Method for preparing phenol-rich tartary buckwheat protein with high solubility and high bioaccessibility by combining pH driving with proteolysis

Abstract

The invention discloses a method for preparing phenol-rich tartary buckwheat protein with high solubility and high bioaccessibility by combining pH driving with proteolysis. According to the method, the degreased tartary buckwheat flour is pretreated by adopting an initial strong alkaline environment for the first time through a pH driving regulation strategy, and the synergistic release of protein and polyphenol is remarkably enhanced by combining protease enzymolysis. And aiming at the compound protease, staged pH regulation is innovatively introduced, so that the co-extraction efficiency of phenols and proteins is further optimized. And after enzymolysis, carrying out low-molecular-weight dialysis and freeze-drying processes to obtain the phenol-rich tartary buckwheat protein powder. According to the method, the phenol-rich protein powder with the total phenol content being larger than 100 mg GAE / g, the rutin content being 25-35 mg / g and the solubility being remarkably improved can be obtained, the protein hydrolysis degree in an in-vitro digestion experiment is improved by 30% or above compared with a traditional alkali-solution and acid-isolation method, the biological accessibility is as high as 55%, and the antioxidant activity is improved by 40% or above.

Description

Technical Field

[0001] The present invention belongs to the field of food science and engineering technology, and specifically relates to a method for preparing phenol-rich buckwheat protein with high solubility and high bioaccessibility by pH-driven combined protein hydrolysis, which is suitable for the preparation of functional food raw materials and the co-extraction process of protein polyphenols. Background Art

[0002] Buckwheat, a pseudo-cereal with both nutritional and medicinal value, is rich in protein and has a superior amino acid profile, making it a key source of plant-based protein. Furthermore, buckwheat is rich in natural polyphenols, such as rutin and quercetin, which possess significant antioxidant, anti-inflammatory, and lipid-lowering properties. Therefore, the simultaneous extraction of buckwheat protein and phenols for their application in functional foods holds great promise.

[0003] Currently, the food industry generally uses the alkali dissolution and acid precipitation method for plant protein extraction. This method uses alkaline solution to increase protein solubility, and then precipitates the protein through acid precipitation to obtain a crude extract. However, this method has obvious drawbacks: first, the alkaline environment easily destroys the molecular structure of the natural polyphenols in buckwheat, causing the phenolic compounds to be oxidized or degraded during the extraction process, ultimately resulting in a significant reduction in the content of active ingredients in the product; second, the strong alkaline treatment and subsequent acid precipitation can cause protein denaturation, resulting in a compact structure, which not only reduces solubility and functionality, but also reduces its bioaccessibility in the gastrointestinal environment.

[0004] Enzymatic hydrolysis, a technique that uses enzymes to hydrolyze macromolecules and release them, is widely used in the extraction of plant and animal proteins due to its gentle, efficient, clean, and safe nature. For example, in their paper "Study on Optimal Conditions for Enzymatic Hydrolysis of Tartary Buckwheat Protein to Prepare Soluble Bioactive Peptides," Cui Xia et al. compared the hydrolysis of buckwheat protein using neutral and alkaline proteases, finding that alkaline proteases were the most effective. While alkaline protease alone effectively extracted buckwheat protein, the yields of protein and polyphenols were still low. The resulting protein powder had a poor, bitter taste, and poor bioaccessibility. While using a combination of proteases to extract buckwheat protein improved its taste, the optimal process conditions for the combination were not conducive to increasing protein and polyphenol yields. Summary of the Invention

[0005] In response to the above-mentioned problems, the present invention aims to overcome the problems of severe polyphenol loss, protein structural denaturation, low solubility, and low bioaccessibility during the traditional alkali dissolution and acid precipitation method for extracting buckwheat protein, and provides a method for preparing phenol-rich buckwheat protein with high solubility and high bioaccessibility by pH-driven combined with proteolysis. This method combines a staged pH-driven strategy with enzymatic hydrolysis technology to achieve synergistic and efficient extraction and activity retention of proteins and polyphenols. The resulting product is significantly superior to existing technologies in terms of solubility, antioxidant properties, digestibility, and in vitro bioaccessibility.

