Soybean protein as well as extraction method and application thereof

Abstract

The invention provides soybean protein as well as an extraction method and application thereof, and relates to the field of plant protein extraction. The extraction method of the soybean protein comprises the following steps: mixing low-temperature soybean meal with a sodium chloride solution and cellulase, adjusting the pH value to 3.5-4.5, carrying out first extraction, then carrying out enzyme deactivation under a heating condition, and carrying out first solid-liquid separation to obtain a first extracting solution and soybean dregs; mixing the bean dregs with water, adjusting the pH value of the solution to 9-11, carrying out second extraction, and then carrying out second solid-liquid separation to obtain a second extracting solution; and mixing the first extracting solution and the second extracting solution, adjusting the pH value of the mixed extracting solution to 3.5-4.5, carrying out third solid-liquid separation after standing, and purifying and drying the obtained precipitate to obtain the soybean protein. According to the method, the extraction rate of the soybean protein is relatively high, and the extracted soybean protein has relatively high purity; in addition, the soybean protein extracted by the invention has an immune regulation function.

Description

Technical Field

[0001] This application relates to the field of plant protein extraction, and more particularly to a soybean protein, its extraction method, and its application. Background Technology

[0002] Soybeans originated in China and are one of the world's most important oilseed and high-protein crops. Soybean protein contains a variety of essential amino acids, possessing high nutritional value; it is a complete protein and is known as the "king of plant proteins." Soybean meal, a byproduct of soybean oil extraction, is inexpensive and abundant, with a protein content as high as 40%-50%, and contains no cholesterol. It is a valuable plant protein alternative to animal protein and is currently mainly used in animal feed. However, with increasing focus on sustainable development and efficient resource utilization, research on extracting high-quality protein from soybean meal has become particularly important. Current protein extraction methods mainly employ the traditional alkali dissolution and acid precipitation method, but this method suffers from problems such as low purity and poor quality of the obtained protein. Therefore, it is necessary to develop a soybean protein extraction method that can improve protein purity and quality, thereby expanding the application range of soybean protein, meeting the needs of different fields, and possessing significant market potential and social benefits. Summary of the Invention

[0003] The purpose of this application is to provide a soybean protein, its extraction method, and its application to solve the above-mentioned problems.

[0004] To achieve the above objectives, this application adopts the following technical solution: A method for extracting soybean protein, comprising: Low-temperature soybean meal was mixed with sodium chloride solution and cellulase, and the pH was adjusted to 3.5-4.5 for the first extraction. Then, the enzyme was inactivated under heating conditions, and the first solid-liquid separation was performed to obtain the first extract and soybean residue. The soybean residue was mixed with water, the pH of the solution was adjusted to 9-11, and a second extraction was performed, followed by a second solid-liquid separation to obtain a second extract. The first and second extracts are mixed, and the pH of the mixed extract is adjusted to 3.5-4.5. After standing, a third solid-liquid separation is performed. The precipitate is purified and dried to obtain soy protein.

[0005] According to an embodiment of this application, the concentration of the sodium chloride solution is 0.1-0.3 mol / L.

[0006] According to an embodiment of this application, the mass of the cellulase accounts for 1.0-2.0% of the mass of the low-temperature soybean meal.

[0007] According to an embodiment of this application, the ratio of the low-temperature soybean meal to the sodium chloride solution is 1:15-1:25 g / mL.

[0008] According to an embodiment of this application, the enzyme inactivation temperature is 75-100°C, and the enzyme inactivation time is 10-15 min.

[0009] According to an embodiment of this application, the first extraction time is 40-80 min; And / or, the temperature of the first extraction is 40-45°C; And / or, the first extraction is performed under ultrasonic conditions with a power of 200-280 W; And / or, the method further includes: adding a first pH adjuster to make the pH during the first extraction 3.5-4.5; the first pH adjuster includes a hydrochloric acid solution.

