A high-calcium, high-phosphorus soybean protein, products containing it, and preparation method thereof

Abstract

This invention discloses a high-calcium, high-phosphorus soybean protein, a product containing the same, and a preparation method thereof. The high-calcium, high-phosphorus soybean protein is obtained by sequentially extracting defatted soybean meal using an alkali-soluble acid precipitation method, followed by heat treatment and enzymatic hydrolysis, and then compounding it with calcium salts and phosphates. The high-calcium, high-phosphorus soybean protein prepared by this invention has a calcium content ≥3.1% and a phosphorus content ≥2%, and exhibits good stability in food systems, fully meeting the calcium and phosphorus intake requirements of plant-based food consumers.

Description

Technical Field

[0001] This invention belongs to the field of food chemistry, and specifically relates to a high-calcium, high-phosphorus soybean protein, products containing the same, and a preparation method thereof. Background Technology

[0002] Soy protein is a high-quality plant protein with excellent nutritional and health properties and functional characteristics, making it widely used in the food industry. Replacing animal protein with plant protein is a future trend in food development. However, compared to animal protein, soy protein still has the disadvantage of lower calcium content, and its calcium content is also lower. 2+ In the presence of certain substances, aggregation and precipitation are likely to occur, leading to changes in the structure and functional properties of soybean protein.

[0003] Previous studies have extensively explored ways to increase the calcium content of soy protein products. Some research has involved directly adding insoluble or slightly soluble calcium salts such as molecular calcium and tricalcium phosphate to soy protein to achieve this goal. However, these calcium salts exhibit low bioavailability and poor heat and pH stability. Another method, using alkali dissolution and acid precipitation to prepare soy protein, involves partially replacing NaOH with Ca(OH)₂, resulting in calcium-enriched soy protein. However, the calcium content only reaches 6.5 mg / g, and the calcium stability remains unsatisfactory. Other studies have employed calcium salt encapsulation to reduce calcium-protein contact and prevent precipitation. However, this requires the addition of emulsifiers and stabilizers and is primarily used in soy milk and instant soy flour systems, not in the preparation of soy protein products. Furthermore, existing technologies focus solely on high calcium content without considering the need for high phosphorus. Calcium ions readily form phosphate precipitates in the presence of phosphates, further complicating the preparation of high-phosphorus, high-calcium soy protein.

[0004] Therefore, the lack of technology for preparing high-calcium and high-phosphorus soybean protein products with high absorbable calcium content and good calcium stability has become an urgent technical problem to be solved. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a high-calcium, high-phosphorus soybean protein, a product containing the same, and a preparation method thereof. The high-calcium, high-phosphorus soybean protein prepared by the method of this invention has a calcium content ≥3.1% and a phosphorus content ≥2%, and exhibits good stability in food systems.

[0006] In a first aspect, the present invention provides a method for preparing high-calcium, high-phosphorus soybean protein, comprising: pulverizing defatted soybean meal and mixing it with water to form a solution; adjusting the pH of the solution to alkaline; stirring; centrifuging and collecting the supernatant; adjusting the pH of the supernatant to acidic; allowing it to stand; centrifuging and collecting the precipitate; reconstituted with water and adjusting the pH to alkaline to obtain a first solution; heating the first solution to 100±10℃ and maintaining it for 30±5 min; cooling and drying it into powder; reconstituted with water and adjusting the pH to neutral to obtain a second solution; adding protease to the second solution for enzymatic hydrolysis; after inactivating the enzyme, drying it into powder; reconstituted with water to obtain a third solution; and sequentially adding calcium salt and phosphate to the third solution and stirring to allow the reaction to proceed.