[0006] To achieve the above objectives, the present invention first provides a method for preparing phenol-rich buckwheat protein with high solubility and high bioaccessibility by pH-driven combined proteolysis, comprising the following steps:

[0007] (1) mixing defatted buckwheat flour and deionized water to form a suspension, and stirring to obtain a mixed system;

[0008] (2) Using NaOH solution, adjust the pH of the mixed system in step (1) to 10-12, and mix and stir;

[0009] (3) adjusting the pH value of the mixed system in step (2) to 7.5-9.5 using HCl solution, and adding protease to the suspension for enzymatic hydrolysis;

[0010] (4) After the enzymatic hydrolysis is completed, centrifugation is performed to obtain the supernatant;

[0011] (5) The pH of the supernatant of step (4) is adjusted to 6.8-7.2, dialyzed through a dialysis bag with a molecular weight cutoff of 100 Da to remove small molecular impurities, and then freeze-dried to obtain phenol-rich buckwheat protein powder.

[0012] In one embodiment of the present invention, the mass ratio of the defatted buckwheat flour to deionized water in step (1) is 1:8-1:12.

[0013] In one embodiment of the present invention, the stirring speed in step (1) is 200 to 600 rpm.

[0014] In one embodiment of the present invention, the pH in step (2) is 10-12, and the stirring time is 0.5-1.5h.

[0015] In one embodiment of the present invention, the protease in step (3) is alkaline protease or composite protease.

[0016] In one embodiment of the present invention, when the protease is alkaline protease, the initial pH of the enzymatic hydrolysis is 8.5-9.5, the enzymatic hydrolysis temperature is 35-40° C., the enzymatic hydrolysis time is 3.5-4.5 h, and the added amount of the enzyme is 500-2000 U / g buckwheat flour.

[0017] In one embodiment of the present invention, when the protease is a composite protease, the initial pH of the enzymatic hydrolysis is 7.5-8.5, the enzymatic hydrolysis temperature is 35-40°C, the enzymatic hydrolysis time is 2.5-3.5h, the enzyme addition amount is 500-2000U / g buckwheat flour, and after the enzymatic hydrolysis is completed, the pH is adjusted to 9.5-10.5, and the duration is 0.5-1.5h.

[0018] In one embodiment of the present invention, the centrifugal speed in step (4) is 8000-10000 rpm, and the centrifugal time is 10-15 min.

[0019] In one embodiment of the present invention, the dialysis treatment time in step (5) is 48-72 hours, and the dialysate is replaced every 2-6 hours.

[0020] In one embodiment of the present invention, the drying method in step (5) is vacuum freeze drying, and the drying time is 72 hours.

[0021] The invention also discloses phenol-rich tartary buckwheat protein powder prepared according to the method.

[0022] In one embodiment of the present invention, the total phenol content of the tartary buckwheat protein powder is greater than 100 mg / g, the solubility is greater than 80%, the protein hydrolysis degree is greater than 65%, and the bioaccessibility is greater than 45%.

[0023] The present invention also discloses an application of the phenol-rich buckwheat protein powder in the fields of functional foods, plant-based protein drinks, sports nutrition formulas and foods for special medical purposes.

[0024] Beneficial effects:

[0025] (1) Compared with the traditional alkali dissolution and acid precipitation method, the present invention prepares phenol-rich buckwheat protein powder through a pH-driven combined enzymatic hydrolysis method. This method can not only significantly shorten the extraction time and improve the extraction rate of protein and polyphenols, but also the prepared phenol-rich buckwheat protein has higher solubility and stronger antioxidant capacity; in vitro simulated digestion experiments show that the protein hydrolysis degree is significantly improved and has higher bioaccessibility. Specifically, the protein extraction rate of the phenol-rich protein powder prepared by the method of the present invention is greater than 85%, the polyphenol extraction rate is greater than 80%, the total phenol content is greater than 100 mg / g, the solubility is greater than 80%, the protein hydrolysis degree is greater than 65%, the bioaccessibility is greater than 45%, and the antioxidant capacity is significantly improved.

[0026] (2) Enzymatic hydrolysis using alkaline protease is a common method for improving the extraction rate of phenol-rich proteins. However, it is difficult to obtain phenol-rich proteins with high extraction rate, high solubility and high bioaccessibility by using only alkaline protease hydrolysis. The present invention adds a pH-driven treatment before enzymatic hydrolysis. The pH-driven treatment can make the protein structure loose, and the protease hydrolyzes the macromolecular protein into small molecular peptides, thereby greatly improving its solubility, bioaccessibility and antioxidant capacity. The pH-driven combined with protease hydrolysis technology can also avoid the long-term strong alkaline environment in traditional alkaline dissolution and acid precipitation extraction, and can fully promote the release of polyphenols, retaining more phenolic substances.

