Selenium tea ethanol dehydrogenase activating peptide composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of health food, in particular to a selenium tea ethanol dehydrogenase activating peptide composition as well as a preparation method and application thereof. A response surface method is adopted to optimize a TSE-PP preparation process, affinity ultrafiltration and LC-MS / MS technologies are utilized to screen ADH binding peptides, and the action mechanism of the ADH binding peptides is researched through molecular docking and animal experiments. Experiments prove that the TSE-PP can promote ethanol metabolism, relieve liver cell injury, inhibit inflammatory response, relieve oxidative stress and improve liver lipid accumulation, the high-dose use effect is more remarkable, and a scientific basis is provided for application of the TSE-PP in the food with the function of dispelling the effects of alcohol and the nutrition fortified product. The invention discloses the preparation process of the TSE-PP and the anti-alcohol and liver protection effects of the TSE-PP, meanwhile, the TSE-PP has good food processing characteristics, and a theoretical basis and an experimental support are provided for production and application of the TSE-PP in functional foods.

Description

Technical Field

[0001] The present invention relates to the technical field of health foods, and particularly relates to a selenium tea ethanol dehydrogenase activating peptide combination, a preparation method thereof, and an application thereof. Background Art

[0002] Proteins are key substances for maintaining the physiological functions, nutritional balance, and overall health of organisms. In recent years, bioactive peptides derived from proteins have received extensive attention due to their various biological activities, especially in aspects such as antioxidant, antibacterial, anti-inflammatory, blood glucose and blood pressure regulation, etc., and have become an important direction in the research of functional foods. Among them, active peptides derived from plant proteins have gradually become a popular research topic in the health field due to their green, sustainable, and low-toxic characteristics.

[0003] Tea is a high-quality source of plant protein, with its protein content reaching 21%-28%. It is rich in various essential amino acids and bioactive peptides. Existing research has shown that tea protein and its derived peptides have multiple health benefits such as antioxidant, lipid metabolism regulation, and cardiovascular protection. In addition, tea can also enrich the trace element selenium (Se) to form selenoprotein or selenium-containing active peptides. The existing technology (Research on the Separation and Preparation Process of Selenium-Enriched Tea Polypeptides, He Hong, Wuhan Polytechnic University) has studied selenium-enriched tea protein in terms of the screening of selenium-enriched tea, protein extraction, enzymatic hydrolysis, and polypeptide separation. Selenium-enriched tea that meets the requirements was screened by measuring the selenium content; then, alkali method was used to extract tea protein, and the effects of factors such as alkali solution concentration, time, and temperature on the protein extraction rate were studied to determine the optimal extraction process conditions. Then, the extracted protein was enzymatically hydrolyzed, the enzymatic hydrolysis effects of different proteases were compared, and the enzymatic hydrolysis conditions of alkaline protease were optimized. Finally, the enzymatic hydrolysis products were separated and purified by ultrafiltration and molecular sieve chromatography to obtain three selenium-enriched tea polypeptides. It provides a reference for improving the extraction rate and purity of selenium-enriched tea protein, as well as how to optimize the enzymatic hydrolysis conditions to obtain selenium-enriched tea polypeptides with high antioxidant activity. The existing technology (Antioxidant and Prebiotic Activities of Selenium-Containing Green Tea, Molan, AL., 2013, Nutrition 29(2), 476-477) mentioned that selenium-enriched tea has attracted wide attention in the fields of antioxidant and functional foods. Compared with ordinary tea protein, selenium tea protein (STP) not only retains the biological activity of tea protein but also shows stronger antioxidant ability and metabolic regulation function due to the presence of organic selenium. The research (Restoring Energy Metabolism by NAD+ Supplementation Prevents Alcohol-Induced Liver Injury and Promotes Liver Regeneration, Food Science and Nutrition 12(7), 5100-5110) has shown that selenium can inhibit the overactivation of the NF-κB pathway and reduce the levels of inflammatory factors, thus alleviating alcoholic hepatitis; another study has shown that enhancing the activities of ADH and ALDH and accelerating the clearance of ethanol and its metabolites help reduce the damage of alcohol to the liver. (The Role of Selenium and Selenoproteins in Health, Biomolecules 13(5)) indicates that selenium-binding proteins or selenium-containing active peptides have unique advantages in antioxidant, immune regulation, and metabolic regulation and may become the core components of new functional foods. However, currently, the application research of selenium tea protein mainly focuses on antioxidant properties, and the functional development of selenium tea protein peptides is still in its infancy, especially the research on alcohol metabolism regulation is not sufficient.

[0004] Acute drunkenness is a severe metabolic disorder caused by excessive alcohol consumption, mainly manifested as physiological and pathological changes such as ethanol metabolism disorders, oxidative stress, free radical generation, and inflammatory responses. Acute drunkenness can induce health problems such as neurological dysfunction and liver damage. Although existing treatment methods can relieve acute drunkenness, their side effects, effectiveness, and affordability still face many challenges. Therefore, developing natural, safe, and efficient anti-hangover functional foods has important practical significance. Summary of the Invention

[0005] In the present invention, ADH-activating peptides are mined from natural selenium tea food sources, and the promoting effects of TSE-PP on ethanol metabolism and liver protection are systematically evaluated by combining in vitro enzymatic screening and animal experiments. At the same time, its food processing characteristics are explored, providing a theoretical basis and technical support for its production and application in functional foods.

[0006] To achieve the above object, the present aspect provides a preparation method of a selenium tea ethanol dehydrogenase-activating peptide combination, comprising the following steps: S1. Dephenolization of selenium-containing tea The selenium-containing tea is dried, crushed, and sieved for later use; The reserved selenium-containing tea is added to ultrapure water and heated thoroughly in a boiling water bath for multiple times. After the residue is dried, defatted tea residue is obtained; S2. Preparation of tea selenium protein peptide The defatted tea residue is mixed with an alkaline solution and extracted by ultrasonic waves. The extraction solutions are combined after multiple extractions; The pH of the extraction solution is adjusted to acidic, and the precipitate is collected by centrifugation. The precipitate is washed with deionized water until neutral and then freeze-dried to obtain tea selenium protein powder; The tea selenium protein powder is fully dissolved in ultrapure water, and protease is added for full hydrolysis, and then freeze-dried to obtain a crude TSE-PP solution; S3. Preparation, separation, and purification of ADH-activating peptide TSE-PP TSE-PP is fully mixed with ADH and incubated at a constant temperature to promote the binding of ADH to the polypeptide receptor, obtaining a mixed solution of ADH-binding peptides; The mixed solution of ADH-binding peptides is separated to obtain a supernatant. The supernatant is added with an acetonitrile-aqueous solution, shaken and mixed evenly, and left standing at room temperature to dissociate the ADH-binding peptides; After standing, the filtrate is collected by centrifugation to obtain a filtrate of the ADH-activating peptide TSE-PP combination.

[0007] Preferably, the protease is one or two of alkaline protease and papain.

[0008] Preferably, the defatted tea residue is mixed with a 0.3 M NaOH solution at a solid-liquid ratio of 1:20 (g / mL), extracted at a ultrasonic power of 100 W and a temperature of 50 °C for 90 min, and extracted three times repeatedly.

[0009] Preferably, the pH of the extract is adjusted to 4.2.

[0010] Preferably, a 10 mg / mL tea selenoprotein peptide solution and a 2 U / mL ADH solution are prepared using a PBS buffer solution at pH 7.4 and are thoroughly mixed.

[0011] Preferably, the water bath incubation is a 37°C constant temperature water bath incubation.

[0012] Preferably, the ADH-binding peptide mixed solution is placed in a 10 kDa ultrafiltration centrifuge tube and centrifuged at 10,000 r / min for 10 min at 4°C to separate the supernatant.

[0013] Preferably, according to the mass percentage, alkaline protease: papain = (1-7): (1-7), ADH enzyme dosage is 500-8000 U / g, extraction pH is 5-10, dephenolized selenium tea eggs: ultrapure water = 1: (10-60) g / mL, and the protease hydrolysis time is 4-6h.

