High-solubility high-antioxidant-activity soybean protein and preparation method and application thereof

Abstract

The application discloses high-solubility high-antioxidant-activity soybean protein and a preparation method and application thereof, and belongs to the technical field of food protein processing. The application takes soybean protein isolate as raw material, and improves the open degree of protein structure through ultrasonic-assisted pretreatment and enzymatic hydrolysis modification technology, promotes the unfolding of protein molecular chains, and releases functional peptide segments with antioxidant activity. The soybean protein product prepared by the method has high water solubility, protein recovery rate and antioxidant capacity, the solubility can reach more than 90%, the protein extraction rate can reach about 90%, the degree of hydrolysis can reach about 25%, and the DPPH and ABTS free radical scavenging capacities can reach more than 45% respectively, and the soybean protein product can be used in the fields of functional food, nutritional fortifier and plant protein beverage.

Description

Technical Field

[0001] This invention relates to the field of plant protein deep processing and functional food technology, and more specifically to a highly soluble soybean protein with high antioxidant activity, its preparation method and application. Background Technology

[0002] Soy protein is an important plant protein resource derived from soybeans. Due to its high protein content, relatively balanced amino acid composition, rich nutritional value, and wide availability, it is widely used in plant-based foods, protein beverages, sports nutrition foods, and functional foods. Among them, soy protein isolate, with its high protein content and certain emulsifying, gelling, and water-holding properties, is one of the most widely used plant protein raw materials in the food industry.

[0003] Natural soy protein is mainly composed of storage proteins such as 7S and 11S globulins. Its molecular structure is relatively stable, with numerous hydrophobic groups within the protein. Furthermore, the highly ordered spatial structure is maintained between molecules through hydrogen bonds, hydrophobic interactions, and disulfide bonds, limiting its dispersion and solubility in aqueous systems. Especially under neutral conditions, soy protein tends to aggregate, leading to reduced protein solution stability and thus limiting its application in high-protein liquid foods and functional foods. In addition, the functional peptides with antioxidant activity in natural soy protein are usually hidden within the protein macromolecule structure, making full release difficult and resulting in limited antioxidant capacity.

[0004] To improve the functional properties of soybean protein, researchers have developed various physical, chemical, and biological modification methods. Among them, ultrasound-assisted modification technology has received widespread attention in recent years due to its green, safe, and simple operation. The cavitation effect, local high temperature and pressure, and mechanical shearing generated during ultrasound treatment can alter the higher-order structure of proteins, promote protein molecule unfolding, and increase the exposure of protein surface active groups, thereby improving protein solubility, emulsification, and interfacial properties. However, ultrasound treatment alone usually only induces conformational changes in proteins and cannot fully achieve the targeted degradation of protein peptide chains, thus its effect on increasing the degree of protein hydrolysis and releasing antioxidant active peptides remains limited.

[0005] Enzymatic hydrolysis is one of the important methods for improving the functional properties of plant proteins. Proteases can selectively cleave peptide bonds in proteins, generating a large number of low-molecular-weight bioactive peptides, thereby improving protein digestibility and bioactivity. Alkaline proteases, neutral proteases, and complex proteases have been widely used in research on the hydrolytic modification of plant proteins. Protein hydrolysis not only improves protein solubility but also releases antioxidant peptides with free radical scavenging capabilities. However, the dense structure of natural soybean protein means that enzyme action sites are partially embedded within the protein, leading to low hydrolysis efficiency. Furthermore, excessive hydrolysis may degrade protein functional properties. Therefore, how to improve hydrolysis efficiency while achieving synergistic improvement in protein functional properties remains a key research challenge.