[0027] (3) Although the traditional alkaline protease enzymatic hydrolysis method can improve the performance of protein powder to a certain extent, its effects in various aspects are still relatively poor, especially the free radical scavenging ability, which will limit the application of the prepared protein powder. In addition, the samples obtained by alkaline protease hydrolysis often do not have a good taste and taste bitter. When using composite protease hydrolysis to prepare protein powder, although the taste of the obtained samples is improved to a certain extent, the suitable pH for the composite protease to work is low, resulting in protein extraction rate and polyphenol extraction rate often difficult to reach a high level. When the present invention uses the composite protease method to prepare samples, in addition to using pH-driven treatment to promote extraction in the early stage, it also innovatively introduces a staged pH control strategy after the enzymatic hydrolysis is completed, that is, accurately adjusting the pH to a level that is moderately alkaline and not too high, while protecting the diversity of polyphenol species, promoting the further dissolution of residual proteins and polyphenols, thereby significantly improving the protein extraction rate and polyphenol extraction rate, and ultimately making them close to the extraction rate level achieved by the alkaline protease method.

[0028] (4) The present invention is green and environmentally friendly, and has gentle operation: it avoids the nutrient loss caused by strong alkali / strong acid, and is conducive to industrial promotion. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0030] The alkaline protease used in the examples of the present invention has an activity of 280,000 U / g and was purchased from Novozymes Enzymes under the model Novozym 37071. The complex protease has an activity of 33,000 U / g and was purchased from Novozymes Enzymes under the model Protamex 1.6.

[0031] Test method:

[0032] 1. Test method for protein extraction rate

[0033] The protein content in buckwheat protein powder and extract was determined by Kjeldahl nitrogen determination method, with specific reference to national standard GB5009.5-2016. The extraction rate was calculated as follows:

[0034]

[0035] 2. Total phenol content test method

[0036] The phenol content was determined using the Folin-Ciocalteu colorimetric method, and the specific steps are as follows:

[0037] Accurately weigh 0.010 g of gallic acid standard, prepare 100 μg / mL gallic acid standard stock solution, and dilute it into 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL and 100 μg / mL gradient working solution. Take 0.5 mL of gradient working solution of different concentrations and the diluted test sample solution (extract or 0.1 mg / mL buckwheat protein powder dispersion) and 2.5 mL of 0.2 M Folin phenol reagent and react at room temperature in the dark for 5 minutes. After sufficient reaction, add 2.5 mL of Na2CO3 solution (7.5%, w / v), shake and mix again, and react at room temperature in the dark for 1.5 hours. Finally, use a microplate reader / UV spectrophotometer to detect its absorbance at 760 nm and draw a gallic acid standard curve. The total phenol content (Y) is calculated as follows:

[0038]

[0039] Where: C is the mass concentration of the polyphenol solution to be tested (μg / mL); N is the dilution factor; V is the volume of the extract (mL); M is the mass of phenol-rich protein in buckwheat (g).

[0040]

[0041] 3. Analysis of phenolic substances (UPLC-MS)

[0042] Free and bound phenolic species were identified by UPLC-Q / TOF-MS mass spectrometry. Chromatographic conditions were as follows: SB-AQ C18 column; mobile phase A: acetonitrile containing 0.1% formic acid; mobile phase B: 0.1% formic acid in water; gradient elution program: 0-20 min, 5% to 50% A; 20-24 min, 50% to 100% A; 24-25 min, 100% to 5% A; 25-30 min, 5% A; flow rate: 0.4 mL / min; column oven temperature: 35°C; injection volume: 5 μL. The information obtained from each mass spectral fragment was compared with an online database to determine the composition of the substance based on the best solution. Online databases included Human Metabolome Database (http: / / www.hmdb.ca), Metlin (http: / / metlin.scriproteins.edu), Massbank (http: / / www.massbank.jp / ), and mzCloud (https: / / www.mzcloud.org). Quantitative analysis was performed using a standard curve generated based on the identified optimal substances.

[0043] 4. Solubility test and calculation method

[0044] Buckwheat protein powder was mixed with phosphate buffer (0.2 mol / L, pH 7.0) to prepare a 5 mg / mL protein solution. After stirring at room temperature for 2 hours, the solution was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and the protein content was determined using the Coomassie Brilliant Blue method. Total protein was determined using the Kjeldahl method using bovine serum albumin as a standard.

