Preparation method of high-absorptivity calcium supplement containing vitamin D3 and casein phosphopeptide chelated calcium
By combining casein phosphopeptide with vitamin D3 to prepare a calcium supplement with high absorption rate, the problem of low calcium absorption rate is solved, and efficient absorption and bioavailability of calcium throughout the intestine are achieved.
Patent Information
- Application Number
- CN202511630861.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-10
- Publication Date
- 2026-01-02
AI Technical Summary
Existing calcium supplements have low absorption rates and are easily affected by dietary factors. Traditional calcium supplements have an absorption rate of only 20% to 30% in the small intestine, making it difficult to effectively improve the bioavailability of calcium.
A calcium supplement with high absorption rate was prepared by combining casein phosphopeptide (CPP) and vitamin D3 (VD3). A soluble phosphate complex was formed through a chelation reaction, which promoted the absorption and utilization of calcium. The preparation process conditions were optimized to improve the chelation rate and binding rate.
It significantly improves calcium absorption and bioavailability, covering calcium absorption throughout the entire intestine, especially achieving higher calcium absorption in the distal small intestine and ileum, thus solving the problem of low absorption rate of traditional calcium supplements.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of food processing, and particularly relates to a preparation method of a high-absorption calcium supplement of vitamin D3 and casein phosphopeptide chelated calcium. BACKGROUND
[0002] Osteoporosis (OP) is a metabolic bone disease characterized by bone mass loss, bone microstructure destruction and bone fragility increase, mainly affecting the middle-aged and elderly population, especially postmenopausal women. Since calcium metabolism imbalance is the core mechanism of osteoporosis, low calcium content leads to the occurrence of osteoporosis, therefore, prevention and treatment of osteoporosis requires improving the absorption and utilization rate of calcium. Traditional calcium supplement strategies face two major challenges of low absorption rate and poor utilization rate. The absorption rate of ordinary calcium agents (such as calcium carbonate) in the small intestine is only 20% to 30%, and is easily affected by dietary factors such as phytic acid and oxalic acid to form insoluble precipitates, therefore, developing efficient and safe calcium absorption promoters has become a research hotspot in the field of osteoporosis prevention and treatment.
[0003] Casein phosphopeptide (CPP) is a natural bioactive polypeptide obtained by hydrolysis of bovine milk casein from the nitrogen end of trypsin or trypsin. The core structure of CPP is: -Ser (P) -Ser (P) -Ser (P) -Glu-Glu- (Ser: serine, Glu: glutamic acid, P: phosphate group). Due to its cluster of phosphoserine residues [-Ser (P) -], it can chelate calcium ions to form a soluble phosphate complex, effectively avoiding the formation of calcium phosphate precipitates under neutral or alkaline conditions, thereby promoting calcium absorption and improving its bioavailability.
[0004] As the precursors of hormones VD2 and VD3, vitamin D3 is successively catalyzed by liver and kidney hydroxylase to transform into active metabolites 1,25(OH)2D2 and 1,25(OH)2, which are biologically active VD3. Among them, 1,25(OH)2 is the highest active form of VD3, which is an important hormone for regulating calcium and phosphorus metabolism together with parathyroid hormone (PTH) and calcitonin (CT), and directly or indirectly participates in bone metabolism. When VD3 is deficient, the serum calcium level decreases, causing the pituitary to secrete PTH, which acts on the kidney to promote calcium reabsorption, reduce phosphate content, and accelerate osteoclast activity, which can also increase the calcium content in the serum. VD3 plays a key role in promoting intestinal absorption of calcium ions, which can increase the absorption and utilization rate of calcium by activating calcium ion channel proteins on the cell membrane surface or inducing the synthesis of calcium binding proteins on small intestinal epithelial cells.
