Torreya grandis protein extract releasing antioxidant peptides in the intestine and a preparation method thereof
By preparing and separating small molecule peptides SFY and YFY from Torreya grandis protein extract, the problem of antioxidant peptides being decomposed in the digestive system was solved, thus maintaining their antioxidant activity in the intestine and enhancing the body's antioxidant capacity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI MEDICAL UNIV
- Filing Date
- 2026-01-13
- Publication Date
- 2026-06-12
AI Technical Summary
Antioxidant peptides are easily destroyed by the digestive system after passing through the gastrointestinal tract, resulting in the loss of their biological activity and making it difficult for them to effectively exert their antioxidant effects.
Using torreya nut protein as raw material, intestinal-release antioxidant peptides were prepared through alkaline protease hydrolysis, nanofiltration, and chromatographic separation technology to ensure that the small molecule peptides SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr) retain their antioxidant activity after digestion by pepsin and trypsin.
The prepared intestinal-release antioxidant peptides still exhibit significant antioxidant activity after simulated digestion, effectively enhancing the body's ability to resist oxidative stress and strengthening cellular antioxidant defense mechanisms by activating the Keap1-Nrf2-ARE pathway.
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Figure CN122189130A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of intestinal-released antioxidant peptide technology, specifically relating to a torreya nut protein extract containing intestinal-released antioxidant peptides and its preparation method. Background Technology
[0002] Bioactive peptides are short-chain amino acid sequences released from proteins through enzymatic hydrolysis or processing. They possess a variety of physiological functions, particularly excelling in antioxidant activity. Recent studies have found that plant-derived bioactive peptides can enhance cellular defense against oxidative stress by regulating the Keap1-Nrf2-ARE signaling pathway. For example, various peptides from mulberry leaves, kudzu root, soybean meal, and walnuts have been shown to activate this pathway, demonstrating their potential as natural Nrf2 activators. Antioxidant peptides are a class of bioactive peptides with free radical scavenging activity. They reduce cellular oxidative damage by blocking oxidative chain reactions or chelating metal ions. Antioxidant peptides are used in anti-aging skincare products to inhibit UV damage and are also used in the preparation of functional foods for the prevention of cardiovascular diseases.
[0003] Proteases are a class of biological enzymes that specifically catalyze the breaking of peptide bonds in proteins or polypeptides, decomposing long-chain polypeptides into shorter peptides or free amino acids through hydrolysis. This function is ubiquitous in living organisms; for example, processes such as food digestion, cell metabolism, and protein renewal all rely on protease activity. The proteases used in human digestion are mainly pepsin and trypsin. When bioactive antioxidant peptides pass through the digestive system, their original peptide chain structure is further broken down by pepsin and trypsin, ultimately becoming smaller peptides or amino acids that are absorbed by the small intestine, thus losing their antioxidant and other biological activities. Therefore, when preparing functional foods using antioxidant peptides, it is necessary to consider the enzymatic breakdown and destruction of antioxidant peptides by digestive proteases.
[0004] Torreya grandis, a highly nutritious dried fruit, contains 10%–16% protein by dry weight, with a rich amino acid composition, making it an ideal raw material for developing functional protein components. Processing byproducts of Torreya grandis also contain a large amount of high-quality protein, which can serve as a sustainable source for preparing bioactive peptides. Despite the abundance of Torreya grandis protein resources, systematic exploration, preparation, and activity evaluation of its antioxidant peptides are still relatively lacking. Therefore, based on its high protein content, an intestinal-release antioxidant peptide was developed from Torreya grandis and its byproducts. After digestion simulation experiments, its antioxidant activity was significantly enhanced. The inventors studied its mechanism and established scientific identification and activity evaluation methods, which have significant application prospects for the preparation of antioxidant peptides that can be orally absorbed by the human body. Summary of the Invention
[0005] To address the technical problem of antioxidant peptides being easily destroyed by the digestive system after passing through the gastrointestinal tract, this invention provides a Torreya grandis protein extract containing intestinal-release antioxidant peptides. The Torreya grandis protein extract is obtained by enzymatic hydrolysis of Torreya grandis protein under the action of alkaline protease, centrifugation, collection of the supernatant, and then processing through a nanofiltration system to retain components with a molecular weight less than 3 kDa. The extract is then separated using a Superdex™ 200 increase10 / 300 GL column, collecting 22.55 mL to 24.69 mL of liquid during separation. This liquid is then freeze-dried to obtain the Torreya grandis protein extract containing intestinal-release antioxidant peptides. This Torreya grandis protein extract retains strong antioxidant properties even after digestion with pepsin and trypsin. Analysis shows that the main antioxidant components after digestion are two small molecule peptides: SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr). The chemical structural formula of YFY is: H2N-CH(CH2C6H4OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH; The chemical structural formula of SFY is: H2N-CH(CH2OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH; The two small molecule peptides, SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr), are used to prepare antioxidant compositions.
