Preparation method of citric acid grafted casein phosphopeptide calcium compound

Abstract

The invention discloses a preparation method of a citric acid grafted casein phosphopeptide calcium compound. The preparation method comprises the following steps: 1, dissolving casein phosphopeptide in a 0.1 mol / L MES buffer solution with the pH value of 5.5; 2, dissolving citric acid according to a mass ratio of the citric acid to the casein phosphopeptides of 1: 1, adding a reagent according to a ratio of CA: EDC: NHS of 1: 1.5: 1.2, and activating in an ice bath at 4 DEG C for 30 minutes; 3, mixing the two solutions, reacting at room temperature for 24 hours, dialyzing, and freeze-drying to obtain CA-CPP; and 4, dissolving CA-CPP in water, adding calcium acetate monohydrate to adjust the pH value to 7.0, carrying out a reaction at 50 DEG C for 60 min, carrying out ethanol precipitation, and carrying out centrifugation and freeze-drying to obtain the product. The calcium chelating ability and stability are improved, the process is simple and safe, and the method is suitable for functional food and calcium supplements.

Description

Technical Field

[0001] This invention belongs to the field of nutrition and food science and technology, specifically a method for preparing a citric acid-grafted casein phosphopeptide calcium complex. Background Technology

[0002] Calcium is an essential macroelement for maintaining human life, playing a crucial role in physiological processes such as bone formation, nerve conduction, muscle contraction, enzyme activity regulation, and cell signal transduction. Long-term insufficient intake or low absorption rates can lead to health problems such as osteoporosis, rickets, muscle cramps, and cardiovascular metabolic abnormalities. Although our daily diet contains abundant calcium sources, the actual proportion of calcium absorbed by the human body remains low due to limitations in solubility, absorption rate, and bioavailability. Therefore, developing highly absorbable and stable organic calcium supplementation systems has become an important direction in current nutritional and food science research, and has significant application value in health promotion and functional food development.

[0003] Currently, commercially available calcium supplements mainly fall into two categories: inorganic calcium and organic calcium. Inorganic calcium, such as calcium carbonate, calcium chloride, and calcium nitrate, has a high calcium content and low cost, but its solubility is limited, especially in people with insufficient stomach acid or the elderly, where absorption is lower and it can easily cause constipation or gastrointestinal discomfort. In contrast, organic calcium, such as calcium lactate, calcium citrate, amino acid chelated calcium, and peptide calcium, is bound to organic ligands, which improves solubility and bioavailability, resulting in higher absorption efficiency and better gastrointestinal tolerance.

[0004] Among organic calcium sources, peptide calcium has attracted widespread attention in recent years due to its natural origin, high safety, good biocompatibility, and the presence of potential coordinating groups such as carboxyl, amino, and hydroxyl groups on its peptide chains. These groups can form stable complexes with calcium ions, enabling efficient transport and absorption. Peptides are rich in potential coordinating groups such as carboxyl, amino, hydroxyl, and amide groups, which can form stable complexes with calcium ions, facilitating calcium transport and absorption. However, the calcium chelation efficiency of natural peptides is limited by peptide chain conformation, functional group distribution, and spatial accessibility, resulting in a limited number of chelation sites. Therefore, the stability and bioavailability of the complexes still need to be improved.

[0005] In recent years, researchers have enhanced the calcium chelating ability of peptides by optimizing enzymatic digestion conditions, purifying peptide sequences with high calcium chelating activity, or modifying peptides (such as phosphorylation and glycosylation). However, existing modification methods still have certain limitations: phosphorylation modification can provide additional negative charge sites, but phosphate ester bonds are easily hydrolyzed in aqueous solutions or gastrointestinal environments, and chemical or enzymatic phosphorylation is complex and site selectivity is difficult to control; glycosylation modification can improve the water solubility and stability of peptides, but the large size of the glycosyl group may create steric hindrance to the chelating site, reducing calcium binding efficiency, and the preparation cost is high and difficult to precisely control; traditional N-terminal amidation modification can improve the stability and resistance to enzyme degradation of peptides, but it will eliminate the N-terminal amino active site, cannot provide multivalent binding function, and has limited contribution to calcium chelation.