[0006] Current research largely focuses on the effects of single ultrasonic treatment or enzymatic hydrolysis on the structure and functional properties of soybean protein. Research on the synergistic mechanism between ultrasound-induced protein structural changes and enzymatic hydrolysis remains insufficient. Furthermore, existing technologies struggle to simultaneously enhance protein solubility, degree of hydrolysis, and antioxidant activity while maintaining protein structural stability. Therefore, developing a simple, safe, and effective modification method that synergistically improves soybean protein solubility and antioxidant activity is crucial for expanding the application of soybean protein in high-value-added food sectors. Summary of the Invention

[0007] The present invention aims to solve the technical problems of existing soybean protein products, such as low solubility, insufficient protein extraction rate, limited hydrolysis efficiency and weak antioxidant activity, and to provide a soybean protein product with high solubility and high antioxidant activity and its preparation method.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A highly soluble soybean protein with high antioxidant activity and its preparation method, characterized in that the method includes the following steps: (1) Preparation of soybean protein dispersion: Soy protein raw materials are added to deionized water and fully dispersed to form a uniform soy protein dispersion. The soy protein raw materials include one or more combinations of soy protein isolate, soy protein concentrate, soy protein powder, soy 7S globulin, and soy 11S globulin. Soy protein isolate is preferably used as the raw material, and the protein dispersion concentration is 1%–15%. The soy protein is extracted using an alkali-soluble acid precipitation method, achieving an extraction rate of 87%.

[0010] (2) Ultrasonic-assisted structural pretreatment: The above soybean protein dispersion was pretreated under ultrasonic conditions. The ultrasonic cavitation effect, mechanical shearing and microfluidic effect promoted the unfolding of the higher-order structure of the protein, destroyed the intermolecular interactions of the protein, exposed the hydrophobic regions and enzyme action sites inside the protein, and improved the hydrolyzability of the protein.

[0011] The ultrasonic treatment conditions are: ultrasonic power of 200–600 W; ultrasonic time of 10–40 min; ultrasonic temperature of 20–50℃. Preferred conditions are: ultrasonic power of 300–500 W; treatment time of 20–30 min; treatment temperature of 30–40℃.

[0012] (3) Synergistic modification by enzymatic hydrolysis: Protease was added to the soy protein dispersion after ultrasonic pretreatment, and enzymatic hydrolysis was carried out under suitable temperature and pH conditions. The protein structure unfolding induced by ultrasound was synergistic with the enzymatic hydrolysis to improve the binding efficiency of protease to substrate, promote the cleavage of protein peptide chains, and release low molecular weight functional peptides with antioxidant activity, thereby improving protein solubility, degree of hydrolysis and antioxidant capacity.

[0013] The protease includes one or more of alkaline protease, neutral protease, papain, flavor protease, trypsin, or complex protease.

[0014] Alcalase, an alkaline protease, is preferably used. The amount of protease added is 0.5% to 5% of the soybean protein content, preferably 1% to 3%. The enzymatic hydrolysis conditions are: reaction temperature 40 to 60°C; reaction time 1 to 5 h; reaction system pH 7.0 to 9.0. Preferred conditions are: temperature 45 to 55°C; time 2 to 4 h; pH 7.5 to 8.5.

[0015] (4) Enzyme inactivation and separation / purification: After enzymatic hydrolysis, the reaction system is subjected to enzyme inactivation treatment, followed by centrifugation to remove insoluble impurities. The supernatant is collected and concentrated or dried to obtain a highly soluble soybean protein product with high antioxidant activity.

[0016] The enzyme inactivation conditions are: temperature 80℃~100℃; time 5~20 min. The drying method includes one of freeze drying, spray drying or vacuum drying.

[0017] Freeze-drying is preferred to preserve the structure and antioxidant activity of the active peptides.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: The highly soluble soybean protein product with high antioxidant activity has the following characteristics: (1) It has high water solubility, with a protein solubility of over 90%, preferably over 95%, and more preferably around 98%; (2) It has high protein utilization efficiency, with a protein extraction rate of over 85%, preferably around 90%; (3) It has a high degree of protein hydrolysis, with a degree of hydrolysis (DH) of more than 20%, preferably around 25%; (4) It has strong antioxidant capacity, with a DPPH free radical scavenging rate of over 40%, preferably over 45%; (5) It has a strong ABTS radical scavenging ability, with an ABTS radical scavenging rate of over 40%, preferably over 45%. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The effect of different treatment methods on the solubility of soybean protein.

