Plukenetia volubilis linneo meal protein peptide, antioxidant polypeptide, preparation method and application

Abstract

The invention belongs to the technical field of active peptide production and application, and discloses a preparation method and application of sacha inchi meal protein peptide with antioxidant activity. According to the method, the sacha inchi meal is used as a raw material, the sacha inchi meal protein peptide is prepared through a single-stage alkaline proteolysis process, the operation process is simple, the cost is low, the excellent effect that the peptide yield reaches up to 62.33% is achieved, and meanwhile the high antioxidant activity of the product is guaranteed. The prepared plukenetia volubilis linneo meal protein peptide shows outstanding small molecule characteristics: the proportion of peptide fragments with the molecular weight of 1500 kDa or below exceeds 60%, and the bioavailability is greatly improved; under the concentration of 1 mg / mL, the ABTS free radical scavenging capacity reaches (91.08 + / -0.38)%, and through synergistic compounding with other plant peptides, not only is the antioxidant activity further enhanced, but also the original bitter taste is effectively improved, and the palatability of the product is improved.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation and application, and specifically relates to a method for preparing and applying sacha inca pulp protein peptides and antioxidant polypeptides. Background Technology

[0002] Sacha inchi is a novel food resource. Sacha inchi pulp, a byproduct of oil extraction, is extremely high in protein and can be used to prepare protein peptides.

[0003] For example, the literature "Optimization of Enzymatic Preparation Process of Sacha Inchi Peptides Combined with Pretreatment Methods" discloses the following: screening pretreatment methods that can promote protein hydrolysis, optimizing the enzymatic peptide preparation process of Sacha Inchi peptides using response surface methodology, and analyzing the relative molecular weight of the peptides obtained by this method. The aforementioned literature focuses on how to improve the extraction rate of Sacha Inchi protein peptides, but does not mention how to obtain protein peptides or antioxidant peptides with excellent antioxidant functions.

[0004] There are also patents that have produced saponin fruit pulp peptides with antioxidant activity through microbial fermentation technology. However, this method requires fermentation, which results in a long production cycle. In addition, the required peptide solution concentration is relatively high to achieve the highest antioxidant activity, which is costly.

[0005] Therefore, further research is needed on the aforementioned Sacha inchi pulp to invent a Sacha inchi pulp protein peptide or antioxidant peptide with excellent antioxidant activity. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a Sacha Inchi pulp protein peptide with antioxidant activity, as well as a method for preparing the aforementioned protein peptide and its applications.

[0007] The *Sapindus mukorossi* pomace protein peptides provided by this invention contain antioxidant polypeptides having the following 81 amino acid sequences: WEF, YRPF, YEF, LPSF, RLLF, KWF, WLE, FRF, LEW, RWE, KFRP, WRL, DFF, LRPY, RPY, LFDL, VFRP, QAW, PKYF, WRPQ, RFP, LRYL, RLY, LPAR, EWL, LRY, RYL, TWR, EMW, VDF, DRVF, VDFL, SDFL, FDR, KFP, FYR. , KPF, FRP, LEF, LGLR, KWL, KLW, RHF, SEDYLAHLL, LFTR, KHGF, FSR, RLF, FRL, DLRL, SRLL, YGR, RQW, SDLF, WVR, LFK, PKF, L KF, KLF, KWP, YRW, LLRP, KFN, YRLL, RFK, MKF, LLR, PLLR, FRS, WRY, YRL, LPR, LRL, RLP, FKL, VRF, GFK, KDW, CPR, MLR, KRF.

[0008] The above-mentioned method for preparing Sacha Inchi pulp protein peptides includes the following steps:

[0009] Step (1): Crush the sacha inchi pulp, sieve, dry, and defatted to obtain defatted sacha inchi pulp powder;

[0010] Step (2): Take defatted saponin fruit meal powder, add distilled water at a material-to-liquid ratio of 1g:10mL~1g:30mL, add alkaline protease at an enzyme-to-saponin fruit meal powder ratio of 7500~8500 U / g, enzymatically hydrolyze at 45~65℃ for 3.5~5.5h, and then inactivate the enzyme at 95~100℃ for 10~15min to obtain the enzymatic hydrolysate;

[0011] Step (3): Centrifuge the enzymatic hydrolysate at 3000~4000r / min for 10~25min, take the supernatant, and obtain the crude peptide solution of Sacha indica;

[0012] Step (4): Filter the crude peptide solution of Sacha indica through a membrane, pass it onto a chromatography column, collect the eluent, concentrate and dry it under vacuum to obtain Sacha indica meal protein peptides.

[0013] The preparation method of the above-mentioned antioxidant peptides includes the following steps:

[0014] S1: The amino acid sequences of the above 81 peptide segments were synthesized using solid-phase synthesis to obtain crude antioxidant peptides;

[0015] S2: The crude antioxidant peptide obtained in S1 was dissolved in a 50% (v / v) acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the purified antioxidant peptide. During RP-HPLC purification, mobile phase A was a 0.1% (v / v) formic acid aqueous solution, and mobile phase B was an acetonitrile aqueous solution containing 0.1% (v / v) formic acid, wherein the acetonitrile concentration in the acetonitrile aqueous solution was 84%.