[0007] According to a specific embodiment of the present invention, defatted soybean meal is pulverized and mixed with water at a ratio of 1g:10-20mL to prepare a solution. The pH of the solution is adjusted to 8.0±0.5, stirred at 25℃ for 2±0.5h, and the supernatant is collected by centrifugation. The pH of the supernatant is adjusted to 4.5±0.5, allowed to stand, and the precipitate is collected by centrifugation. The precipitate is then reconstituted with water at a ratio of 1g:3-5mL and the pH is adjusted to 8.0±0.5 to obtain the first solution. Preferably, the ratio of defatted soybean meal to water is 1g:15mL. Preferably, the pH of the solution of defatted soybean meal and water is adjusted to 8. Preferably, the pH of the supernatant is adjusted to 4.5. Preferably, the pH is adjusted to 8 after reconstitution with water at a ratio of 1g:4mL.

[0008] According to a specific embodiment of the present invention, the first solution is heated to 100°C and held for 30 minutes.

[0009] According to a specific embodiment of the present invention, the concentration of the second solution is 4-6% w / w, preferably 5% w / w.

[0010] According to a specific embodiment of the present invention, the protease is a flavor protease; preferably, 1000±100 U / g of flavor protease is added according to the mass of the protein substrate, the enzymatic hydrolysis temperature is 50±5℃, and the enzymatic hydrolysis time is 180±30 min; more preferably, 1000 U / g of flavor protease is added according to the mass of the protein substrate, the enzymatic hydrolysis temperature is 50℃, and the enzymatic hydrolysis time is 180 min.

[0011] According to a specific embodiment of the present invention, the concentration of the third solution is 5-7% w / v, preferably 6% w / v.

[0012] According to a specific embodiment of the present invention, the calcium salt is calcium chloride, and the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate; preferably, the molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1:1; preferably, the calcium salt and phosphate are added in the form of calcium salt solution and phosphate solution, respectively, with the concentration of calcium salt solution being 1 mol / L and the concentration of phosphate solution being 0.2 mol / L; the mass-volume ratio of the solid solution, calcium salt solution and phosphate solution in the third solution is 1 g:0.9-1.1 mL:1.9-2.1 mL, preferably 1 g:1 mL:2 mL.

[0013] According to a specific embodiment of the present invention, after adding calcium salt and phosphate, the pH is first adjusted to 8.4±0.5, stirred at 37±5℃ for 1±0.5h, and then the pH is adjusted to neutral, and then freeze-dried; preferably, the pH is first adjusted to 8.4, stirred at 37℃ for 1h, and then the pH is adjusted to neutral.

[0014] In a second aspect, the present invention provides a high-calcium, high-phosphorus soybean protein, which is prepared by the aforementioned method.

[0015] In a third aspect, the present invention provides a food product containing the aforementioned high-calcium, high-phosphorus soy protein.

[0016] The beneficial effects of this invention are as follows:

[0017] The preparation method provided by this invention uses defatted soybean meal as raw material, and sequentially performs alkali dissolution and acid precipitation, heat treatment, and enzymatic hydrolysis. Then, calcium salts and phosphates are added to produce high-calcium, high-phosphorus soybean protein. The alkali dissolution and acid precipitation method effectively extracts soybean protein. Through heat-enzyme combined modification treatment, more calcium-binding sites are exposed in the soybean protein, thereby greatly improving its calcium-binding capacity. Adding calcium salts and phosphates on this basis can ultimately produce high-calcium, high-phosphorus soybean protein with a calcium content ≥3.1% and a phosphorus content ≥2%, exhibiting good stability in food systems, which can fully meet the calcium and phosphorus intake requirements of plant-based food consumers. Attached Figure Description

[0018] Figure 1a The percentage of different forms of calcium in soybean protein obtained at different protein concentrations;

[0019] Figure 1b The percentage of different forms of calcium in soybean protein obtained at different pH values;

[0020] Figure 2a The percentage of different forms of calcium in soybean protein obtained at different alkali-soluble pH values;

[0021] Figure 2b The percentage of different forms of calcium in soybean protein obtained at different alkali dissolution temperatures;

[0022] Figure 2c The percentage of different forms of calcium in soybean protein obtained at different neutralized protein concentrations;

[0023] Figure 2d The percentage of different forms of calcium in soybean protein obtained at different neutralization pH;