[0021] Figure 2 The effect of different treatment methods on the degree of hydrolysis (DH) of soybean protein.

[0022] Figure 3 The effects of different treatment methods on the DPPH radical scavenging ability.

[0023] Figure 4 The effects of different treatment methods on the free radical scavenging ability of ABTS. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0025] This invention provides a method for preparing a highly soluble soybean protein product with high antioxidant activity, comprising the following steps: (1) Protein dispersion: Soy protein raw materials are added to deionized water and dispersed thoroughly to form a uniform soybean protein dispersion; (2) Ultrasonic structural pretreatment: The above soybean protein dispersion was subjected to ultrasonic pretreatment. The ultrasonic cavitation effect promoted the unfolding of protein structure, destroyed the intermolecular interactions of protein molecules, exposed the hydrophobic regions and enzyme action sites inside the protein, and improved the hydrolysability of the protein. (3) Enzymatic hydrolysis modification: Add protease to the protein dispersion after ultrasonic pretreatment and carry out enzymatic hydrolysis under suitable temperature and pH conditions. Through the synergistic effect of ultrasonic induction and enzymatic hydrolysis, protein peptide chain breakage is promoted, more low molecular weight active peptides are generated, and protein solubility and antioxidant activity are improved. (4) Post-treatment: After the hydrolysis reaction is completed, the reaction system is subjected to enzyme inactivation treatment, and insoluble impurities are removed by centrifugation. The supernatant obtained is concentrated and dried to obtain a soybean protein product with high solubility and high antioxidant activity.

[0026] To further optimize the above technical solution, the soybean protein raw material includes one or more of the following: soybean protein isolate, soybean protein concentrate, soybean protein powder, soybean 7S globulin, and soybean 11S globulin.

[0027] To further optimize the above technical solution, the soybean protein raw material is soybean protein isolate, and the protein concentration in the soybean protein dispersion is 1% to 15%, preferably 3% to 10%.

[0028] To further optimize the above technical solution, the ultrasonic structure pretreatment conditions are as follows: ultrasonic power is 200-600 W, preferably 300-500 W; ultrasonic time is 10-40 min, preferably 20-30 min; ultrasonic treatment temperature is 20-50 ℃, preferably 30-40 ℃.

[0029] To further optimize the above technical solution, the protease includes one or more of the following: alkaline protease, neutral protease, papain, flavor protease, trypsin, and complex protease. The protease is an alkaline protease. The amount of protease added is 0.5%–5% of the soybean protein content, preferably 1%–3%. The enzymatic hydrolysis conditions are: reaction temperature of 40–60 °C, preferably 45–55 °C; reaction time of 1–5 h, preferably 2–4 h; and pH of the reaction system of 7.0–9.0, preferably 7.5–8.5.

[0030] To further optimize the above technical solution, the enzyme inactivation treatment conditions are: temperature 80–100 °C, treatment time 5–20 min. The drying method includes one of freeze-drying, spray drying, and vacuum drying. Freeze-drying is used to maximize the preservation of the active peptide structure and antioxidant activity.

[0031] Example 1: Ultrasonic-assisted alkaline protease hydrolysis for the preparation of highly soluble soybean protein products with high antioxidant activity Weigh 5 g of soy protein isolate powder, add it to 95 mL of deionized water, and stir thoroughly at room temperature to form a uniform soy protein dispersion.

[0032] The protein dispersion was pretreated in an ultrasonic treatment device with an ultrasonic power of 300 W, an ultrasonic time of 20 min, and a treatment temperature of 35 °C.

[0033] The cavitation effect and mechanical shearing generated during ultrasonic treatment promote the unfolding of protein spatial structure and increase the exposure of active regions on the surface of protein molecules.

