A soybean-based peptide calcium chelate, its preparation method and application

Abstract

This invention discloses a soybean-based peptide-calcium chelate, its preparation method, and its application. The preparation method includes the following steps: S1: Adding a protease to a soybean protein isolate solution and enzymatically hydrolyzing it at 48℃~55℃ for 5h~8h, followed by enzyme inactivation treatment to obtain soybean protein peptides; the protease includes bromelain; S2: Chelating the soybean protein peptides obtained in step S1 with calcium salts to obtain a peptide-calcium chelate reaction solution, subjecting the peptide-calcium chelate reaction solution to alcohol precipitation to obtain a precipitate, and freeze-drying the precipitate to obtain the soybean-based peptide-calcium chelate. This invention uses bromelain to enzymatically hydrolyze soybean protein isolate, obtaining soybean protein peptides with optimal calcium chelating ability through a specific enzymatic hydrolysis process, thereby increasing the calcium chelation content in the peptide-calcium chelate. This invention, through alcohol precipitation combined with freeze-drying, enables the prepared peptide-calcium chelate to have high purity, high chelation rate, and high bioavailability.

Description

Technical Field

[0001] This invention relates to the fields of functional food and biomedicine, and in particular to a soybean-based peptide calcium chelate, its preparation method, and its application. Background Technology

[0002] Calcium is the most abundant mineral element in the human body, accounting for 1.5%-2.2% of body weight. It is a major component of bones and teeth and participates in various physiological activities such as nerve conduction, muscle contraction, and blood clotting. Traditional calcium supplements mainly include inorganic calcium (such as calcium carbonate and calcium phosphate), organic calcium (such as calcium gluconate, calcium lactate, and calcium citrate), and amino acid chelated calcium. Although inorganic calcium has a high calcium content, it has poor solubility and easily forms precipitates in the neutral or slightly alkaline environment of the intestine, resulting in low bioavailability (usually <30%) and a tendency to cause side effects such as constipation and gastrointestinal discomfort. Although organic calcium has improved solubility, its calcium content is low, and its absorption rate is still not ideal. Third-generation calcium supplements, such as amino acid chelated calcium, have improved absorption rates, but their production costs are high, and the calcium-binding capacity of amino acids is limited.

[0003] Peptide-calcium chelates are stable chelates formed by coordination bonds between polypeptides and calcium ions, exhibiting excellent absorption performance and high bioavailability. Compared to amino acid-chelated calcium, small molecule peptides offer multiple advantages as calcium carriers: their carboxyl, amino, and phosphate groups can form coordination bonds with calcium ions, constructing stable, soluble chelates; simultaneously, peptide-calcium chelates achieve efficient absorption through a transmembrane transport mechanism mediated by peptide transporters, a pathway characterized by low energy consumption, rapid transport speed, and a high carrier saturation threshold. More importantly, peptide-calcium chelates demonstrate superior chemical stability in the gastrointestinal environment. In simulated gastrointestinal digestion experiments, the calcium dissolution rate of peptide-calcium chelates in the intestine was significantly higher than that of the calcium chloride control group. This stability effectively avoids the defect of traditional calcium supplements forming precipitation in the alkaline environment of the intestine. Therefore, peptide-chelated calcium has attracted widespread attention as a novel calcium supplement.

[0004] However, existing peptide-calcium chelates still lack systematic technical solutions in terms of peptide chain length control, chelation site modification, and optimization of chelation reaction conditions, resulting in problems such as low calcium chelation, poor thermal stability, and difficulty in large-scale production. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the first objective of the present invention is to provide a method for preparing a soybean-based peptide calcium chelate. The second objective of the present invention is to provide the soybean-based peptide calcium chelate obtained by this preparation method. The third objective of the present invention is to provide the application of this soybean-based peptide calcium chelate.

[0006] The inventive concept of this invention is as follows: soybean protein isolate is enzymatically hydrolyzed under specific enzymes and conditions to obtain soybean protein peptides, and then the soybean protein peptides are chelated with calcium salts, and a peptide-calcium chelate with high calcium chelation and good thermal stability is obtained by alcohol precipitation combined with freeze drying.

[0007] To achieve the first objective mentioned above, the present invention provides the following technical solution.

[0008] In a first aspect, the present invention provides a method for preparing soybean-based peptide calcium chelates, comprising the following steps: S1: Add protease to the soy protein isolate solution, hydrolyze at 48℃~55℃ for 5h~8h, and obtain soy protein peptides after enzyme inactivation treatment; the protease includes bromelain. S2: The soybean protein peptides described in step S1 are chelated with calcium salts to obtain a peptide-calcium chelate reaction solution. The peptide-calcium chelate reaction solution is precipitated with alcohol to obtain a precipitate. The precipitate is freeze-dried to obtain the soybean-based peptide-calcium chelate.

