A method for separating and decoloring protein of Idesia sanguinea Maxim. meal and Idesia sanguinea Maxim. meal decolorized protein powder prepared by the method

Abstract

The application discloses a kind of separation and decolorization method of Idesia polycarpa Maxim meal protein and Idesia polycarpa Maxim meal decolorized protein powder prepared by the method, belong to deep processing technical field.The application uses Idesia polycarpa Maxim meal as raw material, adopts salting-out method to separate Idesia polycarpa Maxim meal protein, and is decolorized by hydrogen peroxide under different pH conditions, systematically explores the influence of salting-out precipitation and hydrogen peroxide decolorization process on the physicochemical properties and functional characteristics of Idesia polycarpa Maxim meal protein, to obtain high-purity, light-colored Idesia polycarpa Maxim meal decolorized protein powder.The protein decolorization treatment of the application significantly improves the rigidity of protein structure, and promotes the recognition of enzyme and contact site, thereby improving the efficiency of pepsin enzymolysis.The obtained Idesia polycarpa Maxim meal decolorized protein powder has good inhibitory activity on alpha-amylase and alpha-glucosidase after continuous digestion in gastrointestinal tract, indicating that it has potential hypoglycemic biological function.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology, and in particular to a method for separating and decolorizing protein from *Vernicia fordii* seed meal, and the decolorized protein powder from *Vernicia fordii* seed meal obtained by this method. Background Technology

[0002] Mountain privet seed meal is a major byproduct of oil extraction from mountain privet fruit. Rich in nutrients such as protein, polyphenols, and polysaccharides, it is currently mainly used for animal feed and compost, with extremely low added value, and its protein resources have not been fully developed and utilized. However, mountain privet seed meal protein suffers from prominent problems such as low purity, poor functionality, and dark color, severely hindering its large-scale application. Therefore, it is urgent to explore the effects of different separation and decolorization methods on the physicochemical properties, functional characteristics, and in vitro digestibility of mountain privet seed meal protein. Previous studies have shown that existing methods still have certain shortcomings. For example, mountain privet seed meal protein obtained by acid precipitation has problems such as high polysaccharide content and dark color, severely limiting its further application.

[0003] Salting-out precipitation refers to the precipitation of proteins in a high-salt-concentration neutral salt solution by disrupting the hydration layer. Salting-out precipitation offers advantages such as ease of operation, low cost, and fractional extraction. It has a weaker impact on the higher-order structure of proteins than methods like isoelectric point precipitation, yielding relatively pure proteins. However, current research has not found studies on the separation of *Vernicia fordii* meal protein using salting-out precipitation, and the effects of different anion salting-out methods on the physicochemical and functional properties of *Vernicia fordii* meal protein remain unclear.

[0004] Decolorization techniques for plant proteins mainly include physical adsorption, chemical oxidation, organic solvent extraction, and biomodification to reduce dark complexes and improve color. However, due to the inherent differences in the pigment composition (such as phenols, flavonoids, and anthocyanins) of different plant raw materials and their binding modes with proteins (non-covalent interaction, covalent cross-linking, or physical encapsulation), the effectiveness and universality of decolorization methods cannot be simply predicted from existing protein systems or directly extended to another protein system. For example, the inventors' preliminary experimental results showed that the decolorization effect of using activated carbon and macroporous resin to decolorize *Vernicia fordii* meal protein was generally poor. Moreover, taking the hydrogen peroxide decolorization method as an example, there are few studies on the reprecipitation of proteins after using hydrogen peroxide to decolorize the extract under different pH conditions, and the impact of the decolorization process on the physicochemical properties, functional characteristics, and digestibility of proteins remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a method for separating and decolorizing protein from *Vernicia fordii* seed meal, and a decolorized protein powder from *Vernicia fordii* seed meal obtained by this method, so as to solve the above-mentioned problems in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a method for separating and decolorizing protein from *Vernicia fordii* seed meal, comprising the following steps: (1) Mix the powder of tung oil seed meal with water, treat it with alkali under alkaline conditions, and then separate the solid and liquid phases to collect the liquid phase components; (2) Add hydrogen peroxide solution to the liquid phase component to obtain the decolorizing solution, and perform decolorization treatment to obtain the decolorized solution; (3) Adjust the pH of the decolorizing solution to neutral, add salting-out agent to obtain the solution to be salted out, precipitate, then add water to dissolve the precipitate, dialyze, and obtain the decolorized protein powder of tung oil seed meal.

[0007] Preferably, in step (1), the ratio of the amount of *Vernicia fordii* seed meal powder to water is 1g:20mL.

[0008] Preferably, the pH value of the alkali treatment is 12.

[0009] Preferably, the alkali treatment is performed at a temperature of 50 °C for 2 h.

[0010] Preferably, in step (2): the concentration of the hydrogen peroxide solution is 4 vol%; the volume ratio of the liquid phase component to the hydrogen peroxide solution is 180:7.2.

[0011] Preferably, before the decolorization treatment, the step further includes adjusting the pH value of the solution to be decolorized to 3-11.

[0012] Preferably, the decolorization treatment is performed at a temperature of 45 °C for 45 min.

[0013] Preferably, adjusting the pH value to neutral means adjusting it to 7.00.

[0014] Preferably, the salting-out agent is ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate, or potassium citrate; the concentration of the salting-out agent in the solution to be salted out is 45 vol.

[0015] Preferably, the salting-out agent is potassium citrate.

[0016] Preferably, the precipitation treatment is performed at a temperature of 4 °C for 2 h.

[0017] Preferably, the dialysis is performed for 2 days using a 3500 Da dialysis bag.

[0018] The second technical solution of the present invention provides a decolorized protein powder of *Vernicia fordii* seed meal prepared according to the above separation and decolorization method.

[0019] The beneficial technical effects of the present invention are as follows: This invention uses *Vernicia fordii* seed meal as raw material, employs salting-out to separate protein from the meal, and then uses hydrogen peroxide for decolorization under different pH conditions (3, 5, 7, 9, 11). The effects of salting-out precipitation and hydrogen peroxide decolorization on the physicochemical properties and functional characteristics of *Vernicia fordii* seed meal protein were systematically investigated to obtain high-purity, light-colored decolorized protein powder. Finally, an in vitro simulated gastrointestinal digestion experiment was conducted to explore the digestibility of the decolorized protein, as well as the peptide profile and biological activity of the products from continuous in vitro simulated gastrointestinal digestion.

[0020] This invention investigates the effects of various salting-out agents at different saturations on the basic composition of *Vernicia fordii* meal protein, screening out ammonium sulfate (NHS group), sodium sulfate (NS group), dipotassium hydrogen phosphate (KP group), and potassium citrate (KN group) and their optimal saturations. Using an acid precipitation group (JS group) as a control, the effects of salting-out on the physicochemical and functional properties of the protein were systematically investigated. Results showed that salting-out treatment significantly improved protein purity; SDS-PAGE electrophoresis indicated that the molecular weight of *Vernicia fordii* meal protein was mainly distributed in the 35-40 kDa, 15-25 kDa, and less than 10 kDa ranges. FTIR analysis showed that salting-out promoted the transformation of the protein's secondary structure from α-helix to β-sheet. Among them, the KN group protein exhibited the lowest particle size and surface hydrophobicity, while reaching the highest levels of solubility, foaming activity, foaming stability, and emulsifying activity. Principal component analysis and thermogram analysis further confirmed that the KN group protein exhibited the best physicochemical and functional properties.

[0021] Under different pH conditions, hydrogen peroxide was used to decolorize the alkaline supernatant of *Vernicia fordii* meal protein, and 45% saturated potassium citrate was used to precipitate the protein in the decolorized extract. The undecolorized group was used as a control to investigate the effects of decolorization treatment on the physicochemical and functional properties of *Vernicia fordii* meal protein. The results showed that hydrogen peroxide decolorization treatment significantly improved the purity and whiteness of the protein. Although decolorization treatment disrupted the ordered spatial structure of the protein, it did not change its molecular weight distribution. Under different pH conditions, the particle size and solubility of the decolorized protein, from largest to smallest, were: KN-11>KN-9>KN-7>KN-3>KN-5>KN, while surface hydrophobicity, enthalpy, and interfacial wettability showed the opposite order. Molecular flexibility, emulsifying activity, and emulsifying stability, from largest to smallest, were: KN-11>KN-9>KN-7>KN-5>KN-3>KN. This study provides a theoretical basis for the separation and decolorization process of protein from *Vernicia fordii* meal.

