Method for preparing polyphenol-protein compound from pecan deastringency wastewater

Abstract

The invention provides a method for preparing a polyphenol-protein compound from pecan deastringency wastewater, which comprises the following steps: adding an alkaline substance into the pecan deastringency wastewater, and adjusting the pH value to 8-13 to obtain alkali-treated deastringency wastewater; dissolving protein in an alkaline solution with the pH value of 8-13 to obtain a protein solution, adding the deastringency wastewater subjected to alkali treatment into the protein solution while stirring, and stirring and reacting for 30-90 minutes at the temperature of 20-60 DEG C; centrifuging the obtained reactant, taking supernate to obtain a polyphenol-protein compound solution, adding an acidic material into the polyphenol-protein compound solution under a stirring condition to adjust the pH value to 2.0-3.5, standing, precipitating, centrifuging, and collecting a precipitate to obtain a polyphenol-protein compound; according to the method disclosed by the invention, efficient recovery and enrichment of polyphenol substances in the pecan deastringency wastewater and high encapsulation efficiency and high load rate of polyphenol are realized, the process is stable, the repeatability is good, the stability and oxidation resistance of polyphenol are enhanced, and the method has a good application prospect in the fields of functional foods, health-care products and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high-value utilization of agricultural product by-products, and particularly relates to a method for recovering polyphenols from hickory debittering wastewater, and especially to a preparation method and application of polyphenol-protein complex with improved antioxidant capacity. BACKGROUND

[0002] Hickory is a deciduous tree of the Juglans genus in the Juglandaceae family, and is a unique high-economic-value woody oil tree species in China, mainly distributed in the subtropical region between Zhejiang Province and Anhui Province. Among the 18 kinds of walnut kernels in the world, the hickory kernel produced in Lin'an, Zhejiang Province, is known as "a gem among fruits" due to its thin shell, high content of unsaturated fatty acids, unique flavor and other characteristics, and is listed as a national geographical indication product, ranking among the "three treasures" of Lin'an (Zhao Yushun. Study on the Nutritional Composition Characteristics of Lin'an Hickory Products[D]. Zhejiang A&F University, 2013.). Studies have shown that hickory is rich in various bioactive components and has antioxidant, anticancer, anti-inflammatory and other effects (Anjum S, Gani A, Ahmad M, et al. Antioxidant and antiproliferative activity of walnut extract (Juglans regia L.) processed by different methods and identification of compounds using GC / MS and LC / MS technique[J]. Journal of Food Processing and Preservation, 2017, 41(1): 12756.). However, polyphenols represented by tannin interact with oral saliva proteins during consumption, causing a significant astringent sensation in the mouth and a bitter taste, thereby affecting the eating quality and consumer acceptance of hickory products. Therefore, hickory products generally need to be debittered during production and processing (Wu Shuidian, Xia Yue, Ma Yangyu, et al. Optimization of Tea-flavored Hickory Processing Technology and Study on Tea Polyphenol Coating Preservation Method[J]. Journal of Food Safety and Quality, 2024, 15(3): 256-264.). From the debittering mechanism, the debittering wastewater should contain a large amount of polyphenols, and the wastewater treatment will not only cause waste of bioactive components, but also increase the cost of wastewater treatment. Hickory debittering wastewater has not been effectively utilized, causing great resource waste and environmental pollution. Recovery of chemical components from debittering wastewater through protein recovery can provide valuable research data for the rational development and utilization of hickory resources.

[0003] Polyphenols are easily oxidized and unstable in free state, and may lead to reduced bioavailability if not protected. Therefore, the interaction between proteins and polyphenols is an important research field in functional food science. The interaction between polyphenols and proteins is mainly achieved through hydrogen bonds, hydrophobic interactions, and the formation of covalent bonds. The phenolic hydroxyl groups in polyphenol molecules can form hydrogen bonds with amino acid residues in proteins, while the hydrophobic part of polyphenols can interact with the hydrophobic region of proteins, promoting the formation of complexes. Under certain conditions, polyphenols can bind to proteins through covalent bonds to form stable complexes (Wang Y, Kang X, Tao Y, et al. Interaction mechanism between proteins and polyphenols and its application[J]. Food Science, 2024, 24(2): 357-372.). These interactions can change the secondary structure of proteins and affect their functional properties.