[0006] Meanwhile, the present invention also provides a method for preparing a Torreya grandis protein extract containing intestinal-release antioxidant peptides, comprising the following steps: ① Mix the dried torreya nut protein powder with pure water to form a hydrolysis system. Add alkaline protease to the system, shake and hydrolyze for 3.5-4 hours. After hydrolysis, inactivate the enzyme, then cool in an ice bath to obtain the cooled hydrolysate. ② After cooling, the hydrolysate is centrifuged and the supernatant is collected. Then, it is processed by a nanofiltration system to retain components with a molecular weight of less than 3 kDa, and the permeate is obtained, which is the crude peptide extract. ③ The permeate was separated using a Superdex™ 200 increase10 / 300 GL column. During the separation process, 22.55 mL to 24.69 mL of liquid was collected and freeze-dried to obtain Torreya grandis protein extract containing intestinal-releasing antioxidant peptides.
[0007] The preparation method of the torreya nut protein powder is as follows: The shells of Torreya grandis are removed and the powder is crushed to pass through a 60-100 mesh sieve to obtain Torreya grandis fine powder. The Torreya grandis fine powder is then degreased to remove the oil components, resulting in degreased Torreya grandis powder. Defatted Torreya grandis powder was suspended in water at a ratio of 8:1 to 10:1 (v / w). The pH was adjusted to 7.0±0.2. After stirring for 1 to 3 hours, the mixture was centrifuged at 10,000 to 15,000 × g for 5 to 20 minutes. The supernatant was collected, and the pH was adjusted to 4.0±0.2. The mixture was centrifuged at 2,000 to 10,000 r / min. The precipitate was collected, and the precipitate was redissolved in water. After stirring for 1 to 3 hours, the mixture was freeze-dried to obtain Torreya grandis protein powder. Torreya protein powder was dissolved in ultrapure water, stirred to reconstitute, and then freeze-dried to obtain dried torreya protein powder.
[0008] The defatting method employs either supercritical CO2 extraction or solvent extraction. Specifically, supercritical CO2 extraction is carried out at 25-40 MPa, 30-50 °C, and a CO2 flow rate of 12-18 L / min for 60-240 min. Solvent extraction involves extraction with diethyl ether, petroleum ether, or No. 6 solvent oil 2-3 times followed by drying.
[0009] The mixing ratio of the torreya nut protein powder to pure water is 1:50~1:30 g / mL.
[0010] The alkaline protease is added at a concentration of 3000-3500 U / g of protein powder.
[0011] Specifically, the centrifugation is performed at 2-4°C and 10,000-12,000 g for 12-15 minutes; the hydrolysis is performed under constant temperature oscillation at 35-37°C and 120-150 rpm; and the freeze-drying temperature is -70 to -18°C.
[0012] The eluent for the chromatographic column was a 0.12% aqueous solution of trifluoroacetic acid.
[0013] The Torreya grandis protein extract containing intestinal-releasing antioxidant peptides prepared by this invention can also be used to prepare an intestinal-absorbable antioxidant oral liquid. Beneficial effects
[0014] This invention first discovered that the antioxidant activity of Torreya grandis polypeptide component X4 after enzymatic hydrolysis was significantly stronger than that of other components. Through layer-by-layer analysis, it was found that the main active ingredients of Torreya grandis polypeptide component X4 are SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr). SFY and YFY are two small molecule peptides, and their antioxidant activity was confirmed after artificial synthesis. Although their in vitro antioxidant activity is not significantly different from other antioxidant peptides, fruit fly model experiments showed that the median survival time of fruit flies pretreated with 0.05% and 0.10% peptides was significantly longer than that of the control group. This proves that the target peptides can effectively enhance the body's ability to resist acute oxidative stress. SFY and YFY showed more significant protective effects against H2O2-induced oxidative stress than other peptides. Further mechanistic studies revealed and confirmed that both SFY and YFY can form stable complexes with Keap1, thereby promoting Nrf2 dissociation and enhancing cellular antioxidant defense mechanisms. SFY and YFY enhance the expression of antioxidant enzymes by activating the Keap1-Nrf2-ARE pathway, thereby reducing oxidative stress levels and having a significant protective effect against H2O2-induced oxidative damage in Drosophila.