[0022] In vitro simulated gastrointestinal digestion experiments were conducted on proteins prepared by hydrogen peroxide decolorization treatment under different pH conditions to analyze the differences in digestive characteristics, peptide profiles of continuous gastrointestinal digestion products, and biological activities among the protein groups. The results showed that decolorization treatment significantly improved the degree of protein hydrolysis and digestibility. After continuous gastrointestinal digestion, the decolorized protein produced a higher number of peptides, a higher proportion of low-molecular-weight peptides, and a higher variety and number of terminal amino acids compared to the undecolorized group, suggesting that these differences may be related to changes in protein structure caused by decolorization treatment. Furthermore, the decolorized protein powder obtained from *Vernicia fordii* seed meal exhibited good inhibitory activity against both α-amylase and α-glucosidase after continuous gastrointestinal digestion, indicating its potential hypoglycemic biological function.

[0023] The protein decolorization treatment of this invention significantly improves the rigid structure of proteins and promotes enzyme recognition at contact sites, thereby enhancing the efficiency of pepsin digestion. The decolorized proteins exhibit high digestibility, promote the generation of small peptides and free amino acids, and the peptide sequences of the decolorized group are rich in Serine (serine) and Arg (arginine) at both ends, resulting in a high α-glucosidase inhibition rate. Arg in the continuous gastrointestinal digestion products of *Vernicia fordii* meal protein of this invention may play an important role in α-amylase inhibition. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a statistical analysis of the basic components of the product obtained from the treatment group where the salting-out agent was ammonium sulfate in Example 1. Wherein, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0026] Figure 2 The basic component content statistics of the products obtained from the sodium sulfate-ammonium sulfate treatment group in Example 1 are shown. Among them, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0027] Figure 3 This is a statistical analysis of the basic components of the product obtained from the group treated with potassium sulfate as the salting-out agent in Example 1. Wherein, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0028] Figure 4 This is a statistical analysis of the basic components of the products obtained from the group treated with magnesium sulfate as the salting-out agent in Example 1. Wherein, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0029] Figure 5 This is a statistical analysis of the basic components of the products obtained from the treatment group where dipotassium hydrogen phosphate was used as the salting-out agent in Example 1. Wherein, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0030] Figure 6 This is a statistical analysis of the basic components of the product obtained from the treatment group where potassium citrate was used as the salting-out agent in Example 1. Wherein, a) is protein, b) is polysaccharide, c) is polyphenol, and d) is ash.

[0031] Figure 7 Fourier transform infrared (a) and FTIR curves (b) of protein from *Vernicia fordii* meal treated with different optimal concentrations of salting-out agent in Example 1.

[0032] Figure 8 Principal component analysis (a) and thermogram analysis (b) of protein from *Vernicia fordii* meal treated with different optimal concentrations of salting-out agent in Example 1.

[0033] Figure 9 This is Example 2, showing the effect of different decolorization treatments on the content of basic components of the product. Wherein, a) represents protein, b) polysaccharide, c) polyphenol, and d) ash.

[0034] Figure 10 This illustrates the effect of decolorization treatment under different pH conditions on the color value of the product in Example 2. Where a) represents L... * Value, b) is a * Value, c) is b * Value, d) is WI * value.

[0035] Figure 11 The images show actual products after decolorization treatment under different pH conditions in Example 2.

[0036] Figure 12 This illustrates the effect of decolorization treatment under different pH conditions on the microstructure of proteins in Example 2. Data for KN, KN-3, KN-5, KN-7, KN-9, and KN-11 are shown in a(a), b), c), d), e), and f), respectively.

[0037] Figure 13 The effect of decolorization treatment under different pH conditions on the degree of protein hydrolysis in Example 2.

[0038] Figure 14 The effect of decolorization treatment under different pH conditions on protein digestibility in Example 2.

[0039] Figure 15 This is a statistical analysis of the molecular weight distribution of the products from the continuous gastrointestinal digestion of each product in Example 2 after digestion experiments.

[0040] Figure 16 a), b), c), d), e), and f) are the terminal amino acid analyses of the products of KN, KN-3, KN-5, KN-7, KN-9, and KN-11 groups after continuous digestion in the gastrointestinal tract, respectively.

[0041] Figure 17 This is a statistical analysis of the DPPH free radical scavenging capacity of the products from the continuous gastrointestinal digestion of each product in Example 2 after digestion tests.

[0042] Figure 18 This is a statistical analysis of the α-glucosidase inhibition rate of the products from continuous gastrointestinal digestion after digestion tests of the products in Example 2.

[0043] Figure 19 This is a statistical analysis of the α-amylase inhibition rate of the products from the continuous gastrointestinal digestion of each product in Example 2 after digestion tests. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0045] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. It should be noted that any aspects of this invention not described in detail are conventional practices in the art and are not the focus of this invention.

[0047] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0048] Degree of hydrolysis and digestibility are indicators of protein hydrolysis sensitivity, directly determining the body's utilization of amino acids. Studies have shown that protein structural modifications, especially changes in secondary structure and aggregate formation, may alter enzyme bioavailability and cleavage sites, affecting protein digestion and leading to decreased degree of hydrolysis and digestibility. Current research has not found in vitro digestion studies of *Vernicia fordii* meal protein decolorized with hydrogen peroxide at different pH levels.

[0049] The main raw materials and reagents used in this invention are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0050] Table 1 Main Raw Materials and Reagents Table 2 Main Instruments and Equipment The basic composition of the high-temperature pressed tung oil meal used in this invention is: crude protein (10.94±0.05%), total sugar (32.4±1.83%), moisture (3.95±0.13%), fat (11.55±0.32%), and total phenols (8.34±0.04%).

[0051] Preparation of defatted ball-milled powder from *Vernicia fordii* seed meal: High-temperature pressed *Vernicia fordii* seed meal was pulverized for 1 min using an ultrafine pulverizer, repeated twice. It was then sieved through a 60-mesh sieve. The sieved *Vernicia fordii* seed meal powder was defatted in hexane using a magnetic stirrer for 2 h, and then air-dried overnight. Subsequently, the defatted meal was ground with ball milling beads (stainless steel, 6 mm in diameter) using a planetary gear ball mill. The parameters were as follows: frequency 40 Hz; total volume of meal and ball milling beads not exceeding 2 / 3 of the ball mill jar volume; mass ratio of meal to ball milling beads 1:8; ball milling for 6 h. The collected powder was the defatted ball-milled powder of *Vernicia fordii* seed meal.

[0052] Unless otherwise specified, room temperature in this invention is 20±10℃. Overnight stays are 12 hours unless otherwise specified.

[0053] All raw materials used in the following embodiments and comparative examples of the present invention are commercially available products.

[0054] The technical solution of the present invention will be further illustrated by the following embodiments.

[0055] Example 1 (Salting out of protein from tung oil seed meal) Defatted powder of *Vernicia fordii* meal was ball-milled and uniformly dispersed in deionized water at a ratio of 1:20 (w / v). The pH was adjusted to 12.00 using 5M NaOH, and the mixture was treated with alkali in a 50 ℃ water bath with stirring for 2 h. Subsequently, the mixture was centrifuged twice to completely remove insoluble matter (4000 rpm, 15 min), and the supernatant was collected. The pH of the supernatant was adjusted to 7.00 using 5M HCl solution, and then a salting-out agent (type and mass concentration shown in Table 3) was added. The mixture was allowed to stand at 4 ℃ for 2 h to precipitate, and then centrifuged at 10000 rpm for 20 min to collect the precipitate. The precipitate was dissolved in deionized water, dialyzed using a 3500 Da dialysis bag for 2 days, and finally freeze-dried to obtain *Vernicia fordii* meal protein powder.

[0056] Table 3 Different salting-out agents and their corresponding saturation Comparative Example 1 (Acid Precipitation Group JS) Defatted powder of *Vernicia fordii* meal was ball-milled and uniformly dispersed in deionized water at a ratio of 1:20 (w / v). The pH was adjusted to 12.00 using 5M NaOH, and the mixture was treated with alkali in a 50 ℃ water bath with stirring for 2 h. Subsequently, the mixture was centrifuged twice (4000 rpm, 15 min) to completely remove insoluble matter, and the supernatant was collected. The pH of the supernatant was adjusted to 3.40 using 5M HCl solution, and the mixture was allowed to stand for acid precipitation for 30 min, followed by centrifugation at 10000 rpm for 20 min to collect the precipitate. The precipitate was dissolved in deionized water, dialyzed using a 3500 Da dialysis bag for 2 days, and finally freeze-dried to obtain *Vernicia fordii* meal protein powder.

[0057] Example 2 (Effects of hydrogen peroxide decolorization treatment under different pH conditions on the physicochemical and functional properties of tung oil seed meal protein) The supernatant from Example 1 was taken, and the pH was adjusted to 3, 5, 7, 9, and 11 respectively. Then, 4 vol% hydrogen peroxide solution was added. The solution was placed in a water bath at 45 ℃ and magnetically stirred for decolorization for 45 min. Afterward, the pH of the decolorized solution was uniformly adjusted to 7.00, and potassium citrate was added as a salting-out agent to obtain the salting-out solution. The saturation of the salting-out solution was made to 45 vol%, and the solution was allowed to stand at 4 ℃ for 2 h to precipitate. The precipitate was collected by centrifugation at 10000 rpm for 20 min. Deionized water was added to dissolve the precipitate, followed by dialyzing using a 3500 Da dialysis bag for 2 days. Finally, the solution was freeze-dried to obtain decolorized protein powder from *Vernicia fordii* meal (the products were sequentially designated as KN-3, KN-5, KN-7, KN-9, and KN-11 depending on the adjusted pH).