[0034] After ultrasonic treatment, the pH of the protein dispersion was adjusted to 8.0, and alkaline protease (Alcalase) was added at a rate of 2% of the soybean protein content.

[0035] It was then placed in a constant temperature water bath at 50 ℃ for enzymatic hydrolysis for 3 h.

[0036] After the reaction was completed, the system was placed in a 90 °C water bath for 10 min to inactivate the enzyme.

[0037] After cooling, the mixture was centrifuged at 10000×g for 20 min, and the supernatant was collected. After freeze-drying for 48 h, the highly soluble and highly antioxidant soybean protein product described in Example 1 was obtained.

[0038] Example 2: Preparation of soybean protein products under different ultrasonic power conditions Soy protein products were prepared according to the method in Example 1, except that the ultrasonic pretreatment conditions were different.

[0039] The ultrasonic power is set as follows: 200 W; 400 W; 500 W. The sonication time was 20 min for all cases, and other conditions remained consistent. Soy protein products obtained in Examples 2-1, 2-2, and 2-3 were obtained respectively.

[0040] By comparing the effects of different ultrasonic powers on the degree of protein structure unfolding and enzymatic digestion, suitable ultrasonic treatment conditions were determined.

[0041] Example 3: Preparation of soybean protein products under different enzyme addition levels The preparation was carried out according to the method of Example 1, except that the amount of alkaline protease added was different.

[0042] The protease addition amounts were set at 1%, 2%, and 3%, respectively. All other ultrasonic treatment and enzymatic hydrolysis conditions remained consistent. The resulting samples were used to evaluate the effects of different enzyme addition amounts on the degree of protein hydrolysis, solubility, and antioxidant activity.

[0043] Comparative Example 1: Untreated soy protein isolate Weigh 5 g of soy protein isolate and add 95 mL of deionized water to disperse it thoroughly.

[0044] It is not subjected to ultrasonic treatment or enzymatic hydrolysis.

[0045] The obtained samples were directly centrifuged and freeze-dried to serve as a control sample for natural soybean protein.

[0046] Comparative Example 2: Soy protein treated with ultrasound alone Weigh 5 g of soy protein isolate and sonicate it according to the conditions in Example 1. The sonication power was 300 W; the time was 20 min; and the temperature was 35 ℃. After sonication, no protease was added; the mixture was simply subjected to enzyme inactivation, centrifugation, and freeze-drying.

[0047] The resulting product was used as a control sample for separate ultrasonic treatment.

[0048] Comparative Example 3: Enzymatic hydrolysis of soybean protein alone Weigh 5 g of soy protein isolate and hydrolyze it with alkaline protease according to the conditions in Example 1. The difference is that ultrasonic pretreatment was not performed. The pH was directly adjusted to 8.0, 2% Alcalase was added, and hydrolysis was carried out at 50 °C for 3 h. After the reaction, enzyme inactivation, centrifugation, and lyophilization were performed. The resulting sample was used as a control sample for enzymatic hydrolysis alone.

[0049] The product performance evaluation method of this invention is as follows: To evaluate the functional properties of the highly soluble and highly antioxidant soybean protein product obtained in this invention, the following methods were used for testing: (1) Protein solubility determination Protein solubility was determined by centrifugation.

[0050] The sample was prepared into a protein solution of a certain concentration, fully hydrated, and then centrifuged at 10000×g for 20 min. The supernatant was then collected.

[0051] The soluble protein content in the supernatant was determined using a protein content assay method.

[0052] Protein solubility is calculated using the following formula:

[0053] Depend on Figure 1It can be seen that the solubility of untreated soy protein isolate (CK) is 68.5%. After ultrasonic treatment (US), the protein solubility increased to 79.6%, indicating that ultrasonic treatment can effectively improve the dispersion performance of soy protein.

