A method for efficiently extracting soybean peptides

Abstract

The application belongs to the field of protein processing, and particularly relates to a method for efficiently extracting soybean peptides. The method comprises the following steps: preparing soybean protein into a suspension, and performing ultrasonic pretreatment, then adding a chlorogenic acid grafted UiO-66 biomimetic catalyst, performing catalytic hydrolysis reaction under weak alkaline conditions, and obtaining soybean peptides after separation and purification. The core of the application is that the catalyst innovatively combines the protein recognition and enrichment function of chlorogenic acid with the Zr 6+ hydrolysis active center of UiO-66, significantly improving the efficiency of catalytic hydrolysis of proteins. The yield of soybean peptides obtained by the method is as high as 88.5%, and the catalyst can be recycled for more than 5 times with an activity retention rate of more than 87.5%, greatly reducing the cost. More outstandingly, the obtained soybean peptides have excellent antioxidant activity, and the DPPH free radical scavenging capacity is improved by about 52% compared with the traditional enzyme product, realizing the overall improvement from the production process to the quality of the terminal product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of protein processing, and particularly relates to a method for efficiently extracting soybean peptides. BACKGROUND

[0002] Soybean peptides are small molecular fragments generated after hydrolysis of soybean proteins. They not only have the nutritional properties of rapid absorption and low allergenicity superior to intact proteins, but also exhibit various physiological activities such as blood pressure reduction, antioxidant activity, and immune enhancement. Therefore, soybean peptides have broad application prospects in the fields of functional foods, special medical use formula foods, and health products.

[0003] Currently, the mainstream method for producing soybean peptides in industry is enzymatic hydrolysis. Although this method has mild conditions and strong specificity, it still has some inherent defects. First, the cost of protease is high, and it is usually inactivated after the reaction and cannot be recycled, resulting in high production cost. Second, the enzymatic hydrolysis process is easily inhibited by products, and the hydrolysis efficiency and yield still have room for improvement. In addition, the enzymatic hydrolysis products often have obvious bitter taste due to the presence of hydrophobic peptide segments, which affects the flavor of the products.

[0004] To overcome the limitations of enzymatic hydrolysis, researchers have also explored alternative methods such as chemical hydrolysis (acid or alkali method) and microbial fermentation. However, chemical methods often have severe conditions, which can damage the structure of amino acids and produce harmful byproducts, and cause environmental pollution. Fermentation methods have complex processes, wide molecular weight distribution of products, and low yield of target small peptides.

[0005] In recent years, artificial enzyme catalysts designed based on metal-organic framework materials have provided a new idea for protein hydrolysis. For example, zirconium-based MOF materials can be used as efficient and stable artificial proteases due to their catalytic activity center similar to phosphatase. However, pure MOF catalysts still face challenges such as low substrate mass transfer efficiency and poor accessibility of catalytic sites when dealing with complex structures and large molecular weight of natural proteins, and their catalytic efficiency is difficult to compare with traditional proteases.

[0006] Therefore, there is an urgent need in the art to develop a new method for preparing soybean peptides that can balance efficiency, economy, greenness, and excellent product quality, in order to break through the bottleneck of existing technology. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a method for efficiently extracting soybean peptides.

[0008] In a first aspect, the present application provides a method for efficiently extracting soybean peptides, comprising the following steps:

[0009] (1) mixing soybean protein isolate and phosphate buffer to prepare a 5wt%-8wt% soybean protein isolate suspension, then ultrasonic treatment at 25-40℃ for 10-20min to obtain a protein suspension;

[0010] (2) adding a chlorogenic acid grafted UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stirring at 50-70℃ and a rotation speed of 100-200r / min for 2-4h, after the reaction, cooling to room temperature, centrifuging at a rotation speed of 8000-10000r / min for 8-10min, taking the supernatant, filtering the soybean peptide solution with a molecular weight cutoff of ≤3 kDa by ultrafiltration membrane, vacuum concentration to a soybean peptide content of 20wt%-30wt%, then spray drying to obtain soybean peptides.

[0011] In step (1), the concentration of the phosphate buffer is 0.05-0.06 mol / L, and the pH is 7.5-8.0.

[0012] In step (1), the ultrasonic power is 300-400W.

[0013] In step (2), the vacuum concentration temperature is 50-60℃.

[0014] In step (2), the inlet temperature of the spray drying is 170-185℃, and the outlet temperature is 80-90℃.