[0058] Comparative Example 2 The only difference from Example 2 is that the pH value of the supernatant in Example 1 was adjusted to 7, the addition of hydrogen peroxide solution was omitted, and an equal volume of deionized water was added (the resulting product is denoted as KN).

[0059] Effect verification I. Testing Methods 1. Testing of physicochemical properties 1.1 Determination of basic components (1) Total sugar: Accurately prepare a 0.1 mg / mL glucose standard solution, and successively add 0.00, 0.02, 0.03, 0.04, 0.05, 0.06, and 0.07 mL of glucose standard solution to make up to 1 mL using deionized water. Then, add an equal volume of 5% phenol solution and 5 times the volume of concentrated sulfuric acid solution. After cooling to room temperature, measure the absorbance value of the solution at 490 nm. Plot a standard curve with the glucose standard solution concentration as the x-axis and the absorbance value as the y-axis. Prepare a solution of the sample to be tested using deionized water and measure the absorbance. Substitute the absorbance into the standard curve to obtain the polysaccharide concentration. (2) Ash: Refer to the 550 ℃ baking method in GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food". (3) Protein: Refer to the combustion method in GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food". (4) Polyphenols: The determination shall be made in accordance with the method of GB / T 44349-2024 "Determination of total polyphenols in bee pollen by Folin-Ciocalteu reagent colorimetric method".

[0060] The polyphenol removal rates of Example 2 and Comparative Example 2 were determined according to the following calculation formula: .

[0061] 1.2 Fourier Transform Infrared Spectroscopy (FTIR) Analysis The *Vernicia fordii* meal protein powder obtained in each example and comparative example was mixed with potassium bromide (KBr) at a ratio of 1:100, and the mixture was ground into powder and then compressed into tablets. Subsequently, the tablets were analyzed using a Nicolet iS50 spectrometer at 4000–400 cm⁻¹. -1 Within the wavelength range of 4 cm -1 The infrared spectrum of protein from *Vernicia fordii* meal was detected at high resolution. The amide I band (1700–1600 cm⁻¹) was selected using OMNIC and Peakfit software. -1 The secondary structure information of the protein in *Vernicia fordii* meal was obtained by fitting, and the content of secondary structure was calculated.

[0062] 1.3 Intrinsic Fluorescence Spectroscopy Analysis The protein from *Vernicia fordii* seed meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 1 mg / mL protein solution, which was then stirred with a magnetic stirrer to promote dissolution. 2 mL of the protein solution was pipetted into a 1.0 cm fluorescence cuvette. Endogenous fluorescence was measured at 298 K with the following parameters: excitation wavelength 280 nm, emission wavelength 300–470 nm, excitation and emission slit widths of 2.5 and 5 nm, respectively, scan rate 240 nm / s, and operating voltage 650 V.

[0063] 1.4 Scanning electron microscopy observation Freeze-dried tung oil protein was attached to a carbon sheet on a cylindrical aluminum base. The sample was then sputter-coated with gold and the microstructure was observed under a scanning electron microscope at magnifications of 500x and 2000x, with a voltage of 5.00 kV.

[0064] 1.5 SDS-PAGE gel electrophoresis Prepare a 2 mg / mL protein solution and mix it with SDS-PAGE loading buffer at a ratio of 4:1 (v / v). Heat the mixture in a boiling water bath for 5 min. Then, centrifuge at 10,000 rpm for 5 min and collect the supernatant as the test solution. Place the pre-cast gel in an electrophoresis tank, add electrophoresis buffer, and use a syringe to add 7 μL of the test solution and indicator marker (10-180 kDa) sequentially to the wells. Cap the wells. The initial voltage is 120 V. When the gel reaches 1 / 3 of its volume, switch to a constant voltage of 150 V and continue electrophoresis until bromophenol blue migrates to the bottom of the gel. After electrophoresis, remove the gel. First, fix it with fixative for 30 min and discard the fixative. Then, stain with Coomassie R-250 for 30 min and discard the staining solution. Finally, destain the gel multiple times on a shaker until the gel background becomes transparent. Observe and photograph the protein bands using a gel imaging system.

[0065] 1.6 particle size The sample was thoroughly dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00), and the protein particle size was determined using a laser particle size analyzer. Before measurement, the protein sample was diluted to 2 mg / ml using the same buffer. The protein solution was slowly added dropwise into the sample cell, with the refractive index set to 1.763 and the refractive index set to 1.33. The test temperature was 25 °C, and the sample particle size was measured.

[0066] 1.7 Surface hydrophobicity The surface hydrophobicity of *Vernicia fordii* meal protein was determined using the bromophenol blue binding method (BPB). *Vernicia fordii* meal protein was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 1 mg / mL protein solution, which was then stirred with a magnetic stirrer to promote dissolution. 2 mL of the protein solution was then bound with 200 μL of BPB (1 mg / mL), and vortexed for 15 min. Subsequently, the solution was centrifuged at 3000 rpm for 15 min at 4 °C, and the supernatant was collected and diluted 10-fold with PBS buffer. The absorbance at 595 nm was measured using a UV spectrophotometer and recorded as A1. PBS was used as a blank control and recorded as A0. The formula for calculating the surface hydrophobicity of the protein is as follows: .

[0067] 1.8 Solubility Protein from *Vernicia fordii* meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 10 mg / mL protein solution. The solution was stirred with a magnetic stirrer to promote dissolution. Subsequently, the solution was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. The protein content in the supernatant was determined using Coomassie Brilliant Blue. The protein solution was mixed with Coomassie Brilliant Blue at a 1:4 volume ratio, vortexed, and allowed to stand for 5 min. The absorbance of the reaction solution at 595 nm was measured. PBS buffer alone was used as a blank control. The protein content in the sample was determined using a Dumas nitrogen analyzer. The solubility calculation formula is as follows: .

[0068] 1.9 Foaming Activity and Foam Stability Protein from *Vernicia fordii* meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 10 mg / mL protein solution, which was then stirred with a magnetic stirrer to promote dissolution. First, 10 mL of the sample was placed in a 50 mL graduated cylinder, and the initial volume V (mL) was recorded. Then, the sample was dispersed at 10,000 rpm for 2 min using a high-speed homogenizer to form foam. After dispersion, the total sample volume V1 (mL) was quickly recorded. Finally, the volume of the foamed sample after standing for 30 min was recorded as V2 (mL). The formulas for calculating foaming ability (FA) and foam stability (FS) are as follows: ; .

[0069] 1.10 Emulsifying activity and emulsifying stability The protein from *Vernicia fordii* meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 10 mg / mL protein solution, which was then stirred with a magnetic stirrer to promote dissolution. 12 mL of the *Vernicia fordii* meal protein solution was mixed with 4 mL of soybean oil and emulsified at 10,000 rpm for 2 min using a high-speed homogenizer. Immediately after the initial emulsion was prepared, 50 μL of sample was aspirated from the bottom of the solution and transferred to 5 mL of 0.1% SDS solution for dilution. Using the 0.1% SDS solution as a blank control, the absorbance value A0 of the solution at 500 nm was measured. After the sample was allowed to stand for 10 min, A0 was measured using the same method. 10 The formulas for calculating emulsifying activity (EAI) and emulsifying stability (ESI) are as follows: ; .

[0070] Where N is the dilution factor; φ is the oil phase ratio; and ω is the concentration of the protein solution (g / mL). 1.11 Principal Component Analysis Principal component analysis was performed on the physicochemical properties (protein content, solubility, particle size, foaming activity, foaming stability, emulsifying activity, and emulsifying stability) of cinnamon seed meal protein precipitated with different precipitants. After data normalization using SPSS statistics, loading plots and score plots were generated using Origin, and heatmaps were generated using Prism to screen for the optimal precipitant for cinnamon seed meal protein.

[0071] 1.12 Color difference measurement and whiteness calculation The color of *Vernicia fordii* meal protein was measured using a CR-400 colorimeter, which was calibrated before each use. The L value of the *Vernicia fordii* meal protein samples was recorded. * (Brightness), a * (Redness), b * (Yellowness), and calculate the WI (Whiteness Index) of the sample. WI * The calculation formula is as follows: .

[0072] 1.13 Ultraviolet-Visible Spectral Scan Protein from *Vernicia fordii* seed meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 0.05 mg / mL protein solution. The solution was stirred with a magnetic stirrer to promote dissolution. 1 mL of the protein solution was transferred to a cuvette, and the sample was scanned using a UV-Vis spectrophotometer in the wavelength range of 200–400 nm, with data intervals of 1 nm and a moderate scan rate. 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) was used as background subtraction.