[0054] The solubility of the enzymatically isolated (ENZ) group was further increased to 87.3%, indicating that protease hydrolysis can reduce the molecular weight of proteins, disrupt some protein aggregation structures, and increase the exposure of hydrophilic groups, thereby promoting protein solubility in water.

[0055] It is worth noting that the ultrasound-assisted enzymatic digestion group (US+ENZ) showed the highest protein solubility, reaching 97.8%, which was significantly higher than that of the ultrasound-only group and the enzymatic digestion group alone.

[0056] The results showed that ultrasonic pretreatment promoted the unfolding of soybean protein structure, improved the binding efficiency of protease and substrate, and further enhanced the enzymatic hydrolysis effect, with a significant synergistic effect between the two. See results below. Figure 1 .

[0057] (2) Determination of degree of hydrolysis by OPA method The degree of protein hydrolysis was determined using the o-phthalaldehyde (OPA) method.

[0058] After the sample reacted with the OPA colorimetric solution, the absorbance was measured at 340 nm.

[0059] A standard curve was established using L-leucine to calculate the free amino content, and the degree of protein hydrolysis was calculated using the formula:

[0060] DH: Degree of protein hydrolysis; h: The amount of free amino groups released by hydrolysis; h tot The total number of peptide bonds when a protein is completely hydrolyzed.

[0061] Depend on Figure 2 It can be seen that the degree of hydrolysis of natural soybean protein (CK) is low, at only 3.2%.

[0062] After ultrasonic treatment, the hydrolysis density (DH) value increased to 6.1%, indicating that the cavitation effect and mechanical shearing action generated by ultrasound can alter the higher-order structure of proteins, exposing some peptide bonds and improving protein hydrolysability. The DH of the enzymatic hydrolysis group alone (ENZ) reached 21.8%, significantly higher than the CK and US groups, indicating that proteases can effectively degrade soybean protein molecules. The ultrasound-assisted enzymatic hydrolysis group (US+ENZ) had the highest DH, reaching 25.6%, a further increase compared to the enzymatic hydrolysis group alone. This demonstrates that ultrasonic treatment can disrupt the stable internal structure of proteins, exposing more enzyme action sites, thereby promoting protein hydrolysis and improving hydrolysis efficiency. Results are shown below. Figure 2 .

[0063] (3) Determination of DPPH free radical scavenging ability Take a sample solution of a certain concentration, mix it with DPPH ethanol solution, react it under light-protected conditions for 30 min, and measure the absorbance at 517 nm.

[0064] The DPPH free radical scavenging rate was calculated based on the change in absorbance.

[0065]

[0066] A s : Absorbance of sample group; A b : The absorbance of the sample's own color against the background; A c : Absorbance of the DPPH blank control group.

[0067] Depend on Figure 3 It can be seen that the DPPH free radical scavenging rate of untreated soybean protein is 18.5%.

[0068] After sonication, the DPPH clearance rate increased to 26.8%, indicating that changes in protein structure can promote the exposure of some antioxidant active groups.

[0069] The DPPH scavenging rate of the enzymatic hydrolysis group was further increased to 40.2%, indicating that the low molecular weight active peptides released during the enzymatic hydrolysis process have a strong free radical scavenging ability.

[0070] Among them, the US+ENZ group had the highest DPPH radical scavenging rate, reaching 46.7%, which was significantly higher than other treatment groups.

[0071] These results demonstrate that ultrasound-assisted enzymatic hydrolysis can promote protein degradation, releasing more peptides with antioxidant activity, thereby enhancing the antioxidant capacity of protein products. See results below. Figure 3 .

[0072] (4) Determination of ABTS free radical scavenging ability The antioxidant capacity of the samples was evaluated using the ABTS radical cation decolorization method. After the samples reacted with the ABTS working solution, the absorbance was measured at 734 nm, and the ABTS radical scavenging rate was calculated based on the change in absorbance.

[0073]

[0074] A s : Absorbance of the ABTS system after adding the sample; A c : Absorbance of the ABTS blank control system.

