Lipophilic short peptide antioxidant based on 3D-QSAR model rational design and its application in edible oil

Abstract

This invention discloses a rationally designed lipophilic short peptide antioxidant based on a 3D-QSAR model and its application in edible oils, belonging to the field of bioactive peptide technology. The method provided by this invention integrates computational chemistry, multivariate statistical analysis, and experimental verification. First, it constructs a library of lipophilic short peptides covering structural diversity and a database of their antioxidant activities. Then, it establishes and verifies a 3D-QSAR model that can quantitatively describe the relationship between the three-dimensional molecular field characteristics of short peptides and their antioxidant activity. Through model analysis, it reveals the key three-dimensional structural factors affecting antioxidant activity and their spatial interaction rules. Finally, based on the structure-activity relationship obtained from the analysis, novel short peptide sequences are rationally designed, and the model is used for high-throughput virtual screening and activity prediction. Furthermore, this invention provides a method for the targeted enzymatic hydrolysis preparation of short peptides with high antioxidant activity. By optimizing precursor proteins and employing specific enzymatic hydrolysis strategies, the target active peptide fragments can be efficiently enriched from natural protein sources.

Description

Technical Field

[0001] This invention relates to a lipophilic short peptide antioxidant rationally designed based on a 3D-QSAR model and its application in edible oils, belonging to the field of bioactive peptide technology. Background Technology

[0002] With increasing health awareness, the consumption of edible oils rich in polyunsaturated fatty acids (such as sunflower seed oil and flaxseed oil) is growing. However, the multiple unsaturated double bonds in polyunsaturated fatty acid molecules make them highly susceptible to auto-oxidation under the influence of environmental factors such as light, heat, oxygen, and metal ions. This produces primary oxidation products such as hydroperoxides, which further degrade into small-molecule volatile secondary oxidation products such as aldehydes, ketones, and acids. This process not only leads to a rancid taste and darkening of the oil's color, but may also generate potentially harmful substances, seriously threatening food safety and consumer health. Therefore, effectively delaying the oxidative rancidity of edible oils is a critical issue that urgently needs to be addressed in the oil processing and storage industry.

[0003] Currently, antioxidants are commonly used in industry to improve the oxidative stability of oils and fats. However, the long-term safety of widely used synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) is increasingly being questioned, and they have been strictly restricted or banned in some countries and regions. While natural antioxidants are generally safe, their antioxidant efficacy in complex oil and fat systems is limited, their cost is high, and they are constrained by solubility and dispersibility, making it difficult to meet the requirements for efficient and stable applications. Therefore, developing novel, efficient, safe, and suitable natural antioxidants for oil and fat systems has become an important research direction in the field of food science and technology.

[0004] Bioactive peptides, especially short peptides derived from edible proteins (typically containing 3-10 amino acid residues), have attracted considerable attention due to their natural origin, high safety, potential for various biological activities (including antioxidant activity), and favorable absorption and metabolic properties. Some short peptides have been shown to exert antioxidant effects through multiple mechanisms, such as hydrogen donation, electron donation, and chelation of metal ions. Nevertheless, the discovery of antioxidant peptides currently relies primarily on empirical screening, isolation, and identification of natural protein enzymatic hydrolysates, or on simple modifications based on limited known active peptide sequences. This approach is inherently prone to bias and chance, lacking a deep understanding and systematic exploration of the intrinsic quantitative relationships between amino acid sequence, three-dimensional spatial structure, and antioxidant activity. This results in long development cycles, high costs, and low efficiency, making it difficult to achieve targeted, rational design and precise activity control for the antioxidant needs of specific oil systems.