[0014] In a second aspect, the present invention provides a selenium-tea alcohol dehydrogenase activating peptide combination, which is prepared by the preparation method of the selenium-tea alcohol dehydrogenase activating peptide combination.

[0015] Preferably, a selenium-tea alcohol dehydrogenase activating peptide combination includes non-selenium-modified peptide APLLFPP, non-selenium-modified PSPPIVVPP2, selenium-modified peptide PSC(Se)PFC(SeMe)T and selenium-modified peptide PNM(Se)PPGS.

[0016] In a third aspect, the present invention provides an application of a selenium-tea alcohol dehydrogenase activating peptide combination, including the following applications: (1) Application in functional foods that regulate ethanol metabolism, relieve liver cell damage, inhibit inflammatory response, relieve oxidative stress, and improve liver lipid accumulation; (2) Application in alkaline foods; (3) Application in functional foods for sobering up; (4) Application in improving food flavor; (5) Application in food in aqueous environment; (6) Application in thermally processed foods; (7) Application in low to medium fat foods.

[0017] The beneficial effects of the invention include at least: 1. The present invention evaluates the potential nutritional value by detecting the main chemical components in tea. The preparation method of the selenium tea ethanol dehydrogenase activating peptide combination removes water-soluble polyphenols through dephenolization treatment, releasing the proteins originally bound to the polyphenols and redepositing them in an insoluble form, thereby increasing the proportion of insoluble proteins in the tea residue and significantly increasing the protein content of the tea residue.

[0018] 2. Based on bioinformatics prediction of the influence of different proteases on the hydrolysis degree of tea proteins and experimental verification, the present invention finds that when papain and alkaline protease are used in combination, the peptide extraction rate reaches 53.99%, which is significantly higher than that when papain or alkaline protease is used alone; in addition, the activation rate of the peptides obtained under this condition for alcohol dehydrogenase (ADH) reaches 56.89%, showing higher biological activity compared with the single enzymatic hydrolysis condition.

[0019] 3. Based on regression model analysis and single-factor experiments, the present invention optimizes the preparation process of TSE-PP, determines the optimal single-factor conditions, so that the protein content and selenium content of TSE-PP under the optimized conditions reach relatively high levels, and the prepared TSE-PP has high nutritional value.

[0020] 4. Through affinity ultrafiltration-liquid chromatography-mass spectrometry technology, the present invention separates, screens and analyzes the structural characteristics of the prepared TSE-PP, screens out 6 peptide segments, and determines 4 bioactive peptides that are non-toxic, non-allergenic, non-carcinogenic, highly stable, suitable for gastrointestinal absorption (HIA) but do not penetrate the blood-brain barrier (BBB).

[0021] 5. By studying the anti-alcoholism activity of TSE-PP on mice and the processing characteristics of TSE-PP, the present invention provides theoretical support for its application in functional foods for ethanol metabolism, alleviating hepatocyte damage and inhibiting inflammatory responses, alleviating oxidative stress, and improving liver lipid accumulation, as well as its application in functional food processing. Description of the Drawings

[0022] Figure 1 It is the analysis diagram of the main chemical components of the tea in the embodiment of the present invention; Figure 1 A is the analysis diagram of the main chemical components of the tea before dephenolization; Figure 1 B is the analysis diagram of the main chemical components of the tea residue after dephenolization; Figure 2 Comparison diagram of the influence of different proteases on the hydrolysis of tea proteins; Figure 2 A is the predicted hydrolysis degree diagram of different proteases on the hydrolysis of tea proteins based on bioinformatics; Figure 2 B is the comparison diagram of the influence of experimental verification of different proteases on the hydrolysis of tea proteins; Figure 3Comparison chart of the effects of different single factors on the ADH activity of tea selenium protein peptide; Figure 3 A is the comparison chart of the effect of enzyme ratio on the ADH activity of tea selenium protein peptide; Figure 3 B is the comparison chart of the effect of enzymatic hydrolysis time on the ADH activity of tea selenium protein peptide; Figure 3 C is the comparison chart of the effect of reaction temperature on the ADH activity of tea selenium protein peptide; Figure 3 D is the comparison chart of the effect of pH on the ADH activity of tea selenium protein peptide; Figure 3 E is the comparison chart of the effect of enzyme addition amount on the ADH activity of tea selenium protein peptide; Figure 3 F is the comparison chart of the effect of solid-liquid ratio on the ADH activity of tea selenium protein peptide; Figure 4 Structural diagram of molecular docking analysis of tea peptide and alcohol dehydrogenase; Figure 4 A is the structural diagram of molecular docking analysis of PSC(Se)PFC(SeMe)T and alcohol dehydrogenase; Figure 4 B is the structural diagram of molecular docking analysis of PNM(Se)PPGS and alcohol dehydrogenase; Figure 4 C is the structural diagram of molecular docking analysis of APLLFPP and alcohol dehydrogenase; Figure 4 D is the structural diagram of molecular docking analysis of PSPPIVVPP and alcohol dehydrogenase; Figure 5 Analysis chart of the effects of tea selenium protein peptide on the drunken behavior and liver tissue injury of mice; Figure 5 A is the experimental design and sample intervention diagram of acute drunkenness experiment in mice; Figure 5 B is the comparison chart of the drunkenness rate and drunkenness time of mice in different experimental groups; Figure 5 C is the comparison chart of the average body weight of mice during the growth period; Figure 5 D is the comparison chart of the liver index of mice; Figure 5 E is the H&E staining diagram of mouse liver tissue under a 40-fold magnifying glass; Figure 5 F is the H&E staining of mouse liver tissue under a 100-fold magnifying glass; Data represent the mean ± SD of 8 independent experiments; Note: Significance was calculated by analysis of variance p<0.01, p<0.001; Figure 6 Effect diagram of the antioxidant effect and lipid metabolism regulation of tea selenium protein peptide on drunken mice; Figure 6 A is the comparison chart of SOD activity in the liver of mice; Figure 6 B is the comparison chart of GSH content in the liver of mice; Figure 6 C is the comparison chart of MDA content in the liver of mice; Figure 6 D is the comparison chart of ADH activity in the liver of mice; Figure 6 E is the comparison chart of ALT level in the serum of mice; Figure 6Comparison of AST levels in the serum of F mice; Figure 6 G is a comparison chart of TG levels in the serum of mice; Figure 6 H is a comparison chart of TC levels in the serum of mice; Data represent the mean ± SD of eight independent experiments. Note: Significance was calculated by analysis of variance p < 0.05, p < 0.01, p < 0.001, p < 0.0001; Figure 7 is an analysis chart of the processing characteristics of TSE-PP; Figure 7 A is an analysis chart of the solubility of TSE-PP in water and ethanol, Figure 7 B is an analysis chart of the solubility of TSE-PP at different temperatures; Figure 7 C is an analysis chart of the solubility of TSE-PP at different pH values; Figure 7 D is an analysis chart of the effect of temperature on the emulsifying property and emulsifying stability of TSE-PP; Figure 7 E is an analysis chart of the effect of pH on the emulsifying property and emulsifying stability of TSE-PP; Figure 7 F is an analysis chart of the effect of oil content on the emulsifying property and emulsifying stability of TSE-PP; Figure 7 G is an analysis chart of the effect of temperature on the foaming property and foam stability of TSE-PP; Figure 7 H is an analysis chart of the effect of pH on the foaming property and foam stability of TSE-PP; Figure 7 I is an analysis chart of the effect of peptide concentration on the foaming property and foam stability of TSE-PP; Figure 7 J is an analysis chart of the effect of different temperatures on the ADH activity of TSE-PP; Figure 7 K is an analysis chart of the effect of different pH values on the ADH activity of TSE-PP; Figure 7 L is an analysis chart of the effect of different storage times on the ADH activity of TSE-PP; Figure 7 M is an analysis chart of the effect of different additives on the ADH activity of TSE-PP. Detailed implementation manners

[0023] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0024] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0025] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0026] Referring to the attached Figures 1 - 7 , a preferred embodiment of the present invention, a preparation method of a selenium tea alcohol dehydrogenase activating peptide combination, comprises the following steps: Step 1: Raw material preparation. The selenium-containing tea was collected from Dongyu Wuliba Tea Garden, Xixiang County, Hanzhong City in August 2023, dried and crushed at 60 °C, and passed through a 60-mesh sieve. All solvents and reagents used in the experiments were purchased from commercial channels. The proteases alkaline protease (EC 3.4.21.62), papain (EC 3.4.22.2), pepsin (EC 3.4.23.1), and trypsin (EC 3.4.21.4) were all food-grade and purchased from Qiansheng Biotechnology (Hefei, China). Alcohol dehydrogenase was purchased from Hefei Bomei Biotechnology. Silymarin was purchased from Wanbond Pharmaceutical Group Co., Ltd. The selenium standard solution was purchased from the National Nonferrous Metals Analysis and Testing Center (Beijing, China), and other reagents were all of analytical grade. The enzyme-linked immunosorbent assay kits were all purchased from Nanjing Jiancheng Bioengineering Institute, China.