[0006] Given the shortcomings of the existing technologies, there is an urgent need to develop a new peptide modification method to address the problems of low peptide-calcium chelation efficiency, insufficient complex stability, and the need to improve bioavailability. Summary of the Invention

[0007] To address the problems of low chelation efficiency, poor stability, and insufficient bioavailability in existing peptide-calcium complex preparation technologies, this invention provides a method for preparing a citric acid-grafted casein phosphopeptide-calcium complex. This method modifies casein phosphopeptides with citric acid, increasing the number of active coordinating groups on the peptide chain, thereby improving its chelation ability with calcium ions and the stability of the complex, ultimately yielding a calcium supplement precursor with high bioavailability.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] A method for preparing a citrate-grafted casein phosphopeptide calcium complex includes the following steps:

[0010] Step 1: Preparation of casein phosphopeptide solution:

[0011] Weigh out casein phosphopeptides and dissolve them in MES buffer solution with a pH of 5.5 and a concentration of 0.1 mol / L. Stir magnetically until completely dissolved.

[0012] Step 2, Preparation of citric acid activation solution:

[0013] Citric acid was weighed at a mass ratio of 1:1 to casein phosphopeptide and dissolved in MES buffer at pH 5.5 with a concentration of 0.1 mol / L. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially at a molar ratio of CA:EDC:NHS=1:1.5:1.2. The mixture was then sealed and reacted in an ice bath at 4°C in the dark for 30 min to activate the carboxyl groups in the citric acid.

[0014] Step 3: Preparation of citric acid-grafted casein phosphopeptide complex:

[0015] The citric acid activation solution was slowly added dropwise to the casein phosphopeptide solution, and the reaction was carried out at room temperature in the dark with the mixture stirred for 24 hours. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 72 hours, pre-frozen at -80℃ for 12 hours, and then freeze-dried under vacuum to obtain a powdered citric acid grafted casein phosphopeptide complex.

[0016] Step 4: Preparation of citric acid-grafted casein phosphopeptide calcium complex:

[0017] The citrate-grafted casein phosphopeptide complex was dissolved in deionized water, and calcium acetate monohydrate was added to adjust the pH to 7.0. The mixture was reacted in a water bath at 50°C for 60 min. Nine volumes of anhydrous ethanol were added to the reaction solution, and the mixture was allowed to stand at 4°C for 60 min. The precipitate was collected by centrifugation at 7500 rpm for 10 min and then freeze-dried to obtain the citrate-grafted casein phosphopeptide calcium complex.

[0018] Preferably, in step 1, the mixture is magnetically stirred at room temperature (25°C) until completely dissolved.

[0019] Preferably, in step 1, the ratio of casein phosphopeptide to MES buffer is 5.0 g: 300 mL.

[0020] Preferably, in step 2, the ratio of citric acid to MES buffer is 5.0 g: 300 mL.

[0021] Preferably, in step 3, the deionized water is replaced every 6 hours during the dialysis process.

[0022] Preferably, in step 4, the citric acid-grafted casein phosphopeptide complex is fully dissolved in deionized water under magnetic stirring.

[0023] Preferably, in step 4, the ratio of citric acid-grafted casein phosphopeptide complex to deionized water is 1.0 g: 100 mL.

[0024] Preferably, in step 4, the mass ratio of the citrate-grafted casein phosphopeptide complex to calcium acetate monohydrate is 1.0 g: 0.5 g.

[0025] The present invention has the following beneficial effects:

[0026] 1. Enhance calcium chelation capacity: Citric acid molecules contain multiple carboxyl groups. After being successfully grafted onto casein phosphopeptide molecules via the coupling method of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, it can provide more coordination sites for calcium ions and significantly enhance the calcium binding capacity of the peptide.