[0015] In step (2), the addition amount of the chlorogenic acid grafted UiO-66 biomimetic catalyst is 1wt%-5wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0016] The preparation method of the chlorogenic acid grafted UiO-66 biomimetic catalyst comprises the following steps:

[0017] S1, mixing 1.0-1.2mmol of zirconium tetrachloride and 1.0-1.2mmol of 2-amino terephthalic acid, adding 20-40mL of anhydrous N,N-dimethylformamide as a solvent, then adding 0.8-1.2mL of concentrated hydrochloric acid, placing in a hydrothermal kettle at 110-120℃ and reacting for 24h, cooling to room temperature, then centrifuging at a rotation speed of 8000-10000r / min for 5-6min, taking the precipitate, washing and centrifuging with anhydrous methanol for 2-3 times, then drying at 100-120℃ under vacuum for 10-12h to obtain activated UiO-66;

[0018] S2, dissolve 0.1-0.2 mmol chlorogenic acid in 20-40 mL anhydrous N,N-dimethylformamide, add 0.15-0.3 mmol 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.15-0.3 mmol N-hydroxysuccinimide, stir at room temperature, avoid light, nitrogen protection, 100-120 r / min for 2-4 h, to obtain an activated chlorogenic acid solution;

[0019] S3, add 90-110 mg of the activated UiO-66 obtained in step S1 to the activated chlorogenic acid solution obtained in step S2, place at 35-40 DEG C, continue to stir for 12-24 h, centrifuge, wash, vacuum dry, to obtain the chlorogenic acid grafted UiO-66 biomimetic catalyst.

[0020] Advantages of the present application:

[0021] The present application stably fixes the naturally extracted chlorogenic acid on the UiO-66 material, wherein the chlorogenic acid can actively capture and enrich soybean protein in the solution, so that they are densely surrounded around the catalyst; at the same time, the zirconium metal active center on the UiO-66 acts as a high-efficiency molecular scissors to accurately cut these fixed proteins. This synergistic effect of first grabbing and then cutting can effectively improve the hydrolysis efficiency of soybean protein. At the same time, the soybean peptide produced by the chlorogenic acid grafted UiO-66 biomimetic catalyst has high antioxidant activity, which means that the efficient extraction method of soybean peptide adopted by the present application not only optimizes the production process, but also produces a high-quality raw material with stronger function, which opens up a new situation for its entry into the high-end health care and cosmetic markets. In addition, the chlorogenic acid grafted UiO-66 biomimetic catalyst prepared by the present application can maintain a catalytic activity of more than 87.5% after being used continuously for 5 times. This completely changes the high-cost mode of traditional enzyme preparation for one-time use, greatly improving the economy. DETAILED DESCRIPTION

[0022] Soybean protein isolate, item number: S832685, Shanghai Macklin Biochemical Science and Technology Co., Ltd.

[0023] Chlorogenic acid, CAS number: 327-97-9.

[0024] N,N-dimethylformamide, CAS number: 68-12-2.

[0025] 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, CAS number: 25952-53-8.

[0026] N-hydroxysuccinimide, CAS number: 6066-82-6.

[0027] Alkaline protease, enzyme activity: 200 U / mg, Shanghai Kanglang Biotechnology Co., Ltd.

[0028] Example 1

[0029] A method for efficiently extracting soybean peptides, comprising the following steps:

[0030] (1) Soybean protein isolate and phosphate buffer are mixed to prepare a 6wt% soybean protein isolate suspension, which is then ultrasonicated at 35℃ for 15min to obtain a protein suspension;

[0031] (2) The protein suspension obtained in step (1) is added with a chlorogenic acid grafted UiO-66 biomimetic catalyst, and stirred at 200r / min for 3h at 60℃. After the reaction, it is cooled to room temperature, centrifuged at 8000r / min for 10min, and the supernatant is taken. The soybean peptide solution with a molecular weight cutoff of ≤3 kDa is filtered through an ultrafiltration membrane, vacuum concentrated to a soybean peptide content of 25wt%, and then spray dried to obtain soybean peptides.

[0032] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0033] The ultrasonic power in step (1) is 400W.

[0034] The vacuum concentration temperature in step (2) is 60℃.

[0035] The inlet temperature of the spray drying in step (2) is 175℃, and the outlet temperature is 85℃.