[0005] In recent years, the rapid development of computational chemistry and molecular simulation technologies has provided new opportunities to address the aforementioned bottlenecks. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models, as a powerful computer-aided drug design tool, can establish and visualize the quantitative mathematical relationship between the three-dimensional structural features of small molecule compounds and their biological activities without relying on complex biological receptor structural information. However, there are few mature and verifiable methodologies reported domestically and internationally for fully introducing this systematic and predictive rational design paradigm into the development of food-derived antioxidant short peptides, especially for lipophilic short peptides suitable for non-aqueous oil systems. Therefore, constructing a closed-loop rational design platform integrating computational design, model prediction, and experimental verification to achieve the efficient and precise creation of edible oil antioxidant peptides not only has significant theoretical innovation value but also has significant practical implications for promoting the green, intelligent, and sustainable development of the food additive industry. Summary of the Invention

[0006] [Technical Issues] The present invention aims to provide a series of lipophilic short peptides with excellent antioxidant activity and to establish a rational design method based on a three-dimensional quantitative structure-activity relationship model, so as to efficiently and purposefully design novel antioxidant peptides for edible oil systems.

[0007] [Technical Solution] The first object of the present invention is to provide a lipophilic short peptide, or a pharmaceutically or food-industrially acceptable salt thereof, said lipophilic short peptide having an amino acid sequence as shown in Formula I: Xaa1-Xaa2-Xaa3-Xaa4 (Formula I). Xaa1 is selected from phenylalanine F, tyrosine Y, tryptophan W, serine S, threonine T, methionine M, valine V, leucine L, proline P, and isoleucine I. Xaa2 is selected from tyrosine Y, phenylalanine F, tryptophan W, histidine H, serine S, valine V, glycine G, leucine L, proline P, arginine R, isoleucine I, and alanine A. The Xaa3 is selected from proline P, tryptophan W, tyrosine Y, phenylalanine F, histidine H, valine V, leucine L, aspartic acid D, histidine H, glutamine Q, isoleucine I, and alanine A. Xaa4 is either absent or selected from tyrosine Y, histidine H, glutamine Q, threonine T, aspartic acid D, tryptophan W, proline P, and isoleucine I.

[0008] In one embodiment of the present invention, the short peptide is selected from the group consisting of: tripeptides: FIH, FIL, FLA, FLY, FYV, HFY, IAV, ILF, IYP, LFA, LFQ, MFQ, PVY, TVL, VFH, VFY, VPF, WFH, WFQ, WYW, YFP, YIH, YPF, YPI, YVH, FYP, FYW, MFY, WYF, YSW; tetrapeptides: FFLK, FHLP, IIVS, IPFG, KIGI, LPFP, LPVP, LRVF, LVIF, TIYP, TLPY, TLVP, TVPY, TVYL, TVYP, TWYW, VTIV, FYLT, FWHD, SWYQ, TGYQ, THYQ, TWYH.

[0009] In one embodiment of the invention, the N-terminus of the short peptide is acetylated, and / or its C-terminus is amidated.

[0010] A second object of the present invention is to provide the use of the lipophilic short peptide in products for inhibiting or delaying the oxidative deterioration of oils or oil-containing compositions.

[0011] In one embodiment of the present invention, the oil or oil-containing composition is selected from food, health products, pharmaceuticals, cosmetics, feed or industrial products.

[0012] In one embodiment of the present invention, the food is selected from at least one of the following: edible oils, baked goods, fried foods, oily dairy products, meat products, confectionery products, infant formula, nuts and their products.

[0013] In one embodiment of the present invention, edible oils include soybean oil, peanut oil, olive oil, corn oil, sunflower oil, animal oil, margarine, shortening, blended oil, and salad dressing.

[0014] In one embodiment of the present invention, baked goods include biscuits, cakes, and bread.

[0015] In one embodiment of the present invention, fried foods include potato chips and instant noodles.

[0016] In one embodiment of the present invention, the oil-containing dairy products include cheese and cream.

[0017] In one embodiment of the present invention, the meat products include sausages, bacon, and luncheon meat.

[0018] In one embodiment of the invention, the confectionery products include chocolate and nut-containing candies.

[0019] In one embodiment of the present invention, the cosmetic is selected from at least one of the following: cream, lotion, serum, skin care oil, essential oil, massage oil, sunscreen product, makeup product, hair care product, and cleansing product.