[0027] 2. Enhanced complex stability: The amide bond formed between citric acid and casein phosphopeptide is highly stable and not easily hydrolyzed in aqueous solution and gastrointestinal environment. At the same time, the incorporation of citric acid can optimize the peptide chain conformation, improve the spatial stability of the calcium complex, and reduce the loss of calcium ions during transport.

[0028] 3. Simple and controllable process: The modification and chelation reaction conditions used in this invention are mild, the operation steps are simple, the reaction parameters are easy to control, and no complex and expensive equipment is required, making it suitable for industrial-scale production.

[0029] 4. High safety: The raw materials used in this invention are all food-grade or analytical grade reagents, which are natural and safe. The modified complex has good biocompatibility and no toxic side effects, and can be directly applied to the development of functional foods and calcium supplements. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] A method for preparing a citrate-grafted casein phosphopeptide calcium complex includes the following steps:

[0032] 1. Preparation of casein phosphopeptide solution: Weigh casein phosphopeptide (CPP), dissolve it in MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L, and stir magnetically at room temperature (25℃) until completely dissolved to obtain casein phosphopeptide solution; wherein, the material-to-liquid ratio of casein phosphopeptide to MES buffer solution is 5.0 g: 300 mL.

[0033] 2. Preparation of citric acid activation solution: Citric acid (CA) was weighed at a mass ratio of 1:1 to casein phosphopeptide (CPP) and dissolved in MES buffer solution with a pH of 5.5 and a concentration of 0.1 mol / L to obtain a citric acid solution. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added sequentially at a molar ratio of CA:EDC:NHS = 1:1.5:1.2. The reaction was carried out in a sealed environment at 4°C in the dark for 30 min to activate the carboxyl groups in the citric acid to obtain the citric acid activation solution. The material-to-liquid ratio of citric acid to MES buffer solution was 5.0 g: 300 mL.

[0034] 3. Preparation of citric acid-casein phosphopeptide complex (CA-CPP): The citric acid activation solution prepared in step 2 was slowly added dropwise to the casein phosphopeptide solution prepared in step 1, while maintaining magnetic stirring, and reacted at room temperature for 24 h under light-protected and sealed conditions; after the reaction was completed, the reaction solution was placed into a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed in deionized water for 72 h, with the dialyzing water being changed every 6 h; after dialysis, the sample was pre-frozen at -80℃ for 12 h, and then freeze-dried under vacuum to obtain powdered citric acid-casein phosphopeptide complex (CA-CPP).

[0035] 4. Preparation of citric acid-grafted casein phosphopeptide calcium complex (CA-CPP-Ca): Weigh the citric acid-casein phosphopeptide complex (CA-CPP) prepared in step 3, dissolve it in deionized water, and fully dissolve it under magnetic stirring to obtain a CA-CPP solution; then add calcium acetate monohydrate (Ca(CH3COO)2·H2O), adjust the pH of the solution to 7.0, and react in a 50℃ water bath for 60 min; after the reaction, add the reaction solution to 9 times its volume of anhydrous ethanol, mix thoroughly, and let it stand at 4℃ for 60 min; collect the precipitate by centrifugation at 7500 rpm for 10 min, and freeze-dry it to obtain the citric acid-grafted casein phosphopeptide calcium complex (CA-CPP-Ca); wherein, the material-to-liquid ratio of citric acid-casein phosphopeptide complex (CA-CPP) to deionized water is 1.0 g: 100 mL, and the mass ratio of citric acid-casein phosphopeptide complex (CA-CPP) to calcium acetate monohydrate is 1.0 g: 0.5 g.

[0036] Example

[0037] A method for preparing a citric acid-grafted casein phosphopeptide calcium complex, comprising the following steps:

[0038] 1. Preparation of casein phosphopeptide solution: Weigh 5.0 g of casein phosphopeptide (CPP) with a purity of 92% and a molecular weight distribution of 1000-3000 Da, and slowly add it to 300 mL of MES buffer with pH 5.5 and a concentration of 0.1 mol / L. Place the mixture on a magnetic stirrer and stir at 300 rpm for 30 min at room temperature (25℃) until the casein phosphopeptide is completely dissolved to obtain a homogeneous and transparent casein phosphopeptide solution.