[0073] 1.14 Molecular flexibility Protein from *Vernicia fordii* seed meal was dissolved in 10 mmol / L phosphate-buffered saline (PBS, pH 7.00) to prepare a 1 mg / mL protein solution, and stirred with a magnetic stirrer to promote dissolution. Separately, trypsin was dissolved in 0.05 M Tris-HCl buffer to prepare a 1 mg / mL trypsin solution. 4 mL of the protein solution was mixed with 250 μL of the trypsin solution and incubated at 37 °C for 10 min. Subsequently, 4 mL of TCA solution (50 mg / mL) was added, mixed thoroughly, and centrifuged at 4000 rpm for 10 min to collect the supernatant. The absorbance of the supernatant was measured at 280 nm.

[0074] 1.15 DSC Analysis Take 3 mg of tung oil seed meal protein powder, place it in an aluminum crucible and test it on the instrument. The scanning temperature is 20-180 ℃, the heating rate is 5 ℃ / min, and the test is carried out in a helium environment. Then, the maximum denaturation temperature is analyzed using Ver1.2N software.

[0075] 1.16 Interface wettability The interfacial wettability of two incompatible substances is represented by the contact angle. The lyophilized protein powder was compressed into tablets with a smooth, flat surface free of noticeable protrusions and cracks. 5 μL of deionized water was dropped onto the tablet surface using a microsyringe, and the contact angle of the sample was determined using the hanging drop method with video optical angles. The hanging drop pattern of the sample was recorded using a camera.

[0076] 1.17 Amino acid composition analysis A certain amount of solid powder was weighed and placed in a hydrolysis tube. 10 mL of 6 M hydrochloric acid solution was added, and nitrogen blowing was applied for 30 min to displace air from the solution. After nitrogen blowing, the hydrolysis tube stopper was tightened, placed on a high-temperature resistant test tube rack, and placed in a 110 ℃ oven for 22 h. After 22 h, the hydrolysis tube was removed, cooled to room temperature, and diluted to 50 mL with deionized water. This was then filtered into a 10 mL centrifuge tube. 1 mL of this solution was then transferred to another new 10 mL centrifuge tube and dried again using a nitrogen blowing apparatus. The dried amino acids were reconstituted with 0.02 N hydrochloric acid and filtered through a 0.22 μm filter membrane into a liquid chromatography sample bottle. The amino acid content in the protein was determined using an automated amino acid analyzer.

[0077] 2. Tests on digestive properties The digestion system was established as follows: an in vitro digestion model experiment was conducted, and the electrolyte solution preparation is shown in Table 4. The operation steps are as follows: Prepare a decolorized protein powder solution with a concentration of 10 mg / mL as in Example 2 and Comparative Example 2. Take 10 mL of the decolorized protein powder solution and add an equal volume of gastric electrolyte solution (simulating gastric digestive fluid). Adjust the pH to 2.00 using 1.0 mol / L HCl. Then, add pepsin (2000 U / mg, based on protein content) and react in a shaker for 2 h at 37 ℃ and 100 rpm. Divide the solution into two parts. Boil one part for 5 min to stop digestion and store at -20 ℃. Mix the other part with an equal volume of intestinal electrolyte solution (simulating intestinal digestive fluid) and adjust the pH to 7.00 using 1.0 M NaOH. Then, add trypsin (250 U / mg, based on protein content) and bile salt (1.6 mg / mL) and react in a shaker for 2 h at 37 ℃ and 100 rpm. After the digestion was completed, the solution was boiled for 5 minutes to stop the digestion and stored at -20 °C to obtain the peptide components after in vitro digestion.

[0078] Table 4 Preparation of Electrolyte Solutions 2.1 Degree of in vitro hydrolysis Prepare an L-leucine standard solution and construct a standard curve. Take 200 μL of the sample solution and mix thoroughly with 1.5 mL of OPA reagent. Incubate at room temperature in the dark for 3 min. Measure the absorbance of the reaction solution at 340 nm. Substitute the absorbance values ​​into the standard curve to obtain the amino content. The formula for calculating the degree of hydrolysis is as follows: .

[0079] 2.2 Determination of digestibility Five mL of undigested *Vernicia fordii* seed meal protein solution (concentration 10 mg / mL, the protein type used was decolorized protein powder prepared at different pH values ​​in Example 2) was taken as a control. Five mL of simulated gastric digestion fluid and simulated intestinal digestion fluid prepared according to "2. Tests on digestive characteristics" were also taken directly for testing. An equal volume of 10% trichloroacetic acid (TCA) was added, mixed well, and centrifuged at 8000 rpm for 15 min. The supernatant was collected. The protein content in the supernatant was determined using a BCA kit. The in vitro digestibility calculation formula is as follows: .

[0080] Note that the five groups of decolorized protein powder with pH values ​​of 3, 5, 7, 9, and 11 were all subjected to in vitro digestion tests. The undigested protein solution of each group was used as a control to determine the differences in the in vitro digestibility of the decolorized protein in each group.

[0081] 2.3 Peptidomics analysis of digestion products DDA analysis was performed on the peptide fractions after in vitro digestion for further peptide analysis. Peptides were separated using a homemade column (15 cm × 100 μm, 1.7 μm) with a Vanquish Neo (Thermo, USA). Mobile phases A and B consisted of 0.1% formic acid, 2% acetonitrile, and 80% acetonitrile, respectively. Gradient elution processes included 0–1 min (8% B, 1 μL / min), 1–5.5 min (17% B, 1 μL / min), 5.5–7 min (55% B, 0.4 μL / min), and 7–8 min (99% B, 0.7 μL / min). Information acquisition and mass spectrometry analysis were performed using an Astral (Thermo, USA) and a Thermo Xcalibur 4.7 (Thermo, USA) mass spectrometer. Parameters were as follows: Mass spectrometry primary scan range: 380–980 m / z.

[0082] Secondary scan range: 150-2000 m / z, acquisition mode: DDA; Top 100 (selecting the 100 strongest signals from the precursor ions for secondary fragmentation); Primary mass spectrometry resolution: 240000, AGC target: 500%, maximum injection time: 3 ms, fragmentation mode: HCD; Secondary resolution: 80000-100000, AGC target: standard, maximum injection time: 10 ms, dynamic exclusion time: 12 s.

[0083] 2.4 DPPH free radical scavenging rate A 1 mg / mL protein solution was prepared from lyophilized *Vernicia fordii* seed meal and stirred with a magnetic stirrer to promote dissolution. 0.5 mL of the solution was mixed thoroughly with 2 mL of DPPH solution and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was measured and recorded as A1. Similarly, 2 mL of anhydrous ethanol solution was used instead of DPPH solution, and the absorbance after the reaction in the dark was recorded as A2. The absorbance of deionized water was measured as A0. The DPPH free radical scavenging rate was calculated using the following formula: .

[0084] Wherein, A1: sample and DPPH solution; A2: DPPH solution replaced by anhydrous ethanol; A0: sample solution replaced by deionized water.

[0085] 2.5 α-glucosidase inhibition rate 50 μL of the solution from section 2.4 was mixed with an equal volume of α-glucosidase (0.2 U / mL, based on protein) and reacted at 37 °C for 10 min. Then, 100 μL of PNPG (5 mmol / L) was added, mixed, and reacted at 37 °C for 10 min. After the reaction, the absorbance at 405 nm was measured. .

[0086] In this context, A1 means replacing the sample solution with PBS solution and mixing it with the enzyme solution; A2 means replacing both the sample and enzyme solution with PBS; B1 means mixing the sample solution with the enzyme solution; and B2 means replacing the enzyme solution with PBS solution and mixing it with the sample.

[0087] 2.6 α-Amylase Inhibition Rate 0.2 mL of the solution from step 2.4 was mixed with an equal volume of α-amylase (10 U / mL, based on protein) and reacted at 37 °C for 10 min. Then, 0.3 mL of 1% starch solution was added, mixed, and reacted with shaking at 37 °C for 10 min. Finally, 0.2 mL of DNS solution was added, the reaction was stopped by boiling for 10 min, and the volume was adjusted to 4 mL. The absorbance at 540 nm was measured. .

[0088] Wherein, A1: Replace the sample solution with PBS solution and mix with the enzyme solution; A2: Replace the sample and enzyme solution with PBS; A3: Mix the sample solution with the enzyme solution; A4: Replace the enzyme solution with PBS solution and mix with the sample.

[0089] Statistical analysis All experimental results were performed in at least three replicates and are expressed as mean ± standard deviation. ANOVA was performed using SPSS 22.0, and the significance of each mean was tested using Duncan's multiple range test. P A value <0.05 indicates a significant difference. Plotting was performed using Prism 9.5.1 and Origin 2021.