[0010] According to an embodiment of this application, the temperature of the second extraction is 40-45°C; And / or, the second extraction time is 40-80 min; And / or, the second extraction is performed under ultrasonic conditions with a power of 200-280 W; And / or, the method further includes: adding a second pH adjuster to make the pH during the second extraction 9-11; the second pH adjuster includes a sodium hydroxide solution; And / or, the method further includes: adding a third pH adjuster to make the pH of the mixed extract 3.5-4.5; the third pH adjuster includes hydrochloric acid solution.

[0011] In some embodiments, the first extract and the second extract are mixed, and after adjusting the pH of the mixed extract to 3.5-4.5, it can be left to stand overnight at 4°C.

[0012] According to an embodiment of this application, the purification includes: redissolving the precipitate obtained from the third solid-liquid separation in water, followed by dialysis with distilled water.

[0013] This application also provides a soybean protein, which is prepared by the soybean protein extraction method described above.

[0014] This application also provides the use of the aforementioned soy protein in health foods.

[0015] Compared with the prior art, the beneficial effects of this application include: The method described in this application has a high extraction rate of soybean protein, and the extracted soybean protein has high purity; moreover, the soybean protein extracted in this application has immunomodulatory functions.

[0016] Specifically, this application uses low-temperature soybean meal as raw material. The introduction of sodium chloride solution accelerates cell wall disruption and protein dissolution, while the addition of cellulase further disrupts the cell wall to promote protein dissolution, yielding a first extract and soybean residue. Next, under specific alkaline conditions, the protein in the soybean residue is fully dissolved, and after solid-liquid separation, a second extract is obtained, significantly improving the protein release efficiency from the soybean meal powder. Subsequently, the first and second extracts are mixed, and the pH of the mixed extract is adjusted to the isoelectric point of soybean protein, allowing soybean protein to precipitate and separating protein from impurities. The collected precipitate is dissolved in distilled water, dialyzed, and dried to obtain high-purity soybean protein, significantly improving the extraction rate. The protein product obtained by this application has high safety, excellent color, and the function of promoting macrophage proliferation and enhancing its phagocytic activity, making it suitable for producing health foods with immunomodulatory functions. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0018] Figure 1 The graph shows the effect of soybean protein on the proliferation of RAW264.7 cells in Example 1 and Comparative Example 1. Figure 2 The graph shows the phagocytic capacity of soybean protein on RAW264.7 cells in Example 1 and Comparative Example 1. Figure 3 The graph shows the effect of soybean protein on NO secretion in RAW264.7 cells in Example 1 and Comparative Example 1. Detailed Implementation

[0019] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0020] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0021] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0022] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0023] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (where K is any number representing a multiplier). It is important to understand that, unlike parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0024] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0025] A method for extracting soybean protein, comprising: Low-temperature soybean meal was mixed with sodium chloride solution and cellulase, and the pH was adjusted to 3.5-4.5 for the first extraction. Then, the enzyme was inactivated under heating conditions, and the first solid-liquid separation was performed to obtain the first extract and soybean residue. The soybean residue was mixed with water, the pH of the solution was adjusted to 9-11, and a second extraction was performed, followed by a second solid-liquid separation to obtain a second extract. The first and second extracts are mixed to obtain a mixed extract. The pH of the mixed extract is adjusted to 3.5-4.5, and after standing, a third solid-liquid separation is performed. The obtained precipitate is purified and dried to obtain soy protein.

[0026] The sodium chloride solution of this application promotes the dissolution of protein in low-temperature soybean meal through salt-dissolving effect, while maintaining the stability of the internal cell structure and preventing protein denaturation in the early stage of extraction. Furthermore, combined with the synergistic effect of the addition of cellulase and ultrasonic treatment, it can effectively destroy the cell wall structure of low-temperature soybean meal, allowing the protein to be released into the solvent. Subsequently, the protein in soybean residue can be further extracted under alkaline conditions. Soybean protein is then obtained through acid precipitation, water washing, and drying.