[0075] Depend on Figure 4 It can be seen that the ABTS free radical scavenging rate of natural soybean protein is 22.3%.

[0076] After sonication, the ABTS scavenging rate increased to 31.5%; after enzymatic hydrolysis alone, it increased to 43.6%. The sonication-assisted enzymatic hydrolysis group showed the highest ABTS free radical scavenging ability, reaching 48.9%. Compared with the CK group, US+ENZ treatment increased the ABTS free radical scavenging ability by approximately 119.3%. Results are shown below. Figure 4 .

[0077] This indicates that the combined treatment can effectively promote the release of antioxidant active peptides and improve the functional activity of soybean protein products.

[0078] In summary, compared with untreated soy protein isolate, both ultrasonic treatment and enzymatic hydrolysis can improve the functional properties of soy protein, with ultrasonic-assisted enzymatic hydrolysis showing the most significant effect. Ultrasonic pretreatment promotes the loosening of protein molecular structure through cavitation, increasing the exposure of protease activation sites; subsequent enzymatic hydrolysis further promotes peptide chain cleavage, releasing short-chain peptides with high antioxidant activity. Therefore, ultrasonic-assisted enzymatic hydrolysis can produce a synergistic effect, resulting in soy protein products with high solubility, high degree of hydrolysis, and high antioxidant activity.

[0079] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing highly soluble soybean protein with high antioxidant activity, characterized in that, Includes the following steps: Soy protein with high solubility and high antioxidant activity was prepared by modifying the structure of soybean protein under ultrasound-assisted conditions and then enzymatically hydrolyzing it with protease.

2. The preparation method according to claim 1, characterized in that, Specifically, the following steps are included: (1) Preparation of soybean protein dispersion: Soy protein raw materials are added to deionized water and dispersed thoroughly to form a uniform soybean protein dispersion. (2) Ultrasonic-assisted structural pretreatment: The above soybean protein dispersion was pretreated under ultrasonic conditions. (3) Synergistic modification by enzymatic hydrolysis: Protease was added to the soybean protein dispersion that had been pretreated by ultrasound to carry out enzymatic hydrolysis. (4) Enzyme inactivation and separation / purification: After enzymatic hydrolysis, the reaction system is subjected to enzyme inactivation treatment, followed by centrifugation to remove insoluble impurities. The supernatant is collected and concentrated or dried to obtain highly soluble soybean protein with high antioxidant activity.

3. The preparation method according to claim 2, characterized in that, The soy protein raw materials include one or more combinations of soy protein isolate, soy protein concentrate, soy protein powder, soy 7S globulin, and soy 11S globulin.

4. The preparation method according to claim 2, characterized in that, The mass concentration of the soybean protein dispersion is 1% to 15%.

5. The preparation method according to claim 2, characterized in that, The ultrasonic processing conditions are as follows: ultrasonic power of 200 W to 600 W, ultrasonic processing time of 10 min to 40 min, and processing temperature of 20℃ to 50℃.

6. The preparation method according to claim 2, characterized in that, The protease includes one or more of alkaline protease, papain, flavor protease, trypsin, neutral protease, or complex protease, and the amount of protease added is 0.5% to 5% of the soybean protein content.

7. The preparation method according to claim 2, characterized in that, The enzymatic hydrolysis reaction conditions are: reaction temperature 40℃~60℃, reaction time 1 h~5 h, and pH value of the reaction system 7.0~9.

0.

8. The preparation method according to claim 2, characterized in that, The enzyme inactivation conditions are: temperature 80℃~100℃; time 5~20 min; The drying process includes one of freeze drying, spray drying, or vacuum drying.

9. A highly soluble soybean protein with high antioxidant activity, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the highly soluble, highly antioxidant soybean protein as described in claim 9 in food, characterized in that, The food products include protein powder, plant-based protein drinks, sports nutrition foods, foods for special medical purposes, or dietary supplements.

Images