[0027] Step 2: Extraction of tea selenium protein: The selenium-containing tea was added to ultrapure water at a solid-liquid ratio of 1:30 (g / mL), heated in a boiling water bath for 1 h, and repeated three times. The residue was dried at 60 °C to obtain defatted tea residue; the defatted tea residue was mixed with 0.3 M NaOH solution at a solid-liquid ratio of 1:20 (g / mL), and extracted at a ultrasonic power of 100 W (Kunshan Ultrasonic Cleaner KQ3200DE, Jiangsu, China), a temperature of 50 °C for 90 min, and the extraction was repeated three times; the extraction solutions were combined, the pH was adjusted to 4.2, and centrifuged at 4000 r / min for 10 min (Hunan Xiangyi Centrifuge H1850R, China), the precipitate was collected, and the precipitate was washed with deionized water until neutral to obtain tea selenium protein precipitate; the tea selenium protein precipitate was freeze-dried under vacuum (Sihuan LGJ-10B, Beijing, China), and stored at -20 °C for later use.

[0028] Step 3: Hydrolysis of tea selenium protein: The tea selenium protein precipitate was dissolved in ultrapure water, and protease was added for full hydrolysis to obtain a crude TSE-PP solution.

[0029] Step 4: Preparation of ADH-activated peptide TSE-PP: In this invention, ultrafiltration affinity method was used to separate the ADH-binding peptide from TSE-PP. 200 μL of 10 mg / mL TSE-PP solution was fully mixed with 2 U / mL ADH solution, and incubated in a constant temperature water bath at 37 °C for 50 min to promote ADH binding.

[0030] Step 5: Dissociation of ADH-binding peptide: The mixture was transferred to a 10 kDa ultrafiltration centrifugal tube, centrifuged at 10000 r / min at 4 °C for 10 min, 250 μL of 35% acetonitrile-aqueous solution was added to the supernatant, shaken and mixed evenly, and left to stand at room temperature for 15 min to dissociate the ADH-binding peptide, centrifuged at 10000 r / min for 10 min, and the filtrate was collected to obtain the ADH-activated peptide TSE-PP solution.

[0031] In a preferred embodiment, the basic components of the selenium tea of the present invention were determined.

[0032] The moisture content was determined using an LSC-60 moisture analyzer (Shenyang Longteng) with reference to the method of GB / T 8304-2013. The total ash content was determined using the method of GB / T 8306-2013. The water extract of the tea was determined with reference to GB / T 8305-2013. The caffeine content of the tea was determined using the basic lead acetate precipitation method with reference to GB / T8312-2013. The polyphenol content in the tea was detected using the Folin-Ciocalteu method with reference to GB / T 8313-2018. The protein content in the tea was determined using the Kjeldahl method with reference to GB5009.5-2016 (Mæhre et al., 2018). With reference to the method of Stibilj et al. (Stibilj et al., 2003), the selenium content of the sample was determined using hydride generation atomic fluorescence spectrometry.

[0033] As attached Figure 1 shown, the main chemical components in the tea were detected in this study, and its potential nutritional value was evaluated. Attached Figure 1 Figure A shows that the protein content of the tea sample was 23.84 ± 1.73%, the polyphenol content was 19.41 ± 3.01%, the selenium content was 0.13 ± 0.03 mg / kg, the caffeine content was 3.55% ± 0.32, the free amino acid content was 3.71 ± 0.53%, the water extract was 38.65 ± 3.19%, and the ash content was 5.33 ± 0.66%. It shows that the tea is rich in a variety of important nutrients and has a high protein content, highlighting the potential of tea as a source of plant protein (Kumar et al., 2023). The protein content of the tea residue after dephenolization increased significantly ( Figure 1 Figure B), reaching 27.76 ± 2.26%, while the selenium content decreased slightly to 0.09 ± 0.01 mg / kg. This phenomenon may be related to the interaction between polyphenols and proteins. Studies have shown that polyphenols can form non-covalent complexes with proteins through hydrogen bonds and hydrophobic interactions, and some of the complexes are soluble in water, relatively reducing the content of free proteins. During the dephenolization process, water-soluble polyphenols are removed, and the proteins originally bound to the polyphenols are released and redeposited in an insoluble form, resulting in an increase in the proportion of insoluble proteins in the tea residue and an increase in the relative content of proteins (Kumaret al., 2023). At the same time, the decrease in selenium content may indicate that part of the selenium exists in the form of selenium polyphenols or selenium polysaccharide complexes, and these complexes are removed during the dephenolization process, resulting in a relatively lower selenium content in the tea residue (Ren et al., 2019).

[0034] The protein content of the tea leaves in this study (23.84 ± 1.73%) was similar to that of green tea protein (21.6%) reported by Wang et al. (2021), but higher than the measured value (19.5%) of ordinary tea leaves by Li et al. (2020). This may be related to the differences in tea varieties, planting environments, and processing methods (Zhang et al., 2020). In addition, the selenium content of the tea leaves (0.13 ± 0.03 mg / kg) was similar to the result (0.12 mg / kg) in the study of selenium-enriched tea by Wu et al. (2018), indicating that the tea leaves in this study have a certain selenium enrichment ability. In contrast, the increase in protein content after dephenolization treatment (to 27.76 ± 2.26%) was higher than the polyphenol-depleted protein enrichment rate (about 24%) in the study by Chen et al. (2017). The results show that tea leaves are not only rich in protein, but also the protein components can be further enriched through dephenolization treatment, improving their utilization value as a source of plant protein (Zhang et al., 2020; Ren et al., 2019).

[0035] In a preferred embodiment, to optimize the enzymatic hydrolysis process of the present invention, the gene sequence of tea glutelin was retrieved using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), translated into an amino acid sequence, and input into the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) to simulate the hydrolysis processes of different proteases (Minkiewicz et al., 2022). By calculating the degree of hydrolysis and the release potential of functional peptide segments under different enzymatic hydrolysis conditions, the optimal protease was screened and experimentally verified.

[0036] Tea protein is mainly composed of glutelin, accounting for more than 80% of the total protein. Therefore, glutelin was selected as a representative substrate to evaluate the effects of different proteases on its hydrolysis effect. As shown in Figure 2 Figure A, the effects of different proteases on the degree of hydrolysis of tea protein were predicted based on bioinformatics. The results show that when papain or alkaline protease was used alone, the degrees of hydrolysis were 41.2% and 31.8% respectively, while when the two were used in combination, the degree of hydrolysis was significantly increased to 55.9%. This result indicates that the composite enzyme system may produce a synergistic effect, making the hydrolysis of proteins more complete and releasing more bioactive peptides. As shown in Figure 2As shown in Figure B, to verify the reliability of the prediction, further experimental verification was carried out. When papain and alkaline protease were used in combination, the peptide extraction rate reached 53.99%, which was significantly higher than that when papain (42.11%) or alkaline protease (35.76%) was used alone. In addition, the activation rate of the peptide obtained under this condition for alcohol dehydrogenase (ADH) reached 56.89%, which had higher biological activity compared to the single enzymatic hydrolysis conditions (activation rates were 40.35% and 33.92% respectively). The hydrolysis efficiency of proteins and the biological activity of the peptides obtained are significantly affected by the specificity of the enzyme, the substrate structure, and the reaction conditions. This may be because papain, as an endopeptidase, mainly acts on the interior of the peptide chain, while alkaline protease has strong exopeptidase activity. The synergistic effect of the two optimized the protein hydrolysis pattern, enabling the release of shorter bioactive peptides.