[0027] This application proposes to add sodium chloride solution and cellulase during the extraction process of soybean protein, which can promote the full dissolution of protein and thus effectively improve the extraction rate of soybean protein.

[0028] According to embodiments of this application, the concentration of the sodium chloride solution is 0.1-0.3 mol / L. When the sodium chloride solution concentration is too low, the low-salt conditions cannot disrupt the binding between proteins and other cellular structures, resulting in the ineffective release of bound proteins and a decrease in protein extraction rate. When the sodium chloride solution concentration is too high, the high ionic strength will damage the hydration layer on the surface of the enzyme protein, leading to cellulase aggregation, conformational changes, decreased enzyme activity, and reduced protein dissolution. At the same time, high salt concentration increases the dielectric constant of the solution, enhancing the interaction between hydrophobic regions of proteins, causing proteins to precipitate due to hydrophobic aggregation, resulting in a decrease in protein yield and consequently a decrease in protein extraction rate.

[0029] For example, the concentration of the sodium chloride solution can be 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, or any value between 0.1 and 0.3 mol / L.

[0030] According to embodiments of this application, the cellulase accounts for 1.0-2.0% of the mass of the low-temperature soybean meal. When the enzyme addition is too low, it is insufficient to effectively disrupt the cell wall structure of the soybean meal, preventing protein release into the solvent and resulting in a decrease in protein yield. When the enzyme addition is too high, the enzyme and substrate become completely saturated, and further enzyme addition will not only fail to increase the protein extraction rate but may even cause a slight decrease in the protein extraction rate due to the enzyme's own physicochemical properties, while also resulting in resource waste.

[0031] For example, the mass of cellulase can be any value between 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or 1.0-2.0% of the mass of low-temperature soybean meal.

[0032] According to embodiments of this application, the material-to-liquid ratio of the low-temperature soybean meal to the sodium chloride solution is 1:15-1:25 g / mL. An appropriate material-to-liquid ratio can maintain good diffusion driving force. However, when the material-to-liquid ratio is too high, the sodium chloride solution content is relatively low, resulting in insufficient solid-liquid contact area. Consequently, the protein in the soybean meal raw material cannot fully diffuse into the solvent, leading to a reduced extraction rate. When the material-to-liquid ratio is too low, i.e., the sodium chloride solution content is relatively high, the protein diffusion driving force is greatly weakened, and the diffusion rate from the raw material to the extraction solvent slows down, resulting in a reduction in the total amount of protein extracted within a predetermined time, thereby reducing the protein extraction rate.

[0033] For example, the ratio of soybean meal to sodium chloride solution at low temperature can be 1:15 g / mL, 1:16 g / mL, 1:17 g / mL, 1:18 g / mL, 1:19 g / mL, 1:20 g / mL, 1:21 g / mL, 1:22 g / mL, 1:23 g / mL, 1:24 g / mL, 1:25 g / mL, or any value between 1:15 and 1:25 g / mL.

[0034] According to an embodiment of this application, the enzyme inactivation temperature is 75-100°C, and the enzyme inactivation time is 10-15 min.

[0035] For example, the enzyme inactivation temperature is any value between 75℃, 80℃, 85℃, 90℃, 95℃, 100℃ or 75-100℃, and the enzyme inactivation time is any value between 10min, 11min, 12min, 13min, 14min, 15min or 10-15min.

[0036] According to an embodiment of this application, prior to the second extraction, the method includes: mixing the soybean residue with water and adjusting the pH of the solution to 9-11. When the solution pH is too low, it approaches the isoelectric point of the protein, resulting in a weak surface charge and low intermolecular electrostatic repulsion. This makes the protein more prone to aggregation through hydrophobic interactions and hydrogen bonds, leading to precipitation and reducing the protein extraction rate. When the solution pH is too high, it disrupts the original structure of the protein, causing denaturation and decreased solubility, thus reducing the protein yield and consequently affecting the protein extraction rate.

