1-3:3-6:3-6.

[0009] In other embodiments, in order to ensure the binding efficiency of the chelating peptide, those skilled in the art also perform purification operations such as desalting on the chelating peptide.

[0010] The aforementioned eutectic solvent includes choline chloride, and also includes one or more of urea, glycerol, and ethylene glycol. In one embodiment of the present invention, the eutectic solvent is obtained by mixing choline chloride and urea at a mass ratio of 1:2.

[0011] In another embodiment, the eutectic solvent is obtained by mixing choline chloride and glycerol in a mass ratio of 1:2.

[0012] In another embodiment, the eutectic solvent is obtained by mixing choline chloride and ethylene glycol in a mass ratio of 1:3.

[0013] The calcium salts mentioned above are preferably inorganic salts, and are inorganic salts with good solubility, such as calcium chloride; in some embodiments of the present invention with better effects, food-grade calcium chloride is used, and the molar ratio of the chelating peptide to the calcium ions in the calcium salt is 1:2~3.

[0014] In some embodiments of the present invention, after the above chelation reaction is completed, alcohol is added to the reaction system to allow the calcium peptide chelate to fully precipitate, and the precipitate is separated by centrifugation. The alcohol is preferably a less toxic alcohol, such as ethanol, and the amount of ethanol added is 2 to 4 times the volume of the reaction system.

[0015] In a third aspect, the present invention provides the application of the calcium peptide chelate described in the first aspect and the method for preparing the calcium peptide chelate described in the second aspect.

[0016] The applications described in the third aspect above include, but are not limited to, applications in the preparation of food, health products, pharmaceuticals, daily chemical products, cosmetics, and agricultural products.

[0017] Specific examples of the aforementioned foods include beverages, dairy products, cereal products, snack foods, and sports-related foods.

[0018] Examples of the aforementioned health products include calcium supplements or compound nutritional preparations.

[0019] Examples of the aforementioned pharmaceuticals include orthopedic adjuvant drugs, medical excipients, and drugs for the prevention and treatment of osteoporosis.

[0020] The aforementioned agricultural products include foliar fertilizers and seedling nutrients.

[0021] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a calcium peptide chelate by combining highly active bovine collagen peptides as chelating peptides with calcium supplements. In the preparation process of this chelate, the invention incorporates a eutectic solvent as the reaction medium to enhance the binding of the peptides and calcium supplements. Verification has shown that bovine bone-derived collagen peptides exhibit good solubility in eutectic solvents, and the prepared calcium peptide chelate also demonstrates good gastrointestinal stability, overcoming the drawbacks of ordinary calcium supplements such as gastrointestinal irritation and easy deposition, thus achieving simultaneous supplementation of calcium and collagen. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 The solubility of chelated peptides from different biological sources in the DES system; Figure 2 The infrared spectra of the chelate peptide and calcium peptide chelate described in Example 1 are compared. Detailed Implementation

[0024] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] As described in the background section, conventional calcium supplements suffer from drawbacks such as low bioavailability and gastrointestinal irritation. To address these technical problems, this invention provides a calcium peptide chelate. Specifically, this invention examines the selection of the chelating peptide and the solvent used in the chelation process. The specific examination is as follows: I. Selection of Chelate Peptides (I) Research Methods 1. Pretreatment of Collagen Peptides in Biological Samples Beef bones: Wash the beef bones to remove impurities and blood, air dry them naturally, then crush them at high speed and pass them through a 40-mesh sieve. Add the crushed beef bone powder to a 1 mol / L hydrochloric acid solution at a material-to-liquid ratio of 1 g: 12 mL, stir at 25°C for 4 h to remove calcium, and then wash with distilled water until neutral to obtain decalcified bone powder.

[0027] Cowhide: Cowhide pieces are rinsed with running water to remove surface hair, blood clots and dirt, then soaked in 40℃ warm water for 20 minutes to soften the epidermal stratum corneum. They are then placed in a high-speed tissue homogenizer, an equal volume of deionized water is added, and the mixture is homogenized for 3 minutes (15000 r / min) to obtain a cowhide homogenate with a particle size ≤5mm. 0.6mol / L hydrochloric acid solution is added at a material-to-liquid ratio of 1g:12mL, and the mixture is stirred at 25℃ for 4 hours to obtain degreased cowhide.