[0045] Protein solubility = supernatant protein concentration / total protein concentration

[0046] 5. Antioxidant test method

[0047] (1) Testing and calculation methods of DPPH free radical scavenging ability

[0048] Take 1 mL of the sample solution to be tested and mix it thoroughly with 4.5 mL of 100 μM DPPH ethanol solution (prepared and used immediately), shake it and react for 30 minutes in the dark. Then use a UV spectrophotometer to measure the absorbance at 517 nm. Use quinoline dimethacrylate (Trolox) as the equivalent to prepare a standard curve and obtain the regression equation y = 0.0031x + 0.2107 (R 2 =0.9992), where y is the free radical scavenging rate, x is the Trolox mass concentration (μmol / L), and the final result is expressed as Trolox equivalent (μmol / g). The DPPH free radical scavenging rate (%) is calculated using the following formula:

[0049]

[0050] Where: A1 is the absorbance of the measuring tube (sample and DPPH ethanol solution) at 517 nm; A2 is the absorbance of the control tube (sample and ethanol solution) at 517 nm; A0 is the absorbance of the blank tube (ethanol and DPPH ethanol solution) at 517 nm.

[0051] (2) Testing and calculation methods of ABTS free radical scavenging ability

[0052] First, prepare the Trolox standard solution and dilute it. Prepare the ABTS working solution - Reagent A: 0.0384g ABTS dissolved in 10 mL deionized water and reagent B: 0.0134g potassium persulfate dissolved in 10 mL deionized water, mix them in a ratio of 2:1, and react at room temperature in the dark for 12-16 hours to form a blue-green ABTS cation radical solution. Before use, dilute the solution appropriately with anhydrous ethanol and adjust its absorbance at 734nm to 0.700±0.02; then add 1mL of sample solution and 4.5mL of ABTS radical working solution to a 10mL test tube; mix well, let it stand for 6 minutes, and then measure the absorbance at 734nm. A standard curve was prepared using quinoline dimethacrylate (Trolox) as the equivalent, and the regression equation y=0.0068x+0.2732(R 2 =0.9989), where y is the free radical scavenging rate and x is the mass concentration of Trolox (μmol / L). The final result is expressed as Trolox equivalent (μmol / g). The ABTS free radical scavenging rate (%) is calculated using the following formula:

[0053]

[0054] Where: A1 is the absorbance of the measurement tube (sample and ABTS ethanol solution) at 517 nm; A2 is the absorbance of the control tube (sample and ethanol solution) at 517 nm; A0 is the absorbance of the blank tube (ethanol and ABTS ethanol solution) at 517 nm.

[0055] (3) FRAP testing and calculation methods

[0056] First, prepare Fe 3+ -TPTZ working solution was prepared by mixing 0.3mol / L acetate buffer (pH 3.6), 10mmol / L TPTZ solution (dissolved in 40mM HCl) and 20mmol / L FeCl3 solution in a ratio of 10:1:1; during the determination, 0.1mL of the sample solution and 3mL of Fe 3+-TPTZ working solution, mix well, let it stand for 5 minutes and measure the absorbance at 593nm. Use Trolox as the equivalent to prepare a standard curve and obtain the regression equation y=0.0014x+0.1994(R 2 =0.9975), where y is the absorbance and x is the Trolox concentration (μg / mL). The final FRAP value is expressed in Trolox equivalents (mg / g).

[0057] 6. In vitro digestion experiment

[0058] 0.1 g of tartary buckwheat phenolic protein was mixed with SGF (20 mL). 0.3 mM CaCl2(H2O)2 was added, and the pH of the solution was adjusted to 2.0 with 4 M HCl. The solution was preheated in a 37°C water bath for 15 minutes. Pepsin was then added to a final activity of 2000 U / mL. The simulated gastric digestion phase was initiated with shaking (150 rpm / min) in a 37°C water bath for 1 hour. Gastric digestion was terminated by adjusting the pH to 7 with 4 M NaOH. The sample solution was collected and stored at -20°C for later analysis. The simulated intestinal fluid (SIF) was adjusted to pH 7.0 and incubated at 37°C for 5 minutes. After the gastric digestion was completed, simulated intestinal fluid was added to the sample solution at a volume ratio of 1:1, and 0.3 M CaCl2(H2O)2 and 10 mM bile salt solution were added. Trypsin was then added to the mixed solution to make the trypsin activity 100 U / mL. The simulated intestinal digestion was carried out for 2 h, and the digestion was terminated by adding trypsin inhibitor. Appropriate sample solutions were collected at 30, 60, 90 and 120 min, respectively, and stored at -20°C for subsequent analysis.

[0059] Another gastric digestion sample was placed in a dialysis bag with sealed ends (molecular weight cutoff 10,000 Da, in 100 mL of normal saline) and placed in a 37°C water bath with shaking (50 rpm / min) for digestion for 2 h. The sample dispersed outside the dialysis bag was the absorbable sample (OUT), and the component retained in the dialysis bag was the non-absorbable sample (IN). The sample was quickly centrifuged at low temperature (8,000 rpm / min), and the supernatant was collected and stored at -20°C for later use.