[0020] In one embodiment of the present invention, the medicine or health product is selected from at least one of the following: oil-containing ointments, creams, drug delivery carriers, nutritional supplements containing unsaturated fatty acids, and vitamin preparations.

[0021] In one embodiment of the present invention, the drug delivery carrier comprises liposomes.

[0022] In one embodiment of the present invention, nutritional supplements containing unsaturated fatty acids include fish oil capsules and flaxseed oil capsules.

[0023] In one embodiment of the present invention, the vitamin preparation includes fat-soluble vitamins, such as vitamin A, D, and E soft capsules.

[0024] The industrial product is selected from at least one of the following: lubricating oil, biodiesel, oil-based coatings, and leather fatliquoring agents.

[0025] A third objective of this invention is to provide a method for inhibiting or delaying the oxidative deterioration of oils or oil-containing compositions, comprising adding the lipophilic short peptide to the oils or oil-containing compositions.

[0026] A fourth object of the present invention is to provide an antioxidant composition comprising: (a) Lipophilic short peptides as antioxidant active ingredients; and, (b) Oils and fats.

[0027] In one embodiment of the present invention, the composition further comprises at least one additive selected from emulsifiers, preservatives, fragrances, pigments, and other antioxidants.

[0028] Preferably, the other antioxidants include tocopherol and BHT.

[0029] The fifth objective of this invention is to provide a method for preparing the lipophilic short peptide, wherein the method uses a natural precursor protein as a substrate and prepares the lipophilic short peptide by enzymatic hydrolysis with a protease.

[0030] In one embodiment of the present invention, the enzymatic hydrolysis method involves adding a protease to a natural precursor protein at a mass fraction of 1-4%, and hydrolyzing for 20-200 min at 20-80°C and pH 4.0-9.0.

[0031] In one embodiment of the present invention, the natural precursor protein includes zein, rice protein, or soy protein.

[0032] In one embodiment of the present invention, the protease includes chymotrypsin or a complex protease.

[0033] Preferably, the co-protease includes alkaline protease and / or flavor protease.

[0034] A sixth object of the present invention is to provide the use of the lipophilic short peptide in screening proteins containing lipophilic short peptide antioxidants.

[0035] The seventh objective of this invention is to provide a method for establishing a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for predicting the antioxidant activity of lipophilic short peptides, comprising the following steps: (1) Obtain a training set, wherein the training set contains the amino acid sequences of multiple lipophilic short peptides and their corresponding measured antioxidant activity values; (2) Construct a minimum energy three-dimensional conformation for each lipophilic short peptide in the training set described in step (1); (3) Select a template molecule and, using the preset atoms of the template molecule as the superposition reference, spatially superimpose the three-dimensional conformation of each lipophilic short peptide obtained in step (2) with the template molecule; (4) Calculate a set of three-dimensional structure descriptors for each lipophilic short peptide after superposition in a preset molecular force field; (5) Using partial least squares regression, a mathematical relationship is established between the three-dimensional structure descriptor and the antioxidant activity value, thereby obtaining the 3D-QSAR model.

[0036] In one embodiment of the present invention, in step (1), the antioxidant activity values ​​are the inhibition rate of primary oxidation products and the inhibition rate of secondary oxidation products.

[0037] In one embodiment of the present invention, in step (3), the template molecule is a tripeptide WYF or YFP, and the superposition reference is the C of the peptide backbone. α atom.

[0038] In one embodiment of the present invention, the method further includes step (6), performing internal and / or external validation on the 3D-QSAR model, wherein the cross-validation correlation coefficient Q of the model is... 2 Greater than 0.5.

[0039] This invention also provides a method for screening or designing lipophilic short peptides with antioxidant activity using a 3D-QSAR model constructed based on the above method, comprising the following steps: (1) Provide the amino acid sequence of at least one candidate lipophilic short peptide; (2) Construct a minimum energy three-dimensional conformation for the candidate lipophilic short peptide and spatially superimpose it with the template molecule; (3) Calculate the three-dimensional structure descriptor for the superimposed candidate lipophilic short peptides; (4) Input the descriptor obtained in step (3) into the 3D-QSAR model to obtain its predicted antioxidant activity value; (5) Based on the predicted antioxidant activity value, determine whether the candidate lipophilic short peptide is a peptide with preset antioxidant activity.