[0090] 3. Results and Analysis 3.1 Effects of different salting-out agents on the physicochemical and functional properties of *Vernicia fordii* meal protein 3.1.1 Basic Composition Analysis of Precipitates with Different Concentrations of Salting-out Agent 3.1.1.1 Basic Composition of Ammonium Sulfate (NHS) Precipitates at Different Saturations Ammonium sulfate is a classic salting-out reagent in the field of protein separation and purification. NH4 + and SO4 2- All of these belong to the kosmotropic ions in the Hofmeister spectroscopy sequence. This invention uses the acid precipitation (JS) group of Comparative Example 1 as a control to investigate the effect of ammonium sulfate with different saturations on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* seed meal. The results are as follows: Figure 1 As shown, with increasing ammonium sulfate saturation, the content of protein and polyphenols in the precipitate showed an upward trend followed by a downward trend, with the two trends being basically consistent, and the maximum values ​​appearing at 35% and 25% saturation, respectively.

[0091] It is noteworthy that the protein content in the ammonium sulfate precipitate at 35% saturation was significantly higher than that at other saturations and in the JS control group. P <0.05%, and its protein content was 54.15 ± 0.05%. Meanwhile, the contents of polysaccharides, ash, and polyphenols in this precipitate were significantly lower than those in the JS control group ( P <0.05). Based on the above results, this invention selects an ammonium sulfate solution with a saturation of 35% for subsequent experiments.

[0092] In this invention, different lowercase letters represent statistically significant differences (P<0.05).

[0093] 3.1.1.2 Basic Composition of Sodium Sulfate (NS) Precipitates with Different Saturations Sodium sulfate dissociates in solution to produce Na + and SO4 2- Hydration occurs, and both belong to the kosmotropicion group in the Hofmeister sequence. Using the JS group as a control, the effect of sodium sulfate saturation at different levels on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* was investigated. The results are as follows: Figure 2 As shown.

[0094] It is noteworthy that the protein content in the sodium sulfate precipitate at 45% saturation was significantly higher than that at other saturations and in the JS control group. P <0.05%, with a content of 48.98 ± 0.01%. The content of polysaccharides and ash was significantly lower than that of the JS control group ( P <0.05%, while the polyphenol content was lower than that of the JS control group ( P >0.05) No significant difference. Therefore, this invention selected a sodium sulfate solution with a saturation of 45% for subsequent experiments.

[0095] 3.1.1.3 Basic Composition of Potassium Sulfate (KS) Precipitates at Different Saturations Potassium sulfate dissociates in solution to produce K + and SO4 2- Both of these compounds combine with water molecules to form an ordered structure and belong to kosmotropicions. Using the JS group as a control, the effects of different saturations of potassium sulfate on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* were investigated. The results are as follows: Figure 3 As shown.

[0096] At all saturation levels, the polyphenol and ash contents of the potassium sulfate precipitate were significantly lower than those of the JS control group. P <0.05. The protein content in the precipitate of potassium sulfate solution with a saturation of 40% was significantly higher than that of other saturations and the JS control group ( P <0.05%, with a content of 43.19 ± 1.35%. However, at this saturation level, the polysaccharide content was not significantly lower than that of the JS control group ( P >0.05). In summary, potassium sulfate salting-out is insufficient to separate proteins from the alkaline extract of *Vernicia fordii* seed meal, and thus fails to achieve the intended separation of proteins and polysaccharides.

[0097] 3.1.1.4 Basic Composition of Magnesium Sulfate (MS) Precipitates with Different Saturations Magnesium sulfate ionizes in aqueous solution to produce Mg 2+ and SO4 2- Mg 2+ Smaller radius and higher charge density, in the Hofmeister sequence with Na + and K + Both belong to the kosmotropicion group. Using the JS group as a control, the effect of magnesium sulfate with different saturations on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* was investigated. The results are as follows: Figure 4 As shown.

[0098] Although high-valence cations exhibit excellent salt-bridging effects, the protein content in the precipitate at all saturation levels is significantly lower than that in the control group. P<0.05). This result indicates that it is difficult to separate proteins from the alkaline extract of *Vernicia fordii* meal using magnesium sulfate salting-out.

[0099] 3.1.1.5 Basic Composition of Dipotassium Hydrogen Phosphate (KP) Precipitates with Different Saturations Dipotassium hydrogen phosphate ionizes in solution to produce K+. + and HPO4 2- HPO4 2- In the Hofmeister sequence with SO4 2- All belong to kosmotropicion. Using the JS group as a control, the effects of different saturations of dipotassium hydrogen phosphate on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* meal were investigated. The results are as follows: Figure 5 As shown.

[0100] It is noteworthy that the protein content in the precipitates from 35% and 40% saturated dipotassium hydrogen phosphate solutions did not differ significantly, but was significantly higher than that from other saturations and the JS control group. There were no significant differences in the contents of polysaccharides, ash, and polyphenols between the two saturations. To select a more economical and environmentally friendly method, a 35% saturated dipotassium hydrogen phosphate solution was used for the next stage of the experiment.

[0101] 3.1.1.6 Basic Composition of Potassium Citrate (KN) Precipitates with Different Saturations Potassium citrate ionizes in solution to produce K+. + and C6H5O7 3- C6H5O7 3- In the Hofmeister sequence with SO4 2- HPO4 2- All belong to kosmotropicion. Using the JS group as a control, the effect of potassium citrate with different saturations on the basic composition of the precipitate from the alkaline extract of *Vernicia fordii* was investigated. The results are as follows: Figure 6 As shown, with increasing potassium citrate saturation, the content of protein and polyphenols in the precipitate first increased and then decreased. The polysaccharide content in the precipitate decreased with increasing saturation. The ash content of each experimental group was significantly lower than that of the JS group (…). P <0.05).

[0102] It is noteworthy that the protein content in the potassium citrate precipitate at 45% saturation was significantly higher than that at other saturations and in the JS control group. P <0.05), with a content of 46.47 ± 0.02%. At this saturation level, the polysaccharide content was significantly lower than that of the JS control group, while the polyphenol content showed no significant difference from the JS control group. Therefore, the potassium citrate precipitate at 45% saturation was selected for the next step of the experiment.

[0103] 3.1.1.7 Establishment of Saturation for Different Salting-Out Agents Based on the above experimental results, salting-out precipitates with significantly high protein content and significantly low impurity content were selected from NHS, NS, KS, MS, KP, and KN. These precipitates, along with the protein precipitate from the JS control group, were used to construct a basic composition table, as shown in Table 5. The protein content in the precipitates, from highest to lowest, was: NHS > NS > KN > KP > JS. SO4 2- The salting-out ability is stronger than that of HPO4. 2- The corresponding SO4 2- The protein content in the precipitates of the precipitates (NHS and NS) was also higher than that of HPO4. 2- This largely coincides with the Hofmeister sequence of the anion, indicating that the experimental results are reliable; that is, the stronger the structure-enhancing ability of the anion, the higher the protein content in the precipitate. Citrate (C6H5O7) 3- As an organic acid anion, it did not participate in Hofmeister sequence sequencing. Protein content results showed that the protein content of the KN group was lower than that of the NHS and NS groups, but higher than that of the KP group. This indicates that C6H5O7... 3- The structural enhancement capability may be between that of SO4 2- and HPO4 2- between.

[0104] The polysaccharide content in the precipitates, from highest to lowest, was: JS > KP > NS > KN > NHS. There was no significant difference in the polyphenol content among the precipitates.

[0105] Table 5. Basic composition of protein precipitates under different treatments Unless otherwise specified, the concentrations of different salting-out agents used to treat samples in the following text are the corresponding concentrations in Table 5.

[0106] 3.1.2 FTIR spectral analysis The effects of different salting-out agents on the Fourier transform infrared spectra of protein in *Vernicia fordii* meal are as follows: Figure 7 As shown in a). The infrared spectrum indicates that the sample is within the range of 4000–400 cm⁻¹. -1 Characteristic absorption peaks are present below. Notably, the FTIR curves of different samples did not show significant differences, indicating that the protein backbone structure of *Vernicia fordii* meal remained unchanged. All samples showed absorption peaks in the range of 3800–3200 cm⁻¹. -1 The presence of broad and large absorption peaks in the vicinity indicates the presence of hydrogen bonding within the sample. (1650 cm⁻¹) -1 The presence of a characteristic absorption peak of the protein nearby is due to the stretching vibration of the amide I band, indicating the presence of the protein structure.