[0037] According to the embodiments of this application, the first extraction time is 40-80 min. Under appropriate ultrasonic extraction time, the solvent fully penetrates the cells, making the cell structure loose, and the internal proteins gradually dissolve and diffuse into the external solvent, thus increasing the extraction rate. However, when the extraction time is too long, the protein molecules will unfold and aggregate, and the solubility will decrease, thereby forming insoluble aggregates, which will cause the extracted protein to precipitate again, resulting in a decrease in protein yield. In addition, with the extension of time, other non-protein impurities such as starch and phytic acid will dissolve, reducing the protein purity and thus reducing the protein extraction rate.

[0038] For example, the first extraction time can be any value between 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, or 40-80 min.

[0039] The first extraction temperature is 40-45℃; for example, the first extraction temperature is 40℃, 41℃, 42℃, 43℃, 44℃, 45℃ or any value between 40-45℃.

[0040] The first extraction is performed under ultrasonic conditions with a power of 200-280 W; for example, the ultrasonic power during the first extraction is 200 W, 210 W, 220 W, 230 W, 240 W, 250 W, 260 W, 270 W, 280 W or any value between 200-280 W.

[0041] The method further includes: adding a first pH adjuster to make the pH during the first extraction 3.5-4.5; the first pH adjuster includes hydrochloric acid solution; for example, the pH during the first extraction is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 or any value between 3.5 and 4.5.

[0042] The second extraction temperature is 40-45℃; for example, the second extraction temperature is 40℃, 41℃, 42℃, 43℃, 44℃, 45℃ or any value between 40-45℃.

[0043] The second extraction time is 40-80 min; for example, the second extraction time is any value between 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min or 40-80 min.

[0044] The second extraction is performed under ultrasonic conditions with a power of 200-280 W; for example, the ultrasonic power during the second extraction is 200 W, 210 W, 220 W, 230 W, 240 W, 250 W, 260 W, 270 W, 280 W or any value between 200-280 W.

[0045] The method further includes adding a second pH adjuster to make the pH during the second extraction 9-11; the second pH adjuster includes a sodium hydroxide solution; for example, the pH during the second extraction is 9, 10, 11 or any value between 9 and 11.

[0046] The method further includes adding a third pH adjuster to adjust the pH of the mixed extract to 3.5-4.5; the third pH adjuster includes a hydrochloric acid solution. For example, the pH of the mixed extract is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, or any value between 3.5 and 4.5.

[0047] According to an embodiment of this application, the purification includes: redissolving the precipitate obtained from the third solid-liquid separation in water, followed by dialysis with distilled water.

[0048] This application also provides a soybean protein, which is prepared by the soybean protein extraction method described above.

[0049] This application also provides the use of the aforementioned soy protein in health foods.

[0050] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0051] The raw material information in the examples and comparative examples is as follows: Low-temperature soybean meal was purchased from Shandong Yuxin Biotechnology Co., Ltd. The cellulase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS No. 9012-54-8.

[0052] I. Extraction of soybean protein Example 1 Example 1 provides a soybean protein, the extraction method of which includes: (1) Take an appropriate amount of low-temperature soybean meal, crush it, and pass it through an 80-mesh sieve to obtain low-temperature soybean meal powder.

[0053] Accurately weigh 4.0000 g of pulverized low-temperature soybean meal powder and place it in an Erlenmeyer flask. Add 0.2 mol / L sodium chloride solution at a material-to-liquid ratio of 1:20 g / mL, dissolve and mix well, then add cellulase, where the mass of cellulase accounts for 1.5% of the mass of the low-temperature soybean meal. Adjust the pH to 4.5 with 1 mol / L hydrochloric acid solution, and sonicate at 45℃ and 240 W for 60 min. Subsequently, inactivate the enzyme at 85℃ for 10 min, centrifuge at 2000 r / min for 10 min, collect the supernatant, and obtain the first filtrate and soybean residue.