[0015] However, SFY and YFY are polypeptides, which are broken down into amino acids when digested alone by pepsin and trypsin. After simulated digestion, the X4 polypeptide component of Torreya grandis showed significantly higher levels of these polypeptides than other Torreya grandis polypeptide components and also significantly higher levels than Torreya grandis protein powder. Preliminary research results indicate that after digestion by pepsin and trypsin, the X4 polypeptide component of Torreya grandis retains more SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr), allowing the antioxidant peptides SFY and YFY with antioxidant activity to be absorbed in the intestines. This effectively solves the problem of antioxidant peptides being broken down in the digestive system. The preparation of antioxidant foods can maximize the antioxidant effect and avoid the further enzymatic breakdown of artificially synthesized SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr) into amino acids after digestion.
[0016] The method provided by this invention enables the systematic and efficient preparation and identification of polypeptides with potent antioxidant activity from Torreya grandis. A multi-level evaluation system combining an in vivo Drosophila model and advanced computer simulations provides solid and reliable experimental data and theoretical support for the antioxidant efficacy of the polypeptides, clarifying their application potential in drug development. Attached Figure Description
[0017] Figure 1 Chromatographic analysis of Torreya grandis protein powder by size group and in vitro antioxidant capacity of each component; Figure 2The effect of antioxidant peptides on the growth curves of a Drosophila model under hydrogen peroxide-induced oxidative stress; Figure 3 The antioxidant effects of SFY and YFY on oxidation indicators and antioxidant enzymes; Figure 4 These are the docking sites for SFY and YFY with the Keap1 Kelch domain; Figure 5 Molecular dynamics diagrams for Keap1, SFY, and YFY; Figure 6 The binding capability calculated for MMGBSA; Figure 7 The scavenging rates of hydroxyl radicals, DPPH radicals, and ABTS radicals were measured after simulated digestion of different protein powders. Detailed Implementation
[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0019] The protein samples used in the examples were commercially available milk protein, chicken protein, soy protein and yeast protein, with a protein content of ≥90%.
[0020] All enzymes required for in vitro digestion were purchased from Sigma-Aldrich, specifically including: pepsin derived from porcine gastric mucosa (catalog number P7000, activity ≥2500 units / mg protein), trypsin derived from porcine pancreas (catalog number P7545, its trypsin activity was calibrated to 31000 U / mg before use), and porcine bile extract (catalog number B8631). Electrolytes required for the simulated digestion solution, such as KCl, KH₂PO₄, NaHCO₃, and NaCl, were all analytical grade reagents.
[0021] In the embodiments, the preparation method of Torreya grandis protein powder is as follows.
[0022] High-quality Torreya grandis nuts were selected, shelled, and mechanically pulverized until they passed through a 60-100 mesh sieve to obtain fine Torreya grandis powder. Subsequently, supercritical CO2 extraction was performed at 25-40 MPa, 30-50℃, and a CO2 flow rate of 12-18 L / min for 60-240 min to degrease the powder, removing oily components and obtaining defatted Torreya grandis powder.
[0023] Defatted Torreya grandis powder can also be obtained by extraction with solvents such as diethyl ether, petroleum ether, and No. 6 solvent oil 2-3 times followed by drying.
[0024] Defatted Torreya grandis powder was suspended in water at a ratio of 8:1 to 10:1 (v / w), and the pH was adjusted to 7.0 ± 0.2. After stirring for 1 to 3 hours, the mixture was centrifuged at 10,000 to 15,000 × g for 5 to 20 minutes. This process was repeated 1 to 5 times, and the supernatants were combined. Subsequently, the pH was adjusted to 4.0 ± 0.2, and the mixture was centrifuged at 2,000 to 10,000 r / min. The precipitate was collected, redissolved in water, stirred for 1 to 3 hours, and then freeze-dried at -70 to -18℃ to obtain Torreya grandis protein powder with a protein content ≥ 90%.
[0025] The method for determining in vitro antioxidant activity is as follows.
[0026] (1) ·OH radical scavenging ability 1 mL of the digestion product solution (1 mg / mL) was added to 1 mL of 6 mM salicylic acid-ethanol solution and 1 mL of 6 mM FeSO4 solution and mixed. Then, 1 mL of H2O2 (5 mM) was added to the reactants and vortexed for 10 s. The mixture was incubated at 37 °C for 60 min, and the absorbance was measured at 520 nm. Each group was measured in triplicate.