[0028] Fish bones: same as beef bones.

[0029] Fish skin: Same as cowhide.

[0030] 2. Collagen peptide extraction methods The pretreated biological sample was added to a compound enzyme and enzymatically hydrolyzed at 40-50℃ for 30 min. Peptides with a molecular weight of 500-3000 Da were screened from the hydrolysate and desalted to obtain collagen peptides. The compound enzyme was obtained by mixing trypsin, alkaline protease, and collagenase in a mass ratio of 2:5:5.

[0031] 3. Selection of eutectic solvent Peptides ranging from 500 to 3000 Da were dissolved at 5 mg / mL in different DES solutions, and the dissolution was observed (25℃, stirring for 30 min). DES systems with a solubility ≥ 20 mg / mL and clear, precipitate-free solutions were selected, while physiological saline was used as the control group.

[0032] Table 1. DES systems and preparation methods to be investigated (II) Results of the Investigation In this embodiment, four chelate peptides from biological samples were dissolved in physiological saline (control group) and three DES media, respectively. The solubility results are as follows: Figure 1As shown: In the control group, chelated peptides derived from fish skin and fish bones had good solubility in physiological saline. However, the solubility was slightly improved after the addition of DES medium. In contrast, chelated peptides derived from bovine bones showed good solubility improvement in all three DES media.

[0033] II. Preparation of calcium peptide chelates (I) Preparation method Chelated peptides derived from bovine bone were fully dissolved in three DES systems, according to the ratio of chelated peptides to Ca... 2+ Food-grade calcium chloride was slowly added at a molar ratio of 1:2.5 and stirred in a constant temperature water bath at 55°C for 1.5 hours. After the reaction was completed, 3 times the volume of anhydrous ethanol was added and stirred to allow the calcium peptide chelate to fully precipitate. The precipitate was then centrifuged and retained to obtain the final product.

[0034] The control group used physiological saline as the reaction solvent.

[0035] (II) Evaluation Indicators 1. Determination of calcium chelation degree by atomic absorption spectrophotometry Total calcium determination: Add 20 mL of 1 mol / L hydrochloric acid, sonicate for 30 min, transfer to a 100 mL volumetric flask, dilute to the mark with deionized water, shake well, and filter through a 0.22 μm filter membrane for later use. Determination of free calcium: Add 20 mL of deionized water, stir magnetically for 30 min, centrifuge at 12000 r / min for 20 min (4℃), transfer the supernatant to a 100 mL volumetric flask, dilute to the mark with deionized water, filter through a 0.22 μm filter membrane, and set aside. Prepare a series of calcium standard stock solutions with different concentrations. Measure and plot standard curves under the conditions of a wavelength of 422.7 nm, a lamp current of 10 mA, a slit width of 0.5 nm, and an air-acetylene flame (air flow rate 10 L / min, acetylene flow rate 2 L / min). Calcium content is calculated as follows: Calcium content (mg / g) = (c × V) / m Where: c = calcium concentration calculated by the regression equation (μg / mL), V = fixed volume (100mL), m = sample mass (g).

[0036] Chelation rate (%) = [(Total calcium content - Free calcium content) / Total calcium content] × 100% 2. Stability Evaluation Gastrointestinal digestion (pH 2.0 → pH 7.5) stability: A calcium peptide chelate sample was weighed and dissolved in deionized water to prepare a 10 mg / mL solution. 2 mL of this solution was added to 8 mL of artificial gastric juice (pH 2.0, containing pepsin, enzyme activity 2000 U / mL), and incubated at 37°C with shaking for 2 h. Subsequently, the pH was adjusted to 7.5, and 10 mL of artificial intestinal juice (containing trypsin, enzyme activity 1000 U / mL) was added, followed by incubation at 37°C for another 4 h. Samples were taken at 0 h, 2 h, and 6 h of digestion, centrifuged (10000 r / min, 15 min), and the supernatant was collected. The free calcium content was determined by atomic absorption spectrophotometry, and the calcium retention rate and chelation rate at different time points were calculated.