[0107] FTIR can not only observe functional groups in proteins but also characterize their secondary structure. Based on the deconvolution results of the FTIR curves, the content of the protein's secondary structure can be obtained, such as... Figure 7 As shown in b). α-helices and β-sheets are ordered structures in proteins, while β-turns and random coils are relatively loose and disordered structures. The results showed that the α-helices of the salted-out group were reduced, the β-sheets were increased, and the overall ordered structure was increased. This may be because salting-out promotes the formation of hydrogen bonds in proteins. In the JS control group, the lower pH during acid precipitation led to the formation of more disordered protein structures. SO4 2- Charge density greater than HPO4 2- and C6H5O7 3- During protein precipitation, a lower charge density may result in slower stripping of the hydration layer and shielding of the electrical double layer, thus promoting orderly protein precipitation and the formation of more ordered structures.

[0108] 3.1.3 Intrinsic Fluorescence Spectroscopy The results of intrinsic fluorescence spectroscopy showed that λmax in the salting-out group was blue-shifted compared to the JS control group, indicating that salting-out precipitation promoted the encapsulation of Trp within the protein, and that salting-out promoted the formation of protein aggregates, resulting in amino acid residues being encapsulated inside the protein molecule.

[0109] 3.1.4 SDS-PAGE electrophoresis analysis SDS-PAGE electrophoresis results showed that the JS control group and the salted-out histones had the same subunit composition, with molecular weights mainly distributed at the loading wells, 35-40 kDa, 15-25 kDa, and less than 10 kDa. Compared with the raw materials, the bands in all experimental groups were darker at the loading wells, indicating that the proteins exhibited varying degrees of thermal aggregation, which may be due to aggregation caused by overheating during ball milling.

[0110] Disulfide bonds exist within the raw material of *Vernicia fordii* meal. Similar bands appeared in the JS control group and the salting-out group at values ​​greater than 10 kDa, with the JS control group showing a darker band color, indicating stronger aggregation in the JS group than in the salting-out group.

[0111] 3.1.5 Scanning electron microscopy analysis The results of the effects of different salting-out agents on the microstructure of protein in *Vernicia fordii* meal showed that all protein samples from *Vernicia fordii* meal exhibited a lamellar structure with varying lamellar sizes. Specifically, the JS control group samples had smooth, flat surfaces and clear edges, while the salting-out group samples had rougher surfaces and edges, with the protein lamellars showing bending and folding.

[0112] 3.1.6 Particle size analysis The results of the effect of different salting-out agents on the average particle size of protein in *Vernicia fordii* meal showed that the average particle size of protein in *Vernicia fordii* meal decreased in the following order: JS > NHS > NS > KP > KN. The decreasing trend in particle size among different salting-out agents was consistent with the weakening trend in the ability of kosmotropic ions to form structures.

[0113] 3.1.7 Surface hydrophobicity analysis The results of the effect of different salting-out agents on the surface hydrophobicity of protein in *Vernicia fordii* meal showed that the surface hydrophobicity of protein in *Vernicia fordii* meal, from strongest to weakest, was: JS, NHS, NS, KP, and KN. The hydrophobicity of protein in the salting-out group was significantly lower than that in the JS control group.

[0114] 3.1.8 Solubility Analysis The results of the effect of different salting-out agents on the solubility of protein in *Vernicia fordii* meal showed that the order from largest to smallest was KN > KP > NS > NHS > JS. The solubility of the protein in the salted-out group was significantly higher than that in the JS control group (P<0.05), and there were significant differences in solubility among the different salting-out groups.

[0115] 3.1.9 Foaming property analysis The foaming activity results of the protein in *Vernicia fordii* meal showed that the foaming activity of the salt-out group was significantly higher than that of the JS control group (P<0.05). The order of foaming activity from high to low was: KN>KP>NS>NHS>JS.

[0116] The foaming stability results of the protein in *Vernicia fordii* meal showed that the foaming stability trend was consistent with the foaming activity trend, and the stability of the protein in the salting-out group was more than double that of the protein in the JS control group.

[0117] 3.1.10 Emulsification Analysis The results of the effects of different salting-out agents on the emulsifying activity and emulsifying stability of *Vernicia fordii* meal protein showed that the emulsifying activity, from high to low, was: KN > KP > NS > NHS > JS. The results of the emulsifying stability of *Vernicia fordii* meal protein showed that the emulsifying stability, from high to low, was: JS > NHS > NS > KP > KN.

[0118] 3.1.11 Principal Component Analysis Principal component analysis of protein in *Vernicia fordii* meal using different salting-out agents, as follows: Figure 8 As shown in the load diagram, the increase in the angle between the two arrows from 0° to 180° indicates a change in the correlation between them from a strong positive correlation to a strong negative correlation. When the angle between the two arrows is 90°, it indicates no correlation between them. Figure 8As shown in section a), principal components 1 (PC1) and 2 (PC2) account for 98.4% of the total variables, with PC1 accounting for 78.8% and PC2 for 19.6%. Protein content, solubility, foaming activity, foaming stability, and emulsifying activity are correlated with PC1, while particle size and emulsifying stability are correlated with PC2. The solubility of *Vernicia fordii* meal protein is positively correlated with foaming activity, foaming stability, and emulsifying activity; the particle size of *Vernicia fordii* meal protein is positively correlated with emulsifying stability; and it is negatively correlated with other functional indicators. Among these, solubility and particle size show a strong negative correlation. The JS group falls in the third quadrant and is negatively correlated with both principal components, indicating poor overall sample integrity. The NS group falls in the first quadrant and is positively correlated with both principal components. The NHS group falls in the second quadrant and is negatively and positively correlated with PC1 and PC2, respectively. The KP and KN groups fall in the fourth quadrant and are positively and negatively correlated with PC1 and PC2, respectively. Subsequently, scores were calculated for each group of samples, and the scores are shown in Table 6. The scores of each group from highest to lowest are: KN>KP>NS>NHS>JS. KN had a score of 0.69, ranking first, indicating that the protein precipitated using potassium citrate has good physicochemical and functional properties and is relatively more worthy of further research.

[0119] Figure 8 (b) allows for a more intuitive representation of the magnitude of data differences through changes in color intensity. The results show that, in addition to emulsification stability, salting out significantly improves the quality of protein from *Vernicia fordii* meal. Comparison of different salting-out agents revealed that reducing the charge density of salting-out ions has a positive effect on the physicochemical properties and functions of proteins, excluding emulsification stability. This indicates that lower charge density leads to milder protein denaturation, resulting in a greater yield of proteins with better functional activity.

[0120] Table 6. Effects of different salting-out agents on the PCA score of protein in *Vernicia fordii* meal. 3.2 Effects of hydrogen peroxide decolorization treatment under different pH conditions on the physicochemical and functional properties of *Vernicia fordii* meal protein 3.2.1 Basic Component Analysis The oxidizing power of hydrogen peroxide varies under different pH conditions. Under acidic conditions, hydrogen peroxide is relatively stable and its decolorizing effect on proteins is weak; under alkaline conditions, its oxidizing power is enhanced, resulting in the extensive oxidation of chromophores and thus protein decolorization. The effect of hydrogen peroxide decolorization on the basic protein composition of *Vernicia fordii* meal under different pH conditions is as follows: Figure 9 As shown.

[0121] The effect of decolorization treatment on the protein content of *Vernicia fordii* meal protein samples is as follows: Figure 9As shown in a), the protein content of the control group was not significantly different from that of the KN-3 and KN-5 groups. This may be because the oxidation of proteins by hydrogen peroxide is not vigorous under acidic conditions. From neutral to alkaline conditions, the protein content showed an increasing trend with increasing pH.

[0122] Figure 9 b) represents the polysaccharide content after hydrogen peroxide decolorization treatment under different pH conditions. The polysaccharide content in the decolorization group was significantly higher than that in the control group. P <0.05), which may be due to the fact that hydrogen peroxide treatment alters the structure of the polysaccharide and exposes hydrophilic groups, making it more susceptible to loss of water molecules and reduced solubility during subsequent salting-out, thus leading to precipitation.

[0123] Figure 9 c) represents the polyphenol content after hydrogen peroxide decolorization treatment under different pH conditions. After hydrogen peroxide decolorization treatment, the polyphenol content decreased significantly in all cases. P <0.05). The binding of plant protein and polyphenols often exhibits non-covalent bonds under acidic and neutral conditions, but covalent bonds under alkaline conditions. This may explain the difference in polyphenol content after hydrogen peroxide decolorization under different pH conditions. When the solution pH is 3, the pH value is lower than the isoelectric point of *Vernicia fordii* meal protein (pI = 3.2), causing the protein to carry a positive charge. The acidic environment leads to increased protonation of polyphenols, which also carry a positive charge. The two exhibit electrostatic repulsion, further exposing the polyphenols to the hydrogen peroxide environment, and the chromophores are oxidized. This may explain the lower polyphenol content in the KN-3 group. Except for the KN-3 group, the polyphenol removal rate gradually increased with increasing pH in the other decolorization groups. Figure 9 d) is the ash content in the sample. Hydrogen peroxide treatment has no significant effect on the ash content in the sample.