[0039] 2. Preparation of citric acid activation solution: Weigh 5.0 g of citric acid (1:1 mass ratio with CPP) and dissolve it in 300 mL of 0.1 mol / L MES buffer solution with pH 5.5. Stir until completely dissolved. Then, according to the molar ratio of CA:EDC:NHS = 1:1.5:1.2, add 6.85 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 3.65 g of N-hydroxysuccinimide (NHS) in sequence. After stirring evenly, place the reaction vessel in an ice bath at 4 °C, wrap it with aluminum foil to protect it from light, and seal it for 30 min to activate the carboxyl group in citric acid, thus obtaining the citric acid activation solution.

[0040] 3. Preparation of CA-CPP complex: The citric acid activation solution prepared in step 2 was slowly added dropwise to the casein phosphopeptide solution from step 1 at a rate of 1 mL / min using a constant pressure dropping funnel, with magnetic stirring maintained during the addition (300 rpm). After the addition was complete, the reaction vessel was wrapped with aluminum foil and sealed for 24 h at room temperature in the dark. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, the bag was tightly sealed, and the bag was placed in a beaker containing 5 L of deionized water. Dialysis was performed at 4 °C for 72 h, with the deionized water replaced every 6 h. After dialysis, the sample in the dialysis bag was transferred to a lyophilization bottle and pre-frozen at -80 °C for 12 h. Then, it was placed in a vacuum freeze dryer and freeze-dried for 24 h under conditions of vacuum ≤10 Pa and cold trap temperature ≤-50 °C to obtain a white powdery citric acid-casein phosphopeptide complex (CA-CPP) with a yield of 89.2%.

[0041] 4. Preparation of the CA-CPP-Ca complex: Weigh 1.0 g of the CA-CPP prepared in step 3 and add it to 100 mL of deionized water. Stir magnetically (400 rpm) for 30 min until completely dissolved. Then add 0.5 g of calcium acetate monohydrate and stir for 10 min. Adjust the pH of the solution to 7.0 with 0.1 mol / L hydrochloric acid or sodium hydroxide solution. Place the reaction vessel in a 50℃ constant temperature water bath and keep it at this temperature for 60 min. After the reaction is complete, slowly add the reaction solution to 900 mL of anhydrous ethanol while stirring. Continue stirring for 10 min after the addition is complete, and then place it in a 4℃ refrigerator and let it stand for 60 min to allow the chelate to precipitate completely. The precipitate was transferred to a centrifuge tube and centrifuged at 7500 rpm for 10 min. The supernatant was discarded and the precipitate was collected. The precipitate was placed in a freeze-drying bottle and freeze-dried under vacuum for 24 h to obtain a white powdery citric acid-grafted casein phosphopeptide calcium complex (CA-CPP-Ca) with a yield of 85.6%.

[0042] Performance testing

[0043] The performance of the CA-CPP and CA-CPP-Ca complexes prepared in the examples was tested. The specific testing methods and results are as follows:

[0044] 1. Grafting rate determination: The content of free amino groups in CPP and CA-CPP was determined by the TNBS method (2,4,6-trinitrobenzenesulfonic acid method), and the grafting rate was calculated. The results showed that the free amino group content in CPP was 391.07±8.41 mg / g, and the free amino group content in CA-CPP was 345.40±19.80 mg / g, with a grafting rate of 13.27±2.01%, indicating that citric acid had been successfully grafted onto the CPP molecule.