[0040] In one embodiment of the present invention, the template molecule is WYF or YFP.

[0041] [Beneficial Effects] This invention is the first to construct a reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) model for lipophilic short peptides in an oil-phase system. The model construction process is as follows: Figure 1 As shown. Based on this 3D-QSAR model, rational design and optimization can be carried out, achieving a shift from "blind screening" to "intelligent design," and designing and screening natural antioxidants with high safety and natural origin with an 80% success rate. This invention further provides a lipophilic short peptide, WYF, with good antioxidant activity, which exhibits superior free radical scavenging ability compared to vitamin E (VE) in electron spin resonance (ESR) experiments. Attached Figure Description

[0042] Figure 1 This is a flowchart of the model construction process for the present invention; Figure 2 For Williams diagram; Figure 3 A is a three-dimensional contour map; B is a hydrophobic field contour map; C is a three-dimensional field contour map; D is a hydrogen bond acceptor field contour map; and E is a hydrogen bond donor field contour map. Detailed Implementation

[0043] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0044] The following embodiments involve test methods: Antioxidant activity assay: (1) Primary oxidation product inhibition rate: The inhibition rate of lipophilic short peptides on primary oxidation products of sunflower seed oil was determined by the ammonium thiocyanate method. Specifically, the oxidized oil sample was mixed with a chloroform-n-butanol-methanol mixed solvent in a certain proportion. The mixture was then added to ammonium thiocyanate solution and ferrous chloride solution in sequence. After reacting at room temperature for 5 minutes, the absorbance was immediately measured at a wavelength of 500 nm. The inhibition rate of primary oxidation products of the lipophilic short peptides was calculated according to the following formula.

[0045]

[0046] Wherein: I inhibition rate; A0 absorbance value of sunflower seed oil; A absorbance value of sunflower seed oil after the addition of lipophilic short peptides; A b Absorbance value without added sunflower seed oil.

[0047] (2) Inhibition rate of secondary oxidation products: The inhibition rate of lipophilic short peptides on secondary oxidation products of sunflower seed oil was determined by the thiobarbituric acid reactant method. Specifically, the oxidized oil sample was mixed with TBA solution and reacted in a 90°C water bath for 30 minutes. After cooling, the absorbance was measured at a wavelength of 532 nm. The inhibition rate of secondary oxidation products of the lipophilic short peptides was calculated according to the following formula.

[0048]

[0049] Wherein: I inhibition rate; A0 absorbance value of sunflower seed oil; A absorbance value of sunflower seed oil after the addition of lipophilic short peptides; A b Absorbance value without added sunflower seed oil.

[0050] Example 1: Construction of a database of antioxidant activities of lipophilic short peptides This embodiment constructs an initial peptide library and measures the antioxidant capacity of each active peptide. It also constructs a database containing 92 lipophilic short peptides, categorized as "structure-primary oxidation product inhibition rate" and "structure-secondary oxidation product inhibition rate," as detailed below: 1. Peptide Library Preparation: A preliminary peptide library of 92 lipophilic short peptides (purity verified by HPLC) was synthesized using a solid-phase synthesis method. The library covers tripeptides to nonapeptides, with a focus on 36 tripeptides and 28 tetrapeptides. Sequence design considered the diversity of amino acid types (aromatic, aliphatic, polar, charged, acidic, basic), hydrophobicity, net charge, and a wide range of hydrogen bond donor / acceptor capabilities.