[0060] (1) Testing and calculation methods for polyphenol bioaccessibility

[0061] The calculation method of the bioaccessibility of polyphenols in tartary buckwheat phenol-rich protein after gastrointestinal digestion is shown in Formula 1:

[0062] Bioaccessibility (%) = (P Out / P T )×100%

[0063] Among them, P Outrepresents the total phenol content in OUT, and PT represents the total phenol content in OUT and IN.

[0064] (2) Changes in protein hydrolysis degree during in vitro digestion:

[0065] The degree of protein hydrolysis is an important indicator of protein digestibility in reaction samples. The OPA method was used to determine changes in the degree of hydrolysis during the digestion of phenolic buckwheat proteins. The OPA reagent was prepared as follows: 2.01 g of anhydrous sodium tetraborate and 100 mg of SDS were completely dissolved in 75 ml of distilled water. 80 mg of o-phthalaldehyde was then dissolved in 2 ml of ethanol. The two solutions were mixed thoroughly, and 88 mg of dithiothreitol (DTT) was added. For the assay, 400 μL of the digested supernatant / water / serine standard solution was mixed with 3 mL of the OPA reagent. After a 2-min reaction, the absorbance was immediately measured at 340 nm.

[0066] The calculation method of DH (degree of protein hydrolysis) is shown in Formulas 1 and 2.

[0067]

[0068] Where: A1, A2, A0 are the absorbance values ​​of the sample, serine standard, and blank, respectively; serine NH2 is the average amino content (mM / g); 0.9516 is the concentration of the serine standard solution (mM); V is the sample volume (L); m is the sample mass (g); Psample is the protein content of the sample (g / 100g); α and β are correction factors, which are used as constants (α and β of buckwheat protein are 1.00 and 0.40, respectively). tot represents the total number of peptide bonds in the protein (for buckwheat protein, h tot is 7.8mM / g).

[0069] 7. Sensory evaluation method

[0070] Bitterness values ​​were determined by a five-person panel. Before evaluating the samples in this study, the panelists were trained using a control standard solution containing caffeine at 0.3, 0.8, and 1.2 g / L, corresponding to bitterness intensities of 2, 5, and 10, respectively. One gram of freeze-dried sample was dissolved in 100 mL of Wahaha purified water and randomly distributed to trained sensory panelists. Each sensory evaluation was conducted with a 5-minute interval between samples. Samples were scored against the standard solution, and the bitterness value of each sample was the average of the individual panelists' assessments. To ensure accuracy during each tasting, each sample was rinsed with 10% concentrated lemon juice and then rinsed twice with deionized water before tasting. If bitterness persists, a salt-free cracker can be used to eliminate it.

[0071] Example 1

[0072] Preparation of phenol-rich buckwheat protein by pH-driven method combined with protease method (alkaline protease method)

[0073] Weigh 100g of defatted buckwheat flour, add deionized water to a solid-liquid ratio of 1:10, and stir thoroughly to form a uniform suspension. First, adjust the pH of the mixture to 12 using 1M NaOH solution and magnetically stir for 1 hour. Then, adjust the pH to 9.0 using 1M NaOH solution, and maintain the temperature at 37°C. Add alkaline protease to the reaction system at a concentration of 1750 IU / g buckwheat flour. Incubate the mixture at 300 rpm in a thermostatic shaker for 4 hours.

[0074] After the reaction, the mixture was immediately centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was collected and adjusted to pH 7. A portion of the supernatant was used for protein and phenol quantification, while the remaining portion was placed in a 100 Da dialysis bag and dialyzed at 4°C for 48 hours, with the dialysate replaced every four hours. The resulting solution was freeze-dried to obtain the enzymatic hydrolysate, PA10000.

[0075] Example 2

[0076] Preparation of phenol-rich buckwheat protein by pH-driven method combined with enzyme method (complex protease method)

[0077] Weigh 100g of defatted buckwheat flour, add deionized water to a solid-liquid ratio of 1:10, and stir thoroughly to form a uniform suspension. First, adjust the pH of the mixture to 12 using 1M NaOH solution and magnetically stir for 1 hour. Then, adjust the pH to 8.0 using 1M HCl solution, and maintain the temperature at 37°C. Add 1750U / g of composite protease to the reaction system. Incubate the mixture at 300 rpm on a thermostatic shaker for 3 hours. After enzymatic hydrolysis, raise the pH to 10.0 and continue the reaction for 1 hour. Centrifugation, separation, dialysis, and drying procedures are performed as in Example 1 to obtain sample PP10000.