[0016] The application of sacha incogenesis protein peptides or antioxidant polypeptides in food processing, including but not limited to their application in solid beverages, is also a key focus of this invention. Solid beverages can be coffee or other types of drinks.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The sacha indica pulp protein peptides / antioxidant peptides prepared by the present invention have significant small molecule characteristics and high antioxidant activity: the proportion of small molecule peptides with a molecular weight of less than 1500kDa is over 60%, and the bioavailability is significantly improved; at a concentration of 1mg / mL, the ABTS free radical scavenging ability reaches (91.08±0.38)%, which is much higher than the 40.55% scavenging efficiency of the existing enzymatic hydrolysis process at 2.0mg / mL for DPPH free radicals;

[0019] Furthermore, the binding characteristics of Sacha Inchi peptides to the key antioxidant receptor protein Keap1 were predicted and analyzed using molecular docking simulation technology. The results showed that the docking energies of the core peptides WEF and YRPF with Keap1 were as low as -10.4 kcal / mol and -10 kcal / mol, respectively, which confirmed their high affinity binding ability to key proteins in the antioxidant pathway at the molecular level.

[0020] (2) The process cost of the present invention is low and the operation is simple. The peptide yield is as high as 62.33%, which is significantly better than the peptide yield of 56.53% of the existing fermentation technology and the peptide yield of about 45% of the traditional single-stage enzymatic hydrolysis process.

[0021] (3) When the Sacha indica pulp protein peptide / antioxidant peptide of the present invention is applied to food, especially coffee solid beverage, the synergistic combination with other plant peptides enhances the antioxidant activity and improves the original bitter taste, thereby improving the palatability of the product. Attached Figure Description

[0022] Figure 1 This is a graph showing the effect of different enzymatic hydrolysis temperatures on peptide yield and antioxidant activity in this invention.

[0023] Figure 2 This is a graph showing the effect of different enzymatic hydrolysis times on peptide yield and antioxidant activity in this invention.

[0024] Figure 3 This is a graph showing the effect of different feed-to-liquid ratios on peptide yield and antioxidant activity in this invention.

[0025] Figure 4 This is a graph showing the effect of different enzyme addition amounts on peptide yield and antioxidant activity in this invention.

[0026] Figure 5 This is a response surface methodology diagram illustrating the interaction between enzymatic hydrolysis temperature and enzymatic hydrolysis time in this invention.

[0027] Figure 6 This is a response surface methodology diagram illustrating the interaction between enzymatic hydrolysis temperature and liquid-to-material ratio in this invention.

[0028] Figure 7 This is a response surface methodology diagram illustrating the interaction between enzymatic hydrolysis time and liquid-to-material ratio in this invention.

[0029] Figure 8 This is a graph showing the DPPH free radical scavenging ability of sacha inca pulp peptides with different polypeptide contents in this invention;

[0030] Figure 9 This is a graph showing the free radical scavenging ability of ABTS peptide from sacha inca pulp with different polypeptide contents in this invention;

[0031] Figure 10 This is a diagram showing the molecular docking results between WEF and Keap1 protein in this invention;

[0032] Figure 11 This is a diagram showing the molecular docking results of YRPF and Keap1 protein in this invention.

[0033] Figure 12 Primary mass spectrum of functional peptides from sacha inchi pulp;

[0034] Figure 13 This is a molecular weight distribution diagram of peptide fragments in Sacha Inchi pulp.

[0035] Figure 14 The results are obtained by high-performance liquid chromatography (HPLC) determination of the synthetic peptide WEF(a);

[0036] Figure 15 The results are from the high-performance liquid chromatography (HPLC) determination of the synthetic peptide YRPF.

[0037] Figure 16 MS assay results for the WEF of the synthetic peptide;

[0038] Figure 17 MS assay results for the synthetic peptide YRPF;

[0039] Figure 18 The results validate the hydroxyl radical scavenging activity of the synthetic peptide. Detailed Implementation

[0040] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0041] Example 1

[0042] The preparation of *Sapindus mukorossi* fruit meal protein peptides includes the following steps:

[0043] Step (1): Grind the sacha indica fruit pulp sample into powder using a food-grade high-speed blender, sieve it through a 100-mesh sieve, spread it evenly on an iron tray, and dry it in an electric heating drying oven at 105℃ for 2 hours; then degrease it by continuously refluxing for 7 hours using Soxhlet extraction, and then place it in a fume hood until the petroleum ether evaporates and it is completely dried to obtain the degreased sacha indica fruit pulp sample, which is then sealed and stored for later use.

[0044] Step (2): Distilled water was added to the defatted saponin fruit meal sample according to the material-liquid ratio of 1:19. Alkaline protease was added at a rate of 8000 U / g saponin fruit meal powder. The temperature was maintained at about 59℃ during the enzymatic hydrolysis process, and food-grade sodium hydroxide was added. The enzymatic hydrolysis time was 4.85 h at pH 8.0. After enzymatic hydrolysis, food-grade citric acid was added to adjust the pH value to 7.0. After the enzymatic hydrolysis was completed, the alkaline protease was inactivated by heating at 100℃ for 15 min to obtain saponin fruit meal hydrolysate.

[0045] Step (3): Take the enzymatic hydrolysate and centrifuge it in a centrifuge at 3500 r / min for 20 min to remove insoluble matter, and obtain the clear upper layer of enzymatic hydrolysate. Repeat the above process twice for the residue. Keep the clear upper layer of peptide solution from multiple times, mix them, and obtain the crude peptide solution of Sacha indica. Vacuum filter the crude peptide solution of Sacha indica and concentrate it under reduced pressure at 50℃ using a rotary evaporator. The vacuum degree is controlled at close to 0 MPa until the volume of the crude peptide solution is reduced to about 1 / 3 of the original volume, and Sacha indica meal protein peptide concentrate is obtained. Store the concentrate in a refrigerator at -20℃ for later use.