[0051] 2. Antioxidant activity assay: (1) Sample preparation: Refined sunflower seed oil was selected as the model oil system to determine the inhibition rates of primary oxidation products and secondary oxidation products of each lipophilic short peptide in the initial peptide library in step 1. The 92 lipophilic short peptides synthesized in step 1 were added to sunflower seed oil to a final concentration of 50 mg / kg. Oil samples without any added antioxidants were used as blank controls, and oil samples with added vitamin E (VE, α-tocopherol) at the same concentration were used as positive controls. All samples were vortexed for 5 min and sonicated for 20 min to ensure uniform dispersion of antioxidants.

[0052] (2) Accelerated oxidation experiment: The sample prepared in step (1) was placed in a transparent glass vial and accelerated oxidized at 90°C for 12 h in an oven. Subsequently, the inhibition rates of primary oxidation products and secondary oxidation products of each lipophilic short peptide were measured.

[0053] 3. Database construction: Based on the inhibition rates of primary oxidation products and secondary oxidation products of each lipophilic short peptide in step 2, a database of "structure-primary oxidation product inhibition rate" and "structure-secondary oxidation product inhibition rate" containing 92 lipophilic short peptides is finally obtained.

[0054] Analysis showed that the 44 peptides exhibited higher inhibitory effects on both oxidation products than the natural antioxidant vitamin E, providing high-quality antioxidant activity data for modeling.

[0055] Table 1. Antioxidant activity data of peptides

[0056] Example 2: Construction and Validation of a 3D-QSAR Model Based on Primary Oxidation Product Suppression Rate This embodiment constructs and verifies the 3D-QSAR model based on the "sequence-primary oxidation product suppression rate" of Example 1, as detailed below: 1. Structure and Overlap: (1) Data selection and preparation: From the database of Example 1, the “sequence-primary oxidation product inhibition rate” data of all 36 tripeptides and 28 tetrapeptides (a total of 64 peptides) were selected as the modeling source data.

[0057] (2) Molecular construction and structure-activity optimization: The initial three-dimensional structure of each lipophilic short peptide was constructed using the "Peptide Builder" module in the Tripos SYBYL-X software. The Gasteiger-Hückel method was used to distribute atomic charges, and the Tripos force field was selected. The lowest energy conformation of each lipophilic short peptide was obtained by minimizing energy (conjugate gradient method, with a termination gradient of 0.005 kcal / (mol·Å)).

[0058] (3) Molecular superposition: The tripeptide YFP, which has the highest inhibition rate of primary oxidation products, was selected as the template molecule. Using the "Align Database" function, the C-axis of the peptide backbone of tripeptide YFP was used as the template molecule. α Using atoms as superposition points, the least squares method is used to spatially superimpose the optimized conformations of other lipophilic short peptide molecules with the template molecule YFP, ensuring that the main chain spatial orientation is consistent.

[0059] 2. 3D-QSAR Model Construction (1) CoMSIA descriptor calculation: Place the superimposed molecular system in an automatically generated grid (grid spacing is usually set to 2 Å). Set a methyl probe with a +1 charge and calculate the interaction energy of the five molecular force fields (stereo field, electrostatic field, hydrophobic field, hydrogen bond donor field, and hydrogen bond acceptor field) at each grid point. Use the default attenuation factor.

[0060] (2) Data partitioning and model training: Using the "Sphere Exclusion" algorithm in the software to ensure structural diversity, the 64 samples were divided into a training set containing 51 samples and a test set containing 13 samples. Partial least squares regression was used, with five molecular force field descriptors as independent variables and the primary inhibition rate as the dependent variable, to fit the model. The optimal number of principal components was determined through cross-validation.

[0061] 3. Validation of the 3D-QSAR model (1) Internal model validation: The training set is analyzed using methods such as leave-one-out cross-validation to obtain the model's goodness-of-fit index: the non-cross-validation correlation coefficient (R²). 2 The cross-validation correlation coefficient (Q) was significantly higher than the acceptable threshold. 2 A value greater than 0.5 indicates that the model has good robustness and internal predictive power. The standard estimation error and F-test value further support the statistical significance of the model.

[0062] (2) External validation and application domain analysis of the model: The structural descriptors of the 13 peptides in the test set were input into the 3D-QSAR model to obtain the predicted inhibition rate. The consistency correlation coefficient between the predicted value and the measured value was calculated. The value was close to 0.9, which proved that the model has good external prediction ability.