[0005] When CPP is combined with VD3, the calcium absorption rate is significantly higher than that of either ingredient alone. The synergistic effect of CPP and VD3 in the field of calcium absorption promotion shows unique advantages, and this synergy is not simply the superposition of effects, but a comprehensive improvement achieved through complementary mechanisms. This synergy comes from the action of the two substances on different links and different segments of calcium absorption, forming a calcium absorption enhancement network covering the entire intestinal tract. From the perspective of the action site, VD3 mainly regulates the active calcium transport in the upper small intestine (duodenum and jejunum), while CPP mainly enhances the passive calcium diffusion in the lower small intestine (lower jejunum and ileum). This spatial complementarity ensures efficient calcium absorption throughout the small intestine. Research data show that when VD3 is used alone, calcium absorption mainly occurs in the proximal small intestine, and after the addition of CPP, the distal small intestine becomes an important site for calcium absorption, making full use of the larger absorption area in this region. This "segmented absorption" mode greatly improves the overall utilization of calcium. SUMMARY
[0006] The technical problem to be solved by the present application is to improve the defects of the prior art and establish a preparation method of vitamin D3 and casein phosphopeptide calcium complex. Based on the optimized preparation conditions, the prepared product is characterized and the binding mechanism is analyzed to obtain a VD3-CPP-Ca complex.
[0007] The purpose of the present application is to provide a preparation method of a calcium supplement containing vitamin D3 and casein phosphopeptide with high absorption rate, which comprises the following steps:
[0008] S1, solution preparation: dissolve casein phosphopeptide in ultrapure water to prepare a casein phosphopeptide solution with a concentration of 10 mg / mL.
[0009] S2, chelation reaction: mix the casein phosphopeptide solution of step S1 with calcium chloride at a mass ratio of 1, and stir in a magnetic stirring water bath for 20-60 minutes.
[0010] S3, product purification: after the reaction is completed, add 9 times the volume of anhydrous ethanol to precipitate CPP-Ca, centrifuge, and freeze-dry to obtain the CPP-Ca product.
[0011] S4, process optimization: based on the influencing factors of the preparation process, taking the chelation rate as the evaluation index, single factor experiments are conducted on reaction temperature, pH value, reaction time and mass ratio, and orthogonal experimental design is used to optimize the chelation conditions;
[0012] S5, complex preparation: prepare the CPP-Ca prepared under the optimized conditions into a solution with a concentration of 1-6 mg / mL, adjust the pH, and add VD3 with a final concentration of 5-25 μg / mL, and react under magnetic stirring.
[0013] S6, complex purification and preservation: after the reaction, centrifugation, freeze-drying at-50℃, 20Pa, to obtain the complex, 4℃ cold storage.
[0014] As preferred, the dissolving temperature in step S1 is 25±2℃; the magnetic stirring is used in the dissolving process, the rotating speed is 400-600rpm; the solution is prepared and used immediately, the storage time is not more than 4 hours.
[0015] Further, the casein phosphopeptide solution in step S1 is mixed with calcium chloride at the mass ratio of 1:0.1-20:0.1, and reacted in a magnetic stirring water bath at 20-60℃ and pH 5-9 for 20-60 minutes.
[0016] As preferred, the mass ratio in step S2 is 10:0.1-15:0.1; the reaction temperature is 40-50℃; the pH value is 7.0-8.0; the reaction time is 40-50 minutes.
[0017] Further, after the reaction in step S2, 9 times the volume of 99% anhydrous ethanol is added to precipitate CPP-Ca, which is centrifuged at 8000rpm and freeze-dried at-50℃ to obtain the casein phosphopeptide chelated calcium product.
[0018] As preferred, the anhydrous ethanol in step S3 is pre-cooled to 4℃; the centrifugation time is 15-20 minutes; the freeze-drying time is 24-48 hours; the product is stored in a desiccator.
[0019] Further, based on the influencing factors of the preparation process, the single-factor experiment is carried out on the reaction temperature (20-60℃), pH value (5-9), reaction time (20-60min) and mass ratio (1:0.1-20:0.1) respectively, and the orthogonal experiment design is used to optimize the chelation conditions.
[0020] As preferred, the single-factor experiment in step S4 is repeated 3 times for each condition; the orthogonal experiment uses an orthogonal table; the chelation rate is determined by atomic absorption spectrometry; the optimization results are verified by variance analysis.