[0078] Comparative Example 1 Alkali dissolution and acid precipitation method

[0079] 100g of defatted buckwheat flour was mixed with deionized water at a ratio of 1:10, adjusted to pH 10.0, and stirred for 6 hours. The mixture was then adjusted to pH 4.0 with 2M HCl, allowed to stand, and centrifuged. The precipitate was collected and redissolved in deionized water, adjusted to pH 7, and dialyzed for 48 hours in a 5000Da dialysis bag to remove small molecule impurities. The dialyzed solution was freeze-dried and designated P0.

[0080] Comparative Example 2

[0081] Weigh 100g of defatted buckwheat flour and add deionized water to a solid-liquid ratio of 1:10. Stir thoroughly to form a uniform suspension. First, adjust the pH of the mixture to 12 using NaOH solution and magnetically stir for 1 hour. Then, adjust the pH to 9.0 using 1M NaOH solution. Control the temperature at 37°C and incubate the mixture at 300 rpm on a thermostatic shaker for 4 hours.

[0082] After the reaction, the mixture was immediately centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was collected and adjusted to pH 7. A portion of the supernatant was used for protein and phenol quantification, while the remaining portion was placed in a 100 Da dialysis bag and dialyzed at 4°C for 48 hours, with the dialysate replaced every four hours. The resulting solution was freeze-dried, and the resulting product was designated P1.

[0083] Comparative Example 3

[0084] Weigh 100g of defatted buckwheat flour and add deionized water to a solid-liquid ratio of 1:10. Stir thoroughly to form a uniform suspension. Adjust the pH of the mixture to 9.0 using NaOH solution, and maintain the reaction temperature at 37°C. Add alkaline protease to the reaction system at a concentration of 1750 IU / g buckwheat flour. Incubate the reaction in a thermostatic shaker at 300 rpm for 4 hours.

[0085] After the reaction, the mixture was immediately centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was collected and adjusted to pH 7. A portion of the supernatant was used for protein and phenol quantification, while the remaining portion was placed in a 100 Da dialysis bag and dialyzed at 4°C for 48 hours, with the dialysate replaced every four hours. The resulting solution was freeze-dried, and the resulting product was designated P2.

[0086] Comparative Example 4

[0087] Weigh 100g of defatted buckwheat flour, add deionized water to a solid-liquid ratio of 1:10, and stir thoroughly to form a uniform suspension. Adjust the pH of the mixture to 8.0 using 1M NaOH solution, and maintain the temperature at 37°C. Add 1750U / g of composite protease to the reaction system, and incubate at 300rpm on a thermostatic shaker for 4 hours. After enzymatic hydrolysis, centrifugation, separation, dialysis, and drying procedures were performed as in Example 1 to obtain Sample P3.

[0088] Comparative Example 5

[0089] Weigh 100g of defatted buckwheat flour, add deionized water to a solid-liquid ratio of 1:10, and stir thoroughly to form a uniform suspension. Adjust the pH of the mixture to 8.0 using 1M NaOH solution, and control the temperature to 37°C. Add 1750U / g of composite protease to the reaction system, and incubate at 300rpm on a thermostatic shaker for 3 hours. After enzymatic hydrolysis, raise the pH to 10.0 and continue the reaction for 1 hour. Subsequently, centrifugation, separation, dialysis, and drying procedures were performed as in Example 1 to obtain sample P4.

[0090] Comparative Example 6

[0091] Weigh 100g of defatted buckwheat flour, add deionized water to a solid-liquid ratio of 1:10, and stir thoroughly to form a uniform suspension. First, adjust the pH of the mixed system to 12 using 1M NaOH solution and stir magnetically for 1 hour. Then, adjust the pH to 8.0 using 1M HCl solution, and control the temperature to 37°C. Add a composite protease to the reaction system at a rate of 175U / g buckwheat flour. The system is continuously reacted at 300rpm in a thermostatic shaker for 3 hours. After the enzymatic hydrolysis is completed, the pH of the system is raised to 10.0, and the reaction is continued for 1 hour. The subsequent centrifugation, separation, dialysis, and drying processes are the same as in Example 1 to obtain sample P5.