[0045] 2. Determination of calcium binding rate and chelation rate: The calcium content in CPP-Ca and CA-CPP-Ca complexes was determined by EDTA complexometric titration, and the calcium binding rate and chelation rate were calculated. Calcium binding rate = (mass of calcium in the complex / mass of the peptide) × 100%; Chelation rate = (mass of calcium in the complex / mass of calcium in the added calcium acetate monohydrate) × 100%. The results showed that the calcium content in CPP-Ca was... 2+ The binding rate was 20.85±0.50 mg / g, and the chelation rate was 18.34±0.44%; while the Ca of CA-CPP-Ca... 2+ The binding rate was 25.03±0.50 mg / g, and the chelation rate was 21.95±0.44%, which were significantly higher than those of the CPP-Ca complex.

[0046] 3. Stability Test: The CA-CPP-Ca complex was placed in simulated gastrointestinal fluid (artificial gastric fluid: pH 1.2, containing 0.3% pepsin; artificial intestinal fluid: pH 7.4, containing 0.1% trypsin) and incubated at 37°C for 2 hours. The calcium dissolution rate before and after incubation was measured. The results showed that the calcium dissolution rate of the CA-CPP-Ca complex after incubation was only 8.2%, while that of the CPP-Ca complex was 15.6%, indicating that the CA-CPP-Ca complex prepared in this invention has higher stability in the gastrointestinal environment.

[0047] The examples provided in this invention are not intended to limit the implementation. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations, and any obvious variations or modifications derived therefrom are still within the scope of this invention.

Claims

1. A method for preparing a citric acid-grafted casein phosphopeptide calcium complex, characterized in that, Includes the following steps: Step 1: Preparation of casein phosphopeptide solution: Weigh out casein phosphopeptides and dissolve them in MES buffer solution with a pH of 5.5 and a concentration of 0.1 mol / L. Stir magnetically until completely dissolved. Step 2, Preparation of citric acid activation solution: Citric acid was weighed at a mass ratio of 1:1 to casein phosphopeptide and dissolved in MES buffer at pH 5.5 with a concentration of 0.1 mol / L. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially at a molar ratio of CA:EDC:NHS=1:1.5:1.

2. The mixture was then sealed and reacted in an ice bath at 4°C in the dark for 30 min to activate the carboxyl groups in the citric acid. Step 3: Preparation of citric acid-grafted casein phosphopeptide complex: The citric acid activation solution was slowly added dropwise to the casein phosphopeptide solution, and the reaction was carried out at room temperature in the dark with the mixture stirred for 24 hours. The reaction solution was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 72 hours, pre-frozen at -80℃ for 12 hours, and then freeze-dried under vacuum to obtain a powdered citric acid grafted casein phosphopeptide complex. Step 4: Preparation of citric acid-grafted casein phosphopeptide calcium complex: The citrate-grafted casein phosphopeptide complex was dissolved in deionized water, and calcium acetate monohydrate was added to adjust the pH to 7.

0. The mixture was reacted in a water bath at 50°C for 60 min. Nine volumes of anhydrous ethanol were added to the reaction solution, and the mixture was allowed to stand at 4°C for 60 min. The precipitate was collected by centrifugation at 7500 rpm for 10 min and then freeze-dried to obtain the citrate-grafted casein phosphopeptide calcium complex.

2. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 1, the mixture is magnetically stirred at room temperature (25°C) until completely dissolved.

3. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 1, the ratio of casein phosphopeptide to MES buffer is 5.0 g: 300 mL.

4. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 2, the ratio of citric acid to MES buffer is 5.0 g: 300 mL.

5. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 3, the deionized water is replaced every 6 hours during the dialysis process.

6. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 4, the citric acid-grafted casein phosphopeptide complex is fully dissolved in deionized water under magnetic stirring.

7. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 4, the ratio of citric acid-grafted casein phosphopeptide complex to deionized water is 1.0 g: 100 mL.

8. The method for preparing a citric acid-grafted casein phosphopeptide calcium complex according to claim 1, characterized in that, In step 4, the mass ratio of the citrate-grafted casein phosphopeptide complex to calcium acetate monohydrate is 1.0 g: 0.5 g.