[0021] Further, in step S4, the CPP-Ca prepared under the optimized conditions is prepared into a solution of 1-6mg / mL, the pH is adjusted to 4-9, and vitamin D3 with a final concentration of 5-25μg / mL is added, and the reaction is carried out under magnetic stirring at 300rpm for 20min.
[0022] As preferred, the CPP-Ca concentration in step S5 is 3 mg / mL; the pH value is 6.0-8.0; the vitamin D3 concentration is 20 μg / mL; and the reaction is carried out in the dark.
[0023] Further, after the reaction in step S5 is completed, centrifugation is carried out at 3000 rpm, and freeze-drying is carried out at -50℃ and 20 Pa to obtain a vitamin D3-CPP-Ca complex, which is stored at 4℃.
[0024] As preferred, the centrifugation time in step S6 is 10-15 minutes; the freeze-drying time is 36-48 hours; the complex is sealed with nitrogen and stored at 4±1℃.
[0025] The present application changes the secondary structure and tertiary structure of the protein by using the interaction between substances. After the calcium ion binds with the phosphate group or carboxyl group of the CPP, the peptide chain is induced to fold or aggregate, the microenvironment of the tryptophan, tyrosine and other fluorescent groups is changed, which indicates that the chelation of casein phosphopeptide and calcium ion occurs, the peptide chain is folded or aggregated, and the peptide structure is changed. When the CPP molecular chain is fully stretched and the negative charge is stable, the hydrophobic microzone is exposed to the appropriate degree, and VD3 can be effectively wrapped. When VD3 is combined with CPPP-Ca, the -CH3 characteristic peak at 3000-2800 cm -1 disappears, indicating that VD3 is successfully encapsulated in the CPP-Ca complex.
[0026] In this study, CPP, VD3 and CaCl2 were selected. Many studies have shown that casein phosphopeptide has good chelation effect, and the addition of VD3 enables the intestinal tract to fully utilize the larger absorption area of the region, thereby improving the intestinal absorption and utilization rate of calcium; CaCl2 is more soluble in water, has good solubility, and CaCl2 is easily dissolved in ethanol, while the peptide calcium chelate is not easily dissolved in ethanol. After the chelation reaction is completed, the obtained polypeptide calcium chelate liquid can be precipitated by ethanol to simultaneously obtain polypeptide calcium chelate and remove residual CaCl2 and free amino acids, which is beneficial to the separation of polypeptide calcium chelate and free calcium, and therefore, CaCl2 is selected as the calcium source. BRIEF DESCRIPTION OF DRAWINGS
[0027] Figure 1 is the influence of different calcium peptide ratios on the CPP-Ca chelation rate in Example 1
[0028] Figure 2 is the influence of different reaction temperatures on the CPP-Ca chelation rate in Example 1
[0029] Figure 3 is the influence of different reaction times on the CPP-Ca chelation rate in Example 1
[0030] Figure 4 Effect of different reaction pH on CPP-Ca chelation rate of Example 1
[0031] Figure 5 Effect of different pH conditions on CPP-Ca particle size and PDI of Example 2
[0032] Figure 6 VD3 binding rate measured under different VD3 addition concentrations of Example 2
[0033] Figure 7 Example 3 VD3, CPP-Ca and CPP-Ca-VD3 particle size and potential diagram
[0034] Figure 8 Example 3 VD3, CPP-Ca and CPP-Ca-VD3 Fourier infrared spectrum
[0035] Figure 9 Example 3 VD3, CPP-Ca and CPP-Ca-VD3 circular dichroism spectrum
[0036] Figure 10 Example 3 VD3, CPP-Ca and CPP-Ca-VD3 fluorescence spectrum
[0037] Figure 11 Example 4 VD3, CPP-Ca and CPP-Ca-VD3 scanning electron microscope DETAILED DESCRIPTION
[0038] Example 1: Preparation of casein phosphopeptide chelated calcium (CPP-Ca) and single factor optimization experiment
[0039] This example details the screening process of the optimal chelation process conditions of CPP-Ca:
[0040] Experimental method: A certain amount of casein phosphopeptide (CPP) powder was weighed and dissolved in deionized water to prepare a CPP solution of a certain concentration. Under constant temperature stirring conditions, calcium chloride (CaCl2) solution was slowly added to the CPP solution, and sodium hydroxide (NaOH) or hydrochloric acid (HC1) solution was used to adjust and maintain the pH value of the system. After a certain reaction time, the reaction solution was centrifuged at 8000 r / min for 15 min, and the supernatant was taken. The free calcium content was determined by using the o-cresol red complex ketone method or atomic absorption spectrometry. The chelation rate of CPP and Ca 2+ was calculated according to the following formula:
[0041] Chelation rate (%) = [(total calcium content-free calcium content) / total calcium content] x 100%
[0042] To explore the effects of various factors on the CPP and calcium ion chelation, we designed the following single-factor experiments:
[0043] (a) Calcium peptide mass ratio: fixed temperature 40 °C, pH 7.0, reaction time 40 min, respectively investigate the influence of calcium peptide ratio of 1:1, 5:1, 10:1, 15:1, 20:1 on the chelation rate.