[0036] The addition amount of the chlorogenic acid grafted UiO-66 biomimetic catalyst in step (2) is 3wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0037] The preparation method of the chlorogenic acid grafted UiO-66 biomimetic catalyst comprises the following steps:

[0038] S1, 1.0mmol of zirconium tetrachloride and 1.0mmol of 2-amino terephthalic acid are mixed, 30mL of anhydrous N,N-dimethylformamide is added as a solvent, followed by 1.0mL of concentrated hydrochloric acid, and then placed in a hydrothermal kettle at 120℃ for reaction for 24h. After cooling to room temperature, centrifugation is performed at 8000r / min for 5min, the precipitate is washed with anhydrous methanol and centrifuged for 3 times, and then dried at 120℃ under vacuum for 12h to obtain activated UiO-66;

[0039] S2, dissolve 0.1 mmol chlorogenic acid in 20 mL of anhydrous N,N- dimethylformamide, add 0.15 mmol 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.15 mmol N-hydroxysuccinimide, stir at room temperature, avoid light, nitrogen protection, 120 r / min for 3 h, to obtain an activated chlorogenic acid solution;

[0040] S3, add 100 mg of the activated UiO-66 obtained in step S1 to the activated chlorogenic acid solution obtained in step S2, place at 40°C, continue stirring for 18 h, centrifuge, wash, vacuum dry to obtain the chlorogenic acid grafted UiO-66 biomimetic catalyst.

[0041] Example 2

[0042] A method for efficiently extracting soybean peptides, comprising the following steps:

[0043] (1) Mix soybean protein isolate and phosphate buffer to prepare a 6wt% soybean protein isolate suspension, then ultrasonic at 35°C for 15 min to obtain a protein suspension;

[0044] (2) Add the chlorogenic acid grafted UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stir at 60°C at a speed of 200 r / min for 3 h, after the reaction is completed, cool to room temperature, centrifuge at a speed of 8000 r / min for 10 min, take the supernatant, pass the soybean peptide solution with a molecular weight of ≤3 kDa through an ultrafiltration membrane, vacuum concentrate to a soybean peptide content of 25wt%, then spray dry to obtain soybean peptides.

[0045] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0046] The ultrasonic power in step (1) is 400W.

[0047] The temperature of the vacuum concentration in step (2) is 60°C.

[0048] The inlet temperature of the spray drying in step (2) is 175°C, and the outlet temperature is 85°C.

[0049] The amount of the chlorogenic acid grafted UiO-66 biomimetic catalyst added in step (2) is 1wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0050] The preparation method of the chlorogenic acid grafted UiO-66 biomimetic catalyst comprises the following steps:

[0051] S1, 1.0 mmol of zirconium tetrachloride and 1.0 mmol of 2-amino terephthalic acid were mixed, 30 mL of anhydrous N,N-dimethylformamide was added as a solvent, then 1.0 mL of concentrated hydrochloric acid was added, and the mixture was placed in a hydrothermal kettle at 120°C and reacted for 24 h. After cooling to room temperature, centrifugation was performed at a speed of 8000 r / min for 5 min. The precipitate was washed with anhydrous methanol and centrifuged for 3 times, and then dried at 120°C under vacuum for 12 h to obtain activated UiO-66;

[0052] S2, 0.1 mmol of chlorogenic acid was dissolved in 20 mL of anhydrous N,N-dimethylformamide, 0.15 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.15 mmol of N-hydroxysuccinimide were added, and the mixture was stirred at room temperature, in the dark, under nitrogen protection, at a speed of 120 r / min for 3 h to obtain an activated chlorogenic acid solution;

[0053] S3, 100 mg of activated UiO-66 obtained in step S1 was added to the activated chlorogenic acid solution obtained in step S2, and the mixture was stirred at 40°C for 18 h. After centrifugation, washing and vacuum drying, the chlorogenic acid grafted UiO-66 biomimetic catalyst was obtained.

[0054] Example 3

[0055] A method for efficiently extracting soybean peptides, comprising the following steps:

[0056] (1) Soybean protein isolate and phosphate buffer were mixed to prepare a 6 wt% soybean protein isolate suspension, and then ultrasonic treatment was performed at 35°C for 15 min to obtain a protein suspension;

[0057] (2) The chlorogenic acid grafted UiO-66 biomimetic catalyst was added to the protein suspension obtained in step (1), and the mixture was stirred at 60°C at a speed of 200 r / min for 3 h. After the reaction was completed, the mixture was cooled to room temperature and centrifuged at a speed of 8000 r / min for 10 min. The supernatant was collected, and a soybean peptide solution with a molecular weight cutoff of ≤3 kDa was obtained by ultrafiltration membrane. The soybean peptide solution was vacuum concentrated to a soybean peptide content of 25 wt%, and then spray dried to obtain soybean peptides.