[0037] In a preferred embodiment, the present invention optimized the enzymatic hydrolysis process of tea selenium-enriched protein peptide (TSE-PP) and investigated the effects of six factors on the ADH activation rate of TSE-PP: the ratio of composite enzymes (3:7, 2:3, 1:1, 3:2, 7:3, 4:1), enzymatic hydrolysis time (1, 2, 3, 4, 5, 6 h), temperature (35, 45, 55, 65, 75 °C), enzymatic hydrolysis pH (5, 6, 7, 8, 9, 10), enzyme dosage (500, 2000, 3500, 5000, 6500, 8000 U / g), and solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50, 1:60 g / mL). The fixed parameters were alkaline protease: papain (1:1), solid-liquid ratio 1:25 (g / mL), enzyme dosage 5000 U / g, pH 8.0, and extraction in a water bath at 55 °C for 4 h.

[0038] Based on the results of the single-factor experiments, with the ADH activation rate as the response value, four factors with greater influence were selected as the optimization variables. According to the BBD design principle, a four-factor three-level response surface model was constructed.

[0039] As shown in the appendix Figure 3 First, the effects of six single factors on the ADH activation rate of TSE-PP were evaluated. As shown in the appendix Figure 3 As shown in Figure A, as the proportion of alkaline protease increased, the ADH activation rate showed a trend of first increasing and then decreasing, reaching the maximum value (59.16%) when alkaline protease: papain = 3:2. This may be because alkaline protease can increase the exposure of hydrophobic amino acid residues in the peptide chain, and studies have shown that hydrophobic amino acids can promote ADH activation. However, excessive alkaline protease may lead to changes in the hydrolysis sites, affecting the final peptide composition. As shown in the appendix Figure 3As shown in Figure B, with the increase of enzymatic hydrolysis time, the activation rate of ADH by the generated TSE-PP increased and reached the maximum value (60.08%) at 5 h, showing no significant difference from that at 4 h and 6 h (p>0.05), indicating that the substrate had been fully hydrolyzed after 4 h. As attached Figure 3 As shown in Figure C, with the increase of enzymatic hydrolysis temperature, the ADH activation rate of TSE-PP first increased and then decreased, reaching the highest value (59.09%) at 55 °C. The enzyme activity might decrease due to thermal denaturation, resulting in a significant decrease in the ADH activation rate. As attached Figure 3 As shown in Figure D, the ADH activation rate of TSE-PP was the highest (59.88%) at pH 8.0. Since the optimal pH ranges of papain and alkaline protease were different, too high or too low pH might affect the enzyme activity and inhibit the reaction efficiency. As attached Figure 3 As shown in Figure E, with the increase of enzyme dosage, the ADH activation rate increased and tended to be stable after 5,000 U / g, indicating that the substrate binding sites had been basically saturated, and further increasing the enzyme dosage had limited effects. When the solid-liquid ratio was 1:30, the ADH activation rate of TSE-PP was the highest (59.64%) and then decreased. This might be because too high a solid-liquid ratio decreased the substrate concentration, affected the enzyme-substrate binding, and led to incomplete enzymatic hydrolysis. In summary, the determined optimal single-factor conditions were an enzyme compound ratio (alkaline protease: papain) of 3:2, an enzymatic hydrolysis time of 5 h, a reaction temperature of 55 °C, a pH value of 8.0, an enzyme dosage of 5,000 U / g, and a solid-liquid ratio of 1:30 (g / mL).

[0040] As shown in Table 1, based on the results of the single-factor experiments, the Box-Behnken response surface experimental design (RSM) was adopted to investigate the effects of four key variables, namely the enzyme compound ratio (A), enzyme dosage (B), pH value (C), and solid-liquid ratio (D), on the ADH activation rate. Taking the ADH activation rate (Y) as the response value, the following quadratic multiple regression equation was established: Y = 61.44 + 0.4275A + 4.42B + 0.4042C + 0.0850D + 0.7275AB + 0.3775AC - 0.0675AD - 0.5925BC - 0.2550BD - 0.0875CD - 1.15A² - 3.13B² - 4.20C² - 2.63D² Among them, Y represents the ADH activation rate, and A, B, C, and D represent the enzyme ratio, enzyme dosage, pH value, and solid-liquid ratio, respectively.

[0041] Table 1 Response surface optimization experimental design and ADH activation rate results As shown in Table 2, analysis of variance (ANOVA) indicated that the model had a strong goodness of fit (R² = 0.9949, adjusted R² = 0.9897), and the statistical significance test result of the regression equation (p<0.001) showed that the model had a strong predictive ability for the ADH activation rate. The enzyme addition amount (B) had the most significant effect on the ADH activation rate (p<0.001), followed by the enzyme compound ratio (A, p<0.01), and pH (C, p<0.01). In addition, the interaction analysis showed that there were significant interaction effects between AB and BC (p<0.01), indicating that their combination could further improve the ADH activation rate.

[0042] Based on the regression model analysis, the optimal preparation conditions were determined as follows: enzyme ratio 3:2, enzyme dosage 5700 U / g, pH 8.0, and solid-liquid ratio 1:29 (g / mL). Under these conditions, the ADH activation rate of the experimentally verified TSE-PP was 61.52±2.16%, which was consistent with the model prediction value (61.44%), indicating that the model had good predictive ability and reliability. In addition, the obtained TSE-PP had a protein content of 67.38±4.81% and a selenium content of 1.07±0.16 mg / kg, indicating that TSE-PP under the optimized conditions had high nutritional value.

[0043] Table 2 Analysis of Variance (ANOVA) for Regression Model Note: Indicates p<0.01, and the result is significant; Indicates that p<0.0001 is extremely significant.

[0044] In a preferred embodiment, the present invention performed LC-MS / MS analysis on the ADH activation peptide TSE-PP solution. Liquid chromatography analysis was carried out using an Easy-nLC 1200 nano liquid chromatography system (Thermo Fisher Scientific, USA) (Bheemanapally et al., 2020). Chromatographic separation was performed using a C18 reversed-phase column (Thermo Hypersil Gold, 2.1 × 150 mm, 1.9 μm). Solvent A: 0.1% formic acid aqueous solution, solvent B: 80% acetonitrile, the flow rate was set at 0.6 mL / min, and gradient elution was performed. Electrospray mass spectrometry (Q Exactive Orbitrap mass spectrometer, Thermo Fisher Scientific, USA), MS1 scan parameters: mass range 300–1800 m / z, resolution: 70,000, C-trap capacity 3 × 10 6, maximum injection time 100 ms, MS2 scan parameters: resolution: 17,500, C-trap capacity 1 × 10 5 , maximum injection time 50 ms, select the top 20 ions with the highest intensity for fragmentation, normalized collision energy 28. The original mass spectrometry data was processed using PEAKS Studio 10.6 (Bioinformatics Solutions Inc., Canada). Protein identification was completed by searching the UniProt and NCBI databases (FASTA format).

[0045] In a preferred embodiment, the present invention performs polypeptide activity scoring and molecular docking on TSE-PP. Use PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) to evaluate the bioactive potential of the peptides. Peptides with a score ≥ 0.5 are considered to have high bioactive potential and are selected for further analysis. Further screening is performed using AllerTOP v.2.0 and ToxinPred to exclude peptides with allergic or toxic properties, while preferentially selecting peptides with high intestinal absorption potential. The three-dimensional structure of the polypeptide is generated using Discovery Studio. The three-dimensional structure of ADH is obtained from the Protein Data Bank (PDB, http: / / www.rcsb.org / pdb / home / home.do, PDB ID: 5ENV). Removal of water molecules, addition of hydrogen atoms and format conversion are completed in AutoDock Tools for molecular docking simulation, and visualization is performed using pymol.