[0009] This invention utilizes bromelain to enzymatically hydrolyze soy protein isolate, obtaining soy protein peptides with optimal calcium chelating ability through a specific enzymatic hydrolysis process (time and temperature), thereby increasing the calcium chelation content in the peptide-calcium chelate. Furthermore, existing technologies typically prepare peptide-calcium chelates by directly spray-drying the peptide-calcium chelate solution, but this method results in the presence of unreacted free calcium ions in the product, leading to a low chelation rate, and the high temperature of spray drying may affect the bioactivity of the peptides. This invention, through alcohol precipitation combined with freeze-drying, produces peptide-calcium chelates with high purity, high chelation rate, and high bioavailability.

[0010] Preferably, in step S1, the method for preparing the soy protein isolate includes the following steps: mixing soybean meal powder with water, adding alkaline solution to adjust the pH of the mixed solution to 7.2-7.8, then adding preservative, stirring, and filtering to obtain a filtrate; centrifuging the filtrate to obtain a supernatant, adding acid solution to the supernatant to adjust the pH to 4.1-4.9, allowing it to stand, centrifuging to collect the precipitate; dissolving the precipitate, adding alkaline solution to adjust the pH to 6.8-7.2, and finally dialyzing and freeze-drying to obtain the soy protein isolate.

[0011] The soy protein isolate of this invention is a high-quality plant protein extracted from soybean meal, with a protein content ≥90%, a balanced amino acid composition, and rich in glutamic acid and aspartic acid (accounting for more than 34% of the total amino acids). This invention uses soybean meal as a base material to prepare peptide-calcium chelates, resulting in low preparation costs and ease of industrial production. While ensuring high calcium chelation capacity, the peptide-calcium chelates also exhibit good economic efficiency, stability, and bioavailability.

[0012] Preferably, the mass ratio of soybean meal powder to water is 1:(5~10); more preferably, the mass ratio of soybean meal powder to water is 1:(5.5~9); and even more preferably, the mass ratio of soybean meal powder to water is 1:(6~8).

[0013] Preferably, the alkaline solution includes at least one of NaOH and KOH solutions.

[0014] Preferably, the concentration of the alkaline solution is 1~3 mol / L; more preferably, the concentration of the alkaline solution is 1.2~2.8 mol / L; and even more preferably, the concentration of the alkaline solution is 1.5~2.5 mol / L.

[0015] Preferably, the acid solution includes at least one of HCl, citric acid, acetic acid, and phosphoric acid solution.

[0016] Preferably, the concentration of the acid solution is 1~3 mol / L; more preferably, the concentration of the acid solution is 1.2~2.8 mol / L; and even more preferably, the concentration of the acid solution is 1.5~2.5 mol / L.

[0017] Preferably, the preservative includes at least one of Proclean 150, Proclean 300, and Proclean 950.

[0018] Preferably, the soy protein isolate solution comprises an aqueous solution of soy protein isolate.

[0019] Preferably, the mass concentration of the soy protein isolate solution is 1-4%; more preferably, the mass concentration of the soy protein isolate solution is 1.2-3.5%; and even more preferably, the mass concentration of the soy protein isolate solution is 1.6-3%.

[0020] Preferably, the enzymatic hydrolysis temperature in step S1 is 48℃~53℃; more preferably, the enzymatic hydrolysis temperature in step S1 is 49℃~52℃.

[0021] Preferably, the enzymatic hydrolysis time in step S1 is 5h~7.8h; more preferably, the enzymatic hydrolysis time in step S1 is 5h~7.6h; and even more preferably, the enzymatic hydrolysis time in step S1 is 5h~7.5h.

[0022] Preferably, the pH of the enzymatic hydrolysis in step S1 is 6.8~7.2; more preferably, the pH of the enzymatic hydrolysis in step S1 is 6.9~7.1.

[0023] Preferably, the amount of protease added in step S1 is 0.2% to 0.8% of the soybean protein isolate; more preferably, the amount of protease added in step S1 is 0.21% to 0.77% of the soybean protein isolate; even more preferably, the amount of protease added in step S1 is 0.23% to 0.76% of the soybean protein isolate; and even more preferably, the amount of protease added in step S1 is 0.25% to 0.75% of the soybean protein isolate. This invention optimizes the type of protease and the enzymatic hydrolysis process, achieving excellent enzymatic hydrolysis effects while maintaining a low amount of protease added.

[0024] Preferably, the enzyme inactivation treatment in step S1 includes heating in a water bath at 90~100℃ for 10~20 minutes.

[0025] Preferably, before the chelation reaction, the soybean protein peptides described in step S1 are subjected to ultrafiltration to obtain soybean protein peptides with a molecular weight > 5 kDa or a molecular weight < 3 kDa. This invention uses ultrafiltration to treat the enzymatically hydrolyzed soybean protein peptides, obtaining soybean protein peptides with a reasonable molecular weight distribution and optimal calcium chelation capacity. This improves the thermal stability and calcium chelation amount of the peptide-calcium chelate while making it easier for the small intestine to absorb.

[0026] Preferably, the ultrafiltration method is to use a 3kDa and / or 5kDa membrane pack for ultrafiltration.