[0058] In step (1), the concentration of the phosphate buffer is 0.05 mol / L, and the pH is 7.5.

[0059] In step (1), the ultrasonic power is 400 W.

[0060] In step (2), the vacuum concentration temperature is 60°C.

[0061] In step (2), the inlet temperature of the spray drying is 175°C, and the outlet temperature is 85°C.

[0062] The amount of chlorogenic acid-grafted UiO-66 biomimetic catalyst added in step (2) is 5 wt% of the amount of soybean protein isolated in the protein suspension obtained in step (1).

[0063] The preparation method of the chlorogenic acid-grafted UiO-66 biomimetic catalyst includes the following steps:

[0064] S1. Mix 1.0 mmol zirconium tetrachloride and 1.0 mmol 2-aminoterephthalic acid, add 30 mL of anhydrous N,N-dimethylformamide as solvent, then add 1.0 mL of concentrated hydrochloric acid, and react in a hydrothermal reactor at 120 °C for 24 h. After cooling to room temperature, centrifuge at 8000 r / min for 5 min, take the precipitate, wash with anhydrous methanol, centrifuge 3 times, and dry under vacuum at 120 °C for 12 h to obtain activated UiO-66.

[0065] S2 Dissolve 0.1 mmol of chlorogenic acid in 20 mL of anhydrous N,N-dimethylformamide, add 0.15 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.15 mmol of N-hydroxysuccinimide, and stir at 120 r / min for 3 h at room temperature, protected from light and under nitrogen protection to obtain an activated chlorogenic acid solution;

[0066] S3 Add 100 mg of the activated UiO-66 obtained in step S1 to the activated chlorogenic acid solution obtained in step S2, place it at 40°C, and stir continuously for 18 h. After centrifugation, washing, and vacuum drying, the chlorogenic acid-grafted UiO-66 biomimetic catalyst is obtained.

[0067] Example 4

[0068] A method for efficiently extracting soybean peptides includes the following steps:

[0069] (1) Mix soy protein isolate and phosphate buffer to prepare a 6 wt% soy protein isolate suspension, and then sonicate at 35°C for 15 min to obtain a protein suspension;

[0070] (2) Add chlorogenic acid-grafted UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stir at 200 r / min for 3 h at 50 °C, after the reaction is completed, cool to room temperature, centrifuge at 8000 r / min for 10 min, take the supernatant, filter through an ultrafiltration membrane to retain soybean peptide solution with a molecular weight cutoff of ≤3 kDa, concentrate under vacuum to a soybean peptide content of 25 wt%, and then spray dry to obtain soybean peptide.

[0071] The concentration of the phosphate buffer in step (1) is 0.05 mol / L and the pH is 7.5.

[0072] The power of the ultrasonic in step (1) is 400 W.

[0073] The temperature of the vacuum concentration in step (2) is 60℃.

[0074] The inlet temperature of the spray drying in step (2) is 175℃, and the outlet temperature is 85℃.

[0075] The amount of the chlorogenic acid grafted UiO-66 biomimetic catalyst added in step (2) is 3wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0076] The method for preparing the chlorogenic acid grafted UiO-66 biomimetic catalyst comprises the following steps:

[0077] S1, 1.0 mmol of zirconium tetrachloride and 1.0 mmol of 2-amino terephthalic acid are mixed, 30 mL of anhydrous N,N-dimethylformamide is added as a solvent, then 1.0 mL of concentrated hydrochloric acid is added, and the mixture is placed in a hydrothermal kettle at 120℃ for reaction for 24 h. After cooling to room temperature, centrifugation is performed at a speed of 8000 r / min for 5 min. The precipitate is washed with anhydrous methanol and centrifuged for 3 times. Then, the precipitate is dried under vacuum at 120℃ for 12 h to obtain activated UiO-66;

[0078] S2, 0.1 mmol of chlorogenic acid is dissolved in 20 mL of anhydrous N,N-dimethylformamide, 0.15 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.15 mmol of N-hydroxysuccinimide are added, and the mixture is stirred at a speed of 120 r / min for 3 h at room temperature, in the dark, and under nitrogen protection to obtain an activated chlorogenic acid solution;

[0079] S3, 100 mg of the activated UiO-66 obtained in step S1 is added to the activated chlorogenic acid solution obtained in step S2, and the mixture is continuously stirred at 40℃ for 18 h. After centrifugation, washing and vacuum drying, the chlorogenic acid grafted UiO-66 biomimetic catalyst is obtained.