[0044] (b) Reaction temperature: fixed calcium peptide ratio 10:1, pH 7.0, reaction time 40 min, respectively investigate the influence of temperature at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C on the chelation rate.
[0045] (c) Reaction time (t): fixed calcium peptide ratio 10:1, temperature 40 °C, pH 7.0, respectively investigate the influence of reaction time 20 min, 30 min, 40 min, 50 min, 60 min on the chelation rate.
[0046] (d) Reaction pH value: fixed calcium peptide ratio 10:1, temperature 40 °C, reaction time 40 min, respectively investigate the influence of pH value 5.0, 6.0, 7.0, 8.0, 9.0 on the chelation rate.
[0047] Through the above single-factor experiments, the chelation rate under each condition was determined and recorded. The results showed that the chelation reaction of CPP and calcium ions was significantly affected by the above factors. Through comparative analysis of the chelation rate, we preliminarily screened out the optimal process condition range: calcium peptide ratio 5:1 ~ 15:1, reaction temperature 30 ~ 50 °C, reaction time 30 ~ 50 min, pH 7.0 ~ 8.0. Under this condition range, the chelation rate of CPP to calcium is the highest.
[0048] Example 2: Preparation of VD3 and CPP-Ca complex and optimization of binding conditions
[0049] On the basis of obtaining high chelation rate of CPP-Ca, this embodiment further complexes vitamin D3 with it.
[0050] (1) Preparation of CPP-Ca stock solution: CPP-Ca chelation solution was prepared according to the optimal chelation conditions determined in Example 1. The chelation solution was appropriately concentrated or diluted to prepare a 3 mg / mL CPP-Ca solution, which was ready for use.
[0051] (2) Preparation of complex and determination of binding rate
[0052] Under the light condition, different amounts of vitamin D3 ethanol stock solution were added to the above solution, so that the final concentration of VD3 in the mixed system was 5, 10, 15, 20, and 25 μg / mL, respectively. The reaction was carried out under constant temperature (e.g. 25°C), light protection, and slow stirring for a period of time. After the reaction was completed, the free VD3 was separated by dialysis or ultrafiltration centrifugation, and the content of free VD3 in the filtrate was determined by high performance liquid chromatography (HPLC), and the binding rate of VD3 was calculated.
[0053] Binding rate (%) = (total amount of added VD3 - amount of free VD3) / total amount of added VD3 x 100%.
[0054] (3) Effect of solution pH on complexation
[0055] With the CPP-Ca concentration (3 mg / mL) and VD3 concentration fixed, the effect of different pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) on the binding rate of VD3 was investigated. The results showed that under near neutral conditions of pH 7.0-8.0, the binding rate of VD3 was the highest. Acidic or strongly alkaline conditions would result in a decrease in the binding rate. This pH range is consistent with the stable pH range of CPP-Ca, which is conducive to the formation and stability of the complex.