[0027] OH radical scavenging rate = [A0 - (A1 - A2)] / A0 × 100% In the formula, A0, A1 and A2 are the absorbance values of the mixtures of water and H2O2, digestion products and H2O2, and digestion products and water, respectively.
[0028] (2) DPPH free radical scavenging activity Add 1 mL of the digest product solution (1 mg / mL) to 0.5 mL of DPPH ethanol solution (0.1 mM ethanol), store at 25°C in the dark for 30 min, and then measure the absorbance at 517 nm. Each group was measured in triplicate.
[0029] DPPH free radical scavenging rate = [A0 - (A1 - A2)] / A0 × 100% In the formula, A0, A1, and A2 are the absorbance values of the mixture of ethanol and DPPH solution, the digestion product and DPPH solution, and the digestion product solution and ethanol, respectively.
[0030] (3) ABTS free radical scavenging ability Equal volumes of ABTS solution (7.0 mM) and K₂O₈S₂ solution (2.45 mM) were stored at 25°C in the dark for 16 h, and then diluted with PBS to an absorbance of 0.70 ± 0.02 at 734 nm. 0.5 mL of the digestion product solution (1 mg / mL) was added to 3 mL of the diluted ABTS solution, and the mixture was incubated at 25°C in the dark for 60 min. The absorbance was then measured at 734 nm. Each group was measured in triplicate.
[0031] ABTS free radical scavenging activity = [A0 - (A1 - A2)] / A0 × 100% Wherein, A0, A1, and A2 are the absorbance values of the mixture of water and ABTS solution, the digestion product and ABTS solution, and the mixture of digestion product solution and water, respectively. Example 1
[0032] In this embodiment, size exclusion chromatography was first used for preliminary separation to study the antioxidant peptide components in Torreya grandis protein powder, target antioxidant peptides were selected and identified, and target polypeptides with high antioxidant activity were screened and identified from the purified components.
[0033] First, the torreya nut protein powder was dissolved in ultrapure water, stirred for 1 hour, and then freeze-dried to obtain torreya nut protein dry powder. The torreya nut protein dry powder was mixed with pure water at a ratio of 1:50 (g / mL) to form a hydrolysis system. Alkaline protease (3000 U / g protein powder) was added to this system, and the reaction was carried out at 37℃ and 150 rpm for 4 hours under constant temperature shaking conditions. After hydrolysis, the reaction system was kept in a boiling water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled in an ice bath. The cooled hydrolysate was centrifuged at 4℃ and 10000 g for 15 minutes, the supernatant was collected, and further processed by a nanofiltration system to retain components with a molecular weight less than 3 kDa. The resulting permeate was the crude polypeptide extract, which was stored at 4℃ for later use.
[0034] The permeate was eluted using a Superdex™ 200 10 / 300 GL column with 0.12% trifluoroacetic acid solution as the mobile phase under constant flow elution at a detection wavelength of 280 nm. Components with different retention times were manually collected based on peak shape and then freeze-dried under vacuum. Next, the freeze-dried component with the best activity was redissolved and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Gradient elution was performed using a C18 column with acetonitrile solution containing 0.1% trifluoroacetic acid as mobile phase A and an aqueous solution containing 0.1% trifluoroacetic acid as mobile phase B. The size-packed chromatogram was obtained by monitoring at a wavelength of 214 nm. Figure 1 As shown in Figure A, four main components were obtained, named X1, X2, X3, and X4 respectively.
[0035] Based on the results of the above size-group chromatographic analysis, the permeate was separated using a Superdex™ 200 increase10 / 300 GL (Cytiva, USA) column. Elution was performed using a 0.12% (v / v) trifluoroacetic acid aqueous solution. Based on the peak time, the following fractions were collected during separation: 14.54 mL - 18.75 mL (X1), 18.76 mL - 20.21 mL (X2), 20.22 mL - 22.54 mL (X3), and 22.55 mL - 24.69 mL (X4). These fractions were then lyophilized, and their in vitro antioxidant capacity was analyzed.