[0092] Table 1 Protein, polyphenol extraction rate, total phenol content and solubility

[0093] Protein extraction rate (%) Polyphenol extraction rate (%) Total phenol content (mg / g) Solubility (%) Example 1 86.21 93.34 102.10 84.75 Example 2 85.06 90.32 113.96 85.70 Comparative Example 1 62.33 59.20 101.70 65.41 Comparative Example 2 60.78 54.30 92.65 62.32 Comparative Example 3 75.88 79.42 96.78 76.64 Comparative Example 4 66.56 69.83 101.98 72.56 Comparative Example 5 74.92 76.16 103.40 75.34 Comparative Example 6 69.21 78.15 93.46 65.24

[0094] Table 2 Degree of protein hydrolysis and bioaccessibility of polyphenols in in vitro digestion experiments

[0095] Degree of protein hydrolysis (%) Bioaccessibility (%) Example 1 70.68 45.61 Example 2 68.08 55.30 Comparative Example 1 51.69 43.62 Comparative Example 2 53.32 38.20 Comparative Example 3 60.74 43.78 Comparative Example 4 58.76 46.13 Comparative Example 5 62.86 47.86 Comparative Example 6 54.36 45.46

[0096] Table 3 Antioxidant capacity

[0097]

[0098]

[0099] Table 4 Sensory evaluation results

[0100] Grouping Bitterness Example 1 3.8 Example 2 1.2

[0101] Table 5 Composition and content of polyphenols in different phenol-rich buckwheat proteins, mg / g

[0102] Example 1 Example 2 Comparative Example 1 1 Rutin 29.6091 25.5541 22.6932 2 Quercetin 9.6057 16.9289 15.6976 3 Quercetin 3-O-rutinoside-glucoside 3.7647 0.3597 0.0000 4 Kaempferol-3-O-rutin 2.0113 0.6981 0.5251 5 Geranin 0.1631 0.0133 0.024 6 Protocatechuic acid 12.0963 17.1055 10.1312 7 Parabens 5.7807 2.3212 6.2606 8 Hyperoside 0.2265 0.1784 0.0309 9 Sinapic acid 0.4323 1.7942 0.2676 10 Myricetin 0.0733 0.2673 0.4012 11 Vanillic acid 0.6959 0.2529 0.8066 12 Kaempferol 0.0000 0.1910 0.0689 13 p-Hydroxycinnamic acid 0.2073 0.0445 0.1232 14 catechins 0.0916 0.0018 0.0000

[0103] The results in Tables 1 to 5 show that compared with the traditional extraction method of alkali dissolution and acid precipitation (Comparative Example 1), the preparation of phenol-rich buckwheat protein using pH-driven combined proteolysis (Examples 1 and 2) can greatly improve the extraction rate of protein and polyphenols, wherein the protein extraction rate is increased from 62.33% to 82.61%, and the polyphenol extraction rate is increased from 59.2% to 93.34%. The total phenol content and solubility are also greatly improved, wherein the total phenol content is increased from 101.70 to 113.96 mg / g, and the solubility is increased from 65.41% to 85.70%. This is mainly due to the pH-driven treatment in the early stage of extraction, which can destroy the binding force between protein, polyphenol and cell wall, releasing some protein and polyphenol, and also provides an environment that is easier for proteolysis to contact the substrate. The subsequent proteolysis can not only hydrolyze the released protein, but also further release the protein and polyphenols in the buckwheat matrix that have not been released after the strong alkali treatment, further improving the extraction rate of protein and polyphenols. Furthermore, the pH-driven treatment loosens the protein structure, and the protease hydrolyzes the macromolecular protein into small peptides, significantly improving its solubility. In in vitro digestion experiments, the degree of proteolysis and the bioaccessibility of polyphenols were also significantly improved, with proteolysis increasing by 36.73% and polyphenol bioaccessibility by 26.77%. This is primarily due to the pH-driven combined proteolysis technique yielding a looser structure and lower molecular weight of tartary buckwheat protein, which facilitates the penetration and action of digestive enzymes, thereby significantly improving its digestibility. Furthermore, the method described in this technology significantly enhanced the antioxidant capacity of phenol-rich tartary buckwheat protein, with increases in DPPH free radical scavenging capacity by 78.14%, ABTS free radical scavenging capacity by 34.89%, and FRAP by 111.94%. This is primarily attributed to two factors: first, the pH-driven combined proteolysis technique avoids the prolonged strong alkaline environment required in traditional alkaline dissolution and acid precipitation extraction, while also promoting the release of polyphenols and retaining more phenolic compounds. As shown in Table 5, a total of 14 polyphenols were identified in the phenol-rich buckwheat protein obtained by the method of this technology, and the types and contents of these phenols were higher than those of the traditional method. On the other hand, proteolysis produced small-molecule polypeptides, and polypeptides with appropriate molecular weight usually have strong antioxidant capacity. These two reasons led to the use of this technology to obtain phenol-rich proteins with significantly improved antioxidant capacity.