[0004] For example, soy protein is a high-quality plant protein source. The complex formed by the combination of soy protein and polyphenols can change the secondary structure, surface hydrophobicity, and thermal stability of proteins, and convert the solution state to a precipitate. In this process, polyphenol molecules promote protein cross-linking and aggregation through "bridging" action, while with increasing polyphenol concentration, the conjugation effect is enhanced, leading to reduced hydrophobicity, and finally forming a functional complex with in vitro digestibility, antioxidant, anti-inflammatory, and antibacterial properties (Manzoor MF, Zeng X A, Waseem M, et al. Soy protein-polyphenols conjugates interaction mechanism, characterization, techno-functional and biological properties: An updated review[J]. Food Chemistry, 2024, 460: 140571.).

[0005] A method for removing astringency from hickory nuts is disclosed in Chinese patent (CNCN119732472A). This method can effectively reduce the content of main astringency components such as condensed tannins, flavonoids, and phenolic compounds in the inner seed coat of hickory nuts, and maintain the original rich nutrition and unique flavor of hickory nuts. Chinese patent (CN119684404A) screened an antioxidant peptide with antioxidant activity from thin-shelled hickory nut protease hydrolysate. This thin-shelled hickory nut antioxidant peptide has the characteristics of safety, non-toxicity, good water solubility, and high biological activity. However, there are few reports on the recovery and application of antioxidant substances in hickory nut astringency removal wastewater.

[0006] However, none of the aforementioned existing technologies address the resource utilization of wastewater generated during the astringency removal process. Currently, there is a lack of effective solutions for the recovery, stabilization, and functional applications of polyphenols in pecan astringency removal wastewater.

[0007] This invention utilizes the deastringency-removing wastewater discarded during pecan processing. A polyphenol-protein complex is prepared using a pH adjustment method to recover polyphenols from the wastewater, a method not previously reported. This research aims to develop a technology for recovering polyphenol compounds from deastringency-removing wastewater, providing data and technical support for the comprehensive utilization of this wastewater and increasing the added value of the pecan industry. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing a simple and low-cost method for preparing polyphenol-protein complexes from pecan deastringency wastewater. This method can efficiently recover and stabilize polyphenols in the deastringency wastewater, and the resulting product has significant antioxidant activity, enabling high-value resource utilization of deastringency wastewater.

[0009] This invention provides a method for preparing polyphenol-protein complexes from pecan deastringency wastewater, the method comprising the following steps: (1) Add alkaline substances to the pecan deastringent wastewater containing 2-10 g / L (preferably 5 g / L) of total polyphenols and adjust the pH to 8-13 (preferably pH=10) to obtain the deastringent wastewater after alkali treatment; (2) Dissolve the protein in an alkaline solution with pH 8-13 (preferably pH=10) to obtain a protein solution with a concentration of 5-40 g / L (preferably 30 g / L); the protein is selected from sodium caseinate, soy protein isolate or whey protein; (3) Under stirring conditions, the deastringent wastewater after alkali treatment in step (1) is added dropwise to the protein solution in step (2), and stirred at 20-60℃ (preferably 30℃) (at 500 r / min) for 30-90 min (preferably 75 min); after the reaction is completed, the resulting reactants are centrifuged at 4-10℃ and 3000-5000 r / min for 5-20 min (preferably at 4℃ and 3500 r / min for 10 min), and the supernatant is taken to obtain a polyphenol-protein complex solution; the mass (g) to volume (L) ratio of the protein to the deastringent wastewater is 5:1 to 40:1 (preferably 30:1). (4) Under stirring conditions, add an acidic substance to the polyphenol-protein complex solution described in step (3) to adjust the pH to 2.0-3.5 (preferably 3.0), let it stand to precipitate, and then centrifuge at 4-10℃ and 10000-15000 r / min for 10-30 min (preferably at 4℃ and 12000 r / min for 15 min) to collect the precipitate, thus obtaining the polyphenol-protein complex.