[0046] Step (4): The above crude peptide solution of Sacha indica is filtered through an ultrafiltration tube with a molecular weight cutoff of 1 kDa, and then centrifuged at 8000 r / min at 4℃ for 15 min to obtain a polypeptide component with a molecular weight of <1 kDa, which is stored at -20℃.

[0047] Example 2

[0048] The specific operation process is the same as in Example 1, except that the enzymatic hydrolysis temperature, enzymatic hydrolysis time, material-liquid ratio and enzyme addition amount are different. The specific parameters are shown in Table 1, and the yield and antioxidant activity under different conditions were measured.

[0049] Table 1. Single-factor level table

[0050] Depend on Figure 1 It can be seen that the yield of peptides and the ABTS free radical scavenging ability of sacha inchi pulp gradually increase with increasing temperature. When the enzymatic hydrolysis temperature reaches 60℃, the peptide yield is 55.98% and the ABTS free radical scavenging rate is 52.38%, indicating the optimal enzymatic hydrolysis effect. When the temperature continues to rise, both indicators decrease. Further response surface methodology experiments were conducted at 55℃ and 65℃.

[0051] Depend on Figure 2 It can be seen that the peptide yield and ABTS free radical scavenging ability gradually increase over time. The peptide yield is approximately at its maximum when the enzymatic hydrolysis time reaches 5 hours; there is no significant difference in peptide yield between 5 hours and 6 hours of hydrolysis. However, when the hydrolysis time is between 4 and 6 hours, the ABTS free radical scavenging rate decreases significantly from 57.73% to 50.92%, indicating a reduction in antioxidant activity. Therefore, subsequent response surface methodology experiments were conducted using 3-hour and 4-hour hydrolysis times.

[0052] Depend on Figure 3 It was found that the highest peptide yield (50.47%) was achieved when the ratio of sacha inoculum pulp to water was 1:20; the ABTS free radical scavenging ability was close to its maximum at ratios of 1:15 and 1:20. When the proportion of sacha inoculum pulp was too high, the powdered solid sample could not fully contact the alkaline protease, resulting in incomplete reaction and poor enzymatic hydrolysis; when the proportion was too low, the peptide concentration was low, and the ABTS free radical scavenging ability also decreased. Therefore, subsequent response surface methodology experiments were conducted using ratios of 1:15 and 1:25.

[0053] Depend on Figure 4 It was found that increasing the amount of alkaline protease added increased the peptide yield and ABTS free radical scavenging ability. When the enzyme addition reached 8000 U / g, the rate of increase for both indicators decreased, and there were no significant differences among the last three groups of data. When the enzyme addition was greater than 8000 U / g, the peptide yield increased to 57.78% and then decreased to 57.51%; while the ABTS free radical scavenging rate gradually increased, reaching 59.74% when the enzyme addition was 12000 U / g. Considering economic and other factors, an enzyme addition of 8000 U / g was selected as the optimal enzymatic hydrolysis condition.

[0054] Example 3

[0055] This embodiment provides a response surface methodology for optimizing the enzymatic hydrolysis conditions of Sacha Inchi pulp to improve yield and antioxidant efficacy, specifically including the following steps:

[0056] Based on the single-factor experimental results obtained in Example 1, a response surface methodology was conducted. The design factors and levels of the response surface methodology are shown in Table 2.

[0057] Table 2 Response Surface Factors and Level Coding Table

[0058] Data analysis using DesignExpert yielded a quadratic multinomial regression equation between peptide yield and the three factors: Peptide yield =

[0059] 0.62-0.013A+0.015B-0.016C+0.002822AB-0.001992AC+0.007636BC-0.022A 2 -0.007520B 2 -0.020C 2 .

[0060] Table 3 Response Surface Experimental Design and Results

[0061] Table 4. Analysis of variance for each term in the regression equation

[0062] Table 4 shows that the p-values ​​for A (enzymatic hydrolysis temperature), B (enzymatic hydrolysis time), and C (liquid-to-solid ratio) in the linear term are all <0.0001, indicating that these three factors have a highly significant effect on the peptide yield of *Sapindus mukorossi* fruit meal. The model p-values ​​are highly significant; the p-values ​​for the lack-of-fit terms are not significant; the correction coefficient R² = 0.9856 differs from the coefficient of determination R²Adj = 0.9672 by less than 0.2, indicating a small fitting error with the actual values. Therefore, the model can well reflect the influence of the three factors on the peptide yield of *Sapindus mukorossi* fruit meal.

[0063] The magnitude of the F-values ​​reflects the degree of influence of individual factors on the yield of sacha inchi pulp peptides. C > B > A, meaning that the liquid-to-solid ratio has the greatest impact on the yield of sacha inchi pulp peptides, followed by enzymatic hydrolysis time, while enzymatic hydrolysis temperature has the least impact.

[0064] By analyzing the experimental results using software, the interactive effects of the yield of sacha inchi pulp peptides on the three selected factors can be obtained. The results are shown in [Figure 1]. Figure 5 , 6 And 7.