[0046] A total of 319 peptides were identified from TSE-PP by affinity ultrafiltration-liquid chromatography-mass spectrometry, among which 26 were selenium or selenomethyl-modified peptides and 240 were small molecule oligopeptides (<15AA). Small molecule peptides are considered potential functional molecules due to their strong target organ permeability and high binding ability. Further analysis of the amino acid composition of the peptides found that hydrophobic amino acids (such as Ala, Val, Leu, Ile, Pro, Met) were enriched in the peptides, which helped to enhance their interaction with lipid targets. In addition, aromatic and charged amino acids (Phe, Tyr, Glu, Arg) may promote ADH activity (Guo-Cai et al., 2011). Considering that the steric hindrance effect on peptide stability <0.55, 6 peptides were screened out, and 4 bioactive peptides with no toxicity / allergenicity / carcinogenicity / high stability, suitable for gastrointestinal absorption (HIA) but not penetrating the blood-brain barrier (BBB) were determined from them.

[0047] To investigate the interaction between TSE-PP and ADH, the binding energy was calculated by molecular docking. As shown in Table 4 below, the results indicate that the binding energy of the selenium-modified peptide is significantly lower than that of the non-selenium-modified peptide, suggesting a higher affinity for ADH. As shown in Figure 4 Appendix A, the selenium-modified peptide PSC(Se)PFC(SeMe)T forms 4 hydrogen bonds (2.5 Å, 2.3 Å, 2.4 Å, and 2.2 Å) at Glu333; as shown in Figure 4 Appendix B, PNM(Se)PPGS mainly acts on Arg298 and Arg302 to form 4 hydrogen bonds (2.0 Å, 2.2 Å, 2.4 Å, 2.1 Å). In contrast, as shown in Figures 4C and 4D of the attached drawings, the non-modified peptides APLLFPP and PSPPIVVPP form only 2-3 hydrogen bonds, and the hydrogen bond lengths are longer (>2.3 Å).

[0048] The number and length of hydrogen bonds are key factors affecting the stability of the receptor-ligand complex (Majewski et al., 2019). When the number of hydrogen bonds is large and the hydrogen bond distance is short (less than 3.5 Å), the interaction between the ligand and the receptor is more stable. The selenium-modified peptide can not only form more hydrogen bonds with ADH, but also has shorter hydrogen bond lengths, enhancing its binding stability with ADH. The selenium-modified peptide improves the binding stability with ADH by optimizing the spatial conformation, providing theoretical support for the potential activation of ADH.

[0049] In a preferred embodiment, the present invention analyzed the amino acid composition of TSE-PP.

[0050] The present invention found that TSE-PP contains 17 kinds of amino acids, and the composition ratio and content of various amino acids are shown in Table 3. The results show that TSE-PP is rich in amino acids such as glutamic acid, aspartic acid and leucine, accounting for 14.35%, 10.35% and 8.29% of the total amino acids, respectively. Among them, Glu and Asp, as acidic amino acids, create negatively charged and structurally flexible regions in the protein structure, which are crucial for protein stability and function (Chou and Wang, 2015). The ratio of essential amino acids (EAA) to non-essential amino acids (NEAA) is 62.50%, meeting the recommended standards of FAO / WHO for high-quality proteins (Romano et al., 2019), indicating that TSE-PP has high nutritional value. In addition, according to the amino acid composition of the FAO / WHO standard pattern, the sum of phenylalanine and tyrosine in TSE-PP is relatively high, approaching the whole egg pattern. The amino acid ratio coefficient (SRCAA) of the polypeptide is 73.61, significantly higher than 67.39 of soy protein, indicating that the amino acid composition of TSE-PP is more balanced, superior to soy protein, and meets the high-quality protein standard. In addition, the total content of umami amino acids (aspartic acid, glutamic acid) and sweet amino acids (serine, proline, glycine, phenylalanine, alanine, threonine) reaches 41.02 g / 100g, accounting for 58.37% of the total amino acids. It shows that TSE-PP may help to improve the food flavor.

[0051] Note: Essential Amino Acids (EAA) are marked with †.

[0052] Table 3 Amino acid composition and relative content of tea selenium protein peptide In a preferred embodiment, the present invention studied the anti-alcoholism effect of TSE-PP on acutely intoxicated mice.

[0053] Experimental design: Forty male mice (body weight 20 ± 2 g) were purchased from the Experimental Animal Center of the School of Medicine, Xi'an Jiaotong University (Shaanxi, China). Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th Edition, ISBN-10: 0-309-15396-4). During the experiment, the mice were housed at a temperature of 21 ± 2 °C and a relative humidity of 50 ± 10%, with a 12-hour light / dark cycle. The mice in each group had free access to food and water. After one week of adaptive feeding, the mice were randomly divided into 5 groups (n = 8 / group): control group CK (0.2 mL of normal saline), alcohol model group AM (17.5 mL / kg·bw of 52° Maotai-flavor liquor), low-dose treatment group TL (TSE-PP 0.25 g / kg·bw), high-dose treatment group TH (TSE-PP 1 g / kg·bw), and positive control group PC (silymarin 0.05 g / kg·bw). Except for the CK group, all the other mice were intragastrically administered 17.5 mL / kg·bw of 52° Maotai-flavor liquor for three consecutive days starting from the 19th day. During the experiment, the body weight of the mice was recorded every two days. After intragastric administration on the 21st day, the mice were fasted for 12 hours and then sacrificed by cervical dislocation.

[0054] Within 0.5 to 1 hour after the mice were given alcohol, their motor status was observed. When the mice showed slow movement or unsteady walking, the mice were turned over so that their backs were facing up, and the recovery time of the righting reflex was recorded. If the mice could not turn back to the normal posture twice within 15 seconds, they were judged to be drunk.

[0055] The blood samples were centrifuged at 4 °C and 3000 r / min for 10 minutes to obtain serum samples. After weighing the liver, it was divided into two parts. One part was used for histopathological analysis, and the other part was homogenized with pre-cooled normal saline to form 10% liver homogenate, and the supernatant was collected. According to the manufacturer's instructions, an enzyme-linked immunosorbent assay kit was used to measure the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC) in the serum, and superoxide dismutase (SOD), alcohol dehydrogenase (ADH), glutathione (GSH), and malondialdehyde (MDA) levels in the liver homogenate.

[0056] Fresh liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-5 μm thin sections, and stained with hematoxylin-eosin (H&E). The pathological structural changes of the liver tissue were observed using an optical microscope (×40, ×100 magnification) (Olympus, Tokyo, Japan).

[0057] As shown in Figure 5 the attachment, the effects of TSE-PP on the righting reflex behavior and liver tissue of acutely drunk mice. As shown in Figure 5 Attachment A shows the experimental design and sample intervention of acute mouse drunkenness experiment. As shown in Figure 5B shows the changes in the drunkenness rate and sobering-up time of mice in different experimental groups. The drunkenness rate of mice in the alcohol model group was 87.5%. The intake of silymarin and TSE-PP could significantly reduce the drunkenness of mice (p < 0.05) and shorten the sobering-up time of mice. Alcohol intake caused the inhibition of the central nervous system and the disorder of neuromuscular connections in mice, resulting in the weakening or disappearance of the righting reflex. Studies have shown that the administration of peptides from collagen and walnuts can rapidly reduce the blood alcohol concentration in rodent models.

[0058] As shown in the appendix Figure 5 C shows the effect of alcohol intake on the body weight of mice. The body weight of mice in the alcohol model group decreased significantly during the drunken stage and recovered slowly. This is consistent with Huang's study on peripubertal mice. Alcohol can cause metabolic disorders, leading to weight loss and energy utilization disorders (Huang et al., 2012). In contrast, the body weight of mice in the TSE-PP intervention group recovered significantly faster after alcohol intake, indicating that TSE-PP may promote weight recovery by regulating energy metabolism, but the specific mechanism still needs further study.