[0080] Example 5

[0081] A method for efficiently extracting soybean peptides, comprising the following steps:

[0082] (1) Soybean protein isolate and phosphate buffer solution are mixed to prepare a 6wt% soybean protein isolate suspension, and then ultrasonic treatment is performed at 35℃ for 15 min to obtain a protein suspension;

[0083] (2) adding the caffeic acid grafted UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stirring at 200 r / min at 70°C for 3 h, after the reaction, cooling to room temperature, centrifuging at 8000 r / min for 10 min, taking the supernatant, and removing soybean peptides with a molecular weight of ≤3 kDa by ultrafiltration membrane, vacuum concentration to a soybean peptide content of 25 wt%, and then spray drying to obtain soybean peptides.

[0084] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0085] The power of the ultrasonic in step (1) is 400 W.

[0086] The temperature of the vacuum concentration in step (2) is 60°C.

[0087] The inlet temperature of the spray drying in step (2) is 175°C, and the outlet temperature is 85°C.

[0088] The amount of the caffeic acid grafted UiO-66 biomimetic catalyst added in step (2) is 3 wt% of the mass of the soybean protein isolate obtained in step (1).

[0089] The preparation method of the caffeic acid grafted UiO-66 biomimetic catalyst comprises the following steps:

[0090] S1, mixing 1.0 mmol of zirconium tetrachloride and 1.0 mmol of 2-amino terephthalic acid, adding 30 mL of anhydrous N,N-dimethylformamide as a solvent, then adding 1.0 mL of concentrated hydrochloric acid, placing in a hydrothermal kettle at 120°C and reacting for 24 h, cooling to room temperature, then centrifuging at 8000 r / min for 5 min, taking the precipitate, washing and centrifuging with anhydrous methanol for 3 times, and drying at 120°C under vacuum for 12 h to obtain activated UiO-66;

[0091] S2, dissolving 0.1 mmol of caffeic acid in 20 mL of anhydrous N,N-dimethylformamide, adding 0.15 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.15 mmol of N-hydroxysuccinimide, stirring at 120 r / min at room temperature, avoiding light, and under nitrogen protection for 3 h to obtain an activated caffeic acid solution;

[0092] S3, adding 100 mg of the activated UiO-66 obtained in step S1 to the activated caffeic acid solution obtained in step S2, placing in a 40°C water bath, and continuously stirring for 18 h, centrifuging, washing, and vacuum drying to obtain the caffeic acid grafted UiO-66 biomimetic catalyst.

[0093] Comparative Example 1

[0094] A method for efficiently extracting soybean peptides, comprising the following steps:

[0095] (1) mixing soybean protein isolate and phosphate buffer to prepare a 6wt% soybean protein isolate suspension, then ultrasonicating at 35°C for 15 min to obtain a protein suspension;

[0096] (2) stirring the protein suspension obtained in step (1) at 60°C at a stirring speed of 200 r / min for 3 h, after the reaction, cooling to room temperature, centrifuging at a speed of 8000 r / min for 10 min, taking the supernatant, passing the soybean peptide solution with a molecular weight of ≤3 kDa through an ultrafiltration membrane, vacuum concentrating to a soybean peptide content of 25wt%, then spray drying to obtain soybean peptides.

[0097] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0098] The power of the ultrasonication in step (1) is 400W.

[0099] The temperature of the vacuum concentration in step (2) is 60°C.

[0100] The inlet temperature of the spray drying in step (2) is 175°C, and the outlet temperature is 85°C.

[0101] Comparative Example 2

[0102] A method for efficiently extracting soybean peptides, comprising the following steps:

[0103] (1) mixing soybean protein isolate and phosphate buffer to prepare a 6wt% soybean protein isolate suspension, then ultrasonicating at 35°C for 15 min to obtain a protein suspension;

[0104] (2) adding UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stirring at 60°C at a stirring speed of 200 r / min for 3 h, after the reaction, cooling to room temperature, centrifuging at a speed of 8000 r / min for 10 min, taking the supernatant, passing the soybean peptide solution with a molecular weight of ≤3 kDa through an ultrafiltration membrane, vacuum concentrating to a soybean peptide content of 25wt%, then spray drying to obtain soybean peptides.