[0027] Preferably, the mass ratio of soybean protein peptides to calcium salts in step S2 is 1:(0.5~2); more preferably, the mass ratio of soybean protein peptides to calcium salts in step S2 is 1:(0.6~1.8); and even more preferably, the mass ratio of soybean protein peptides to calcium salts in step S2 is 1:(0.8~1.2).

[0028] Preferably, the calcium salt in step S2 includes at least one of calcium chloride, calcium lactate, calcium gluconate, calcium citrate, and calcium acetate.

[0029] Preferably, the chelation reaction time in step S2 is 30-80 min; more preferably, the chelation reaction time in step S2 is 40-75 min; and even more preferably, the chelation reaction time in step S2 is 50-70 min.

[0030] Preferably, the chelation reaction temperature in step S2 is 35~40℃; more preferably, the chelation reaction temperature in step S2 is 36~38℃.

[0031] Preferably, the pH of the chelation reaction in step S2 is 6.8 to 7.2; more preferably, the pH of the chelation reaction in step S2 is 6.9 to 7.0.

[0032] Preferably, the alcohol precipitation in step S2 uses anhydrous ethanol; more preferably, the volume ratio of the anhydrous ethanol to the peptide calcium chelate is (2~6):1; even more preferably, the volume ratio of the anhydrous ethanol to the peptide calcium chelate is (2.5~4):1.

[0033] Preferably, the separation method in step S2 is centrifugation; more preferably, the centrifugation speed is 6000~10000g; even more preferably, the centrifugation speed is 7000~9000g.

[0034] Preferably, in step S2, the precipitate is washed multiple times with anhydrous ethanol before freeze-drying.

[0035] To achieve the second objective mentioned above, the present invention provides the following technical solution.

[0036] Secondly, the present invention provides a soybean-based peptide calcium chelate, which is prepared by the preparation method described in the first aspect.

[0037] Preferably, the calcium chelation amount of the soybean ground-state calcium chelate is ≥1.25 mmol / g; more preferably, the calcium chelation amount of the soybean ground-state calcium chelate is ≥1.30 mmol / g; and even more preferably, the calcium chelation amount of the soybean ground-state calcium chelate is 1.30~1.50 mmol / g.

[0038] To achieve the third objective mentioned above, the present invention provides the following technical solution.

[0039] Thirdly, the present invention provides the use of the peptide-calcium chelate described in the second aspect in functional foods, nutritional fortifiers, pharmaceutical preparations, or calcium supplements.

[0040] The beneficial effects of this invention are: (1) This invention uses bromelain to enzymatically hydrolyze soy protein isolate. Through a specific enzymatic hydrolysis process (enzyme type, hydrolysis time, and hydrolysis temperature), soy protein peptides with optimal calcium chelating ability are obtained. After chelating with calcium ions, the amount of calcium chelated in the peptide-calcium chelate and its thermal stability are improved. In addition, this invention uses alcohol precipitation combined with freeze drying, which avoids the problems of unreacted free calcium ions in the product, low product chelation rate, and the impact of high temperature on peptide bioactivity caused by direct spray drying in the prior art. This results in a peptide-calcium chelate with high purity, high chelation rate, and high bioavailability.

[0041] (2) The present invention performs ultrafiltration on the enzymatically hydrolyzed soybean protein peptides to obtain soybean protein peptides with reasonable molecular weight distribution and optimal calcium chelation ability, thereby improving the thermal stability and calcium chelation amount of the peptide calcium chelate and making it easier for the small intestine to absorb.

[0042] (3) The soybean-based peptide calcium chelate prepared by this invention has high calcium chelation capacity, high stability and high bioavailability. The calcium chelation capacity of the soybean-based peptide calcium chelate is as high as 1.373 mmol / g as determined by a calcium ion meter, which can be well applied in functional foods, nutritional fortifiers, pharmaceutical preparations or calcium supplements. Attached Figure Description

[0043] Figure 1 The graphs show the protein content, degree of hydrolysis, and calcium chelation capacity of the enzymatic hydrolysates after enzymatic hydrolysis in Examples 1, 4, and Comparative Examples 4-6. Figure 2 The graphs show the protein content, degree of hydrolysis, and calcium chelation capacity of the enzymatic hydrolysates after enzymatic hydrolysis in Examples 1-3 and Comparative Examples 1-3. Figure 3 The graph shows the calcium chelating capacity of soybean protein peptides S1-S4. Figure 4 The graph shows the calcium chelating capacity test results for soybean protein peptides S3 and F1~F3. Figure 5 The graph shows the difference between the zeta potentials of the peptide calcium chelates S3-Ca, F1-Ca, F2-Ca, and F3-Ca and the zeta potentials of soybean protein peptides S3, F1, F2, and F3. Figure 6 Scanning electron microscope images of soybean protein peptide F3 and chelate F3-Ca at different magnifications; Figure 7 Infrared spectra of CaCl2, soybean protein peptide F3, and chelate F3-Ca; Figure 8 The UV-Vis spectra of soybean protein peptide F3 and its chelate F3-Ca are shown. Figure 9 The circular dichroism chromatograms of soybean protein peptide F3 and its chelate F3-Ca, as well as the percentage of various secondary structures of F3 and F3-Ca; Figure 10 Thermogravimetric analysis diagram of soybean protein peptide F3 and chelate F3-Ca; Figure 11 Differential scanning calorimetry (DSC) spectra of soybean protein peptide F3, chelate F3-Ca, and CaCl2; Figure 12 The diagram shows the digestibility analysis of CaCl2 and several peptide calcium chelates, namely S3-Ca, F1-Ca, F2-Ca, and F3-Ca. Detailed Implementation

[0044] To enable those skilled in the art to better understand this application, the present invention will be further described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims.