[0056] (4) Determination of optimal conditions
[0057] By comprehensively comparing the turbidity, particle size, Zeta potential, and VD3 complexation rate of the complex under different VD3 concentrations and different pH conditions, the optimal conditions for forming a stable and high-encapsulation complex were determined. The experimental results showed that when the CPP-Ca concentration was 3 mg / mL, the VD3 concentration was 15-20 μg / mL, and the pH environment was neutral to weakly alkaline (7.0-8.0), the VD3-CPP-Ca complex solution formed was clear and transparent, with the best stability and the highest VD3 complexation rate.
[0058] Example 3: Structure characterization and force analysis of the complex
[0059] This example characterizes the optimal complex prepared in Example 2 to confirm its formation and explore its mechanism of action.
[0060] (1) Measurement of particle size and potential
[0061] Freshly prepared VD3, CPP-Ca, and CPP-Ca-VD3 solutions of certain concentrations were prepared, and the pH was adjusted to 2-8. Three parallel samples were prepared for each sample, and a Malvern laser particle size analyzer was used to detect the particle size and potential of the samples.
[0062] (2) Fourier infrared spectroscopy
[0063] Accurately weigh 1 mg of sample VD3, CPP-Ca and CPP-Ca-VD3, mix with 100 mg of potassium bromide, and then tablet by tablet machine, and then scan in the range of 4000 cm-1-400 cm-1 by Fourier infrared transform spectrometer, the instrument resolution is 4 cm -1 , scan 64 times.
[0064] (3) Circular dichroism spectrum
[0065] Accurately weigh a certain mass of sample CPP, CPP-Ca and CPP-Ca-VD3, each two, and prepare a 0.2 mg / mL solution. Use ultrapure water as a blank control group, and use a circular dichroism spectrometer to detect the structural changes.
[0066] (4) Scanning electron microscope
[0067] The freeze-dried samples CPP, VD3, CPP-Ca-VD3 and CPP-Ca solid powder are evenly attached to the sample column with carbon conductive glue, and a layer of metal film is plated on the vacuum sputtering instrument. The scanning conditions are: acceleration voltage 15.0 kV, beam current 6.9×10−2mA, working distance 6.7mm, the image becomes clear by adjusting the focus, and the magnification is 3000x. The morphology is observed and the image is obtained.
[0068] (5) Fluorescence spectrum
[0069] Due to the inherent endogenous fluorescence characteristics of proteins, fluorescence spectrum is used to determine the changes in endogenous fluorescence spectrum between VD3 and CPP-Ca chelate, to reflect the interaction between the two. The concentration of CPP-Ca solution is fixed, and the concentration of VD3 is 0-25 μg / ml. At an excitation wavelength of 280 nm, the emission spectrum between 300-450 nm is recorded. The concentration of VD3 is fixed at 20 μg / ml, the concentration of CPP-Ca is 0-5 mg / ml, the excitation wavelength is 265 nm, and the emission spectrum at 290-450 nm is measured.
[0070] Example 4: Preparation of high-absorption calcium supplement powder
[0071] The optimal complex solution obtained in Example 2 is post-processed to prepare a solid powder preparation for easy storage and consumption.
[0072] Take the same CPP-Ca-VD3 complex solution 500 mL, and divide it into culture dishes. Place it in a -80°C ultra-low temperature refrigerator for pre-freezing for more than 4 hours to completely solidify into a solid. Then transfer the sample to a freeze-drying chamber, and freeze-dry it for 24 hours under the conditions of a cold trap temperature of -50°C and a vacuum degree of 0.1 mBar or below. After the process is completed, a white freeze-dried powder block with full and porous morphology is obtained, which can be slightly ground into powder. The product has excellent reconstitution and is more conducive to retaining the activity of heat-sensitive components.
[0073] The method optimizes the chelation process of CPP-Ca and the complexing conditions of VD3, and the process parameters are clear. The final product organically combines calcium, CPP for promoting calcium absorption, and VD3, significantly improving the bioavailability of calcium.