[0059] Liver injury is one of the important physiological responses to alcoholism. As shown in the appendix Figure 5 D shows the liver specific gravity coefficients of mice in different experimental groups. The liver specific gravity coefficient of mice in the alcohol model group was significantly higher than that of the blank group (p < 0.05), suggesting that alcohol intake led to liver enlargement. Compared with the model group, the liver specific gravity coefficients of the silymarin group and the high TSE-PP treatment group decreased significantly (p < 0.05).

[0060] As shown in the appendix Figure 5 As shown in E and F, through HE staining analysis of liver tissue morphology observation, the hepatocytes of mice in the alcohol model group were swollen, the hepatic cords were disordered, the hepatic sinusoids were dilated, and inflammatory cell infiltration was obvious. The arrangement of hepatocytes in the TSE-PP treatment group and the silymarin group was relatively regular, and the dilation of hepatic sinusoids and inflammatory reactions were significantly alleviated. TSE-PP may play a role in relieving alcohol intoxication and protecting the liver by alleviating hepatocyte damage and inhibiting inflammatory reactions.

[0061] In a preferred embodiment, the present invention studied the antioxidant protection and improvement of lipid accumulation of TSE-PP.

[0062] As shown in the appendix Figure 6 As shown, the regulatory effect of TSE-PP on antioxidant protection of liver tissue and liver lipid accumulation in drunken mice. As shown in the appendix Figure 6Figure A shows that the SOD activity in the livers of mice in the alcohol model group decreased significantly (p < 0.05), indicating that alcohol intake exacerbated liver oxidative stress. The SOD activity in the low-dose TSE-PP intervention group increased, but there was no significant difference (p > 0.05). In contrast, the silymarin group and the high-dose TSE-PP group significantly increased the SOD activity (p < 0.05). As an important antioxidant enzyme, SOD can scavenge free radicals and reduce oxidative damage. The generation of a large amount of reactive oxygen species (ROS) during alcohol metabolism can trigger liver oxidative stress response and exacerbate liver damage (Wu and Cederbaum, 2003). The significant increase in SOD activity by high-dose TSE-PP indicates that it effectively alleviated the oxidative stress induced by alcohol, thereby protecting the liver from damage. This result is consistent with the research on the antioxidant effect of plant-derived protein peptides

[120] .

[0063] Appendix Figure 6 Figure B shows that the GSH content in the livers of mice in the alcohol model group was significantly lower than that in the blank group (p < 0.05), indicating that alcohol intake led to a decrease in GSH content, thereby exacerbating oxidative stress. In contrast, the GSH content in the high-dose TSE-PP intervention group increased significantly (p < 0.05). As an important antioxidant molecule, GSH can scavenge excess ROS in the body by directly reacting with free radicals and participating in enzymatic catalytic reactions (Keen et al., 1976). This indicates that TSE-PP played an important role in alleviating the oxidative stress induced by alcohol and restored the body's antioxidant defense mechanism.

[0064] Appendix Figure 6 Figure C shows that the MDA content in the livers of mice in the alcohol model group was significantly higher than that in the blank group (p < 0.05), suggesting that alcohol caused liver lipid peroxidation. As the end product of lipid peroxidation, MDA is an important marker reflecting cell oxidative damage. There was no significant difference in the MDA level between the high-dose TSE-PP group and the blank group (p > 0.05), indicating that TSE-PP had a significant effect on reducing lipid oxidative damage. Lipid peroxidation damage caused by alcohol can interfere with lipid metabolism and promote liver fat accumulation, thereby exacerbating liver damage. The antioxidant effect of TSE-PP may inhibit lipid peroxidation and reduce alcohol-induced liver damage, further supporting its potential protective effect in alcoholic liver disease.

[0065] Appendix Figure 6Group D showed a significant decrease in ADH activity in the livers of mice in the alcohol model group (p < 0.05), with a decrease of 32.82%. As a key enzyme in the alcohol metabolism process, the decrease in ADH activity will lead to a slowdown in the ethanol metabolism rate. The decrease in ADH activity during alcohol metabolism may be related to the competitive binding of alcohol to the active site of ADH. In addition, alcohol exposure will also reduce the level of coenzyme NAD+, further affecting ADH activity. Compared with the alcohol model group, the ADH activity of mice in the high-dose TSE-PP group and the silymarin group was significantly increased, and there was no significant difference from the blank group (p > 0.05). This indicates that TSE-PP can enhance ADH activity to a certain extent, thereby promoting alcohol metabolism and reducing liver injury.

[0066] The utility of ALT and AST as liver health indicators in alcohol model studies. ALT and AST are key biomarkers for evaluating liver injury (Tan et al., 2017). Attached Figure 6 Figures E and 6F show that the levels of ALT and AST in the serum of mice in the alcohol model group were 2.92 times and 2.83 times that of the blank group (p < 0.05), indicating that alcohol-induced hepatocyte damage led to the leakage of these two transaminases, thereby increasing their serum levels. In contrast, the ALT and AST of mice in the low-dose and high-dose TSE-PP groups were significantly reduced, and were dose-dependent, with decreases of 26.77% and 43.13% (ALT) and 29.63% and 42.05% (AST), respectively. The results of this example show that TSE-PP has a significant liver protection effect and can reduce alcohol-induced hepatocyte damage. The decrease in ALT and AST further reflects the potential of TSE-PP in alleviating acute liver injury caused by alcohol.

[0067] The levels of TG and TC are significantly affected by alcohol consumption, and there is a complex relationship between alcohol and lipid metabolism (Clugston et al., 2011). Attached Figure 6 Figure G and attached Figure 6 Figure H show that the levels of TG and TC in the serum of mice in the alcohol model group were significantly higher than those in the blank group (p < 0.05), indicating that alcohol exposure led to lipid metabolism disorders. The TG in the serum of mice in the low-dose and high-dose TSE-PP groups decreased by 10.43% and 19.45%, respectively, and the TC decreased by 10.73% and 21% (P < 0.05). This result shows that TSE-PP can effectively regulate blood lipid levels, improve lipid accumulation, and reduce alcohol-induced liver damage.

[0068] In a preferred embodiment, the present invention studied the processing characteristics of tea selenium protein peptide.

[0069] Solubility Weigh 0.1 g of tea selenium protein peptide, add 10 mL of deionized water or ethanol, adjust the pH to 7.0 (or 2.0, 4.0, 6.0, 8.0, 10.0), stir magnetically for 30 min at 25 °C (or 4, 37, 65, 100 °C), centrifuge the mixture at 3000 r / min for 15 min, take the supernatant, and measure the protein content.

[0070] Figure 7A shows that the solubility of TSE-PP in deionized water is significantly higher than that in ethanol (p < 0.05). This may be because the high dielectric constant of water can shield electrostatic interactions and enhance solubility; while ethanol reduces the dielectric constant, enhances electrostatic attraction, causes polypeptide aggregation, and reduces solubility (Feng et al., 2021). This trend is consistent with the research result that soy protein peptides have better solubility in the aqueous phase (Zhang et al., 2019), indicating that the aqueous phase environment is more suitable for the food processing application of TSE-PP.

[0071] Figure 7B shows that the solubility of TSE-PP first increases and then decreases with the increase of temperature, reaching the maximum value at 65 °C, but decreasing at 100 °C. This may be because moderate heating destroys hydrogen bonds, unfolds the peptide chain, and improves solubility, while too high temperature induces polypeptide denaturation, exposes hydrophobic groups, enhances aggregation, and reduces solubility (Kleemann et al., 2020). This trend is similar to that of whey protein peptides and fish skin collagen peptides (Wang et al., 2022). It is recommended that the food processing temperature be controlled within 65 °C to maintain high solubility.

[0072] Appendix Figure 7 Figure 7C shows that the solubility of TSE-PP changes in a "V" shape with pH, being the lowest at the isoelectric point (pH 4.0 - 6.0) and rising when pH > 8.0. At the isoelectric point, the electrostatic repulsion weakens, molecules aggregate, and solubility decreases, while in an alkaline environment, the electrostatic repulsion increases, the peptide chain stretches, and solubility increases (Kleemann et al., 2020). This trend is similar to that of whey protein peptides and corn germ protein peptides (Li et al., 2021), indicating that TSE-PP is suitable for alkaline food systems to improve dispersibility and processing stability.