[0065] like Figure 5 , 6 In the interaction term BC (enzymatic hydrolysis time versus liquid-to-solid ratio) shown in Figure 7, the surface slope on the liquid-to-solid ratio side is larger, indicating that the liquid-to-solid ratio has a greater impact. The contour lines are densely arranged and their shape is more similar to an ellipse, indicating that the two interactions have a significant impact on the yield of saponin fruit meal peptides. In the interaction term AB (enzymatic hydrolysis temperature versus enzymatic hydrolysis time), the surface slope on the enzymatic hydrolysis time side is larger, indicating that the enzymatic hydrolysis time has a greater impact. The contour lines and Figure 6Compared to a more circular shape, the interaction between the two factors has no significant effect on the yield of peptides from sacha inchi pulp. In the interaction term AC (enzymatic hydrolysis temperature and liquid-to-solid ratio), the surface on the liquid-to-solid ratio side has a larger inclination, indicating that the liquid-to-solid ratio has a greater impact. The contour lines are closer to a circle in shape, and the interaction between the two factors has no significant effect on the yield of peptides from sacha inchi pulp.

[0066] Based on model calculations, the optimal process parameters for enzymatic hydrolysis of *Gynostemma pentaphyllum* peptides are determined to be: a hydrolysis temperature of 58.89℃, a hydrolysis time of 4.84h, and a material-to-liquid ratio of 1:18.87. Under these conditions, the predicted peptide content is 63.4%. Considering actual production needs, the hydrolysis conditions were adjusted to: a hydrolysis temperature of 59℃, a hydrolysis time of 4.85h, a material-to-liquid ratio of 1:19, and an enzyme dosage of 8000 U / g.

[0067] Example 4

[0068] A method for preparing sacha indica pulp protein peptides with antioxidant activity, the specific steps of which are as follows:

[0069] Step (1): Same as in Example 1;

[0070] Step (2): Distilled water was added to the defatted saponin fruit meal sample according to the material-liquid ratio of 1:10. Alkaline protease was added at a rate of 8500 U / g saponin fruit meal powder. The temperature was maintained at 65℃ during the enzymatic hydrolysis process, and food-grade sodium hydroxide was added to maintain the pH at 8.0. The enzymatic hydrolysis time was 3.5h. After enzymatic hydrolysis, food-grade citric acid was added to adjust the pH to 7.0. After the enzymatic hydrolysis was completed, the alkaline protease was inactivated by heating at 100℃ for 15min to obtain saponin fruit meal hydrolysate.

[0071] Step (3): Centrifuge the sacha inophyllum meal enzymatic hydrolysate at 4000 r / min for 10 min, remove the insoluble matter after enzymatic hydrolysis, and leave the upper clear polypeptide solution to obtain sacha inophyllum crude peptide solution;

[0072] Step (4): Same as in Example 1.

[0073] Test Example 1

[0074] Weigh out the sacha inophyllum protein peptide powder prepared in Example 1, and calculate that the yield of sacha inophyllum protein peptide is 62.33%.

[0075] Test Example 2

[0076] The molecular weight distribution of the sacha inchi pulp protein peptides obtained in Example 1 was determined by high performance liquid chromatography. The results are shown in Table 5. Small molecule peptides with a molecular weight of less than 1500 Da accounted for 70%, indicating that the enzymatic hydrolysis effect was good.

[0077] Table 5. Peptide molecular weight distribution under optimal enzymatic hydrolysis conditions Molecular weight range / Da content / % >12200 0.01 12200 ~6511.44 7.26 6511.44~1421.69 22.22 1421.69~451.48 34.37 451.48~189.17 6.78 <189.17 29.36

[0078] Test Example 3

[0079] Single-factor experiments were conducted using the peptide powder obtained in Example 1 to determine the DPPH free radical scavenging rate when the peptide concentrations were 2, 4, 6, 8, and 10 mg / mL. Figure 8 The DPPH free radical scavenging rate was approximately 80% when the polypeptide content of Sacha in Sacha inchi pulp was 10 mg / mL.

[0080] Test Example 4

[0081] Single-factor experiments were conducted using the peptide powder obtained in Example 1 to determine the scavenging rate of ABTS free radicals when the peptide content was 0.2, 0.4, 0.6, 0.8, and 1 mg / mL. Figure 9 The ABTS free radical scavenging rate was approximately 90% when the polypeptide content of Sacha in Sacha inchi pulp was 1 mg / mL.

[0082] Test Example 5

[0083] This test case uses molecular docking simulation technology to predict the binding site and docking energy between the antioxidant peptide of Example 1 and the receptor protein Keap1.

[0084] First, the 2D structure of the peptide from Example 1 was drawn, then converted to a 3D structure, and its energy was minimized. The crystal structure (PDBID: 2FLU) of the complex of the Keap1 protein Kelch region and the Nrf2 16-mer peptide was obtained from a relevant protein database. Water, solvent molecules, Nrf2 16-mer peptide, and other ligands were removed from the protein structure, and hydrogen was added to the protein. Using molecular docking technology, the obtained peptide was used as a ligand and docked with the receptor protein Keap1. The receptor protein was set as rigid, and the ligand molecule as flexible. The center coordinates of the active pocket were x:5, y:9, and z:1, with a radius of 15 Å. Molecular docking was evaluated based on the docking energy. The larger the absolute value of the docking binding energy, the stronger the interaction between the receptor and ligand, indicating a stronger potential antioxidant effect. The docking results were finally used for visualization analysis.

[0085] The results showed that the docking energies of WEF and YRPF with Keap1 were -10.4 and -10 kcal / mol, respectively. The molecular docking results of the antioxidant peptide WEF with the Keap1 receptor are shown below. Figure 10 The molecular docking results of YRPF with the Keap1 receptor are shown in [the table below]. Figure 11 .

[0086] The binding modes and binding sites of the two antioxidant peptides and the receptor protein Keap1 reveal that the interaction between the antioxidant peptides and Keap1 includes intermolecular hydrogen bonds, electrostatic interactions, and hydrophobic interactions. The positional structure of the interaction between WEF and Keap1 protein is shown below. Figure 10 As shown, the positional structure of the interaction between YRPF and Keap1 protein is as follows: Figure 11 As shown.