[0045] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] The membrane packaging used in this invention is manufactured by Sartorius, and the model is Vivaflow.

[0047] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0048] Example 1 This embodiment provides a soybean-based peptide calcium chelate, prepared by the following method: Step 1: Extraction of soy protein isolate: Low-temperature defatted soybean meal is ground into powder using a high-speed grinder. The soybean meal powder is mixed with deionized water at a ratio of 1:7 (mass ratio). After thorough mixing, 2 mol / L NaOH solution is added dropwise to adjust the pH of the mixture to 7.5. Then, Proclean 950 preservative is added dropwise at a liquid-to-mass ratio of 0.1%. After stirring for 4 hours, the mixture is filtered through 4 layers of gauze. The filtrate is centrifuged at 8000g and 4℃ for 20 minutes. The supernatant is collected in a beaker. While stirring, 2 mol / L HCl is slowly added dropwise to adjust the pH to 4.5. The mixture is allowed to stand at 4℃ for 30 minutes, and then centrifuged at 8000g and 4℃ for 20 minutes. The precipitate is collected and washed with deionized water. After washing, deionized water was added to the precipitate and stirred thoroughly. The pH was adjusted to 7.0 with 2 mol / L NaOH and stirred until the precipitate was fully dissolved. The resulting solution was then placed in a 10 kDa dialysis bag and dialyzed at 4°C for 48 h. The deionized water was changed every 8-12 h. The dialyzed solution was pre-frozen and then freeze-dried using a vacuum freeze dryer to obtain soy protein isolate. The isolate was then sealed and stored in a freezer at -20°C.

[0049] Step 2: Enzymatic hydrolysis: Dissolve 2g of the soy protein isolate prepared in Step 1 in 100 mL of deionized water to obtain a soy protein isolate solution. Adjust the pH of the soy protein isolate solution to 7.0 using 1 mol / L NaOH and 1 mol / L HCl. Then add bromelain at a concentration of 0.5% (w / w) of the soy protein isolate mass and hydrolyze in a 50℃ constant temperature water bath for 7 hours.

[0050] Step 3: Enzyme inactivation treatment: After the enzymatic hydrolysis is completed, the temperature of the constant temperature water bath is quickly raised to 100℃ and heated for 15 minutes to obtain the enzymatic hydrolysate.

[0051] Step 4: Centrifugation and freeze-drying: Cool the enzymatic hydrolysate to room temperature, adjust the pH to 7.0, centrifuge at 4℃ and 8000g for 20 minutes, collect the supernatant, and freeze-dry the supernatant to obtain soybean protein peptides.

[0052] Step 5: Chelation reaction: Dissolve the soybean protein peptides obtained in Step 4 with calcium chloride solid in 100 mL of water at a mass ratio of 1:1, with each concentration being 50 g / L. Then adjust the pH to 7.0 with 1 mol / L NaOH and 1 mol / L HCl, and stir the reaction in a 37℃ water bath for 60 minutes.

[0053] Step 6: Separation and purification: After the chelation reaction is completed, add 3 times the volume of anhydrous ethanol to the reaction solution for alcohol precipitation. After standing for 20 minutes, centrifuge at 8000g for 20 minutes, collect the precipitate after centrifugation, and wash it twice with anhydrous ethanol. After each washing, centrifuge and discard the supernatant. Finally, freeze-dry the precipitate to obtain soybean-based peptide calcium chelate.

[0054] Example 2 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the amount of bromelain added in this embodiment is 0.25% of the soybean protein isolate solution, while the rest is the same as in Example 1.

[0055] Example 3 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the amount of bromelain added in this embodiment is 0.75% of the soybean protein isolate solution, while the rest is the same as in Example 1.

[0056] Example 4 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the enzymatic hydrolysis time in this embodiment is 5 hours, while the rest is the same as in Example 1.

[0057] Example 5 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the enzymatic hydrolysis time in this embodiment is 7.5 hours, while the rest is the same as in Example 1.

[0058] Example 6 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 2 in that, before the chelation reaction, the soybean protein peptides obtained in step 4 are subjected to ultrafiltration using a 5kDa membrane to obtain soybean protein peptides with a molecular weight >5kDa. All other aspects are the same as in Example 1.