[0024] Figure 3a The percentage of different forms of calcium in soybean protein obtained at different heating protein concentrations;

[0025] Figure 3b The percentage of different forms of calcium in soybean protein obtained at different heating temperatures;

[0026] Figure 3c The percentage of different forms of calcium in soybean protein obtained at different heating times;

[0027] Figure 4 The percentage of different forms of calcium in soybean protein obtained at different enzymatic hydrolysis times;

[0028] Figure 5a Precipitation rate of soybean protein obtained by different preparation methods after calcium addition;

[0029] Figure 5b Yields of calcium-bound soybean protein from different preparation methods;

[0030] Figure 5c The percentage of calcium in different forms after calcium addition to soybean protein obtained by different preparation methods;

[0031] Figure 6 The soluble calcium content of the high-calcium, high-phosphorus soybean protein prepared in Example 1 and Comparative Examples 4-5;

[0032] Figure 7 The soluble calcium content of the high-calcium, high-phosphorus soybean protein prepared in Example 1 and Comparative Examples 1-3;

[0033] Figure 8 The results show the pH stability of the high-calcium, high-phosphorus soybean protein prepared in Example 1 and Comparative Examples 1-3.

[0034] Figure 9 The results show the thermal stability of the high-calcium, high-phosphorus soybean protein prepared in Example 1 and Comparative Examples 1-3. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0036] Example 1

[0037] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0038] (1) Preparation of modified soybean protein

[0039] Defatted soybean meal was pulverized and dissolved in deionized water at a material-to-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl and allowed to stand at room temperature for 1 hour. Then, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-to-liquid ratio of 1:4, and the pH was adjusted to 8 with NaOH solution. Then, it was heated at 100°C for 30 minutes, immediately cooled in an ice-water bath, and freeze-dried to obtain heat-treated soybean protein. The heat-treated soybean protein was dissolved in deionized water to obtain a 5% (w / w) protein solution. The pH was adjusted to 7.0, and 1000 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 50°C for 180 minutes, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled in an ice-water bath for 20 minutes, and freeze-dried to obtain enzyme-heat-treated soybean protein, denoted as H-SPHs.

[0040] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0041] 1g of enzyme-thermal co-treated soybean protein was dissolved in 14mL of deionized water, and 1mL of 1mol / L CaCl2 and 2mL of pH 7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) were added sequentially to adjust the pH to 8.4. The mixture was stirred in a water bath at 37℃ for 1h, and then the pH was adjusted to 7. The mixture was then freeze-dried to obtain high-calcium and high-phosphorus soybean protein.

[0042] Example 2

[0043] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0044] (1) Preparation of modified soybean protein

[0045] Defatted soybean meal was pulverized and dissolved in deionized water at a material-to-liquid ratio of 1:10. The pH was adjusted to 7.5 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 1.5 h. Then, it was centrifuged at 4000 rpm for 20 min, and the supernatant was collected. The pH was adjusted to 4.0 with HCl and allowed to stand at room temperature for 1 h. Then, it was centrifuged at 2000 rpm for 10 min to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-to-liquid ratio of 1:3, and the pH was adjusted to 7.5 with NaOH solution. Then, it was heated at 90°C for 35 min, immediately cooled in an ice-water bath, and freeze-dried to obtain heat-treated soybean protein. The heat-treated soybean protein was dissolved in deionized water to obtain a 4% (w / w) protein solution. The pH was adjusted to 7.0, and 900 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 45°C for 210 min, the enzyme was inactivated in a boiling water bath for 10 min, cooled in an ice-water bath for 20 min, and freeze-dried to obtain enzyme-heat-treated soybean protein.

[0046] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0047] 1g of enzyme-thermal co-treated soybean protein was dissolved in 14mL of deionized water, and 1mL of 1mol / L CaCl2 and 2mL of pH 7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) were added sequentially to adjust the pH to 8. The mixture was stirred in a water bath at 32℃ for 1.5h, and then the pH was adjusted to 7. The mixture was then freeze-dried to obtain high-calcium and high-phosphorus soybean protein.