[0104] As can be seen from the experimental data of Examples and Comparative Examples, on the basis of traditional alkali-soluble acid precipitation method, if only pH driving strategy is adopted, it is difficult to improve the extraction yield and the solubility of protein and polyphenols, more it is impossible to obtain the protein powder with high nutrient content. Although traditional alkaline protease enzymolysis method can be adopted to improve the performance of protein powder to a certain extent, the effect of its various aspects is still relatively poor, especially free radical scavenging ability, which will limit the application of the protein powder prepared. In addition, due to the sample obtained by alkaline protease hydrolysis, often do not possess good mouthfeel, and taste is bitter, see Table 4. Utilize composite protease hydrolysis to prepare protein powder, although gained sample mouthfeel improves to a certain extent, due to the suitable pH that composite protease plays a role is lower, cause protein extraction yield and polyphenol extraction yield to be often difficult to reach higher levels. In view of this, when preparing samples using the composite protease method, in addition to the initial pH-driven treatment to promote extraction, an innovative phased pH control strategy is introduced after the enzymatic hydrolysis is completed. This precisely adjusts the pH to a level that is moderately alkaline but not too high. While protecting the diversity of polyphenol species, it promotes the further dissolution of remaining proteins and polyphenols, thereby significantly improving the protein and polyphenol extraction rates, ultimately bringing them close to the extraction rate level achieved by the alkaline protease method. In addition, under the three effects of pH-driven, proteolysis, and phased pore control and pH control, it has significantly improved the total phenol content, solubility, antioxidant capacity, and polyphenol bioaccessibility, and the effect is better than that of alkaline protease.

[0105] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing phenol-rich buckwheat protein with high solubility and high bioaccessibility by pH-driven combined proteolysis, characterized in that: The steps include: (1) mixing defatted buckwheat flour and deionized water to form a suspension, and stirring to obtain a mixed system; (2) Using NaOH solution, adjust the pH of the mixed system in step (1) to 10-12, and mix and stir; (3) adjusting the pH value of the mixed system in step (2) to 7.5-9.5 using HCl solution, and adding protease to the suspension for enzymatic hydrolysis; (4) After the enzymatic hydrolysis is completed, centrifugation is performed to obtain the supernatant; (5) The pH of the supernatant of step (4) is adjusted to 6.8-7.2, dialyzed through a dialysis bag with a molecular weight cutoff of 100 Da to remove small molecular impurities, and then freeze-dried to obtain phenol-rich buckwheat protein powder.

2. The method according to claim 1, characterized in that The mass ratio of the defatted buckwheat flour to deionized water in step (1) is 1:8-1:12, and the stirring speed is 200-600 rpm.

3. The method according to claim 1, characterized in that The pH in step (2) is 10-12, and the stirring time is 0.5-1.5h.

4. The method according to claim 1, wherein The protease in step (3) is alkaline protease or composite protease.

5. The method according to claim 1, wherein When the protease is alkaline protease, the initial pH of the enzymatic hydrolysis is 8.5-9.5, the enzymatic hydrolysis temperature is 35-40° C., the enzymatic hydrolysis time is 3.5-4.5 hours, and the added amount of the enzyme is 500-2000 U / g of tartary buckwheat flour.

6. The method according to claim 1, characterized in that When the protease is a composite protease, the initial pH of the enzymolysis is 7.5-8.5, the enzymolysis temperature is 35-40°C, the enzymolysis time is 2.5-3.5 hours, the enzyme addition amount is 500-2000U / g buckwheat flour, and after the enzymolysis is completed, the pH is adjusted to 9.5-10.5, and the duration is 0.5-1.5 hours.

7. The method according to claim 1, characterized in that The centrifugal speed in step (4) is 8000-10000 rpm, and the centrifugal time is 10-15 min.

8. The method according to claim 1, characterized in that The dialysis treatment time in step (5) is 48-72 hours, the dialysate is replaced every 2-6 hours, and the drying method is vacuum freeze drying, and the drying time is 72 hours.

9. Phenol-rich buckwheat protein powder prepared according to the method according to any one of claims 1 to 8.

10. Use of the phenol-rich buckwheat protein powder according to claim 9 in the fields of functional foods, plant-based protein drinks, sports nutrition formulas and foods for special medical purposes.

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