[0087] The results showed that the interactions between the two peptides and the receptor protein Keap1 included intermolecular hydrogen bonds, electrostatic interactions, and hydrophobic interactions. Both peptides formed new, stable complexes with Keap1 and interacted with the six active pockets of the Keap1-Nrf2 ligand. All four peptides were in contact with residues involved in the Keap1-Nrf2 interaction, potentially competitively binding to Keap1 and releasing Nrf2, thereby exerting antioxidant activity by activating the Keap1-Nrf2 / ARE pathway. These data indicate that the four peptides <1 kDa can block the Keap-Nrf2 interaction, promote Nrf2 accumulation, and potentially induce the expression of antioxidant enzyme genes.

[0088] Test Example 6

[0089] In this test example, the amino acid content of the peptide powder obtained in Example 1 was determined using an amino acid analyzer, and the results are shown in Table 6.

[0090] Table 6. Amino acid determination results of sacha inca pulp peptides.

[0091] Glutamic acid and aspartic acid, as acidic amino acids, are widely used in food flavor enhancers due to their ability to enhance food flavor. Table 6 shows that glutamic acid and aspartic acid constitute the largest proportion of the hydrolyzed amino acids in the *Sapindus mukorossi* pomace peptides, with contents of 88.685 mg / g and 66.795 mg / g, respectively. This indicates that the *Sapindus mukorossi* pomace peptides in this test example have a certain flavor-enhancing effect, further demonstrating the influence of hydrolysis on the peptides. The most abundant free amino acids are tyrosine, phenylalanine, and arginine, with contents of 4.239 mg / g, 3.887 mg / g, and 3.233 mg / g, respectively. Tyrosine is the catalytic substrate for the monophenolase function of tyrosinase and is the main raw material for the final formation of eumelanin and pheomelanin. Research on synthesizing tyrosinase structural analogs that compete with tyrosine can effectively inhibit melanin production. Therefore, the *Sapindus mukorossi* pomace peptides in this test example, as an antioxidant peptide, have application potential not only in the food industry but also in the field of cosmetic research and development.

[0092] Test Example 7

[0093] (II) Identification of the polypeptide component sequences in (I)

[0094] (1) Sample preparation

[0095] The supernatant after ultrafiltration was desalted and then analyzed by mass spectrometry.

[0096] (2) Chromatographic separation

[0097] The liquid chromatography (LC) analysis conditions were as follows: Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an aqueous solution containing 0.1% formic acid and acetonitrile (84% by volume). A reversed-phase C18 column (0.15 mm × 150 mm, model RP-C18, Column Technology Inc.) was used. The column was first equilibrated with 95% (by volume) of mobile phase A. The sample was then injected via an autosampler into a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) and separated by the LC column. The relevant LC gradient settings were as follows:

[0098] 0 min–50 min, 4% to 50% B solution;

[0099] 50 min–54 min, 50% to 100% solution B;

[0100] 54 min–60 min, 100% B solution.

[0101] (3) Mass spectrometry identification

[0102] Enzymatic hydrolysates were separated by high-performance liquid chromatography (HPLC), and mass spectrometry analysis was performed for 60 min using a Q Exactive HF-X mass spectrometer (Thermo Fisher). Detection mode: positive ion. The mass-to-charge ratio of peptides and peptide fragments was acquired as follows: 10 fragment spectra were acquired after each full scan (MS2 scan). MaxQuant 1.5.5.1 software was used to search the raw mass spectrometry test files and relevant databases to obtain protein identification and quantitative analysis results.

[0103] The amino acid sequence of small molecule peptides was analyzed using LC-MC / MC technology, and the primary mass spectrum of the identification results is shown below. Figure 12 As shown in the figure. A total of 681 peptide sequences with lengths of 3–25 amino acid residues were identified. Molecular weight determination results showed that 28.3% of the peptide fragments had molecular weights between 200 and 400 Da; 59.9% of the peptide fragments had molecular weights between 400 and 600 Da. The specific distribution is shown in the figure. Figure 13As shown, the lengths of these peptides are mainly concentrated between 2 and 7 kDa, accounting for 88% of the total number of peptides. The above results all indicate that the functional peptides <1 kDa are mainly composed of short peptides.

[0104] (4) Peptide performance testing (antioxidant bioactivity, water solubility and toxicity)

[0105] Antioxidant bioactivity:

[0106] Peptide Ranker; http: / / distilldeep.ucd.ie / PeptideRanker / Peptides are classified based on predicted scores, with a score threshold of 0.5 as the cutoff value; peptides with a score greater than 0.5 are considered to be active.

[0107] Water solubility of peptides:

[0108] Innovagen; http: / / www.innovagen.com / proteomics-tools .

[0109] Verification of antioxidant activity:

[0110] ① Hydroxyl radical scavenging rate

[0111] Prepare 6.0 mmol / L salicylic acid and FeSO4 solutions separately. Add 0.25 mL of each solution to test tubes, mix thoroughly, and then add 0.25 mL of the 0.25 mg / mL sample solution and 0.5 mL of 6 mmol / L H2O2 solution sequentially. Mix well and react in a 37℃ water bath for 30 min. The entire experiment was conducted in the dark. The absorbance was measured at 510 nm. The hydroxyl radical scavenging rate of the sample was calculated using the following formula:

[0112] In the formula: A0 is the absorbance of the sample group; A1 is the absorbance of the control group without H2O2; A is the absorbance of the blank group without sample.