[0124] 3.2.2 Color Difference Analysis The effect of hydrogen peroxide decolorization treatment on the color value of proteins under different pH conditions is as follows: Figure 10 As shown in the image, the decolorized protein product is shown in the picture below. Figure 11 .exist Figure 10 In the middle, L * L represents brightness or darkness. * The higher the value, the brighter the sample; the range is 0 to 100. * and b * The positive and negative values ​​represent different chromaticities, with positive values ​​indicating that the sample leans towards red and yellow, respectively, and negative values ​​indicating that the sample leans towards green and blue, respectively; WI * This indicates whiteness; the higher the value, the closer the sample color is to white.

[0125] Figure 10 a) represents the L of mangosteen seed meal protein *Value. Compared with the control group, L in all decolorization groups * The values ​​all increased significantly ( P <0.05), indicating that hydrogen peroxide treatment significantly improves the brightness of protein in *Vernicia fordii* meal. The lower the polyphenol content after hydrogen peroxide decolorization, the lower the protein's L... * The higher the value, the higher the L content in the protein of *Vernicia fordii* seed meal. * The value is affected by the polyphenol content.

[0126] Figure 10 b) represents a of Mangosteen meal protein * Value. Compared with the control group, a value was higher in all decolorization groups. * The values ​​all decreased significantly ( P <0.05), indicating that hydrogen peroxide treatment can significantly reduce the redness of protein in *Vernicia fordii* meal. * The significant difference in values ​​was directly proportional to the difference in polyphenol content; that is, the lower the polyphenol content after hydrogen peroxide decolorization, the lower the protein a value. * The smaller the value, the more likely it is that hydrogen peroxide decolorization can significantly reduce the redness value of protein in tung oil meal.

[0127] Figure 10 c) represents the protein content of *Vernicia fordii* meal. * Value. Compared with the control group, the b value of all decolorization groups was higher. * The values ​​all increased significantly ( P <0.05), indicating that the yellow color in the protein of *Vernicia fordii* seed meal became more intense. * The upward trend in values ​​may be related to the significant increase in polysaccharide content in the protein. Studies have shown that *Vernicia fordii* meal contains a relatively high amount of pectin, and the yellowness of the samples in this invention may be due to the presence of a small amount of pectin in the samples.

[0128] Figure 10 d) represents the WI of Mangosteen meal protein * Value. Compared with the control group, the whiteness of all decolorization groups was significantly increased ( P <0.05). The whiteness trend is consistent with the polyphenol removal rate trend, indicating that the main melanin in the protein of *Vernicia fordii* seed meal is produced by the oxidation of polyphenols.

[0129] 3.2.3 Amino acid composition analysis The effects of hydrogen peroxide decolorization treatment on the amino acid composition of proteins under different pH conditions showed that, from an amino acid classification perspective, there were differences in amino acid content among different groups. The amino acid content of the decolorized groups decreased to varying degrees. The increase in cysteine ​​and arginine content may be due to the disruption of the protein-polyphenol complex, exposing amino acid residues.

[0130] 3.2.4 Ultraviolet-Visible Spectroscopic Analysis The effects of hydrogen peroxide decolorization treatment on the UV-Vis spectra of proteins under different pH conditions showed that the peak intensities, from strongest to weakest, were: KN > KN-5 > KN-3 > KN-7 > KN-9 > KN-11. The salting-out group samples exhibited a redshift at the 279 nm absorption peak, indicating a decrease in the compactness of the protein's tertiary structure. 3.2.5 FTIR spectral analysis FTIR spectral analysis showed that all samples were within the range of 3450–3300 cm⁻¹. -1 The system exhibits strong and broad absorption peaks, with enhanced absorption peaks in the decolorized group, which can be explained by an increase in the number of hydroxyl groups in the system. (3000~2750 cm⁻¹) -1 The double peaks and 1250~1000 cm -1 The change in absorption peak may be caused by polysaccharides.

[0131] The results of protein secondary structure analysis showed that, compared with the control group, the ordered structure of proteins generally decreased after hydrogen peroxide decolorization treatment under different pH conditions. The ordered structure of decolorized histones decreased continuously compared with the control group, specifically manifested as a decrease in α-helices and an increase in disordered structures represented by β-turns and random coils.

[0132] 3.2.6 SDS-PAGE electrophoresis analysis Non-reduction electrophoresis showed that destaining treatment did not alter the subunit composition of the protein. Reduction electrophoresis showed that the bands near the comb teeth at the top of the lanes were lighter in color, while bands smaller than 10 kDa were darker, indicating that the disulfide bonds of the protein aggregates in these bands were disrupted by β-mercaptoethanol. Compared with the tung oil meal raw material, the destaining group showed weaker macromolecular aggregation than the control group, and destaining did not change the subunit composition of the protein.

[0133] 3.2.7 Scanning electron microscopy observation Figure 12 This illustrates the effect of decolorization treatment under different pH conditions on the microstructure of proteins in Example 2. Data for KN, KN-3, KN-5, KN-7, KN-9, and KN-11 are shown in a(a), b), c), d), e), and f), respectively.

[0134] Depend on Figure 12 It can be seen that after decolorization with hydrogen peroxide, the protein still exhibits a sheet-like structure with relatively rough edges, and the protein still shows bending and folding phenomena. It is worth noting that the size of the decolorized group increased, there were fewer broken sheet-like proteins, and the protein structure appeared as large sheet-like structures.

[0135] 3.2.8 Particle size analysis The results of the effect of hydrogen peroxide decolorization treatment on the average particle size of proteins under different pH conditions showed that the decolorization treatment increased the average particle size of proteins, and the order of particle size from largest to smallest was: KN-11>KN-9>KN-7>KN-3>KN-5>KN.

[0136] 3.2.9 Surface hydrophobicity analysis The results of hydrogen peroxide decolorization treatment under different pH conditions on the surface hydrophobicity of proteins showed that the hydrophobicity of the decolorized proteins was significantly lower than that of the control group, and the surface hydrophobicity decreased with increasing decolorization pH, from largest to smallest: KN>KN-5>KN-3>KN-7>KN-9>KN-11.

[0137] 3.2.10 Molecular flexibility analysis The results of hydrogen peroxide decolorization treatment under different pH conditions on the flexibility of protein molecules showed that after hydrogen peroxide treatment, the flexibility of protein molecules was significantly enhanced (P<0.05), and the molecular flexibility from largest to smallest was: KN-11>KN-9>KN-7>KN-5>KN-3>KN.

[0138] 3.2.11 Solubility Analysis The results of hydrogen peroxide decolorization treatment on protein solubility under different pH conditions showed that the increasing trend of protein solubility was positively correlated with the decrease of surface hydrophobicity. The solubility of proteins from highest to lowest was: KN-11>KN-9>KN-7>KN-3>KN-5>KN.

[0139] 3.2.12 DSC Analysis The results of hydrogen peroxide decolorization treatment on the thermal stability of proteins under different pH conditions showed that hydrogen peroxide decolorization significantly reduced the thermal denaturation initiation temperature and enthalpy of proteins (P<0.05). Except for the KN-3 treatment, which had no difference in thermal denaturation peak temperature from the control group, the decolorization treatment also significantly reduced the thermal denaturation peak temperature of other treatment groups (P<0.05).

[0140] 3.2.13 Emulsification Analysis The results of the effect of hydrogen peroxide decolorization treatment on protein emulsifying properties under different pH conditions showed that hydrogen peroxide treatment significantly improved the emulsifying activity of proteins. P <0.05), the order from high to low is: KN-11 >KN-9 >KN-7 >KN-5 >KN-3 >KN.

[0141] The emulsification stability trend is consistent with the emulsification activity: KN-11>KN-9>KN-7>KN-5>KN-3>KN.

[0142] 3.2.14 Interface wettability analysis The results of hydrogen peroxide decolorization treatment on the wettability of protein interfaces under different pH conditions showed that the smaller the contact angle of the droplets resting on the sample pellet, the more hydrophilic the sample. The hydrophilicity order of different samples was KN>KN-5>KN-3>KN-7>KN-9>KN-11, which is consistent with the surface hydrophobicity results, indicating that hydrogen peroxide decolorization significantly reduced the hydrophobicity of the samples. The decolorization treatment significantly affected the contact angle between proteins and water molecules.

[0143] 3.3 Study on the digestive properties, peptide profile, and biological activity of decolorized proteins 3.3.1 Degree of hydrolysis during protein digestion The degree of protein hydrolysis can evaluate the extent of peptide bond hydrolysis. Proteins are broken down into smaller peptides and free amino acids by pepsin and trypsin, promoting absorption in the small intestine. The effect of hydrogen peroxide decolorization treatment on the degree of protein hydrolysis under different pH conditions is shown below. Figure 13 As shown in the figure, decolorization treatment significantly improved the gastric and gastrointestinal digestibility of proteins. P <0.05). This may be because decolorization significantly increases protein solubility, allowing for better protein-enzyme contact. Significant differences were observed between the decolorized groups, possibly because decolorization alters the protein structure, facilitating the recognition of enzyme hydrolysis sites. The degree of hydrolysis during enteric digestion was greater than that during gastric digestion. This is because pepsin treatment exposes protein peptide bonds, which facilitates trypsin's endoplasmic reaction.