[0010] Furthermore, the pecan deastringency wastewater in step (1) is the wastewater discarded after pecan kernels are soaked in water at 90-100℃ for 15-60 min and then filtered, and concentrated to contain 2-10 g / L of total polyphenols.

[0011] Furthermore, the protein concentration is 5-40 g / L (preferably 30 g / L).

[0012] Furthermore, the protein concentration in the protein solution described in step (2) is 30 g / L.

[0013] Furthermore, the pH of the alkaline solution in step (2) is 10.

[0014] Further, in step (3), the alkaline-treated deastringent wastewater is mixed with the protein solution under stirring conditions, and the mixture is stirred at 30°C for 75 min.

[0015] Furthermore, the acidic substance mentioned in step (4) is an aqueous solution of HCl.

[0016] Furthermore, the preparation method includes the following steps: (1) Alkaline substances were added to the pecan deastringency wastewater containing 2-10 g / L of total polyphenols to adjust the pH to pH=10 to obtain alkali-treated deastringency wastewater. Whey protein was dissolved in an alkaline solution with pH=10 to obtain a protein solution of 30 g / L. The alkali-treated deastringency wastewater was added dropwise to the protein solution under stirring conditions, and the mixture was stirred at 30°C and 500 r / min for 75 min. After the reaction was completed, the resulting reactants were centrifuged at 4°C and 3500 r / min for 10 min, and the supernatant was taken to obtain a polyphenol-protein complex solution. The mass (g) to volume (L) ratio of the whey protein to the deastringency wastewater was 30:1. (2) Under stirring conditions, add HCl to the polyphenol-protein complex solution described in step (1) to adjust the pH to 3.0, let it stand to precipitate, and then centrifuge at 4°C and 12000 r / min for 15 min. Collect the precipitate to obtain the polyphenol-protein complex.

[0017] The beneficial effects of this invention are as follows: 1) It has achieved efficient recovery and enrichment of polyphenols in the astringency removal wastewater of hickory, and transformed waste into high value-added products through polyphenol-protein complex technology, which has significantly improved the value of resource utilization. 2) The pH adjustment method was applied for the first time to the preparation of polyphenol-protein complex in pecan astringency removal wastewater, and a quantifiable evaluation system for encapsulation rate and loading rate was established, providing a reliable basis for process optimization; 3) By optimizing key process parameters, high encapsulation efficiency and high loading rate of polyphenols were achieved, and the process was stable and had good repeatability; 4) The prepared polyphenol-protein complex not only significantly enhances the stability of polyphenols, but also exhibits superior antioxidant capacity compared to polyphenols in the original desulfurization waste liquid, showing promising application prospects in functional foods, health products and other fields. Attached Figure Description

[0018] Figure 1 The images show the deastringent wastewater and the polyphenol-protein complex. From left to right, they are: deastringent wastewater, freeze-dried deastringent wastewater powder, supernatant obtained by centrifugation after precipitation of the polyphenol-protein complex, and the polyphenol-protein complex.

[0019] Figure 2 Ultra-high performance liquid chromatogram of pecan astringency removal wastewater.

[0020] Figure 3 This is an ultra-high performance liquid chromatogram of the supernatant obtained by centrifugation after precipitation of the polyphenol-protein complex.

[0021] Figure 4 The graph shows the comparison of the scavenging rates of DPPH free radicals by polyphenol-protein complex, freeze-dried deacidification wastewater powder, whey protein, and vitamin C.

[0022] Figure 5 The graph shows the comparison of the scavenging rates of ABTS free radicals by polyphenol-protein complex, freeze-dried deacidification wastewater powder, whey protein, and vitamin C.

[0023] Figure 6 The graph shows the comparison of the thermal stability of polyphenol-protein complex and freeze-dried powder of deacidification wastewater. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0025] Example 1: Preparation of polyphenol-protein complex

[0026] Take 5.0 mL of pecan deastringency wastewater containing 5 g / L total polyphenols, and adjust its pH to 10.0 with 1.0 M NaOH solution to obtain alkalized deastringency wastewater. Separately weigh 150.0 mg of whey protein, dissolve it in 5.0 mL of pH 10.0 NaOH aqueous solution, and stir magnetically until completely dissolved to obtain a protein solution.