[0059] Example 7 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 2 in that, before the chelation reaction, the soybean protein peptides obtained in step 4 are subjected to ultrafiltration using 5kDa and 3kDa membranes to obtain soybean protein peptides with a molecular weight of 3kDa to 5kDa. All other aspects are the same as in Example 1.

[0060] Example 8 This embodiment provides a soybean-based peptide calcium chelate, which differs from Example 2 in that, before the chelation reaction, the soybean protein peptides obtained in step 4 are subjected to ultrafiltration using a 3kDa membrane to obtain soybean protein peptides with a molecular weight <3kDa. All other aspects are the same as in Example 1.

[0061] Comparative Example 1 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that no bromelain is added in this comparative example.

[0062] Comparative Example 2 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the amount of bromelain added in this comparative example is 0.1% of the soybean protein isolate solution.

[0063] Comparative Example 3 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the amount of bromelain added in this comparative example is 1% of the soybean protein isolate solution.

[0064] Comparative Example 4 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the enzymatic hydrolysis time in this comparative example is 9 hours.

[0065] Comparative Example 5 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the enzymatic hydrolysis time in this comparative example is 3 hours.

[0066] Comparative Example 6 This comparative example provides a soybean-based peptide calcium chelate, which differs from Example 1 in that the enzymatic hydrolysis time in this comparative example is 1 hour.

[0067] Performance testing 1. The protein content, degree of hydrolysis, and calcium chelation capacity of the enzymatic hydrolysate were tested under different enzymatic hydrolysis times and different enzyme addition amounts.

[0068] Protein content determination: The total nitrogen content of each enzymatic hydrolysis product (inactivated enzymatic hydrolysate) was determined using a semi-automatic Kjeldahl nitrogen analyzer. Based on the average nitrogen content of approximately 16% in protein, the conversion relationship for protein content was obtained, i.e., protein content = nitrogen content × 6.25, and the protein content was calculated accordingly.

[0069] Degree of hydrolysis determination: The degree of hydrolysis was determined by formaldehyde titration. An appropriate amount of the inactivated enzyme hydrolysate (obtained in step 3) was mixed with deionized water to prepare a sample of 80g. The control group consisted of 80g of deionized water. The solution was titrated with 0.10 mol / L NaOH solution to pH 8.2. After adding 10 mL of formaldehyde, titration continued to pH 9.2. The volumes of NaOH consumed by the control group and the sample were recorded as V0 and V, respectively. s The unit is mL. Free amino acid nitrogen (FAAN, g / g) and degree of hydrolysis (DH, %) were calculated using Formula I and Formula II, respectively, where m EH This refers to the mass of the enzyme hydrolysate used, expressed in grams (g); M EH It is the total mass of the enzymatic hydrolysate, in grams (g) and chromium (C). NaOH The concentration of the NaOH solution is 0.10 mol / L.

[0070] FAAN (g / g) = (V s V0) / 1000 × C NaOH × 14 × 100% / m EH Formula I.

[0071] DH (%) = FAAN × M EH × 100% / (MSHM, tN) Formula II.

[0072] Calcium chelating capacity: Precisely prepared CaCl2 solutions with concentrations of 0.1, 0.5, 1, 10, and 100 mmol / L were used as a series of standard solutions. The ionic strength was adjusted to 0.1 mol / L with 0.1 M KCl solution. The potential values ​​(mV) corresponding to the series of standard solutions were measured using a calcium ion electrode. 2+ The logarithm of the concentration (log [Ca) 2+[ ] is used as the x-axis and the potential value as the y-axis, and a linear equation is fitted. Based on the protein content determination results, different enzymatic hydrolysates were prepared into solutions with a protein concentration of 2 g / L as sample groups. 76 mL of each solution was placed in a 100 mL reagent bottle, and 4 mL of 0.25 mol / L CaCl2 solution was added. The pH was adjusted to 7.0 ± 0.2 at 25 ℃ using KOH and HCl solutions. The volume V of the solution after pH adjustment was recorded. The reagent bottle was then placed in a constant temperature water bath and stirred at 37 ℃ for 1 h. After being removed and allowed to return to room temperature, the initial conductivity was measured and recorded using a conductivity meter. Finally, the conductivity was adjusted to be close to that of the calcium standard solution using 3 mol / L KCl solution, and the calcium ion concentration of the sample group solution was measured. The blank group consisted of 74 mL of water, with 2 mL of 3 mol / L KCl and 4 mL of 0.25 mol / L CaCl2 solution added to make its conductivity close to that of the calcium standard solution. The potential values ​​of the blank group and each sample group solutions were obtained using a calcium ion selective electrode, and the calcium ion concentration was calculated by comparing them with a standard curve. The difference in calcium ion concentration between the blank group and the sample group represents the calcium ions chelated by soybean protein peptides in different enzymatic hydrolysates. The calcium ion chelating capacity (mg / g or mmol / g) was calculated based on the solution volume V and protein content.