[0054] (2) The soybean residue was reconstituted with the same volume of distilled water and the pH was adjusted to 10 with 1 mol / L sodium hydroxide solution. The mixture was then sonicated at 45℃ and 240 W for 60 min. After centrifugation at 2000 r / min for 10 min, the supernatant was collected to obtain the second filtrate.

[0055] (3) Combine the first and second filtrates, adjust the pH to 4.0, and let stand overnight at 4°C. Centrifuge at 2000 r / min for 10 min and collect the protein precipitate. After reconstitution, dialyze with distilled water for 24 hours, changing the dialysate every 3 to 4 hours, and freeze-dry under vacuum for 24 hours to obtain soybean protein powder.

[0056] Example 2 The difference between Example 2 and Example 1 is that the concentration of the sodium chloride solution in step (1) is 0.3 mol / L. Everything else is the same as in Example 1.

[0057] Example 3 The difference between Example 3 and Example 1 is that the concentration of the sodium chloride solution in step (1) is 0.1 mol / L. Everything else is the same as in Example 1.

[0058] Example 4 The difference between Example 4 and Example 1 is that the concentration of the sodium chloride solution in step (1) is 0.4 mol / L. Everything else is the same as in Example 1.

[0059] Example 5 The difference between Example 5 and Example 1 is that the concentration of the sodium chloride solution in step (1) is 0.5 mol / L. Everything else is the same as in Example 1.

[0060] Example 6 The difference between Example 6 and Example 1 is that the mass of cellulase in step (1) accounts for 1.0% of the mass of the low-temperature soybean meal. Everything else is the same as in Example 1.

[0061] Example 7 The difference between Example 7 and Example 1 is that the mass of cellulase in step (1) accounts for 2.0% of the mass of the low-temperature soybean meal. Everything else is the same as in Example 1.

[0062] Example 8 The difference between Example 8 and Example 1 is that the mass of cellulase in step (1) accounts for 2.5% of the mass of the low-temperature soybean meal. Everything else is the same as in Example 1.

[0063] Example 9 The difference between Example 9 and Example 1 is that the ratio of low-temperature soybean meal to sodium chloride solution in step (1) is 1:25 g / mL. Everything else is the same as in Example 1.

[0064] Example 10 The difference between Example 10 and Example 1 is that in step (2), the soybean residue is reconstituted with the same volume of distilled water and the pH is adjusted to 9 with a 1 mol / L sodium hydroxide solution. Everything else is the same as in Example 1.

[0065] Example 11 The difference between Example 11 and Example 1 is that the extraction time in steps (1) and (2) is adjusted to 40 minutes. Everything else is the same as in Example 1.

[0066] Example 12 The difference between Example 12 and Example 1 is that the extraction time in steps (1) and (2) is adjusted to 80 minutes. Everything else is the same as in Example 1.

[0067] Comparative Example 1 Comparative Example 1 illustrates a traditional protein extraction method, the specific process of which is as follows: Accurately weigh 4.0000 g of low-temperature soybean meal powder and place it in an Erlenmeyer flask. Add distilled water at a ratio of 1:20 g / mL and mix well. Adjust the pH of the solution to 10 and sonicate at 45℃ and 240 W for 60 min. Centrifuge at 2000 r / min for 10 min, collect the precipitate, and repeat the above steps. Combine the two supernatants and adjust the pH to 4.0. Let stand overnight at 4℃, and collect the precipitate by centrifugation. After redissolving, dialyze with distilled water for 24 hours, changing the dialysate every 3-4 hours. Freeze-dry under vacuum for 24 hours to obtain soybean protein powder extracted by alkali dissolution and acid precipitation.

[0068] II. Performance Testing The soybean protein from the examples and comparative examples was analyzed using the following specific detection methods: 1. Protein yield The formula for calculating protein yield is: Protein yield = Soybean protein content / Raw material low-temperature soybean meal mass × 100%.

[0069] 2. Protein purity The protein content of soybean protein is determined by the BCA method. Protein purity = (protein content of soybean protein / protein content of soybean protein) × 100%.