[0105] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0106] The power of the ultrasonication in step (1) is 400W.

[0107] The temperature of the vacuum concentration in step (2) is 60°C.

[0108] The inlet temperature of the spray drying in step (2) is 175 DEG C, and the outlet temperature is 85 DEG C.

[0109] The addition amount of the UiO-66 biomimetic catalyst in step (2) is 3 wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0110] The preparation method of the UiO-66 biomimetic catalyst comprises the following steps: 1.0 mmol of zirconium tetrachloride and 1.0 mmol of 2-amino terephthalic acid are mixed, 30 mL of anhydrous N,N-dimethylformamide is added as a solvent, then 1.0 mL of concentrated hydrochloric acid is added, and the mixture is placed in a hydrothermal kettle at 120 DEG C and reacted for 24 h; after cooling to room temperature, centrifugation is performed at a speed of 8000 r / min for 5 min; the precipitate is washed with anhydrous methanol and centrifuged for 3 times; and the product is dried under vacuum at 60 DEG C for 2 h to obtain the UiO-66 biomimetic catalyst.

[0111] Comparative Example 3

[0112] A method for efficiently extracting soybean peptides comprises the following steps:

[0113] (1) Soybean protein isolate and phosphate buffer are mixed to prepare a 6 wt% soybean protein isolate suspension, and then ultrasonic treatment is performed at 35 DEG C for 15 min to obtain a protein suspension;

[0114] (2) Alkaline protease is added to the protein suspension obtained in step (1), and reaction is performed at 60 DEG C with stirring at a speed of 200 r / min for 3 h; after the reaction is completed, the mixture is cooled to room temperature, centrifuged at a speed of 8000 r / min for 10 min, and the supernatant is taken; a soybean peptide solution with a molecular weight of less than 3 kDa is obtained by ultrafiltration membrane; vacuum concentration is performed until the content of the soybean peptide is 25 wt%; and then spray drying is performed to obtain the soybean peptide.

[0115] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0116] The power of the ultrasonic treatment in step (1) is 400 W.

[0117] The temperature of the vacuum concentration in step (2) is 60 DEG C.

[0118] The inlet temperature of the spray drying in step (2) is 175 DEG C, and the outlet temperature is 85 DEG C.

[0119] The addition amount of the alkaline protease in step (2) is 3 wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0120] Comparative Example 4

[0121] A method for efficiently extracting soybean peptides comprises the following steps:

[0122] (1) mixing soybean protein isolate and phosphate buffer to prepare a 6wt% soybean protein isolate suspension, then ultrasonic treatment at 35℃ for 15min to obtain a protein suspension;

[0123] (2) adding a composite catalyst to the protein suspension obtained in step (1), stirring at 200r / min at 60℃ for 3h, after reaction, cooling to room temperature, centrifuging at 8000r / min for 10min, taking the supernatant, passing the soybean peptide solution with a molecular weight cutoff of ≤3 kDa through an ultrafiltration membrane, vacuum concentration to a soybean peptide content of 25wt%, then spray drying to obtain soybean peptide.

[0124] The concentration of the phosphate buffer in step (1) is 0.05 mol / L, and the pH is 7.5.

[0125] The power of the ultrasonic treatment in step (1) is 400W.

[0126] The temperature of the vacuum concentration in step (2) is 60℃.

[0127] The inlet temperature of the spray drying in step (2) is 175℃, and the outlet temperature is 85℃.

[0128] The addition amount of the composite catalyst in step (2) is 3wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1), and the composite catalyst is mixed by chlorogenic acid and UiO-66 biomimetic catalyst at a mass ratio of 35.4:100.

[0129] The preparation method of the UiO-66 biomimetic catalyst comprises the following steps: mixing 1.0mmol of zirconium tetrachloride and 1.0mmol of 2-amino terephthalic acid, adding 30mL of anhydrous N,N-dimethylformamide as a solvent, then adding 1.0mL of concentrated hydrochloric acid, placing in a hydrothermal kettle at 120℃ and reacting for 24h, cooling to room temperature, then centrifuging at 8000r / min for 5min, taking the precipitate, washing and centrifuging with anhydrous methanol for 3 times, then drying at 60℃ under vacuum for 2h to obtain the UiO-66 biomimetic catalyst.