[0073] Emulsifying property and foaming property Dissolve 50 mg of tea selenium protein peptide in 15 mL of deionized water, stir evenly in a water bath at 25 °C (or 5, 45, 65, 85 °C), add 5 mL of rapeseed oil, and adjust the oil volume fraction to 5% (or 10%, 20%, 30%, 40%). Adjust the pH value to 7 (or 2.0, 4.0, 6.0, 8.0, 10.0), and homogenize at a high speed of 13000 rpm for 1 min to form an emulsion. Take 50 μL of the emulsion and add it to 5 mL of 0.1% SDS solution, shake evenly, measure the absorbance at 500 nm, and calculate the emulsifying activity index (EAI), emulsifying stability index (ESI), foaming capacity (FC), and foam stability (FS): EAI = 2×T×A0×N / C×φ×104 In the formula: T is the turbidity constant (2.303), A0 is the initial absorbance, N is the dilution factor, C is the solution concentration (g / mL), and φ is the oil phase ratio.

[0074] ESI = An / A0×100% In the formula: An is the absorbance after 20 min, and A0 is the initial absorbance.

[0075] FC = (V1 - V0) / V0×100% FS = (V2 - V1) / (V1 - V0)×100% In the formula: V0 is the initial volume, V1 is the foam volume after homogenization, and V2 is the foam volume after 20 min As can be seen from Figure 7D, the emulsifying property and emulsifying stability of TSE-PP first increase and then decrease with the increase of temperature, and reach the peak at 65 °C (emulsifying property 58.1 m² / g, emulsifying stability 71.99%). Appropriate heating can break hydrogen bonds, unfold the polypeptide structure, and enhance interfacial adsorption (Farjami et al., 2021). However, too high temperature (>65 °C) may cause polypeptide denaturation, enhance hydrophobic interaction, and reduce emulsifying ability (Wang et al., 2022). This trend is similar to that of whey protein peptide, indicating that TSE-PP has the best emulsifying performance below 65 °C and is suitable for thermally processed foods.

[0076] As can be seen from Figure 7E, the emulsifying property and stability of TSE-PP are the lowest at the isoelectric point (pH 4-6) and reach the maximum value under alkaline conditions (pH 10) (emulsifying property: 63.37 m² / g, emulsifying stability: 79.43%). The solubility is low at the isoelectric point, weakening the interfacial adsorption ability, while the electrostatic repulsion is enhanced in an alkaline environment, the peptide chain unfolds, and the emulsifying property is improved (Tang et al., 2023). This trend is similar to that of pea protein peptides, indicating that TSE-PP is suitable for alkaline food systems (such as protein beverages).

[0077] As Figure 7 can be seen from Figure 7F, the emulsifying property and emulsifying stability of TSE-PP first increase and then decrease with the increase of oil volume fraction. The emulsifying property is the highest (60.28 m² / g) at an oil volume fraction of 10%, and the emulsifying stability is the best (80.05%) at 20%. At high oil content (>40%), the emulsifying particles decrease, the interfacial adsorption is insufficient, and the emulsifying property decreases (Olsmats and Rennie, 2024). This trend is consistent with that of fish skin collagen peptides (Lima et al., 2023), suggesting that TSE-PP is suitable for low to medium oil content foods, such as emulsified protein drinks.

[0078] Environmental stability Prepare a 5 mg / mL tea selenium protein peptide solution, place it in a water bath at 25°C (or 4, 37, 80, 100°C), and adjust the pH to 7 (or 3, 5, 9, 11). Add 0% (or 2%, 4%, 6%, 8%, 10%) NaCl, glucose, and citric acid solutions to the solution, and let it stand for 2 h (or 1, 2, 4, 8, 16, 30 d), and measure its ADH activation rate.

[0079] As can be seen from Figure 7G, the foaming property of TSE-PP first increases and then decreases with the increase of temperature, and reaches the maximum value (137.4%) at 45°C. Moderate heating enhances the hydrophobic interaction and swelling rate. At the same time, partial thermal denaturation promotes the interfacial adsorption of polypeptides, improving the foaming property. However, when the temperature further increases, the polypeptide molecules aggregate, too many hydrophobic groups are exposed, and the interfacial adsorption ability decreases, resulting in a decrease in the foaming property. In addition, the foam stability is inversely proportional to the foaming property. The former is affected by hydrophilicity, while the latter is related to hydrophobicity (Li et al., 2019). This phenomenon is consistent with the research results of whey protein peptides, that is, moderate heating can improve the foaming property, but too high a temperature will reduce the foam stability (Wang et al., 2021).

[0080] As can be seen from the attached Figure 7It can be seen that the foaming ability of TSE-PP first decreases and then increases with the change of pH, reaching the maximum value at pH 10. This may be because when far from the isoelectric point, the solubility of the polypeptide increases, enhancing the water-air interface interaction and improving the foaming ability. However, high pH also weakens the foam stability, probably due to the decrease in solution viscosity, thinning of the foam film, and easier rupture (Tang et al., 2023). This trend is similar to the research on pea protein peptides, indicating that an alkaline environment is more conducive to the foaming ability of polypeptides (Zhang et al., 2020).

[0081] As shown Figure 7 It can be seen that the foaming ability of TSE-PP increases with the increase of concentration and reaches the maximum value (157.57%) at 40 mg / mL, but the foam stability decreases to 24.5%. Higher concentrations increase the number of surface-active molecules and enhance bubble formation, but excessive polypeptides may cause foam coarsening and reduce its stability (Jackman et al., 2018). This trend is consistent with the research results of whey protein peptides and soy protein peptides, that is, high concentrations contribute to foaming but may reduce foam persistence (Wang et al., 2022). As shown Figure 7 It can be seen that the ADH activation rate of TSE-PP first increases and then decreases with the increase of temperature, reaching the highest (63.67%) at 37 °C. However, the activity decreases significantly after 80 °C and is only 34.04% at 100 °C, probably because high temperature causes changes in the secondary structure of the polypeptide, weakening its binding ability to ADH (Masson and Lushchekina, 2022). This phenomenon is consistent with the research on whey protein peptides by Wang et al. (2020), that is, high temperature easily causes protein structure denaturation, thereby reducing its functional activity. Therefore, during food processing, it is recommended to control the temperature not to exceed 37 °C and avoid long-term high-temperature treatment to maintain the biological activity of TSE-PP.

[0082] As shown Figure 7 It can be seen that the ADH activation rate of TSE-PP first increases and then decreases with pH, reaching the highest (59.55%) at pH 7.0 and the lowest (37.72%) at pH 3.0. This may be related to the fact that TSE-PP is mainly composed of alkali-soluble proteins and is prone to denaturation in an acidic environment, affecting ADH activity (Zhang et al., 2021). Therefore, TSE-PP is suitable for processing and storage in a neutral or weakly alkaline environment to maintain its optimal biological activity. The solubility of soy protein isolate is poor at neutral pH values, but its solubility increases significantly under acidic and alkaline conditions (TD et al., 2021).

[0083] As shown in Figure 7 Figure L, when stored at 25 °C for 16 days, the ADH activation rate of TSE-PP only decreased by 4.63%, and decreased by 10.39% after 30 days, still remaining above 50.47%. This indicates that it has good stability under normal temperature conditions and can be used as a functional food ingredient for a long time. This result is similar to the research on collagen peptides by Zhou et al. (2022), that is, appropriate storage conditions can effectively maintain the biological activity of polypeptides.

[0084] Food additives may affect the stability of TSE-PP by regulating pH and ionic strength. As shown in Figure 7M, citric acid, sodium chloride, and glucose all decreased the ADH activation rate of TSE-PP, and the influence intensity was in the order of: citric acid > sodium chloride > glucose. When the addition amount of citric acid reached 10%, the ADH activation rate decreased by 26.06%, probably because citric acid significantly decreased the pH, affecting the structure of TSE-PP. This trend is consistent with the research of Liu et al. (2020) on the interaction between food proteins and acidic additives. Therefore, citric acid should be avoided in food processing, and the dosage of sodium chloride should be controlled to maintain the functional activity of TSE-PP.