[0036] First, in vitro activity screening was performed. The ABTS radical scavenging rate, hydroxyl radical scavenging rate, and DPPH radical scavenging rate of the four components X1-X4 obtained above were measured respectively. The results are detailed in [link to results]. Figure 1 As shown in B. By comparing the half-maximal scavenging concentrations of each component at different concentrations, it was found that component X4 exhibited significantly better antioxidant capacity than other components, and therefore it was identified as the target for subsequent structural analysis.
[0037] The component corresponding to the X4 peak exhibited significantly higher scavenging activity against -OH, DPPH, and ABTS radicals than other components. Figure 1 B). Therefore, the X4 component was further purified by reversed-phase high-performance liquid chromatography, yielding two main peaks (XH1 and XH2); Figure 1 C). The XH2 component exhibits superior in vitro antioxidant capacity compared to XH1 ( Figure 1 D).
[0038] Molecular docking is an important computational method applied to the screening, design, and synthesis of bioactive compounds, including peptides. In particular, the Keap1-Nrf2 pathway represents a key endogenous antioxidant signaling mechanism. This study used LC-MS / MS analysis to detect 76 potential antioxidant peptides in the XH2 fraction. These peptides were then used as ligands for molecular docking evaluation targeting the Kelch domain of the Keap1 protein. Based on the binding energy calculations, 10 peptides with the lowest docking scores (indicating the highest affinity for Keap1) were selected as potential antioxidant candidates. Lower docking energies indicate higher binding affinity between the peptide and the protein. The selected 10 peptides, YFN, SFY, YFY, RLY, PIH, FSY, DLY, NLY, ELI, and VYK, have binding energies ranging from -9.2 kcal / mol to -7.6 kcal / mol. All identified peptides were tripeptides with molecular weights ranging from 365.43 to 494.35 Da. Toxicity predictions based on the ToxinPred database confirmed that all peptides were non-toxic. Amino acid composition analysis using Innovagen proteomics tools classified peptides YFN, SFY, YFY, PIH, FSY, and NLY as hydrophobic peptides, while RLY, DLY, ELI, and VYK were classified as hydrophilic peptides.
[0039] The X4 fraction was then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Chromatographic separation was performed on an EASY-nLC1200 system, while mass spectrometry was performed on an Orbitrap Exploris 480 mass spectrometer in positive ion mode. The primary full scan resolution was set to 60,000, with a scan range of m / z 200–1500. The preferred precursor ion was fragmented using HCD, with a secondary resolution of 15,000. The acquired high-precision mass spectrometry data were imported into PEAKS DB software and compared with the protein sequence library of Torreya grandis in the UniProt database. Ultimately, the core amino acid sequences of two novel antioxidant peptides were identified: SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr).
[0040] The chemical structural formula of YFY is: H2N-CH(CH2C6H4OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH.
[0041] The chemical structural formula of SFY is: H2N-CH(CH2OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH.
[0042] Example 2 This embodiment uses a combination of in vivo models and computer simulations to systematically evaluate the antioxidant functions of the target SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr) peptides screened in Example 1.
[0043] H₂O₂ is a potent but unstable oxidant that readily decomposes to produce hydroxyl radicals, causing severe oxidative damage to cellular components and tissues. This study established an H₂O₂-induced oxidative damage model in Drosophila melanogaster, using *Drosophila melanogaster* as the model organism. The study investigated the protective effects of the six most abundant antioxidant peptides (SFY, YFY, RLY, ELI, DLY, and FSY) among the X4 group against oxidative stress. The experiment included a control group, a hydrogen peroxide model group, and a peptide intervention group. Three-day-old male Drosophila were randomly assigned to these groups. The treatment groups were continuously fed 5% sucrose solutions containing 0.01%, 0.05%, and 0.10% of the target peptides, respectively, using the paper disc method for 7 days. Afterward, all Drosophila were transferred to a stress culture medium containing 30% hydrogen peroxide, and mortality was continuously observed and recorded. Survival curve analysis showed that the median survival time of Drosophila pretreated with 0.05% and 0.10% peptides was significantly longer than that of the control group, demonstrating that the target peptide can effectively enhance the body's ability to resist acute oxidative stress. (See details...) Figure 2 As shown in the figure, the results indicated that the average lifespan of fruit flies exposed to H2O2 was shortened compared to the control group. Supplementation with these six antioxidant peptides significantly enhanced the tolerance of fruit flies to H2O2, and this protective effect increased with increasing dosage. Compared to the H2O2 model group, supplementation with 0.10% concentrations of SFY, YFY, RLY, ELI, DLY, and FSY prolonged the median survival time of male fruit flies by 39.59%, 54.54%, 25.44%, 2.58%, 11.92%, and 16.46%, respectively. Among them, YFY and SFY showed more significant protective effects against H2O2-induced oxidative stress than the other peptides.