[0113] ② Determination of the reducing power of iron ions

[0114] Add 1 mL of 0.25 mg / mL sample solution to a test tube, followed by 2.5 mL each of 0.2 mmol / L phosphate buffer (pH 6.6) and 1% potassium ferricyanide solution. Mix well and react at 50°C for 20 min, then rapidly cool. Add 2.5 mL of 10% trichloroacetic acid solution and centrifuge at 3000 r / min for 10 min. Pipette 2.5 mL of the supernatant into a test tube and dilute to 5 mL with distilled water. Add 0.5 mL of 0.1% ferric chloride solution and react at room temperature for 10 min. Measure the absorbance at 700 nm. The reducing power is expressed as absorbance. Each sample has three replicates. Ascorbic acid solution (0.25 mg / mL) is used as a control.

[0115] Example 5

[0116] A method for preparing an antioxidant peptide includes the following steps:

[0117] S1: The following 81 peptide segments (see the invention content section) were synthesized using a solid-phase synthesis method to obtain antioxidant crude polypeptides;

[0118] S2: The crude antioxidant peptide obtained in S1 was dissolved in 50% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then purified by preparative reversed-phase high-performance liquid chromatography to obtain the purified antioxidant peptide.

[0119] The liquid chromatography column was an RP-C18 (4.6 mm × 250 mm, Column Technology luc.). The chromatographic conditions were as follows: mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B was an aqueous solution of 84% (v / v) acetonitrile containing 0.1% (v / v) formic acid. The gradient elution setup was as follows:

[0120] Elution was performed with solution B in a linear gradient from 8% to 33% for 0 to 25 min. The eluent was collected at 13 to 14 min and freeze-dried to obtain antioxidant peptides with a purity ≥90%.

[0121] Analysis of antioxidant mechanisms:

[0122] Docking antioxidant peptides with Keap1 will help in further analysis of the antioxidant mechanism. Molecular docking analysis was performed on 81 identified and screened peptides with Keap1 protein. A higher absolute value of the binding energy indicates a stronger binding affinity between the peptide and Keap1 protein. Among them, 25 peptides had a binding energy ≤ -0.9 kcal / mol, as shown in Table 7.

[0123] Table 7 lists 25 peptides with binding energies ≤ -0.9 kcal / mol. Serial Number peptide sequence peptide chain length Binding energy (kcal / mol) Serial Number peptide sequence peptide chain length Binding energy (kcal / mol) 1 WEF 3 -10.4 14 LRPY 4 -9.1 2 YRPF 4 -10 15 RPY 3 -9.1 3 RLLF 4 -9.4 16 LFDL 4 -9.1 4 KWF 3 -9.4 17 VFRP 4 -9.1 5 YEF 3 -9.3 18 QAW 3 -9.1 6 WLE 3 -9.3 19 PKYF 4 -9.1 7 FRF 3 -9.3 20 WRPQ 4 -9.1 8 LPSF 4 -9.2 21 RFP 3 -9.1 9 LEW 3 -9.2 22 LRYL 4 -9 10 RWE 3 -9.2 23 RLY 3 -9 11 KFRP 4 -9.2 24 LPAR 4 -9 12 WRL 3 -9.2 25 EWL 3 -9 13 DFF 3 -9.2

[0124] Considering the physical and chemical properties of 25 peptides, this study selected two peptides with the lowest binding energies, WEF and YRPF, for further investigation of their antioxidant mechanisms. The binding energies and physical properties of these two peptides are shown in Table 8. A higher overall score indicates a more reliable identification result. Peak intensity, as a relative quantitative result, is typically used to screen for differentially expressed peptides between different samples.

[0125] Table 8. Characteristics of WEF and YRPF peptide sequences peptide sequence peptide length affinity (kcal / mol) PeptideRanker score Mass Score Intensity WEF 3 -10.4 0.944321 480.20088 23.444 12830000 YRPF 4 -10 0.957392 581.29618 8.0791 10903000

[0126] The results showed that WEF forms seven conventional hydrogen bonds with the active sites of Keap1, namely LEU-365, VAL-465, ILE-556, VAL-418, and VAL-561, and forms van der Waals forces, C-H bonds, and π-alkyl interactions with the active sites of ALA-556, ALA-366, ALA-466, VAL-467, GLY-367, GLY-417, and GLY-605; YRPF interacts with the active sites of Keap1. The residues ALA-510, VAL-463, THR-560, VAL-606, VAL-608, and GLY-367 at the site form 8 conventional hydrogen bonds, and form van der Waals forces, C-H bonds, π-alkyl groups, and alkyl interactions with the residues ILE-559, ALA-366, ALA-466, CYS-513, VAL-561, VAL-465, VAL-512, GLY-511, and ARG-415.

[0127] Solid-phase synthesis plays an important role in the preparation of peptide compounds due to its high efficiency and controllability. This study successfully prepared peptides WEF and YRPF using solid-phase synthesis. The purity of the two peptides was analyzed using high-performance liquid chromatography (HPLC). The results showed that the purity of peptide WEF was 97.79%, and the purity of peptide YRPF was 96.09%. Figure 3-4 As shown in the figure. Mass spectrometry was then used to determine the molecular weight of the two peptides. Mass spectrometry, as a precise molecular weight detection method, can provide accurate mass information of compounds. The results showed that the actual molecular weight of peptide WEF was 481.20 Da, and the actual molecular weight of peptide YRPF was 582.35 Da.