[0037] Thermal stability evaluation: Accurately weigh the calcium peptide chelate sample and prepare a 10 mg / mL solution with deionized water. Take 2 mL of each solution and place it in a sealed centrifuge tube. Heat at 60℃, 80℃, and 100℃ for 1 h, 2 h, and 4 h, respectively. After heating, cool to room temperature, centrifuge (10000 r / min, 15 min), collect the supernatant, and determine the free calcium content by atomic absorption spectrophotometry. Calculate the calcium retention rate and chelation rate at different temperatures and time points.

[0038] Calcium retention rate (%) = Bound calcium / Total calcium × 100%; Chelation rate residual rate = chelation rate after heating / initial chelation rate × 100%.

[0039] 3. Structural verification FT-IR spectroscopy analysis confirmed the binding of calcium ions to peptides.

[0040] III. Research Results The chelation rate and gastrointestinal stability of the above-mentioned calcium peptide chelates are shown in Table 2 below: Table 2. Chelation rate and gastrointestinal stability of calcium peptide chelates in different DES media. As shown in Table 2, using a eutectic solvent as the reaction medium effectively improves the binding efficiency of calcium and chelate peptides. Furthermore, the calcium peptide chelates prepared by the above method have better stability in the gastrointestinal environment, effectively avoiding the concentrated release of calcium in the stomach.

[0041] The thermal stability results are shown in Table 3 below: Table 3. Thermal stability results of calcium peptide chelates FT-IR spectra such as Figure 2 As shown, 3420 cm of chelate peptide -1 A broad and strong absorption peak (the strongest peak), covering the stretching vibrations of amino and hydroxyl groups; 1655 cm⁻¹ -11548 cm -1 1335 cm -1 Strong to moderate intensity amide I, II, and III bands (characteristic peaks of peptide bonds) are the hallmark peaks of collagen peptides; After chelation: 3438 cm -1 A broad and strong absorption peak (strongest peak), with a peak shape slightly narrower than that of chelate peptides; 1722 cm⁻¹ -1 Complete disappearance (all carboxyl groups are deprotonated), new Ca-O coordination bond peak (580 cm⁻¹). -1 ).

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1 In this embodiment, a calcium peptide chelate based on bovine bone collagen is provided, the preparation method of which includes the following steps: (1) Preparation of decalcified bone meal: Wash the bovine bones to remove impurities and blood, air dry them naturally, then crush them at high speed and pass them through a 40-mesh sieve. Add the crushed bovine bone meal to a 1 mol / L hydrochloric acid solution at a material-to-liquid ratio of 1 g: 12 mL. Stir at 25°C for 4 h to remove calcium. Then wash with distilled water until neutral to obtain decalcified bone meal.

[0044] (2) Enzymatic hydrolysis: The decalcified bone powder obtained in the step is dissolved in 0.9wt% physiological saline, and 0.2% trypsin, 0.5% alkaline protease and 0.5% collagenase by mass of the system are added for compound enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 40-50℃ for 30min, and the enzyme is inactivated at 80-100℃ to obtain the enzymatic hydrolysate.

[0045] (3) Chelated peptide screening: The enzymatic hydrolysate obtained in step (2) was first centrifuged at high speed (12000 r / min, 20 min, 4℃) to remove insoluble impurities (undigested bone meal particles, denatured proteins); the supernatant was filtered through a 0.22 μm filter membrane to remove small suspended solids and colloids, resulting in a clear enzymatic hydrolysate. 500-3000 Da peptides were retained by ultrafiltration membrane, and after dialysis and desalting using a dialysis bag (MWCO 500 Da) for 24 h, the chelated peptides were concentrated.