[0027] Under magnetic stirring at 500 rpm, the above-mentioned alkalized deastringent wastewater was added dropwise to the protein solution (at this time, the mass (g) to volume (L) ratio of deastringent wastewater to protein was 30 g / L). The reaction was carried out at 30℃ for 75 min. After the reaction was completed, the solution was centrifuged at 4℃ and 3500 r / min for 10 min, the insoluble residue was discarded, and the supernatant was collected, which is the polyphenol-protein complex solution.

[0028] Add 1.0 M HCl solution dropwise to the above polyphenol-protein complex solution to adjust the pH to 3.0; a large amount of precipitate will be observed to form. The acid-precipitated mixture is centrifuged at 12000 r / min for 15 min at 4℃, and the precipitate is collected. The supernatant is used for qualitative comparative analysis of the polyphenol-encapsulated product. The precipitate is transferred to a 40℃ oven and dried for 2-8 h to obtain the product shown below. Figure 1 The dark brown polyphenol-protein complex shown weighed 135.8 mg. The total phenol content was determined using the Folin-Ciocalteu method, yielding an encapsulation efficiency of 90.2% and a loading rate of 40.7%.

[0029] Qualitative analysis of polyphenol encapsulation: The deastringent wastewater and the supernatant obtained by centrifugation after co-precipitation in Example 1 were diluted 10 times with pure water and detected by ultra-high performance liquid chromatography. The obtained spectra are shown below. Figure 2 and Figure 3 As shown. By Figure 2 and Figure 3 It is evident that polyphenols retained for more than 3 minutes were virtually undetectable in the supernatant after co-precipitation, indicating that these polyphenols were successfully encapsulated in the complex by whey protein.

[0030] Example 2: The mass (g) to volume (L) ratio of protein to deacidification wastewater was changed to 5:1, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 35.4 mg, an encapsulation efficiency of 77.3%, and a loading rate of 54.1%.

[0031] Example 3: The mass (g) to volume (L) ratio of protein to deacidification wastewater was changed to 10:1, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 44.4 mg, an encapsulation efficiency of 68.3%, and a loading rate of 23.6%.

[0032] Example 4: The mass (g) to volume (L) ratio of protein to deacidification wastewater was changed to 20:1, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 66.05 mg, an encapsulation efficiency of 86.2%, and a loading rate of 37.0%.

[0033] Example 5: The mass (g) to volume (L) ratio of protein to deacidification wastewater was changed to 40:1, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 183.60 mg, an encapsulation efficiency of 85.8%, and a loading rate of 27.4%.

[0034] Comparing Examples 1-5, it can be seen that when the mass (g) to volume (L) ratio of protein to deacidification wastewater is 30:1, the encapsulation efficiency and loading rate of the polyphenol-protein complex are the highest.

[0035] Example 6: The pH of the deastringency wastewater was changed to 8, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 111.2 mg, with an encapsulation rate of 81.5% and a loading rate of 44.3%.

[0036] Example 7: The pH of the deacidification wastewater was changed to 9, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 136.9 mg, with an encapsulation rate of 83.1% and a loading rate of 35.2%.

[0037] Example 8: The pH of the deacidification wastewater was changed to 11, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 126.6 mg, with an encapsulation rate of 79.7% and a loading rate of 34.0%.

[0038] Example 9: The pH of the deacidification wastewater was changed to 12, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 119.6 mg, with an encapsulation rate of 77.8% and a loading rate of 31.3%.

[0039] Example 10: The pH of the deacidification wastewater was changed to 13, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 96.6 mg, with an encapsulation rate of 83.7% and a loading rate of 42.0%.

[0040] Comparing Examples 1, 6-10, it can be seen that the encapsulation rate of the polyphenol-protein complex is the highest when the pH of the deacidification wastewater is 10.

[0041] Example 11: The reaction temperature of the alkalized deastringency wastewater dropwise with the protein solution was changed to 20°C, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 125.7 mg, an encapsulation rate of 87.8%, and a loading rate of 40.9%.