[0073] Figure 1 The graphs show the protein content, degree of hydrolysis, and calcium chelation capacity of the enzymatic hydrolysates after enzymatic hydrolysis in Examples 1, 4, and Comparative Examples 4-6. Figure 1 The 0h value represents a blank control that is immediately inactivated after enzyme addition. Figure 1 It can be seen that the enzymatic hydrolysis time of 5-8 hours all have good calcium chelation ability, and the calcium chelation ability is strongest when the enzymatic hydrolysis time is 7 hours.

[0074] Figure 2 The graphs show the protein content, degree of hydrolysis, and calcium chelation capacity of the enzymatic hydrolysates after enzymatic hydrolysis in Examples 1-3 and Comparative Examples 1-3. Figure 2 The 0% indicates that Comparative Example 1 did not contain bromelain. Figure 2 It can be seen that bromelain added at concentrations ranging from 0.25% to 0.75% exhibits good calcium chelation ability. Considering cost savings, an addition concentration of 0.25% of bromelain is the optimal amount.

[0075] II. Testing the calcium chelating ability of soybean protein peptides obtained from different proteases.

[0076] This invention also investigated and tested the enzymatic hydrolysis effects of papain, flavor protease, and fig protease. The optimal enzymatic hydrolysis conditions for different proteases and the resulting soybean protein peptides were determined as follows: Papain: The enzyme addition amount was 0.75%, and the enzyme was hydrolyzed at pH 7.0 and 55℃ for 7 hours. The soybean protein peptide obtained after hydrolysis was denoted as S1. Flavor protease: The enzyme addition amount was 0.75%, and the enzyme was hydrolyzed at pH 7.0 and 50℃ for 7 hours. The soybean protein peptide obtained after hydrolysis was denoted as S2. Bromelain: The amount added was 0.25%, and the enzyme was hydrolyzed at pH 7.0 and 50°C for 7 hours (i.e., Example 2). The soybean protein peptide obtained after enzymatic hydrolysis was designated as S3. Fig protease: Added at 0.50%, enzymatically hydrolyzed at pH 7.0 and 65℃ for 7 hours. The resulting soybean protein peptide is designated as S4.

[0077] The calcium chelation capacity of soybean protein peptides S1-S4 was tested using the above-mentioned calcium chelation capacity test method. The sample group solutions were prepared by dissolving soybean protein peptides S1-S4 in water to prepare solutions with a protein concentration of 2 g / L. Figure 3 This is a graph showing the calcium chelating capacity of soybean protein peptides S1-S4. Figure 3 In the figure, abcd represents the significance level of differences among different soybean protein peptides. Figure 3 It can be seen that the soybean protein peptide S3 obtained after enzymatic hydrolysis by bromelain has the best calcium chelation ability.

[0078] The effect of ultrafiltration on the calcium chelating ability of soybean protein peptides was investigated. The calcium chelating ability of soybean protein peptide S3 and the soybean protein peptides obtained in Examples 6-8 were tested respectively. The soybean protein peptides obtained in Examples 6-8 were designated as F1, F2, and F3, respectively. The test results are as follows: Figure 4 As shown, Figure 4 In the figure, abcd represents the significance level of differences between soybean protein peptides of different molecular weights. Figure 4 This is a graph showing the calcium chelating capacity of soybean protein peptides S3 and F1~F3. (Source: [Insert source here]) Figure 4 It can be seen that the soybean protein peptide F3 with a molecular weight of <3kDa obtained in Example 8 has the best calcium chelating ability.

[0079] III. Structural Characterization of Peptide-Calcium Chelates (1) Measurement of ζ potential Using deionized water as the dispersion medium, soybean protein peptides S3, F1, F2, F3, and peptide calcium chelates S3-Ca, F1-Ca, F2-Ca, and F3-Ca were prepared into solutions with a concentration of 0.1 mg / mL. The zeta potentials of soybean protein peptides S3, F1, F2, F3, and peptide calcium chelates S3-Ca, F1-Ca, F2-Ca, and F3-Ca were measured using a dynamic light scattering instrument. Figure 5This is a graph showing the zeta potential of the peptide calcium chelates S3-Ca, F1-Ca, F2-Ca, and F3-Ca, and the difference in zeta potential between them and the soybean protein peptides S3, F1, F2, and F3. Figure 5 In the figure, abc represents the significance level of the differences between different peptide calcium chelates. Figure 5 In this context, S3 represents the zeta potential of the peptide calcium chelate S3-Ca minus the zeta potential of the soybean protein peptide S3, and so on. Figure 5 It can be seen that after different soybean protein peptides chelate with calcium ions, the zeta potential of the peptide-calcium chelate formed shows a positive increasing trend. This indicates that the negatively charged groups on the surface of soybean protein peptides and the positively charged calcium ions chelate to form a stable coordination structure.

[0080] (2) Scanning electron microscopy analysis Soybean protein peptide F3 sample powder and its chelate F3-Ca with calcium ions were uniformly placed on a stage coated with conductive carbon adhesive. A metal thin film was sputtered onto the surface using an ion sputtering instrument in a vacuum environment to ensure conductivity. The microstructure of the samples was obtained using ultra-high resolution field emission scanning electron microscopy. The results are as follows: Figure 6 As shown. Figure 6 Scanning electron microscope (SEM) images of soybean protein peptide F3 and its chelate F3-Ca at different magnifications. Figure 6 It can be seen that after F3 undergoes a chelation reaction with calcium ions, its microstructure changes, exhibiting a distinct spherical granular morphology, increased surface roughness, and a more compact overall structure.