[0044] To evaluate the protective effects of SFY and YFY against H2O2-induced oxidative stress in Drosophila, we examined the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), which are regulated by the Keap1-Nrf2-ARE pathway. Figure 3As shown in AC, after 12 hours of H2O2 exposure, pre-supplementation with SFY or YFY significantly increased the activities of all three enzymes in fruit flies. Specifically, compared with the stress control group, SOD activity increased by 293.18% and 369.36%, respectively; CAT activity increased by 114.99% and 175.91%, respectively; and GSH-Px activity increased by 33.43% and 52.79%, respectively. Furthermore, both peptides effectively reduced the level of reactive oxygen species (ROS) in the fruit fly gut, with reductions of 17.68% and 28.19%, respectively. Figure 3 (D, 3E). The results of this study indicate that SFY and YFY enhance the expression of antioxidant enzymes by activating the Keap1-Nrf2-ARE pathway, thereby reducing oxidative stress levels and providing significant protection against H2O2-induced oxidative damage in Drosophila.
[0045] Key residues in the Keap1-Kelch domain are known to bind to Nrf2 via hydrogen bonds, such as Tyr334, Ser363, Arg380, Asn382, Arg415, Arg483, Ser508, Gln530, Ser555, and Ser602. Figure 4 As shown in Figure A, the complexes formed by SFY and YFY with Keap1 maintain stability through both hydrogen bonding and hydrophobic interactions. Specifically, YFY forms hydrogen bonds with Arg380, Arg415, Ser508, and Ser555, while SFY interacts with Arg380, Ser508, Gln530, and Ser555. Notably, Arg380, Ser508, and Ser555 in the Kelch domain are common binding residues for both peptides, indicating that these sites are crucial in mediating the interaction between the peptides and Keap1. These findings are consistent with previous reports on antioxidant peptides from other sources. For example, bovine milk peptides (DAF, QAF, DGGY) have been found to bind to Ser508 of Keap1, thereby interfering with the binding of Keap1 to Nrf2. The cheese tofu peptide (AAWGKVGGGQAGAHGAEALER) forms hydrogen bonds with Ser555 and Arg380 of Keap1. The results in summary suggest that SFY and YFY likely disrupt the interaction between Nrf2 and Keap1 by competitively binding to the Nrf2 recognition site in the Keap1-Kelch domain.
[0046] Molecular dynamics simulations are important computational tools for studying the interaction mechanisms between bioactive peptides and target receptors. This study characterized the binding properties of SFY and YFY to the Keap1 protein using molecular dynamics simulations: Root mean square bias analysis showed that the Keap1-SFY complex remained stable after fluctuations in the first 60 ns, eventually converging to around 1.1 Å; while the Keap1-YFY complex rapidly reached equilibrium after 2 ns, with fluctuations of approximately 1 Å, indicating that both complexes possess high structural stability. Figure 5 A). Radius of gyration analysis showed that the Rg values of the two complexes changed very little, indicating that peptide binding did not cause significant swelling or contraction of Keap1. Figure 5 B). Root mean square fluctuation analysis revealed that most residues in both complexes had low RMSF values (mainly below 2.3 Å), indicating that the residues exhibited limited flexibility and high stability during simulation. Figure 5 C). Solvent-accessible surface area analysis showed that peptide binding did not cause significant changes in SASA, suggesting that SFY and YFY do not significantly alter the surface morphology of Keap1. Figure 5 D). Hydrogen bond analysis showed that the number of hydrogen bonds between Keap1 and SFY ranged from 0 to 10 (mostly 5), while the number between Keap1 and YFY ranged from 0 to 6 (mostly 3), indicating that both maintain good hydrogen bond interactions with Keap1. Figure 5 E). Binding free energy calculated using the MM / PBSA method: the binding free energy of the Keap1-SFY complex is -22.51 Kcal / mol, and that of the Keap1-YFY complex is -25.31 Kcal / mol. Figure 6 A), in which van der Waals forces are the main contributing factor. Analysis of the binding mode of the steady-state complex shows that hydrogen bonding and hydrophobic interactions play a key role in the stability of the complex (A). Figure 6 This is consistent with molecular dynamics results. Specific key binding sites in the Keap1-YFY complex are: Tyr334, Arg415, Tyr525, Tyr572; in the Keap1-SFY complex: Tyr334, Arg380, Asn414, Ser602, Gly603. These findings confirm that both SFY and YFY can form stable complexes with Keap1, thereby promoting Nrf2 dissociation and enhancing cellular antioxidant defense mechanisms. Example 3
[0047] This embodiment provides a method for preparing a torreya protein extract containing intestinal-release antioxidant peptides, and measures its in vitro antioxidant activity after simulating gastrointestinal digestion.