[0042] Example 12: The reaction temperature of the alkalized deastringency wastewater dropwise with the protein solution was changed to 40°C, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 128.7 mg, an encapsulation rate of 84.4%, and a loading rate of 38.7%.

[0043] Example 13: The reaction temperature of the alkalized deastringency wastewater dropwise with the protein solution was changed to 50°C, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 137.4 mg, an encapsulation rate of 84.0%, and a loading rate of 36.0%.

[0044] Example 14: The reaction temperature of the alkalized deastringency wastewater dropwise with the protein solution was changed to 60°C, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 128.7 mg, an encapsulation rate of 80.3%, and a loading rate of 40.0%.

[0045] Comparing Examples 1 and 11-14, it can be seen that the encapsulation efficiency of the polyphenol-protein complex is the highest when the reaction temperature is 30°C.

[0046] Example 15: The reaction time was changed to 30 minutes, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 99.8 mg, an encapsulation efficiency of 85.5%, and a loading rate of 49.4%.

[0047] Example 16: The reaction time was changed to 45 minutes, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 101.2 mg, with an encapsulation efficiency of 78.8% and a loading rate of 41.3%.

[0048] Example 17: The reaction time was changed to 60 minutes, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 120.9 mg, with an encapsulation efficiency of 88.2% and a loading rate of 33.3%.

[0049] Example 18: The reaction time was changed to 90 minutes, while other conditions and operating procedures remained the same as in Example 1. Analysis showed that the polyphenol-protein complex weighed 139.7 mg, with an encapsulation efficiency of 87.2% and a loading rate of 37.7%.

[0050] Comparing Examples 1 and 15-18, it can be seen that the encapsulation efficiency of the polyphenol-protein complex is the highest when the reaction time is 75 minutes.

[0051] Example 19: The protein was changed to sodium caseinate, while other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 57.9 mg, an encapsulation efficiency of 84.7%, and a loading rate of 88.2%.

[0052] Example 20: The protein was changed to soy protein isolate, and other conditions and operating procedures were the same as in Example 1. Analysis showed that the polyphenol-protein complex had a mass of 52.9 mg, an encapsulation efficiency of 72.0%, and a loading rate of 72.9%.

[0053] Comparing Examples 1 and 19-20, it can be seen that the encapsulation efficiency of the polyphenol-protein complex is the highest when the reactive protein is whey protein.

[0054] Example 21: Take 5.0 mL of deacidification wastewater containing 5 g / L of total polyphenols, weigh 150.0 mg of whey protein, and mix directly. React in a 30℃ constant temperature water bath for 75 min under continuous magnetic stirring (500 rpm). After the reaction, centrifuge the mixture at 3500 r / min for 10 min at 4℃, carefully collect the supernatant, and obtain the precipitate. The total phenol content was determined using the Folin-Ciocalteu method, and the encapsulation efficiency was found to be 55.8%.

[0055] Example 22: Take 5.0 mL of deacidification wastewater containing 5 g / L of total polyphenols, weigh 150.0 mg of whey protein, and mix directly. React in a 30℃ constant temperature water bath for 75 min under continuous magnetic stirring (500 rpm). After the reaction, centrifuge the mixture at 12000 r / min for 10 min at 4℃, carefully collect the supernatant, and obtain the precipitate. The total phenol content was determined using the Folin-Ciocalteu method, and the encapsulation efficiency was found to be 54.9%.

[0056] Comparing Examples 1, 21, and 22, it can be seen that when the deacidification wastewater is directly mixed and reacted with whey protein, the encapsulation rate of the polyphenol-protein complex is low and the encapsulation effect is poor; when the pH adjustment method is used for preparation, the encapsulation rate of the polyphenol-protein complex is higher.

[0057] Example 23: Take 5.0 mL of deastringent wastewater containing 5 g / L of total polyphenols, weigh 150.0 mg of whey protein, mix them directly, then adjust the pH to 10.0 with 1.0 M NaOH solution, and stir magnetically until completely dissolved to obtain an alkalized deastringent wastewater-protein solution.