[0081] (3) Fourier transform infrared spectroscopy analysis Figure 7 The infrared spectra are of CaCl2, soybean protein peptide F3, and the chelate F3-Ca. Figure 7 It can be seen that soybean protein peptide F3 is located at 3386 cm⁻¹. - ¹ (NH stretching vibration), 1659 cm - A characteristic absorption peak is observed at ¹ (C=O stretching vibration, amide I band). After chelation with calcium ions to form F3-Ca, a peak is observed at 3386 cm⁻¹. - The peak at position ¹ shifted blue to 3466 cm⁻¹ - ¹, 1659 cm - The peak at point ¹ has redshifted to 1633 cm⁻¹ - ¹ This indicates that the carboxyl oxygen atom and amino nitrogen atom on the soybean protein peptide participate in the coordination of calcium ions.

[0082] (4) Ultraviolet-visible spectroscopic analysis Soybean protein peptide F3 and peptide calcium chelate F3-Ca were dissolved in deionized water to prepare solutions with a concentration of 0.5 mg / mL. The ultraviolet absorption spectra of the solutions were measured in the range of 190-480 nm using a UV-Vis spectrophotometer. Figure 8 The image shows the UV-Vis spectra of soybean protein peptide F3 and its chelate F3-Ca. Figure 8 It can be seen that after F3 chelates with calcium ions, the absorption peak of the sample shifts to the longer wavelength direction and the absorption intensity is enhanced. This indicates that the carboxyl oxygen and carbonyl group of the amide bond of soybean protein peptide F3 participate in the chelation with calcium ions. The addition of calcium ions may cause a conformational change in the peptide chain, exposing the aromatic residues that were originally buried inside the molecule, thus leading to a change in the ultraviolet absorption spectrum.

[0083] (5) Circular dichroism analysis Soybean protein peptide F3 and peptide calcium chelate F3-Ca were dissolved in deionized water to prepare solutions with a concentration of 0.5 mg / mL. The solutions were then scanned at 1 nm / s in the 190-260 nm range using a circular dichroism chromatograph under nitrogen purging conditions at room temperature. The secondary structure of the samples was calculated using CDNN. Figure 9 Circular dichroism chromatogram of soybean protein peptide F3 and peptide calcium chelate F3-Ca ( Figure 9 a) and the proportion of various secondary structures of F3 and F3-Ca (a) Figure 9 b). Figure 9 The results showed that β-sheet increased from 7.97% in the free state to 24.09% after chelation; random coil increased from 41.41% to 46.91%; helical structure decreased from 6.98% to 5.01%; and β-turn decreased from 43.63% to 24.09%. Figure 9 This indicates that the presence of calcium ions drives the transformation of the peptide chain from random coil to a more ordered β-sheet structure, inducing peptide chain folding and forming a more compact secondary structure.

[0084] IV. Thermal Stability Study (1) Thermogravimetric analysis Soybean protein peptide F3 and peptide calcium chelate F3-Ca (sample mass 5~10 mg) were heated from 40℃ to 600℃ at a heating rate of 10℃ / min under a nitrogen atmosphere to obtain thermogravimetric analysis chromatograms. Figure 10 Thermogravimetric analysis (TGA) chromatograms of soybean protein peptide F3 and its chelate F3-Ca. (From...) Figure 10 It can be seen that, compared with soybean protein peptide F3, the peptide calcium chelate sample F3-Ca has a higher decomposition temperature, lower weight loss rate, and higher high-temperature residual mass. This indicates that the peptide calcium chelate F3-Ca has stronger thermal stability. This enhanced thermal stability originates from the coordination bonds formed between calcium ions and coordinating groups such as carboxyl, amino, and carbonyl groups in the peptide chain, which effectively stabilizes the spatial conformation of the peptide.

[0085] (2) Differential scanning calorimetry Using an empty crucible as a reference, soybean protein peptide F3, peptide calcium chelate F3-Ca, and CaCl2 were heated from 20 ℃ to 200 ℃ at a heating rate of 10 ℃ / min under a nitrogen atmosphere (flow rate 20 mL / min), and the heat flux was recorded as a function of temperature. Figure 11 Differential scanning calorimetry (DSC) spectra of soybean protein peptide F3, peptide-calcium chelate F3-Ca, and CaCl2. Figure 11 It can be seen that, compared with F3 and CaCl2, the endothermic peak of the peptide calcium chelate F3-Ca shifts significantly to the high-temperature region, and the peak shape is sharper. This indicates that calcium chelation can significantly improve the thermal stability of soybean protein peptides, while making the molecular arrangement more regular and increasing the crystallinity. The core reason for this is that calcium ions combine with carboxyl oxygen and amino nitrogen groups in soybean protein peptides to form a stable chelate structure, enhancing the intermolecular interaction, making the spatial structure of the chelate more stable, and increasing the heat required to destroy its crystal structure or intermolecular forces. Therefore, the changes in the spectrum of peptide calcium chelate F3-Ca, such as increased temperature, sharper peak shape, and decreased peak width, all reflect the improved uniformity of molecular arrangement in peptide calcium chelate F3-Ca.