[0070] 3. Protein extraction rate The total protein mass in soybean meal was determined by the Kjeldahl method. Based on the protein mass of soybean protein, the protein extraction rate was calculated. The formula for calculating the protein extraction rate is: Protein extraction rate = (Soybean protein mass / Total soybean meal protein mass) × 100%.

[0071] The efficacy data of soy protein in the examples and comparative examples are shown in Table 1.

[0072] Table 1 Performance test results for Examples 1-12 and Comparative Example 1

[0073] As can be seen from Table 1, compared with Comparative Example 1, Examples 1-12 simultaneously have higher protein yield, higher protein purity, and higher protein extraction rate. Their overall effect is significantly better than that of Comparative Example 1, indicating that the method of this application is superior to traditional extraction methods.

[0074] The comparison of Examples 1-5 shows that Examples 1-3 are better than Examples 4-5, indicating that the concentration of sodium chloride solution is preferably 0.1-0.3 mol / L.

[0075] The comparison of Examples 1, 6-7 and 8 shows that Examples 1, 6 and 7 are better than Example 8, indicating that the optimal amount of cellulase is 1.0-2.0% of the mass of low-temperature soybean meal.

[0076] The immunological activity of the soybean proteins from Examples 1 and Comparative Example 1 was analyzed using the following methods: Amino acid content determination: The amino acid content in USPHG was determined according to GB5009.124-2016 "Determination of Amino Acids in Food". An appropriate amount of sample was placed in a hydrolysis tube, and 10 mL of 6 mol / L hydrochloric acid solution was added for hydrolysis. The solution was then diluted to volume in a 50 mL volumetric flask. 25 mL of the hydrolysate was dried under reduced pressure using a rotary evaporator. The solution was dissolved in 1.0 mL of pH 2.2 sodium citrate buffer solution, shaken to mix, and then filtered through a 0.22 μm filter to obtain the test solution. Analysis was performed using a fully automated amino acid analyzer. The chromatographic column was LCA K06 / Na; the mobile phase was A = 0.12 N, pH 3.45 sodium citrate, and B = 0.2 N, pH 10.85 sodium citrate; the flow rate was 0.25 mL / min; the detection wavelengths were 570 nm and 440 nm, and the temperature was controlled gradient from 58-74℃. Seventeen amino acids in the sample were determined.

[0077] Cell culture: RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 0.1% penicillin-streptomycin-amphoteric B at 37°C in a 5% CO2 incubator. Cells were passaged or cryopreserved when confluence reached 80%. Cells in the logarithmic growth phase were used for experimental research.

[0078] Macrophage proliferation activity: RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 h. After adhesion, the old culture medium in each well was discarded, and intervention was performed under aseptic conditions. The zero group contained no cells and only culture medium. The blank control group was given 100 μL of complete culture medium, and the positive control group was given 100 μL of LPS solution at a concentration of 1 μg / mL. The experimental groups were given different concentrations (25, 50, 100, 200, 400 μg / mL) of soybean protein solution from Example 1 and Comparative Example 1 (n=5). After 24 h of intervention, the old culture medium was discarded, and serum-free culture medium containing 10% CCK-8 was added. The cells were incubated in an incubator for 0.5–1 h, and the OD value was recorded at a wavelength of 450 nm. Cell viability was calculated.

[0079]

[0080] NO content secreted by macrophages: RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 h. After adhesion, the old culture medium in each well was discarded, and intervention was performed under aseptic conditions. The blank control group was given 100 μL of complete culture medium, the positive control group was given 100 μL of 1 μg / mL LPS solution, and the experimental groups were given different concentrations (25, 50, 100, 200, 400 μg / mL) of soybean protein solution from Example 1 and Comparative Example 1 (n = 5). After 24 h of intervention, the NO content in the supernatant was measured strictly according to the NO kit instructions.