[0058] The above-mentioned alkalized deastringent wastewater-protein solution was reacted in a 30℃ constant temperature water bath for 75 min under continuous magnetic stirring (500 rpm). After the reaction, the mixture was centrifuged at 3500 r / min for 10 min at 4℃, and a large amount of insoluble precipitate residue was observed. The supernatant was filtered through medium-speed filter paper, the insoluble precipitate residue was discarded, and the filtrate (i.e., the polyphenol-protein complex solution) was collected.

[0059] Other conditions and operating procedures were the same as in Example 1. The obtained polyphenol-protein complex had a mass of 147.2 mg, an encapsulation efficiency of 70.5%, and a loading rate of 24.5%.

[0060] Comparing Examples 1 and 23, it can be seen that when the deacidification wastewater and whey protein are directly mixed and then the pH is adjusted for reaction, the encapsulation rate and loading rate are both low. However, when the pH is adjusted separately for alkalization before mixing and reaction, the encapsulation rate and loading rate of the resulting polyphenol-protein complex are higher.

[0061] Example 24: The procedure was changed so that the protein solution was added dropwise to the alkalized deastringency wastewater, while other conditions and procedures remained the same as in Example 1. The obtained polyphenol-protein complex had a mass of 119.6 mg, an encapsulation efficiency of 77.8%, and a loading rate of 31.3%.

[0062] Comparing Examples 1 and 24, it can be seen that the order in which the protein solution and the deastringent wastewater are added affects the encapsulation efficiency and loading rate. When the protein solution is added dropwise to the alkalized deastringent wastewater, the encapsulation efficiency and loading rate of the polyphenol-protein complex are both lower than those obtained by the reverse addition method.

[0063] Example 25: Preparation of freeze-dried powder from pecan astringency removal wastewater

[0064] 5 mL of pecan deastringency wastewater was first frozen at -20°C and then transferred to a freeze dryer for vacuum freeze-drying for 72 h to obtain the following result: Figure 1 The brownish-yellow freeze-dried powder of deacidification wastewater shown was weighed to be 73.8 mg.

[0065] Compared with the preparation of freeze-dried powder from deacidified wastewater, the polyphenol-protein complex method significantly shortens the drying time and greatly reduces energy consumption.

[0066] Example 26: Determination of antioxidant activity of polyphenol-protein complex by DPPH free radical scavenging method

[0067] The polyphenol-protein complex prepared in Example 1 was dissolved in 200 μL of hydrochloric acid (pH=3). Using anhydrous ethanol as the solvent, polyphenol-protein complex solutions with concentrations of 800, 1000, 2000, 3000, 4000, and 5000 μg / mL were prepared in parallel for three groups. Separately, lyophilized deacidification wastewater powder was dissolved in 200 μL of hydrochloric acid solution (pH=3) using the same method. Using anhydrous ethanol as the solvent, a series of deacidification wastewater solutions with varying concentrations were prepared to ensure that the total phenol content was comparable to that of the complex group. Separately, whey protein was dissolved in 200 μL of hydrochloric acid solution (pH=3) using the same method. Using ultrapure water as the solvent, a series of whey protein solutions with varying concentrations were prepared to ensure that the protein content was comparable to that of the complex group. DPPH powder was accurately weighed, dissolved in anhydrous ethanol solution, and diluted to a 50 mL amber volumetric flask to prepare a 0.3 mM DPPH working solution.

[0068] The DPPH free radical scavenging experiment was set up with the following four parallel determinations: Sample group: 20 μL of polyphenol-protein complex solution, deacidification waste water and whey protein solution were respectively placed in 96-well plates, 180 μL of 0.3 mM DPPH solution was added, and the reaction was carried out at room temperature in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm. Positive control group: Vitamin C was used as the positive control. Anhydrous ethanol and water were used as solvents. A series of concentration solutions were prepared and measured according to the same method as above. Blank group: Replace the sample solution with 20 μL of anhydrous ethanol, add 180 μL of DPPH solution, react and determine in the same way; Control group: Take 20 μL of polyphenol-protein complex solution and deacidification waste water solution respectively, add 180 μL of anhydrous ethanol, react and measure in the same way, and use it to correct the absorbance of the sample itself.