[0048] First, the torreya nut protein powder was dissolved in ultrapure water, stirred for 2 hours to reconstitute, and then freeze-dried at -70 to -18°C to obtain torreya nut protein dry powder. The torreya nut protein dry powder was mixed with pure water at a ratio of 1:30 (g / mL) to form a hydrolysis system. Alkaline protease (3500 U / g protein powder) was added to this system, and the reaction was carried out at 35°C and 120 rpm under constant temperature shaking conditions for 3.5 hours. After hydrolysis, the reaction system was kept in a boiling water bath for 10 minutes to completely inactivate the enzyme, and then rapidly cooled in an ice bath. The cooled hydrolysate was centrifuged at 2°C and 12000 g for 12 minutes, the supernatant was collected, and further processed by a nanofiltration system to retain components with a molecular weight less than 3 kDa. The resulting permeate was the crude polypeptide extract, which was stored at 4°C for later use.
[0049] The permeate was separated using a Superdex™ 200 increase10 / 300 GL (Cytiva, USA) column. Elution was performed using a 0.12% (v / v) trifluoroacetic acid aqueous solution. Based on the peak time, the following fractions were collected during separation: 14.54 mL - 18.75 mL (X1), 18.76 mL - 20.21 mL (X2), 20.22 mL - 22.54 mL (X3), and 22.55 mL - 24.69 mL (X4). These fractions were then lyophilized at -70 to -18°C to obtain Torreya grandis polypeptide fractions X1, X2, X3, and X4, respectively.
[0050] The in vitro antioxidant properties of various protein powders and polypeptide components after simulated human digestion were determined using the following experimental methods.
[0051] Add 1 g of protein powder or peptide component to 20 mL of phosphate-buffered saline (PBS, pH 7.0), adjust the pH to 2.0 with 6 M HCl, and add pepsin (enzyme activity 400,000 U / g, 126 mg / mL, 1 mL per group) to simulate gastric digestion. The mixture is reacted in a constant temperature shaking water bath at 150 rpm and 37°C for 2 h. Gastric digestion is terminated by adjusting the pH to 7.5 with 2 M NaOH, and then trypsin (protease activity 31,000 U / g, 140 mg / mL, 1 mL per group) is added to begin simulating intestinal digestion. The mixture is then reacted in a constant temperature shaking water bath at 150 rpm and 37°C for 2 h. After the reaction, the mixture is heated in a 95°C water bath for 15 min to terminate the reaction. The resulting mixture is centrifuged for 20 min (12,000 g, 4°C), the supernatant is separated, and stored at -40°C for later use. The hydroxyl radical scavenging rate, DPPH radical scavenging rate, and ABTS radical scavenging rate of different protein powders after simulated digestion are shown in the figure. Figure 7 As shown. (Through) Figure 7 It can be seen that the X4 component of Torreya grandis polypeptide after simulated digestion is significantly higher than other Torreya grandis polypeptide components and also significantly higher than Torreya grandis protein powder. According to preliminary research results, after enzymatic hydrolysis by pepsin and trypsin, the X4 component of Torreya grandis polypeptide can retain more SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr), which allows the antioxidant peptides SFY and YFY with antioxidant activity to be absorbed in the intestine. This effectively solves the problem of antioxidant peptides being decomposed in the digestive system. The preparation of antioxidant foods can maximize the antioxidant effect and avoid the further enzymatic hydrolysis of artificially synthesized SFY (Ser-Phe-Tyr) and YFY (Tyr-Phe-Tyr) into amino acids after passing through the digestive system. Example 4
[0052] This embodiment provides an oral liquid for releasing antioxidant peptides from the intestine, the oral liquid for releasing antioxidant peptides includes 4 Torreya grandis polypeptide components, sucrose and potassium sorbate.
[0053] The preparation method involves adding Torreya grandis polypeptide component X4 at a concentration of 10-100 g / L, sucrose at a concentration of 5-50 g / L, and potassium sorbate at a concentration of 0-50 mg / L to sterile water to obtain a mixed solution that is an oral solution for releasing antioxidant peptides from the intestines.