[0048] Example 3

[0049] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0050] (1) Preparation of modified soybean protein

[0051] Defatted soybean meal was pulverized and dissolved in deionized water at a material-to-liquid ratio of 1:20. The pH was adjusted to 8.5 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2.5 h. After centrifugation at 4000 rpm for 20 min, the supernatant was collected, the pH was adjusted to 5 with HCl, and the mixture was allowed to stand at room temperature for 1 h. Then, it was centrifuged at 2000 rpm for 10 min to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-to-liquid ratio of 1:5, the pH was adjusted to 8.5 with NaOH solution, and then heated at 110°C for 25 min. The mixture was immediately cooled in an ice-water bath and freeze-dried to obtain heat-treated soybean protein. The heat-treated soybean protein was dissolved in deionized water to obtain a 6% (w / w) protein solution. The pH was adjusted to 7.0, and 1100 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 55°C for 150 min, the enzyme was inactivated in a boiling water bath for 10 min, cooled in an ice-water bath for 20 min, and freeze-dried to obtain enzyme-heat-treated soybean protein.

[0052] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0053] 1g of enzyme-thermal co-treated soybean protein was dissolved in 14mL of deionized water, and 1mL of 1mol / L CaCl2 and 2mL of pH 7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) were added sequentially to adjust the pH to 8.9. The mixture was stirred in a water bath at 42℃ for 0.5h, and the pH was adjusted to 7. The mixture was then freeze-dried to obtain high-calcium and high-phosphorus soybean protein.

[0054] Comparative Example 1

[0055] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0056] (1) Soy protein preparation

[0057] Defatted soybean meal was pulverized and dissolved in deionized water at a solid-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl, and the mixture was allowed to stand at room temperature for 1 hour. Finally, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-liquid ratio of 1:4, the pH was adjusted to 8 with NaOH solution, and the mixture was freeze-dried to obtain soy protein, denoted as SPI.

[0058] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0059] Dissolve 1g of soy protein in 14mL of deionized water, then add 1mL of 1mol / L CaCl2 and 2mL of pH7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) sequentially. Adjust the pH to 8.4, stir in a 37℃ water bath for 1h, adjust the pH to 7, and freeze-dry to obtain high-calcium and high-phosphorus soy protein.

[0060] Comparative Example 2

[0061] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0062] (1) Preparation of modified soybean protein

[0063] Defatted soybean meal was pulverized and dissolved in deionized water at a solid-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl and allowed to stand at room temperature for 1 hour. Then, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-liquid ratio of 1:4, and the pH was adjusted to 8 with NaOH solution. Then, it was heated at 100°C for 30 minutes, immediately cooled in an ice-water bath, and freeze-dried to obtain heat-treated soybean protein, denoted as SPI-H.

[0064] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0065] Dissolve 1g of heat-treated soybean protein in 14mL of deionized water, then add 1mL of 1mol / L CaCl2 and 2mL of pH 7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) sequentially. Adjust the pH to 8.4, stir in a water bath at 37℃ for 1h, adjust the pH to 7, and freeze-dry to obtain high-calcium and high-phosphorus soybean protein.

[0066] Comparative Example 3

[0067] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0068] (1) Preparation of modified soybean protein

[0069] Defatted soybean meal was pulverized and dissolved in deionized water at a solid-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl and allowed to stand at room temperature for 1 hour. Then, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-liquid ratio of 1:4, and the pH was adjusted to 8 with NaOH solution. The mixture was then freeze-dried to obtain soy protein. The soy protein was dissolved in deionized water to obtain a 5% (w / w) protein solution. The pH was adjusted to 7.0, and 1000 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 50°C for 180 minutes, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled in an ice water bath for 20 minutes, and then freeze-dried to obtain enzyme-treated soy protein, denoted as SPHs.