Claims
1. The technical problem to be solved by the present application is to overcome the technical defects of low bioavailability, single functional component and lack of synergistic effect among components of existing calcium supplements, and to establish a preparation method of vitamin D3 (VD3) and casein phosphopeptide-chelated calcium complex (CPP-Ca). Based on the optimization of the preparation process parameters, the structure characteristics, stability and binding mechanism of the product are studied in depth by modern analysis techniques, and finally a new type of high-efficiency calcium nutritional supplement with synergistic effect is obtained.
2. The present application adopts a systematic research strategy: first, the chelation process conditions of casein phosphopeptide (CPP) and calcium ions are optimized by single factor experiment combined with orthogonal experiment design; on this basis, the complex process of CPP-Ca and VD3 is further studied, and the interaction mechanism is clarified by various characterization methods, so as to establish a stable preparation method of VD3-CPP-Ca ternary complex.
3. The technical scheme of the present application is: a preparation method of a high-absorption calcium supplement of vitamin D3 and casein phosphopeptide chelated calcium, characterized in that, Comprising the following steps: S1, solution preparation: dissolve CPP in ultrapure water, disperse uniformly, and prepare a casein phosphopeptide solution with a concentration of 10 mg / mL; S2, chelation reaction: add calcium chloride (CaCl2) to the casein phosphopeptide solution described in step S1, control the mass ratio of casein phosphopeptide to calcium chloride to be 1:0.1-20:0.1, adjust the pH value of the reaction system to 5.0-9.0 with sodium hydroxide (NaOH) or hydrochloric acid (HCl) solution, stir the reaction system at different temperatures of 20-60℃, and freeze-dry the precipitate at-50℃ to obtain CPP-Ca chelate powder; S3, product purification: after the reaction is completed, add 9 times the volume of 99% anhydrous ethanol to the system to precipitate CPP-Ca; S4, process optimization: based on the influencing factors of the preparation process, taking the chelation rate as the evaluation index, single factor experiments are carried out on reaction temperature (20-60℃), pH value (5-9), reaction time (20-60min) and mass ratio (1:1-20:1), and orthogonal experiment design is used to optimize the chelation conditions; S5, complex preparation: the CPP-Ca powder prepared under the optimal conditions obtained in step S4 is dissolved again to prepare a CPP-Ca solution with a concentration of 1-6 mg / mL, the solution is adjusted to different pH values (4.0-9.0) with NaOH or HCl solution, under the condition of magnetic stirring at 300 rpm, VD3 anhydrous ethanol solution is slowly added dropwise, the final concentration of VD3 in the system is controlled to be 5-50 μg / mL, and VD3 and CPP-Ca are fully complexed at room temperature for 20 minutes. S6, composite condition optimization and preservation: the solution obtained in step S5 is used to optimize the CPP-Ca concentration, pH value and VD3 concentration in step S5, with the encapsulation rate of VD3 and the stability of the composite solution as indexes, to determine the optimal conditions for preparing VD3-CPP-Ca composite: CPP-Ca concentration 3 mg / mL, pH value 6.0-8.0, VD3 concentration 20 μg / mL, under which stable and high-encapsulation-rate composite can be formed, after reaction, centrifugation at 3000 rpm, freeze-drying at-50℃, 20Pa, to obtain VD3-CPP-Ca composite, which is stored at 4℃.
4. The present application successfully prepares a novel VD3-CPP-Ca ternary composite through the above-mentioned step-by-step optimization strategy, in which calcium ions are effectively chelated by CPP, and VD3 is efficiently encapsulated in the hydrophobic region of CPP-Ca, and the three form a stable structure through intermolecular forces, which not only solves the water solubility and stability of VD3, but more importantly, realizes the synergistic delivery of calcium and VD3, can significantly promote the absorption of calcium in the intestine, and improve the bioavailability of calcium, is a highly efficient new type of calcium supplement.
5. The benefits of the present application include: The ethanol precipitation method is used to realize the purification and separation of the product simultaneously, and the process is simple and efficient; The final product combines CPP and VD3, which promote calcium absorption, with calcium source, synergistically, and has high bioavailability; through in-depth research on the formation conditions and mechanism of the composite, a theoretical basis is provided for product quality control.