[0128] Based on the composition and sequence of the identified peptides, WEF and YRPF were selected as the most potentially active peptides for further chemical synthesis. The purity of the synthesized peptides was above 90%. The hydroxyl radical scavenging rate and total iron reducing power of the two synthetic peptides, WEF and YRPF, were determined, and the results are as follows: Figure 3-6As shown, the hydroxyl radical scavenging rate of WEF was 44.49 ± 6.47%, and that of YRPF was 66.49 ± 17.28%. Both WEF and YRPF exhibited certain reducing power, showing the same trend as their hydroxyl radical scavenging rates (YRPF > WEF). This confirms the antioxidant properties of the two peptides, indicating that Sacha inchi pulp has the potential as a natural antioxidant and can be used in the development of functional foods.

[0129] Example 6

[0130] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline protein peptides obtained in Example 1:

[0131] Accurately weigh 120 parts of complex polypeptide (30 parts of Sacha inchi protein peptide, 30 parts of bird's nest oligopeptide, 30 parts of jujube peptide, and 30 parts of corn oligopeptide), 100 parts of instant coffee powder, 30 parts of maltodextrin, 3 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0132] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0133] Example 7

[0134] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0135] 120 parts of complex polypeptide (45 parts of sacha inchi protein peptide, 35 parts of bird's nest oligopeptide, 20 parts of jujube peptide, 20 parts of corn oligopeptide), 50 parts of instant coffee powder, 20 parts of maltodextrin, 1 part of milk powder flavoring, 1 part of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0136] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0137] Example 8

[0138] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0139] 120 parts of complex polypeptide (35 parts of sacha inchi protein peptide, 35 parts of bird's nest oligopeptide, 20 parts of jujube peptide, and 30 parts of corn oligopeptide), 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0140] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0141] Sensory evaluations were performed on the products obtained according to Examples 6-8, and the results are shown in Table 9.

[0142] Table 9 Sensory Evaluation Form

[0143] Table 10 Sensory Evaluation Results Example Color Flavor Organizational Form taste Total Score 6 26 13 18 24 81 7 27 15 18 27 87 8 25 16 18 20 79

[0144] As can be seen from Table 10, the solid beverage of the present invention has a good sensory score, indicating that it has good color, flavor, texture and taste, and is highly accepted by consumers.

[0145] Comparative Example 1

[0146] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0147] 50 parts of compound polypeptide (19 parts of saponin peptide, 15 parts of bird's nest oligopeptide, 8 parts of jujube peptide, 8 parts of corn oligopeptide), 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0148] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0149] Comparative Example 2

[0150] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0151] 100 parts of complex polypeptide (38 parts of saponin peptide, 30 parts of bird's nest oligopeptide, 16 parts of jujube peptide, 16 parts of corn oligopeptide), 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0152] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0153] Comparative Example 3

[0154] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0155] 150 parts of complex polypeptide (56 parts of saponin peptide, 44 parts of bird's nest oligopeptide, 25 parts of jujube peptide, 25 parts of corn oligopeptide), 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0156] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0157] Comparative Example 4

[0158] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0159] 200 parts of complex polypeptide (75 parts of saponin peptide, 59 parts of bird's nest oligopeptide, 33 parts of jujube peptide, 33 parts of corn oligopeptide), 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycoside, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0160] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0161] Comparative Example 5

[0162] A method for preparing a polypeptide coffee solid beverage with antioxidant effects using the sacha inophylline peptides obtained in Example 1:

[0163] 120 parts of Sacha indica pulp protein peptides, 100 parts of instant coffee powder, 20 parts of maltodextrin, 4 parts of milk powder flavoring, 3 parts of coffee flavoring, 1 part of steviol glycosides, 1 part of CMC, and 2 parts of silicon dioxide; all the above parts are by weight.

[0164] Mix the powders, steep them in hot water, stir thoroughly until homogeneous, sterilize, vacuum concentrate, and freeze dry to obtain the product.

[0165] Table 11 Data on Solid Beverage Formulation and Related Effects Comparative Example Complex polypeptide / serving Sensory rating DPPH removal ability 1 50 66 35% 2 100 75 58% 3 150 80 60% 4 200 68 64% 5 Only 120 portions of *Sedum morganianum* pomace protein peptides from Example 1 were added; no complex polypeptides were used. 60 73%

[0166] Table 11 shows that the amount of compound peptides added has a significant regulatory effect on the sensory quality and antioxidant activity of solid beverages. Sensory scores initially increased and then decreased with the amount of compound peptides added, peaking at 80 points at 150 points, indicating a harmonious flavor and rich taste. However, excessive addition at 200 points caused the score to drop to 68 points due to increased peptide viscosity and a more pronounced fishy and bitter taste. DPPH scavenging ability steadily increased with the amount of compound peptides added, rising from 35% at 50 points to 64% at 200 points. However, the antioxidant activity of single sacha inchi pulp protein peptides (120 points) was significantly better than that of the compound peptide system, demonstrating the functional and sensory advantages of single highly active peptides versus compound peptides.

[0167] In summary, the method for preparing sacha indica fruit pulp protein peptides using a single-stage alkaline enzymatic hydrolysis process provided by this invention achieves an excellent peptide yield of up to 62.33% by simplifying the operation process and reducing production costs, while ensuring high antioxidant activity of the product. The prepared sacha indica fruit pulp protein peptides exhibit outstanding small molecule characteristics—peptides with a molecular weight below 1500 kDa account for more than 60%, significantly improving bioavailability; at a concentration of 1 mg / mL, the ABTS free radical scavenging capacity reaches (91.08±0.38)%. Through synergistic compounding with other plant peptides, not only is the antioxidant activity further enhanced, but the original bitter taste is also effectively improved, enhancing the palatability of the product.