[0070] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0071] 1g of enzyme-treated soybean protein was dissolved in 14mL of deionized water, and 1mL of 1mol / L CaCl2 and 2mL of pH7.4 0.2M phosphate buffer (81% 0.2M Na2HPO4 + 19% 0.2M NaH2PO4) were added sequentially. The pH was adjusted to 8.4, and the mixture was stirred in a water bath at 37℃ for 1h. The pH was then adjusted to 7, and the mixture was freeze-dried to obtain high-calcium and high-phosphorus soybean protein.

[0072] Comparative Example 4

[0073] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0074] (1) Preparation of modified soybean protein

[0075] Defatted soybean meal was pulverized and dissolved in deionized water at a material-to-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl and allowed to stand at room temperature for 1 hour. Then, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-to-liquid ratio of 1:4, and the pH was adjusted to 8 with NaOH solution. Then, it was heated at 100°C for 30 minutes, immediately cooled in an ice-water bath, and freeze-dried to obtain heat-treated soybean protein. The heat-treated soybean protein was dissolved in deionized water to obtain a 5% (w / w) protein solution. The pH was adjusted to 7.0, and 1000 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 50°C for 180 minutes, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled in an ice-water bath for 20 minutes, and freeze-dried to obtain enzyme-heat-treated soybean protein.

[0076] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0077] 1g of enzyme-thermal co-treated soybean protein was added to 14.4mL of 0.75% KH2PO4 solution, the pH was adjusted to 8.0, and the mixture was stirred in a 40℃ water bath for 1h. Then, 1.6mL of deionized water and 1mL of 1mol / L CaCl2 were added, the pH was adjusted to 8.4, and the mixture was stirred in a 37℃ water bath for 1h. The pH was then adjusted to 7, and the mixture was freeze-dried to obtain high-calcium and high-phosphorus soybean protein.

[0078] Comparative Example 5

[0079] A method for preparing high-calcium, high-phosphorus soybean protein is as follows:

[0080] (1) Preparation of modified soybean protein

[0081] Defatted soybean meal was pulverized and dissolved in deionized water at a material-to-liquid ratio of 1:15. The pH was adjusted to 8 with NaOH solution, and the mixture was stirred and extracted in a 25°C water bath for 2 hours. Then, it was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with HCl and allowed to stand at room temperature for 1 hour. Then, it was centrifuged at 2000 rpm for 10 minutes to obtain acid-precipitated protein. The acid-precipitated protein was dissolved in deionized water at a solid-to-liquid ratio of 1:4, and the pH was adjusted to 8 with NaOH solution. Then, it was heated at 100°C for 30 minutes, immediately cooled in an ice-water bath, and freeze-dried to obtain heat-treated soybean protein. The heat-treated soybean protein was dissolved in deionized water to obtain a 5% (w / w) protein solution. The pH was adjusted to 7.0, and 1000 U / g of flavor protease was added according to the protein substrate mass. The mixture was stirred and reacted at 50°C for 180 minutes, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled in an ice-water bath for 20 minutes, and freeze-dried to obtain enzyme-heat-treated soybean protein.

[0082] (2) Preparation of high-calcium and high-phosphorus soybean protein

[0083] Add 0.11g Ca3(PO4)2 to 17mL of deionized water and 1g of enzyme-thermal co-treated soybean protein to adjust the pH to 8.4. Stir in a water bath at 37℃ for 1h, adjust the pH to 7, and freeze-dry to obtain high-calcium and high-phosphorus soybean protein.

[0084] Test case

[0085] 1. Optimization of preparation conditions for high-calcium and high-phosphorus soybean protein

[0086] This invention investigates the effects of the concentration of soybean protein solution (i.e., enzyme-treated soybean protein solution) and the pH value after the addition of calcium salt on the proportion of various forms of calcium in the finished product.

[0087] from Figure 1a It can be seen that the content of bound calcium increases significantly with increasing protein concentration, reaching its peak at a protein concentration of 6%. Subsequent increases in protein concentration do not significantly increase the content of bound calcium, but the calcium content in the precipitate does increase. Figure 1b It can be seen that the proportion of bound calcium first increases and then decreases with increasing pH, reaching its highest value at pH 8.4.