[0069] Each experiment consisted of three replicates. The DPPH radical scavenging rate was calculated using the formula: DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] 100%.

[0070] Where A1 is the absorbance result measured in the sample group or positive control group; A2 is the absorbance of the control group; and A0 is the absorbance of the blank group.

[0071] The comparison results of the scavenging rates of DPPH free radicals by polyphenol-protein complex, deacidification wastewater, whey protein, and vitamin C are shown in the figure. Figure 4 .

[0072] Example 27: Determination of antioxidant activity of polyphenol-protein complex by ABTS free radical scavenging method.

[0073] Accurately weigh ABTS powder, dissolve it in ultrapure water, and dilute to a 10 mL amber volumetric flask to prepare a 7.4 mM ABTS solution. Separately, accurately weigh potassium persulfate powder and prepare a 2.6 mM potassium persulfate solution using the same method. Mix the two solutions at a 1:1 volume ratio and react at room temperature in the dark for 12–16 h to obtain an ABTS stock solution.

[0074] The polyphenol-protein complex prepared in Example 1 was dissolved in 200 μL of 1 mM sodium hydroxide solution (pH=11). Using ultrapure water as the solvent, polyphenol-protein complex solutions with concentrations of 100, 200, 250, 333.3, 400, and 500 μg / mL were prepared in parallel. Separately, lyophilized deacidification wastewater powder was added to 200 μL of sodium hydroxide solution (pH=11) using the same method, and a series of deacidification wastewater aqueous solutions of varying concentrations were prepared with ultrapure water, ensuring that the total phenol content was consistent with that of the complex group. Separately, whey protein was added to 200 μL of sodium hydroxide solution (pH=11) using the same method, and a series of whey protein ABTS sample solutions of varying concentrations were prepared with ultrapure water, ensuring that the protein content was comparable to that of the complex group. The ABTS stock solution was diluted to 0.37 mM with ultrapure water to serve as the ABTS working solution.

[0075] The ABTS free radical scavenging experiment was set up with the following four parallel determinations: Sample group: Take 20 μL of polyphenol-protein complex solution, deacidification waste water and whey protein solution into 96-well plate respectively, add 180 μL of 0.37 mM ABTS working solution, react at room temperature in the dark for 30 min, and measure the absorbance at 734 nm wavelength. Positive control group: Vitamin C was used as a positive control. A series of concentration solutions were prepared and measured using the same method as described above. Blank group: Take 20 μL of ultrapure water, add 180 μL of ABTS working solution, react and determine in the same way; Control group: Take 20 μL of polyphenol-protein complex solution, deacidification waste water and whey protein solution respectively, add 180 μL of ultrapure water, react and measure in the same way, and use it to correct the original color of the sample.

[0076] Each experiment was conducted in triplicate. The ABTS radical scavenging rate was calculated using the following formula: Clearance rate (%) = [1 - (A1 - A2) / A0] 100%.

[0077] Where A1 is the absorbance of the sample group or positive control group; A2 is the absorbance of the control group; and A0 is the absorbance of the blank group.

[0078] The comparison results of the scavenging rates of polyphenol-protein complex, lyophilized powder of deacidification wastewater, whey protein, and vitamin C on ABTS free radicals are shown in the figure. Figure 5 .

[0079] Example 28: Thermal stability experiment of polyphenol-protein complex.

[0080] Take 10 mg of the polyphenol-protein complex prepared in Example 1, dissolve it in 200 μL of 1 mM sodium hydroxide solution (pH=11), and dilute to 10 mL in a volumetric flask using ultrapure water to prepare a polyphenol-protein complex solution. Prepare three parallel groups. Separately, take the lyophilized powder of deacidification wastewater, add 200 μL of sodium hydroxide solution (pH=11) in the same manner, and prepare a deacidification wastewater aqueous solution with ultrapure water to ensure that its total phenol content is consistent with that of the complex group.

[0081] The constant temperature water bath was adjusted to 80℃, and the sample solution was placed inside. Samples were taken at 0, 15, 45, 60, 75, and 90 min. After cooling, the A content of the solution was measured using the Folin-Ciocalteu method. 760nm Calculate the retention rate.