Claims

1. A type of Sacha indica pulp protein peptide, characterized in that, The protein peptide contains an antioxidant polypeptide having at least one of the following 81 amino acid sequences: WEF, YRPF, YEF, LPSF, RLLF, KWF, WLE, FRF, LEW, RWE, KFRP, WRL, DFF, LRPY, RPY, LFDL, VFRP, QAW, PKYF, WRPQ, RFP, LRYL, RLY, LPAR, EWL, LRY, RYL, TWR, EMW, VDF, DRVF, VDFL, SDFL, FDR, KFP, FYR, KP F, FRP, LEF, LGLR, KWL, KLW, RHF, SEDYLAHLL, LFTR, KHGF, FSR, RLF, FRL, DLRL, SRLL, YGR, RQW, SDLF, WVR, LFK, PKF, LK F, KLF, KWP, YRW, LLRP, KFN, YRLL, RFK, MKF, LLR, PLLR, FRS, WRY, YRL, LPR, LRL, RLP, FKL, VRF, GFK, KDW, CPR, MLR, KRF.

2. The *Sapindus mukorossi* pomace protein peptide as described in claim 1, characterized in that, The protein peptide contains an antioxidant polypeptide having at least one of the following two amino acid sequences: WEF and YRPF.

3. The method for preparing Sacha Inchi pulp protein peptides as described in claim 1, characterized in that, Includes the following steps: Step (1): Crush the sacha inchi pulp, sieve, dry, and defatted to obtain defatted sacha inchi pulp powder; Step (2): Take defatted saponin fruit meal powder, add distilled water at a material-to-liquid ratio of 1g:10mL~1g:30mL, add alkaline protease at an enzyme-to-saponin fruit meal powder ratio of 7500~8500 U / g, enzymatically hydrolyze at 45~65℃ for 3.5~5.5h, and then inactivate the enzyme at 95~100℃ for 10~15min to obtain the enzymatic hydrolysate; Step (3): Centrifuge the enzymatic hydrolysate at 3000~4000r / min for 10~25min, take the supernatant, and obtain the crude peptide solution of Sacha indica; Step (4): Filter the crude peptide solution of Sacha indica through a membrane, pass it onto a chromatography column, collect the eluent, concentrate and dry it under vacuum to obtain Sacha indica meal protein peptides.

4. The method for preparing Sacha Inchi pulp protein peptides as described in claim 3, characterized in that, In step (1), the blocky sacha inchi pulp is crushed into powder, passed through an 80-200 mesh sieve, dried at 100-110℃ for 1-3 hours, and defatted to obtain defatted sacha inchi pulp powder; In step (2), the material-to-liquid ratio is 1g:19mL; the enzymatic hydrolysis temperature is 59℃ and the time is 4.85 h; In step (4), after vacuum concentration, the concentrate is passed through an ultrafiltration tube with a molecular weight cutoff of 1 kDa and centrifuged at a speed of 6000-10000 r / min and a temperature of 2-6℃ for 10-20 min to obtain polypeptide components with a molecular weight <1 kDa. After drying, the protein peptides of Sacha Inchi are obtained.

5. An antioxidant polypeptide, characterized in that, The antioxidant polypeptide has at least one of the 81 amino acid sequences as described in claim 1.

6. The antioxidant polypeptide as described in claim 5, characterized in that, The antioxidant polypeptide has any one of the following sequences: WEF, YRPF.

7. The method for preparing an antioxidant polypeptide as described in claim 5, characterized in that, Includes the following steps: S1: The amino acid sequences of the 81 peptide segments described in claim 1 were synthesized using a solid-phase synthesis method to obtain an antioxidant crude polypeptide; S2: Take the crude antioxidant peptide obtained in S1, dissolve it in an aqueous solution of 50% acetonitrile, filter it through a 0.45 μm membrane, and purify it by preparative reversed-phase high-performance liquid chromatography to obtain the purified antioxidant peptide.

8. The method for preparing the antioxidant polypeptide as described in claim 7, characterized in that, In S2, during the reversed-phase high-performance liquid chromatography separation and purification, mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.1%, and mobile phase B is an aqueous solution of acetonitrile containing 0.1% formic acid, wherein the volume concentration of acetonitrile in the aqueous solution of acetonitrile is 84%.

9. The application of the sacha inophylline pomace protein peptide as described in claim 1 or the antioxidant polypeptide as described in claim 5 in food processing.

10. A method for preparing a solid beverage using the sacha inchi pulp protein peptide of claim 1 or the antioxidant polypeptide of claim 5, characterized in that, The steps include the following: (1) Take the following raw materials by mass parts: The ingredients are: 105-130 parts of complex polypeptide, 40-105 parts of coffee powder, 20-30 parts of maltodextrin, 2-10 parts of flavoring, 0.5-2 parts of steviol glycosides, 0.5-2 parts of thickener, and 1-3 parts of stabilizer. The composite polypeptide contains sacha indica pulp protein peptides / antioxidant peptides and other peptides; the other peptides are selected from at least one of bird's nest oligopeptides, jujube peptides, and corn oligopeptides; the ratio of sacha indica pulp protein peptides / antioxidant peptides to other peptides is (6~9):(15~18). (2) Mix the powdered raw materials in (1), brew with hot water at 25-50 ℃, stir thoroughly, sterilize, vacuum concentrate, and dry to obtain a solid beverage.

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