[0088] 2. Optimization of preparation conditions for modified soybean protein

[0089] 2.1 Optimization of Extraction Conditions

[0090] Soy protein with different alkali-soluble pH, different alkali-soluble temperature, different neutralized protein concentration, and different neutralized pH was prepared and formulated into a 6% (w / v) soy protein solution. The solution was then reacted with CaCl2 at pH 8.4, and the proportion of each form of calcium in the system after the reaction was measured.

[0091] like Figure 2a , 2d As shown, with increasing alkali-soluble pH and neutralization pH, the proportion of bound calcium in the post-reaction system first increases and then decreases, with the strongest calcium binding capacity observed at pH 8. Figure 2b As shown, with increasing alkali dissolution temperature, the proportion of bound calcium in the system gradually decreases, while the proportion of calcium in the precipitate increases, indicating that the proportion of insoluble protein in soybean protein increases and the number of calcium binding sites decreases. Figure 2c As shown, the calcium-binding capacity in the system with a neutralizing protein concentration of 1:3 was significantly higher than that in the systems with neutralizing protein concentrations of 1:4 and 1:5. This may be because at higher concentrations, the soybean protein structure is not fully unfolded, resulting in weaker interactions with water molecules, and the resulting spatial structure is more conducive to binding with calcium ions.

[0092] Although the calcium-binding capacity of SPI prepared under the condition of neutralizing protein concentration of 1:3 is the strongest, considering that subsequent heat treatment is required, and the optimal protein concentration for heat treatment is 1:4, in order to ensure the consistency of extraction conditions used in different preparation methods, the optimized preparation conditions for SPI extraction are determined as follows: alkaline dissolution pH 8, alkaline dissolution temperature 25℃, neutralizing protein concentration of 1:4 and neutralization pH 8.

[0093] 2.2 Optimization of heat treatment conditions

[0094] like Figure 3a As shown, with increasing protein concentration upon heating, the resulting SPI and Ca... 2+ The proportion of bound calcium in the reaction system initially increases and then decreases. For example... Figure 3b As shown, with increasing heating temperature, the proportion of bound calcium in the reaction system first decreases and then increases, with the weakest calcium binding capacity at 80℃. Figure 3c As shown, with the extension of heating time, the proportion of bound calcium in the reaction system first increases and then decreases. The optimized preparation conditions for SPI after heat treatment are: alkaline dissolution at pH 8, alkaline dissolution temperature at 25℃, protein concentration neutralization at 1:4, neutralization at pH 8, heating temperature at 100℃, and heating for 30 min.

[0095] 2.3 Optimization of enzymatic hydrolysis conditions

[0096] like Figure 4 As shown, with the extension of enzymatic hydrolysis time, the degree of hydrolysis of soybean protein continuously increases, and the calcium binding capacity shows a trend of first increasing and then decreasing. SPHs with 1000 U / g flavor protease hydrolysis for 180 min have the strongest calcium binding capacity.

[0097] 3. Differences in calcium binding capacity of soybean protein prepared using different methods

[0098] Soy protein solutions of different concentrations (5%, 6%, 7%) were prepared and adjusted to pH 6.4, 7.0, 7.8, 8.4, and 9.8, respectively. A 1 mol / L CaCl2 solution was added at a soybean protein to calcium ratio of 1:1 (g / mmol). The solutions were shaken at 37℃ for 1 h and centrifuged at 8000 rpm for 15 min to obtain a precipitate. The mass of the dried precipitate, M1, was measured. Three volumes (v / v) of anhydrous ethanol were added to the supernatant, and the solutions were allowed to stand for 30 min before centrifugation to obtain calcium-bound compounds and a supernatant containing free calcium. The mass of the calcium-bound compounds, M2, was measured. The total mass of soybean protein and calcium was M0.

[0099]

[0100]

[0101] The calcium content in the precipitate, calcium bounds, and supernatant is determined by EDTA titration. The percentage of the total added calcium content is the proportion of calcium in the precipitate, bound calcium, and free calcium.