[0144] 3.3.2 Digestibility of protein during digestion The effect of hydrogen peroxide decolorization treatment on protein digestibility under different pH conditions is as follows: Figure 14 As shown, decolorization treatment significantly improved the gastric and gastrointestinal digestibility of protein in *Vernicia fordii* seed meal. P <0.05). Existing research indicates that the cleavage site of pepsin is a peptide bond formed by hydrophobic amino acids, and increased surface hydrophobicity promotes pepsin cleavage. However, the gastric digestibility of this invention shows an inverse relationship with hydrophobicity, possibly because the protein decolorization treatment of this invention significantly improves the rigid structure of the protein and promotes enzyme recognition at the contact site, thereby enhancing the efficiency of pepsin hydrolysis.

[0145] 3.3.3 Peptidomics studies of in vitro digested products The differences in peptide composition of the serially digested gastrointestinal products are shown in Table 7. A total of 3331 unique peptides were identified in the serially digested gastrointestinal products. Among them, the control group (KN), KN-3, KN-5, KN-7, KN-9, and KN-11 produced 115, 699, 526, 535, 702, and 754 peptides, respectively. The results indicate that the serially digested gastrointestinal products of the decolorized treatment group in Example 2 contained a greater number of peptides, which may be related to the significant improvement in in vitro digestibility due to decolorization. Protein structure unfolding not only facilitates the exposure of cleavage sites but also promotes their interaction with enzymes. Therefore, KN-11 produced the most newly added peptides (648) and the fewest deleted peptides (79).

[0146] Table 7 Changes in peptide composition in samples Note: New peptides refer to new peptides identified only in the digestion products of the decolorized samples (KN-3, KN-5, KN-7, KN-9, KN-11); missing peptides refer to peptides identified only in the digestion products of the control sample (KN).

[0147] The peptides were divided into 5 groups according to molecular weight (500-700 Da, 700-1200 Da, 1200-1800 Da, 1800-2500 Da, and 2500-5000 Da), and the results are as follows: Figure 15 As shown in the figure, the results indicate that the decolorized gastrointestinal digests contained fewer high-molecular-weight peptides and more low-molecular-weight peptides. This suggests that decolorization promoted the cleavage of high-molecular-weight peptides into lower-molecular-weight peptides. Specifically, over 80% of the peptides in the KN, KN-3, KN-5, and KN-7 groups were concentrated in the 700-1800 Da range, while over 80% of the peptides in the KN-9 and KN-11 groups were concentrated in the 500-1200 Da range. The concentration of these peptides in the KN-9 and KN-11 groups within the low molecular weight range may be related to their higher degree of hydrolysis.

[0148] The biological activity of bioactive peptides depends on the amino acid composition of their primary structure. The different charge distributions and polarities at the two ends of the peptide chain result in different binding capacities between the N-terminal and C-terminal amino acid residues when exerting biological activity. Figure 16Images a), b), c), d), e), and f) represent the terminal amino acid analyses of the products from sequential gastrointestinal digestion of groups KN, KN-3, KN-5, KN-7, KN-9, and KN-11, respectively. All groups were analyzed using the colorimetric scale on the right; an increase in the content of terminal amino acid residues resulted in a darker color in the corresponding amino acid square in the heatmap. Images b through f show a significant darkening of color, indicating a significant increase in the diversity of terminal amino acids in the proteins after decolorization treatment. Arginine (R) showed the most significant increase and was the most abundant C-terminal residue, followed by isoleucine (L) and phenylalanine (F), ranking second and third in C-terminal residue content, respectively. The increased variety and quantity of exposed terminal amino acids reduced the steric hindrance for these amino acids to exert their biological activities, providing conditions for the peptides to exert their biological activities.

[0149] 3.3.4 Bioactivity assay of in vitro digested products Proteins are digested into peptides and amino acids in the gastrointestinal tract and absorbed by the body. Specific conformations and terminal residues of short peptides can exert biological activities, such as antioxidant, hypoglycemic, and antibacterial activities. Peptidomics can serve as a result of digestive properties; however, the exertion of biological activity depends in part on the primary structure of the peptide, therefore, some aspects of peptidomics can support biological activity. This invention further explores the biological activity of products of continuous gastrointestinal digestion.

[0150] 3.3.4.1 DPPH free radical scavenging power Figure 17 The effect of hydrogen peroxide decolorization treatment on the DPPH free radical scavenging capacity of protein products from continuous gastrointestinal digestion under different pH conditions was investigated. The DPPH free radical scavenging rate of the decolorized group was significantly lower than that of the undecolorized protein. P The value was <0.05%, which may be due to differences in the basic composition of the samples. Hydrogen peroxide decolorization significantly reduced the polyphenol content in the samples and destroyed some hydrophobic amino acids, resulting in a decrease in the protein's ability to capture free radicals and a decrease in DPPH free radical scavenging rate. Notably, no significant differences were observed between the decolorization groups, which may be because hydrolysis produced more peptides, leading to increased amino acid exposure.

[0151] 3.3.4.2 α-Glucosidase Inhibition Rate Figure 18 The effect of hydrogen peroxide decolorization treatment under different pH conditions on the inhibition rate of α-glucosidase in protein products of continuous gastrointestinal digestion was investigated. The results showed that decolorization significantly increased the inhibition rate of α-glucosidase in proteins. P<0.05). This may be because the decolorized product of the present invention is rich in peptides containing serine (Ser), tyrosine (Tyr), and threonine (Thr), and certain basic amino acids, including lysine (Lys) and arginine (Arg), can form hydrogen bonds or electrostatic interactions with α-glucosidase through the hydroxyl groups of the amino acid side chains, thereby inhibiting the activity of α-glucosidase. The decolorized protein has a high digestibility, promotes the generation of small molecule peptides and free amino acids, and the peptide sequences of the decolorized group are rich in Ser and Arg at both ends, thus resulting in a high α-glucosidase inhibition rate.

[0152] 3.3.4.3 α-Amylase Inhibition Rate Figure 19 The effect of hydrogen peroxide decolorization treatment under different pH conditions on the inhibition rate of α-amylase in protein products of continuous gastrointestinal digestion was investigated. The results showed that decolorization significantly increased the α-amylase inhibition rate of proteins (…). P <0.05). Studies have shown that Arg plays an important role in α-amylase inhibition. The trend of α-amylase inhibition rate is the same as that of α-glucosidase inhibition rate, indicating that Arg in the gastrointestinal sequential digestion product of *Vernicia fordii* meal protein of the present invention may play an important role in α-amylase inhibition.

[0153] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for separating and decolorizing protein from *Vernicia fordii* seed meal, characterized in that, Includes the following steps: (1) Mix the powder of tung oil seed meal with water, treat it with alkali under alkaline conditions, and then separate the solid and liquid phases to collect the liquid phase components; (2) Add hydrogen peroxide solution to the liquid phase component to obtain the decolorizing solution, and perform decolorization treatment to obtain the decolorized solution; (3) Adjust the pH of the decolorizing solution to neutral, add salting-out agent to obtain the solution to be salted out, precipitate, then add water to dissolve the precipitate, dialyze, and obtain the decolorized protein powder of tung oil seed meal.

2. The separation and decolorization method according to claim 1, characterized in that, In step (1): the ratio of the powder of *Vernicia fordii* seed meal to water is 1g:20mL.

3. The separation and decolorization method according to claim 1, characterized in that, The pH value of the alkali treatment is 12.

4. The separation and decolorization method according to claim 1, characterized in that, The alkali treatment was performed at a temperature of 50 °C for 2 h.

5. The separation and decolorization method according to claim 1, characterized in that, In step (2): the concentration of the hydrogen peroxide solution is 4 vol%; the volume ratio of the liquid phase component to the hydrogen peroxide solution is 180:7.

2.

6. The separation and decolorization method according to claim 1, characterized in that, Before the decolorization process, the pH value of the solution to be decolorized is adjusted to 3-11.

7. The separation and decolorization method according to claim 1, characterized in that, The decolorization process was carried out at a temperature of 45 °C for 45 min.

8. The separation and decolorization method according to claim 1, characterized in that, The salting-out agent is ammonium sulfate, sodium sulfate, dipotassium hydrogen phosphate, or potassium citrate; the concentration of the salting-out agent in the solution to be salted out is 45 vol.

9. The separation and decolorization method according to claim 1, characterized in that, The precipitation treatment was carried out at a temperature of 4 ℃ for 2 h.

10. A decolorized protein powder made from *Vernicia fordii* seed meal prepared by the separation and decolorization method according to any one of claims 1-9.

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