[0046] (4) Preparation of calcium peptide chelates: Add 20 mL of pretreated choline chloride-urea (1:2) solvent to a 50 mL reaction flask, and add the above chelated peptide at a peptide concentration of 15 mg / mL. Stir magnetically at 25 °C for 30 min to ensure complete dissolution of the peptide. The chelated peptide and Ca... 2+ Food-grade calcium chloride was slowly added at a molar ratio of 1:2.5, with continuous stirring until completely dissolved. The mixture was then reacted in a 55°C water bath with stirring for 1.5 hours. After the reaction was complete, the system was cooled to room temperature, and three volumes of anhydrous ethanol were added and stirred to allow the calcium peptide chelate to precipitate completely. The precipitate was collected by centrifugation at 10,000 rpm for 15 min, washed twice with anhydrous ethanol (to remove residual DES), and then dissolved in a small amount of deionized water. The solution was filtered through a 0.22 μm filter membrane and then dialyzed with a dialysis bag (MWCO 500 Da) for 24 h, during which the deionized water was replaced at least three times. The dialyzed solution was then vacuum dried (60 °C, 0.08 MPa) for 12 h to obtain the calcium peptide chelate.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A calcium peptide chelate based on bovine bone collagen, characterized in that, The chelate is combined with calcium salt by using bovine bone collagen with molecular weight of 500-3000 Da as chelating peptide.

2. The method for preparing the calcium peptide chelate according to claim 1, characterized by, The method comprises the following steps: dissolving chelating peptide with molecular weight of 500-3000 Da in a eutectic solvent, adding calcium salt, and reacting at 40-60 ℃ for 1-3 h to prepare; the eutectic solvent comprises at least choline chloride.

3. The method for preparing the calcium peptide chelate as described in claim 2, characterized in that, The chelating peptide is derived from the enzymatic hydrolysate of bovine bone, bovine skin, fish bone or fish skin. Preferably, the chelating peptide is the enzymatic hydrolysate of bovine bone, and the preparation method is as follows: adding decalcified bone powder to a complex enzyme and enzymatically hydrolyzing at 40-50 ℃ for 20-40 min; after the enzymatic hydrolysis reaction is completed, the part with molecular weight of 500-3000 Da is selected by ultrafiltration.

4. The method for preparing the calcium peptide chelate as described in claim 3, characterized in that, The complex enzyme is a mixture of trypsin, alkaline protease and collagenase in a mass ratio of 1-3:3-6:3-6.

5. The method of claim 2, wherein the calcium peptide chelate is prepared by the steps of: a) dissolving the calcium peptide chelate in a solvent; b) filtering the solution; c) evaporating the solvent; and d) drying the calcium peptide chelate. The eutectic solvent comprises choline chloride and one or a combination of urea, glycerol and ethylene glycol.

6. The method of claim 5, wherein the calcium peptide chelate is prepared by the steps of: a) dissolving the calcium peptide chelate in a solvent; b) filtering the solution; c) evaporating the solvent; and d) drying the calcium peptide chelate. The eutectic solvent is a mixture of choline chloride and urea in a mass ratio of 1:

2. Or, the eutectic solvent is a mixture of choline chloride and glycerol in a mass ratio of 1:

2. Or, the eutectic solvent is a mixture of choline chloride and ethylene glycol in a mass ratio of 1:

3.

7. The method of claim 2, wherein the calcium peptide chelate is prepared by the steps of: a) dissolving the calcium peptide chelate in a solvent; b) filtering the solution; c) evaporating the solvent; and d) drying the calcium peptide chelate. The calcium salt is calcium chloride; further, it is food-grade calcium chloride, and the molar ratio of the chelating peptide to calcium ions in the calcium salt is 1:2-3.

8. The method for preparing the calcium peptide chelate as described in claim 2, characterized in that, After the chelation reaction is completed, alcohol is added to the reaction system to fully precipitate the calcium peptide chelate, and the precipitate is separated by centrifugation; the alcohol is preferably ethanol, and the amount of ethanol added is 2-4 times the amount of the reaction system.

9. The calcium peptide chelate of claim 1, or the method for preparing the calcium peptide chelate of any one of claims 2-8.

10. Use according to claim 9, wherein The application includes but is not limited to the application in the preparation of food, health products, medicines, daily chemical products, beauty products, agricultural products. The food is a beverage, a dairy product, a cereal product, a snack food or a sports influencing food. The health product is a calcium supplement or a compound nutritional preparation. The medicine is an orthopedic auxiliary drug, a medical auxiliary material or a drug for preventing and treating osteoporosis. The agricultural product is a foliar fertilizer or a seedling nutrient agent.

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