[0102] Figures 5a-5c The calcium-binding capacity of soybean protein prepared by four different methods (SPI, SPI-H, SPHs, and SPHs-H) was analyzed. It was found that both heat treatment and enzymatic hydrolysis significantly improved the calcium-binding capacity of soybean protein by increasing the yield of calcium-binding compounds and the proportion of bound calcium (P<0.05). Enzymatic hydrolysis was more effective than heat treatment. Furthermore, the heat-enzyme combined treatment significantly improved the precipitation of soybean protein upon contact with calcium and significantly reduced the protein precipitation rate after calcium addition (P<0.05). The highest calcium-binding compound yield and the proportion of bound calcium reached 20%, indicating that it had the strongest calcium-binding capacity.

[0103] 4. Experimental Study on High-Calcium and High-Phosphorus Soybean Protein

[0104] The high-calcium, high-phosphorus soybean protein samples prepared in Example 1 and Comparative Examples 1-5 were subjected to experimental studies on soluble calcium content, pH stability, and thermal stability, as detailed below:

[0105] 4.1 Test Methods

[0106] 4.1.1 Soluble calcium content

[0107] The obtained high-calcium, high-phosphorus soybean protein was dissolved in deionized water to prepare a 1% protein solution. (4000)

[0108] Centrifuge at rpm for 10 min, then collect the supernatant and determine the calcium content by EDTA titration. Soluble calcium content (%)

[0109] = Calcium content in supernatant (mg / mL) / Total calcium content (mg / mL) × 100%.

[0110] 4.1.2 pH stability

[0111] The prepared high-calcium, high-phosphorus soybean protein was dissolved in deionized water to prepare a 1% protein solution. The pH was adjusted to 2, 4, 6, 8, and 10, respectively. After shaking at 37℃ for 1 hour, the solution was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and the calcium content was determined by EDTA titration. Ca retention rate (%) = calcium content in supernatant (mg / mL) / total calcium content (mg / mL) × 100%.

[0112] 4.1.3 Thermal stability

[0113] The prepared high-calcium, high-phosphorus soybean protein was dissolved in deionized water to prepare a 1% protein solution. The solution was shaken at 80℃ for 0, 15, 30, and 60 min, then immediately cooled and centrifuged at 4000 rpm for 10 min. The supernatant was then used to determine the calcium content by EDTA titration. Ca retention rate (%) = Calcium content in supernatant (mg / mL) / Total calcium content (mg / mL) × 100%.

[0114] 4.2 Test Results

[0115] like Figure 6 As shown, the soluble calcium content of the high-calcium and high-phosphorus soybean protein prepared by the method in Example 1 is significantly higher than that of other soybean proteins (P<0.05).

[0116] like Figure 7 As shown, the soluble calcium content of the high-calcium and high-phosphorus soybean protein prepared by the three methods has significant differences (P<0.05), and the high-calcium and high-phosphorus soybean protein prepared in Example 1 has the highest soluble calcium content.

[0117] like Figure 8 As shown, high-calcium, high-phosphorus soy protein is more stable under acidic conditions, with a calcium retention rate exceeding 80%, while it easily forms calcium hydroxide precipitate under alkaline conditions. The high-calcium, high-phosphorus soy protein prepared in Example 1 exhibits better pH stability, with higher Ca retention rates than other soy proteins under various pH conditions.

[0118] like Figure 9 As shown, all samples exhibited good thermal stability within 15 minutes of heat treatment at 80℃, but their stability was compromised to varying degrees after 15 minutes. Although the high-calcium, high-phosphorus soybean protein prepared in Example 1 showed a significant decrease in Ca retention after heating, it still remained at a higher level than other soybean proteins.

[0119] Comprehensive analysis revealed that the high-calcium and high-phosphorus soybean protein prepared by the method in Example 1 of this invention not only meets the requirements of calcium content ≥3.1% and phosphorus content ≥2%, but also contains more soluble calcium and has good pH stability and thermal stability.