[0054] Through simulated digestion experiments, the oral liquid showed a hydroxyl radical scavenging rate of 80.085-92.250%, a DPPH radical scavenging rate of 86.335-92.050%, and an ABTS radical scavenging rate of 78.785-80.360%, demonstrating excellent antioxidant effects.
[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A Torreya grandis protein extract containing intestinal-released antioxidant peptides, characterized in that, The Torreya grandis protein extract was prepared by enzymatic hydrolysis of Torreya grandis protein under the action of alkaline protease, centrifugation, collection of supernatant, and then processing by a nanofiltration system to retain components with a molecular weight less than 3 kDa. The components were then separated using a Superdex™ 200 increase10 / 300 GL column, collecting 22.55 mL to 24.69 mL of liquid during the separation process. The liquid was then freeze-dried to obtain the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides.
2. Uses of two small molecule peptides, namely SFY and YFY; The chemical structural formula of YFY is: H2N-CH(CH2C6H4OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH; The chemical structural formula of SFY is: H2N-CH(CH2OH)-CONH-CH(CH2C6H5)-CONH-CH(CH2C6H4OH)-COOH; Its features are, The small molecule peptides are used to prepare antioxidant compositions.
3. A method for preparing a Torreya grandis protein extract containing intestinal-released antioxidant peptides, characterized in that, Includes the following steps: ① Mix the dried torreya nut protein powder with pure water to form a hydrolysis system. Add alkaline protease to the system, shake and hydrolyze for 3.5-4 hours. After hydrolysis, inactivate the enzyme, then cool in an ice bath to obtain the cooled hydrolysate. ② After cooling, the hydrolysate is centrifuged and the supernatant is collected. Then, it is processed by a nanofiltration system to retain components with a molecular weight of less than 3 kDa, and the permeate is obtained, which is the crude peptide extract. ③ The permeate was separated using a Superdex™ 200 increase10 / 300 GL column. During the separation process, 22.55 mL to 24.69 mL of liquid was collected and freeze-dried to obtain Torreya grandis protein extract containing intestinal-releasing antioxidant peptides.
4. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The preparation method of the Torreya grandis protein powder is as follows: The shells of Torreya grandis are removed and the powder is crushed to pass through a 60-100 mesh sieve to obtain Torreya grandis fine powder. The Torreya grandis fine powder is then degreased to remove the oil components, resulting in degreased Torreya grandis powder. Defatted Torreya grandis powder was suspended in water at a ratio of 8:1 to 10:1 (v / w). The pH was adjusted to 7.0±0.
2. After stirring for 1 to 3 hours, the mixture was centrifuged at 10,000 to 15,000 × g for 5 to 20 minutes. The supernatant was collected, and the pH was adjusted to 4.0±0.
2. The mixture was centrifuged at 2,000 to 10,000 r / min. The precipitate was collected, and the precipitate was redissolved in water. After stirring for 1 to 3 hours, the mixture was freeze-dried to obtain Torreya grandis protein powder. Torreya protein powder was dissolved in ultrapure water, stirred to reconstitute, and then freeze-dried to obtain dried torreya protein powder.
5. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The defatting method employs either supercritical CO2 extraction or solvent extraction. Specifically, supercritical CO2 extraction is carried out at 25-40 MPa, 30-50 °C, and a CO2 flow rate of 12-18 L / min for 60-240 min. Solvent extraction involves extraction with diethyl ether, petroleum ether, or No. 6 solvent oil 2-3 times followed by drying.
6. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The mixing ratio of the torreya protein powder to pure water is 1:50~1:30 g / mL.
7. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The alkaline protease is added at a rate of 3000-3500 U / g of protein powder.
8. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The centrifugation is specifically performed at 2-4°C and 10,000-12,000 g for 12-15 minutes; the oscillating hydrolysis is performed at 35-37°C and 120-150 rpm under constant temperature oscillation; and the freeze-drying temperature is -70 to -18°C.
9. The method for preparing the Torreya grandis protein extract containing intestinal-releasing antioxidant peptides according to claim 3, characterized in that: The eluent for the chromatographic column was a 0.12% aqueous solution of trifluoroacetic acid.
10. The use of a Torreya grandis protein extract containing intestinal-releasing antioxidant peptides as described in claim 1, characterized in that, The torreya protein extract is used to prepare an intestinal-absorbable antioxidant oral liquid.