[0082] Retention rate (%) = / C0 × 100%

[0083] in, C represents the total phenol mass concentration in the deastringent wastewater and polyphenol-protein complex at different heating time points; C0 represents the total phenol mass concentration in the deastringent wastewater and polyphenol-protein complex at 0 min of heating.

[0084] The comparison results of the thermal stability of polyphenol-protein complex and deacidification wastewater freeze-dried powder are shown in the figure. Figure 6 .Depend on Figure 6 It is evident that at 80℃, the thermal stability of the polyphenol-protein complex significantly improves over time compared to the freeze-dried powder of deacidification wastewater.

[0085] The above embodiments are only used to illustrate specific implementations of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A method for preparing polyphenol-protein complexes from pecan deastringency wastewater, characterized in that, The method is performed according to the following steps: (1) Take the pecan deastringency wastewater containing 2-10 g / L of total polyphenols, add alkaline substances to it, adjust the pH to 8-13, and obtain the deastringency wastewater after alkali treatment; (2) Dissolve the protein in an alkaline solution with pH 8-13 to obtain a protein solution with a concentration of 5-40 g / L; the protein is selected from plant protein or animal protein; (3) Under stirring conditions, the deastringent wastewater after alkali treatment in step (1) is added dropwise to the protein solution in step (2), and the mixture is stirred at 20-60℃ for 30-90 min. After the reaction is completed, the resulting reactants are centrifuged at 4-10℃ and 3000-5000 r / min for 5-20 min, and the supernatant is taken to obtain a polyphenol-protein complex solution. The mass (g) to volume (L) ratio of the protein to the deastringent wastewater is 5:1 to 40:

1. (4) Under stirring conditions, add an acidic substance to the polyphenol-protein complex solution described in step (3) to adjust the pH to 2.0-3.

5. After standing and precipitation, centrifuge at 4-10℃ and 10000-15000 r / min for 10-30 min, collect the precipitate, and obtain the polyphenol-protein complex.

2. The method as described in claim 1, characterized in that, The wastewater for removing astringency from pecans in step (1) comes from the wastewater discarded after soaking pecan kernels in water at 90-100℃ for 15-60 min and then filtering it, and then concentrating it to contain 2-10 g / L of total polyphenols.

3. The method as described in claim 1, characterized in that, The plant protein mentioned in step (2) is soy protein isolate, and the animal protein is sodium caseinate or whey protein.

4. The method as described in claim 1, characterized in that, The protein concentration in the protein solution described in step (2) is 30 g / L.

5. The method as described in claim 1, characterized in that, In step (3), the deastringent wastewater after alkali treatment is mixed with the protein solution under stirring conditions, and the mixture is stirred at 30°C for 75 min.

6. The method as described in claim 1, characterized in that, The pH of the alkaline solution in step (2) is 10.

7. The method as described in claim 1, characterized in that, The acidic substance mentioned in step (4) is an aqueous solution of HCl.

8. The method as described in claim 1, characterized in that, The preparation method includes the following steps: (1) Alkaline substances were added to the pecan deastringency wastewater containing 5 g / L of total polyphenols to adjust the pH to pH=10 to obtain alkali-treated deastringency wastewater. Whey protein was dissolved in an alkaline solution with pH=10 to obtain a protein solution of 30 g / L. The alkali-treated deastringency wastewater was added dropwise to the protein solution under stirring conditions, and the mixture was stirred at 30°C and 500 r / min for 75 min. After the reaction was completed, the resulting reactants were centrifuged at 4°C and 3500 r / min for 10 min, and the supernatant was taken to obtain a polyphenol-protein complex solution. The mass (g) to volume (L) ratio of the whey protein to the deastringency wastewater was 30:

1. (2) Under stirring conditions, add HCl to the polyphenol-protein complex solution described in step (1) to adjust the pH to 3.0, let it stand to precipitate, and then centrifuge at 4°C and 12000 r / min for 15 min. Collect the precipitate to obtain the polyphenol-protein complex.

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