[0130] Test Example 1

[0131] Soybean peptide yield calculation: soybean peptide yield (%) = soybean peptide (mg) / soybean protein isolate (mg) x 100%;

[0132] The results are shown in Table 1.

[0133] Table 1: Soybean peptide yield

[0134]

[0135] Test Example 2

[0136] Cyclic performance determination of the caffeic acid grafted UiO-66 biomimetic catalyst;

[0137] A method for efficiently extracting soybean peptides, comprising the following steps:

[0138] (1) Soybean protein isolate and phosphate buffer solution are mixed to prepare a 6wt% soybean protein isolate suspension, which is then ultrasonicated at 35℃ for 15min to obtain a protein suspension;

[0139] (2) Caffeic acid grafted UiO-66 biomimetic catalyst is added to the protein suspension obtained in step (1), and stirred at 200r / min for 3h at 60℃. After the reaction, it is cooled to room temperature, centrifuged at 8000r / min for 10min, and the supernatant is taken. The soybean peptide solution with a molecular weight cutoff of ≤3 kDa is filtered through an ultrafiltration membrane, vacuum concentrated to a soybean peptide content of 25wt%, and then spray dried to obtain soybean peptides.

[0140] The concentration of the phosphate buffer solution in step (1) is 0.05 mol / L, and the pH is 7.5.

[0141] The ultrasonic power in step (1) is 400W.

[0142] The vacuum concentration temperature in step (2) is 60℃.

[0143] The inlet temperature of the spray drying in step (2) is 175℃, and the outlet temperature is 85℃.

[0144] The addition amount of the caffeic acid grafted UiO-66 biomimetic catalyst in step (2) is 3wt% of the mass of the soybean protein isolate in the protein suspension obtained in step (1).

[0145] The precipitate left after the supernatant is taken after centrifugation at 8000r / min for 10min in step (2) is washed with deionized water and anhydrous ethanol alternately for 3 times to remove the adsorbed peptide fragments and impurities on the surface, and vacuum dried at 60℃ overnight to obtain regenerated caffeic acid grafted UiO-66 biomimetic catalyst.

[0146] The regenerated caffeic acid grafted UiO-66 biomimetic catalyst is used again to hydrolyze soybean protein isolate to produce soybean peptides, and the cyclic use is 5 times to test the stability of the caffeic acid grafted UiO-66 biomimetic catalyst.

[0147] Catalytic activity decline rate (%) = fifth soybean peptide yield / first soybean peptide yield x 100%.

[0148] Table 2: Cyclic performance determination results

[0149]

[0150] Test Example 3

[0151] According to GB / T 39100-2020 "Determination of antioxidant activity of polypeptides DPPH and ABTS method", the antioxidant activity of the soybean peptide products obtained in each example and comparative example was determined.

[0152] Table 3: Antioxidant activity determination results

[0153]

[0154] As can be seen from Table 1, Example 1 as the optimal process of the present application, the extraction rate of soybean peptide reached 88.5%, compared with Comparative Example 1 without catalyst, the efficiency was improved by more than 16 times, which showed that the green acid grafted UiO-66 biomimetic catalyst prepared by the present application had excellent ability to catalyze hydrolysis of soybean protein to obtain soybean peptide. Compared with Comparative Example 2 using only UiO-66, the efficiency was improved by 35%, which strongly indicated that the grafted green acid could first enrich the protein around the catalyst, greatly improving the cutting efficiency of UiO-66. Even compared with Comparative Example 3, the most commonly used alkaline protease method, the soybean peptide yield of Example 1 was about 6.5% higher, which proved that the new method was efficient enough to rival or even surpass the existing process. Compared with Comparative Example 4 simply mixing green acid and UiO-66 together, the efficiency of our chemical grafting method was improved by more than 50%, which showed that simple physical mixing could not achieve the performance of green acid grafted UiO-66, indicating that green acid itself did not have the function of catalyzing soybean protein hydrolysis to produce soybean peptide, but by grafting first enriching the protein around the UiO-66 catalyst, thereby improving the cutting efficiency of UiO-66.