[0120] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing high calcium and high phosphorus soy protein, characterized in that, The preparation method comprises: The defatted soybean meal is crushed and mixed with water to form a solution, the pH of the solution is adjusted to be alkaline, stirring, centrifugal collection of supernatant; the pH of the supernatant is adjusted to be acidic, standing, centrifugal collection of precipitate, water is added to re-dissolve and the pH is adjusted to be alkaline to obtain a first solution; The first solution is heated to 100±10 ℃, maintained for 30±5 min, dried into powder after cooling, water is added to re-dissolve and the pH is adjusted to be neutral to obtain a second solution; Protease is added to the second solution for enzymolysis, after enzyme inactivation, dried into powder, water is added to re-dissolve to obtain a third solution; Calcium salt and phosphate are sequentially added to the third solution, the pH is first adjusted to 8.4±0.5, stirred at 37±5 ℃ for 1±0.5 h, then the pH is adjusted to be neutral, and then freeze-dried; the calcium salt is calcium chloride, and the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate.

2. The production method according to claim 1, characterized by, The defatted soybean meal is crushed and mixed with water at a solid-liquid ratio of 1 g:10-20 mL to form a solution, the pH of the solution is adjusted to be 8.0±0.5, stirring at 25 ℃ for 2±0.5 h, centrifugal collection of supernatant; the pH of the supernatant is adjusted to be 4.5±0.5, standing, centrifugal collection of precipitate, water is added to re-dissolve at a solid-liquid ratio of 1 g:3-5 mL, and the pH is adjusted to be 8.0±0.5 to obtain a first solution.

3. The production method according to claim 2, characterized by, The solid-liquid ratio of the defatted soybean meal to water is 1 g:15 mL.

4. The preparation method according to claim 2, characterized in that, The pH of the solution of the defatted soybean meal mixed with water is adjusted to be 8.

5. The preparation method according to claim 2, characterized in that, The pH of the supernatant is adjusted to be 4.

5.

6. The preparation method according to claim 2, characterized in that, Water is added to re-dissolve at a solid-liquid ratio of 1 g:4 mL, and the pH is adjusted to be 8.

7. The preparation method according to claim 1, characterized in that, The first solution is heated to 100 ℃, maintained for 30 min.

8. The method of claim 1, wherein, The concentration of the second solution is 4-6% w / w.

9. The production method according to claim 8, characterized by, The concentration of the second solution is 5% w / w.

10. The method of claim 1, wherein, The protease is flavourzyme.

11. The method of claim 10, wherein, Flavourzyme is added at 1000±100 U / g of protein substrate, the enzymolysis temperature is 50±5 ℃, and the enzymolysis time is 180±30 min.

12. The method of claim 11, wherein, Flavourzyme is added at 1000 U / g of protein substrate, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 180 min.

13. The method of claim 1, wherein, The concentration of the third solution is 5-7% w / v.

14. The method of claim 13, wherein, The concentration of the third solution is 6% w / v.

15. The method of claim 1, wherein, The molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1:

1.

16. The method of claim 1, wherein, The calcium salt and the phosphate are added in the form of a calcium salt solution and a phosphate salt solution respectively, the concentration of the calcium salt solution is 1 mol / L, and the concentration of the phosphate salt solution is 0.2 mol / L; the mass-volume ratio of the solid solution in the third solution, the calcium salt solution and the phosphate salt solution is 1 g:0.9-1.1 mL:1.9-2.1 mL.

17. The method of claim 16, wherein, The mass-volume ratio of the solid solution in the third solution, the calcium salt solution and the phosphate salt solution is 1 g:1 mL:2 mL.

18. The method of claim 1, wherein; The pH is first adjusted to 8.4, stirred at 37 ℃ for 1 h, then the pH is adjusted to be neutral.

19. A high calcium, high phosphorus soy protein, characterized in that, It is prepared by the method of any one of claims 1-18.

20. A food product, characterized by, It contains the high-calcium and high-phosphorus soybean protein of claim 19.

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