[0085] In the above specific implementation, the Adh activity was measured with reference to the method of Wang et al. (Wang et al., 2020) and slightly modified. Briefly, 1.5 mL of 32 mmol Na4P2O7 buffer (pH 8.8), 1.0 mL of 27 mM NAD+, 0.5 mL of 11.5% ethanol (v / v), and 0.1 mL of the sample solution were thoroughly mixed, placed in a 25 °C water bath for 5 minutes, then 0.1 mL of 0.25 U / mL ADH was added, mixed evenly, and the absorbance was measured at 340 nm (in the control group, 0.1 mL of distilled water was used instead of the sample, and in the blank group, 0.5 mL of distilled water was used instead of 11.5% ethanol). The ADH activation rate was calculated using the equation: ADH activation rate = [(Vs - Vb) - (Vc - Vb)] / (Vc - Vb) × 100% where Vs is the absorbance value of the sample group, Vb is the absorbance value of the blank group, and Vc is the absorbance value of the control group In the above specific implementation, the amino acid composition analysis referred to the research of Mant et al. (Mant et al., 2007), and the amino acid composition was determined using an automatic amino acid analyzer (Biochrom, Cambridge, UK). After the sample was hydrolyzed with acid, it was dissolved and filtered, and then the hydrolyzate was subjected to cation exchange chromatography and ninhydrin derivatization.

[0086] In the above specific implementation, the trichloroacetic acid method was used to determine the extraction rate of polypeptides, and the extraction rate of TSE-PP was calculated (Fan et al., 2023). Bovine serum albumin (BSA) was used as a standard product to prepare a series of standard solutions with a concentration range of 0–4 mg / mL, and a standard curve was plotted. Take 1 mL of 1 mg / mL sample aqueous solution, add an equal volume of 0.15 mg / mL trichloroacetic acid solution, mix well, let stand for 20 min, and centrifuge the mixture at 4000 r / min at 4 °C for 10 min. Take 1 mL of the supernatant and add 4 mL of biuret reagent, incubate in a water bath at 60 °C for 5 min, and measure its OD value at 540 nm. Calculate the polypeptide mass through the standard curve, and calculate the polypeptide extraction rate: Polypeptide extraction rate (%) = total polypeptide mass / total tea protein mass × 100% In the above specific implementation, all experimental data were obtained through at least three independent experiments, and the results were expressed as mean ± standard deviation (SD). SPSS statistical software (IBM SPSS Statistics, Version 26.0) was used for data analysis, and one-way analysis of variance (ANOVA) was used to test the differences between groups. For results with significant differences, Tukey's post hoc test was used for further comparison. A p value less than 0.05 was considered statistically significant. GraphPad Prism 9.4 software was used for drawing charts.

[0087] In summary, the present invention optimized the preparation process of tea selenium protein peptides in selenium-enriched tea and systematically evaluated their potential nutritional value and processing characteristics. The optimized preparation conditions were papain and alkaline protease (3:2), enzyme dosage 5700 U / g, pH 8.0, and solid-liquid ratio 1:29 (g / mL). The weight-average molecular weight of the obtained TSE-PP was 1214 Da, the ADH activation rate was 61.52±2.16%, the protein content was 67.38±4.81%, and the selenium content was 1.07±0.16 mg / kg.

[0088] Amino acid analysis showed that TSE-PP contained 17 kinds of amino acids, of which essential amino acids accounted for 38.46%, and the amino acid ratio coefficient score was 73.61, which was better than that of soy protein and met the FAO / WHO ideal protein standard, showing high-quality nutritional value. Selenium-modified peptides PSC(Se)PFC(SeMe)T and PNM(Se)PPGS enhanced the binding force of ADH by forming 4 hydrogen bonds, providing a molecular mechanism support for its anti-alcohol effect.

[0089] Animal experiments have shown that TSE-PP significantly promotes ethanol metabolism, reduces oxidative stress, restores the activity of alcohol-metabolizing enzymes, decreases the ALT / AST levels, improves liver lipid accumulation, and exerts the effects of relieving hangover and protecting the liver, especially with significant effects in the high-dose group. The processing characteristics show that TSE-PP has good solubility (≥60%), emulsifying property (≥50 m² / g), and foaming property (≥100%) in the ranges of 25-121 °C and pH 6.0-10.0, indicating good industrial adaptability and food processing potential.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A method for preparing a selenium tea alcohol dehydrogenase activating peptide combination, characterized in that: The steps include: S1. Dephenolization of selenium-containing tea The selenium-containing tea leaves are dried, crushed, sieved and set aside; Adding the reserved selenium-containing tea leaves to ultrapure water, fully heating in a boiling water bath, repeating for multiple times, and drying the residue to obtain dephenolized tea residue; S2. Preparation of tea selenoprotein peptides Mixing the dephenolized tea residue with an alkaline solution, performing ultrasonic extraction, and combining the extracts after multiple extractions; The pH of the extract is adjusted to acidic, the precipitate is collected by centrifugation, the precipitate is washed with deionized water until neutral, and then freeze-dried to obtain tea selenium protein powder; The tea selenoprotein powder was fully dissolved in ultrapure water, and protease was added to fully hydrolyze it, and then freeze-dried to obtain a crude TSE-PP solution; S3. Preparation and purification of ADH-activating peptide TSE-PP The TSE-PP and ADH are fully mixed and co-cultured at a constant temperature to promote the binding of ADH with the polypeptide receptor to obtain an ADH-binding peptide mixed solution; The ADH binding peptide mixed solution was separated to obtain a supernatant, and the supernatant was added with an acetonitrile-water solution, shaken and mixed, and allowed to stand at room temperature to dissociate the ADH binding peptide; After standing, the filtrate was collected by centrifugation to obtain the ADH activated peptide TSE-PP combined filtrate.

2. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 1, characterized in that: The protease is one or both of alkaline protease and papain.

3. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 1, characterized in that: The dephenolized tea residue was mixed with 0.3 M NaOH solution at a solid-liquid ratio of 1:20 (g / mL), and extracted at an ultrasonic power of 100 W and a temperature of 50°C for 90 min. The extraction was repeated three times.

4. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 3, characterized in that: Adjust the pH of the extract to 4.

2.

5. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 1, characterized in that: Use PBS buffer at pH 7.4 to prepare 10 mg / mL tea selenoprotein peptide solution and 2 U / mL ADH solution and mix them thoroughly.

6. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 1, characterized in that: The water bath incubation was carried out at a constant temperature of 37°C. The ADH-binding peptide mixed solution was placed in a 10 kDa ultrafiltration centrifuge tube and centrifuged at 10,000 r / min for 10 min at 4°C to separate the supernatant.

7. The method for preparing a selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 2, characterized in that: According to the mass percentage of alkaline protease: papain = (1-7): (1-7), ADH enzyme dosage 500-8000 U / g, extraction pH 5-10, dephenolized selenium tea eggs: ultrapure water = 1: (10-60) g / mL, and protease hydrolysis time is 4-6h.

8. A selenium tea alcohol dehydrogenase activating peptide combination, characterized in that: The method for preparing a selenium-tea alcohol dehydrogenase activating peptide combination according to claims 1-7 is used.

9. A selenium tea alcohol dehydrogenase activating peptide combination as claimed in claim 8, characterized in that: Including non-selenium-modified peptide APLLFPP, non-selenium-modified PSPPIVVPP2, selenium-modified peptide PSC(Se)PFC(SeMe)T and selenium-modified peptide PNM(Se)PPGS.

10. An application of a selenium tea alcohol dehydrogenase activating peptide combination, characterized in that: Includes the following applications: (1) Application in functional foods that regulate ethanol metabolism, relieve liver cell damage, inhibit inflammatory response, relieve oxidative stress, and improve liver lipid accumulation; (2) Application in alkaline foods; (3) Application in functional foods for sobering up; (4) Application in improving food flavor; (5) Application in food in aqueous environment; (6) Application in thermally processed foods; (7) Application in low to medium fat foods.

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