[0155] In addition, as can be seen from Table 2, the green acid grafted UiO-66 biomimetic catalyst prepared by the present application can be recycled and reused, and after 5 cycles, its catalytic activity still remains above 87.5%, with very small loss. In contrast, the ungrafted UiO-66 of Comparative Example 2 quickly lost its performance after recycling, while the traditional enzyme method of Comparative Example 3 could not be recycled at all, and new enzymes had to be added each time, with high cost and waste disposal pressure. The present application has made a big step in sustainability in the efficient extraction of soybean peptides.

[0156] Finally, as can be seen from Table 3, the soybean peptides produced by the high-efficiency soybean peptide extraction method of the present application have unexpectedly high antioxidant activity. Compared with the traditional enzyme method product of Comparative Example 3, the core antioxidant index (DPPH clearance rate) is increased by about 52%. This means that the high-efficiency soybean peptide extraction method adopted by the present application not only optimizes the production process, but also obtains a product with stronger function. This makes soybean peptides upgrade from basic nutritional raw materials to high-value functional ingredients that can be used in high-end health products and cosmetics, and have greater market potential.

[0157] In summary, the present application provides a new way of extracting soybean peptides with significant advantages in efficiency, cost, environmental protection and product value through fundamental innovation in catalyst design.

Claims

1. A method for efficiently extracting soybean peptides, characterized in that, Includes the following steps: (1) Mix soy protein isolate and phosphate buffer to prepare a soy protein isolate suspension of 5wt%-8wt%, and then sonicate at 25-40℃ for 10-20 min to obtain a protein suspension; (2) Add chlorogenic acid-grafted UiO-66 biomimetic catalyst to the protein suspension obtained in step (1), stir at 100-200 r / min at 50-70℃ for 2-4 h. After the reaction is completed, cool to room temperature, centrifuge at 8000-10000 r / min for 8-10 min, take the supernatant, filter through an ultrafiltration membrane to retain soybean peptide solution with a molecular weight ≤3 kDa, concentrate under vacuum to a soybean peptide content of 20wt%-30wt%, and then spray dry to obtain soybean peptide; The preparation method of the chlorogenic acid-grafted UiO-66 biomimetic catalyst described in step (2) is characterized by comprising the following steps: S1 Mix 1.0-1.2 mmol zirconium tetrachloride and 1.0-1.2 mmol 2-aminoterephthalic acid, add 20-40 mL of anhydrous N,N-dimethylformamide as a solvent, then add 0.8-1.2 mL of concentrated hydrochloric acid, and react in a hydrothermal reactor at 110-120℃ for 24 h. After cooling to room temperature, centrifuge at 8000-10000 r / min for 5-6 min, take the precipitate, wash with anhydrous methanol and centrifuge 2-3 times, and dry under vacuum at 100-120℃ for 10-12 h to obtain activated UiO-66; S2 Dissolve 0.1-0.2 mmol of chlorogenic acid in 20-40 mL of anhydrous N,N-dimethylformamide, add 0.15-0.3 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.15-0.3 mmol of N-hydroxysuccinimide, and stir at 100-120 r / min for 2-4 h at room temperature, protected from light, and under nitrogen protection to obtain an activated chlorogenic acid solution; S3 Add 90-110 mg of the activated UiO-66 obtained in step S1 to the activated chlorogenic acid solution obtained in step S2, place it at 35-40℃, and stir continuously for 12-24 h. After centrifugation, washing, and vacuum drying, the chlorogenic acid-grafted UiO-66 biomimetic catalyst is obtained.

2. The method for efficiently extracting soybean peptides as described in claim 1, characterized in that, The amount of chlorogenic acid-grafted UiO-66 biomimetic catalyst added in step (2) is 1-5 wt% of the dry weight of soy protein isolate raw material.

3. The method for efficiently extracting soybean peptides as described in claim 2, characterized in that, The amount of the chlorogenic acid-grafted UiO-66 biomimetic catalyst added is 3 wt% of the dry weight of the soybean protein raw material.

4. The method for efficiently extracting soybean peptides as described in claim 1, characterized in that, The reaction temperature of the protein suspension and the chlorogenic acid-grafted UiO-66 biomimetic catalyst in step (2) is 60°C.

5. The method for efficiently extracting soybean peptides as described in claim 1, characterized in that, The inlet temperature of the spray dryer in step (2) is 170-185℃, and the outlet temperature is 80-90℃.

6. The method for efficiently extracting soybean peptides as described in claim 1, characterized in that, The concentration of the phosphate buffer solution in step (1) is 0.05-0.06 mol / L, and the pH is 7.5-8.0.

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