[0086] V. Digestive Stability Study CaCl2 and several peptide calcium chelates, namely S3-Ca, F1-Ca, F2-Ca and F3-Ca, were subjected to in vitro gastrointestinal simulated digestion experiments according to the INFOGEST 2.0 standard method. Figure 12 The diagram shows the digestibility and stability of CaCl2 and several peptide-calcium chelates, including S3-Ca, F1-Ca, F2-Ca, and F3-Ca. A horizontal comparison of the free calcium ion concentrations of the four peptide-calcium chelates reveals that F3-Ca has the lowest free calcium ion concentration, indicating that it has the strongest calcium chelating ability and contains the fewest unchelated calcium ions. A vertical comparison of the peptide-calcium chelates and their respective in vitro digestion products shows that the free calcium ion concentrations of samples S3-Ca, F1-Ca, and F2-Ca all decreased slightly after digestion, indicating a reduction in absorbable calcium ions in the intestine after digestion. However, for sample F3-Ca, the free calcium ion concentration increased by approximately 100% after digestion, indicating an increase in absorbable calcium ions, and some small-molecule peptide-calcium chelates were still present. Combined with the analysis of calcium ion absorption mechanisms, F3-Ca has higher calcium ion accessibility, indicating that the bioavailability of peptide-calcium chelate F3-Ca is relatively high.

[0087] In summary, this invention utilizes bromelain to enzymatically hydrolyze soy protein isolate. Through a specific enzymatic hydrolysis process (enzyme type, hydrolysis time, and hydrolysis temperature), soy protein peptides with optimal calcium chelating ability are obtained. After chelating with calcium ions, the amount of calcium chelated in the peptide-calcium chelate and its thermal stability are improved. Furthermore, in this embodiment, the enzymatically hydrolyzed soy protein peptides are subjected to ultrafiltration to obtain soy protein peptides with a reasonable molecular weight distribution and optimal calcium chelating ability. This improves the thermal stability and calcium chelating amount of the peptide-calcium chelate while making it easier for the small intestine to absorb, making it suitable for use in functional foods, nutritional fortifiers, pharmaceutical preparations, or calcium supplements.

[0088] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing soybean-based peptide calcium chelates, characterized in that, Includes the following steps: S1: Add protease to the soy protein isolate solution, hydrolyze at 48℃~55℃ for 5h~8h, and obtain soy protein peptides after enzyme inactivation treatment; the protease includes bromelain. S2: The soybean protein peptides described in step S1 are chelated with calcium salts to obtain a peptide-calcium chelate reaction solution. The peptide-calcium chelate reaction solution is precipitated with alcohol to obtain a precipitate. The precipitate is freeze-dried to obtain the soybean-based peptide-calcium chelate.

2. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, In step S1, the preparation method of the soy protein isolate includes the following steps: mixing soybean meal powder with water, adding alkaline solution to adjust the pH to 7.2-7.8, then adding preservative, stirring, and filtering to obtain a filtrate; centrifuging the filtrate to obtain a supernatant, adding acid solution to the supernatant to adjust the pH to 4.1-4.9, allowing it to stand, centrifuging to collect the precipitate; dissolving the precipitate, adding alkaline solution to adjust the pH to 6.8-7.2, and finally dialyzing and freeze-drying to obtain the soy protein isolate.

3. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, The mass concentration of the soy protein isolate solution in step S1 is 1-4%.

4. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, The amount of protease added in step S1 is 0.2% to 0.8% of the soybean protein isolate.

5. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, Before the chelation reaction, the soybean protein peptides described in step S1 are subjected to ultrafiltration to obtain soybean protein peptides with a molecular weight >5kDa or a molecular weight <3kDa.

6. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, The mass ratio of soybean protein peptides to calcium salts in step S2 is 1:(0.5~2).

7. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, The calcium salt mentioned in step S2 includes at least one of calcium chloride, calcium lactate, calcium gluconate, calcium citrate, and calcium acetate.

8. The method for preparing soybean-based peptide calcium chelate according to claim 1, characterized in that, The chelation reaction in step S2 is carried out at a temperature of 35~40℃; And / or, the chelation reaction takes 30 to 80 minutes; And / or, the pH of the chelation reaction is 6.8 to 7.

2.

9. A soybean-based peptide calcium chelate, characterized in that, The soybean-based peptide calcium chelate was prepared using the method described in any one of claims 1 to 8.

10. The use of the soybean-based peptide calcium chelate according to claim 9 in functional foods, nutritional fortifiers, pharmaceutical preparations or calcium supplements.

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