[0081] Phagocytic capacity of macrophages: RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 h. After cell adhesion, the cells were treated according to the instructions. After 24 h, the phagocytic index of the cells was calculated strictly according to the instructions of the neutral red reagent kit.

[0082]

[0083] Depend on Figure 1 , Figure 2 , Figure 3 It can be seen that the data of Example 1 at each test concentration are higher than those of Comparative Example 1, indicating that the soybean protein prepared in Example 1 is better than the soybean protein prepared in Comparative Example 1 in terms of promoting the number of immune cells, activating cell function, and enhancing cell clearance ability. That is, the soybean protein prepared in Example 1 has immunomodulatory function, and its immunomodulatory function is better than that of Comparative Example 1.

[0084] Table 2. Amino acid composition analysis of Example 1 and Comparative Example 1

[0085] As shown in Table 2, compared with Comparative Example 1, the soybean protein obtained in Example 1 has a richer amino acid content, at 81.73 ± 2.78 g / 100g. The bioactivity of food proteins is determined by the composition and sequence of specific amino acids. Among them, the immunomodulatory activity of proteins is closely related to the content of hydrophobic amino acids. The content of hydrophobic amino acids in Example 1 is significantly higher than that in Comparative Example 1, indicating that the soybean protein prepared in this application has high immunomodulatory activity and can be applied to health foods with the effect of improving immunomodulatory activity.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0087] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for extracting soybean protein, characterized in that, include: Low-temperature soybean meal was mixed with sodium chloride solution and cellulase, and the pH was adjusted to 3.5-4.5 for the first extraction. Then, the enzyme was inactivated under heating conditions, and the first solid-liquid separation was performed to obtain the first extract and soybean residue. The soybean residue was mixed with water, the pH of the solution was adjusted to 9-11, and a second extraction was performed, followed by a second solid-liquid separation to obtain a second extract. The first and second extracts are mixed, and the pH of the mixed extract is adjusted to 3.5-4.

5. After standing, a third solid-liquid separation is performed. The precipitate is purified and dried to obtain soy protein.

2. The method for extracting soybean protein according to claim 1, characterized in that, The concentration of the sodium chloride solution is 0.1-0.3 mol / L.

3. The method for extracting soybean protein according to claim 1, characterized in that, The cellulase accounts for 1.0-2.0% of the mass of the low-temperature soybean meal.

4. The method for extracting soybean protein according to claim 2, characterized in that, The ratio of the low-temperature soybean meal to the sodium chloride solution is 1:15-1:25 g / mL.

5. The method for extracting soybean protein according to claim 2, characterized in that, The enzyme inactivation temperature is 75-100℃, and the enzyme inactivation time is 10-15 min.

6. The method for extracting soybean protein according to claim 1, characterized in that, The first extraction time is 40-80 min; And / or, the temperature of the first extraction is 40-45°C; And / or, the first extraction is performed under ultrasonic conditions with a power of 200-280 W; And / or, the method further includes: adding a first pH adjuster to make the pH during the first extraction 3.5-4.5; the first pH adjuster includes a hydrochloric acid solution.

7. The method for extracting soybean protein according to claim 1, characterized in that, The second extraction temperature is 40-45℃; And / or, the second extraction time is 40-80 min; And / or, the second extraction is performed under ultrasonic conditions with a power of 200-280 W; And / or, the method further includes: adding a second pH adjuster to make the pH during the second extraction 9-11; the second pH adjuster includes a sodium hydroxide solution; And / or, the method further includes: adding a third pH adjuster to make the pH of the mixed extract 3.5-4.5; the third pH adjuster includes hydrochloric acid solution.

8. The method for extracting soybean protein according to any one of claims 1-7, characterized in that, The purification process includes: redissolving the precipitate obtained from the third solid-liquid separation in water, followed by dialysis with distilled water.

9. A soybean protein, characterized in that, The soybean protein is prepared by the soybean protein extraction method according to any one of claims 1-8.

10. The application of the soybean protein according to claim 9 in health food.

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