[0063] Draw the Williams plot. Calculate the leverage value (h) for all samples (including the training and test sets). i ) and standardized residuals. Set critical leverage values. (where p is the principal component number and n is the number of training set samples). Figure 2 The data shows that the absolute value of the standardized residuals for the vast majority of samples is less than 3, and the leverage value is lower than 3. This indicates that the model's application domain is clearly defined, and only a few samples (such as sequences FIT and VLY) are identified as outliers, making the model generally stable and reliable.

[0064] Example 3: 3D-QSAR Model Analysis and Structure-Activity Relationship Visualization This embodiment performs a quantitative analysis of the contribution of each molecular field to antioxidant activity in the 3D-QSAR model, as detailed below: 1. Contribution Analysis: The percentage contribution of each molecular force (stereoscopic field, electrostatic field, hydrophobic field, hydrogen bond donor field, and hydrogen bond acceptor field) to antioxidant activity was extracted from the 3D-QSAR model constructed in Example 2. Analysis showed that the hydrogen bond donor field had the highest contribution, followed by the hydrophobic field and the electrostatic field, while the contributions of the stereoscopic field and the hydrogen bond acceptor field were relatively low. This quantitatively reveals that in this lipid system, the hydrogen-donating capacity of lipophilic short peptides and their compatibility with the lipid environment are the most critical factors determining their primary oxidation inhibition rate.

[0065] 2. Generation and analysis of 3D contour maps ( Figure 3 ): Based on the training set, generate three-dimensional contour maps of the stereo field, electrostatic field, hydrophobic field, hydrogen bond donor field and hydrogen bond acceptor field of the CoMSIA model, and superimpose them on the template molecule YFP.

[0066] In the three-dimensional contour plots, large yellow contour areas are observed around the N-terminus and middle sites of the lipophilic short peptide, indicating that introducing / enhancing hydrophobicity at these locations is beneficial to activity. A white area appears at the C-terminus, suggesting that hydrophilic groups may be more favorable at this position. In the hydrogen bond donor field contour plot, distinct cyan contour areas are visible near positions R1 and R2, consistent with contribution analysis results, strongly indicating these positions as key hydrogen atom donor sites. In the electrostatic field contour plot, blue areas (preferring negative charges) are visible around the amide nitrogen atom in the peptide backbone and at position R1, while a red area (preferring positive charges) is visible near position R3, providing spatial guidance for side chain charge design. In the stereo field contour plot, a coexistence of green (favorable for bulky groups) and yellow (unfavorable for bulky groups) areas is observed at position R3, suggesting a precise spatial requirement for group volume at this location.

[0067] Example 4: Iterative Optimization of 3D-QSAR Model This embodiment updates and iterates the 3D-QSAR model to obtain a design system that more accurately predicts the antioxidant activity of bioactive peptides, as detailed below: The antioxidant activity of newly designed and synthesized short peptides was determined using the same steps as in Examples 1-3. The measured antioxidant activity was then systematically compared with the predicted values ​​from the 3D-QSAR model. If the antioxidant activity trends of most (e.g., over 80%) of the newly designed peptides are highly consistent with the results predicted by the 3D-QSAR model, this constitutes the strongest empirical support for the effectiveness of the entire rational design methodology.

[0068] Subsequently, the novel peptide amino acid sequence and measured antioxidant activity data were used as new samples and integrated back into the "structure-primary oxidation product inhibition rate" database of Example 1. Using the expanded database, the 3D-QSAR model was retrained and optimized. Through continuous data feedback and model updates, with each round of "design-prediction-synthesis-validation" cycle training, the predictive accuracy and chemical space exploration capabilities of the 3D-QSAR model were continuously enhanced, ultimately forming an intelligent design system with self-learning and improvement capabilities.

[0069] Example 5: Design of novel lipophilic short peptides based on structure-antioxidant activity relationship and verification of their antioxidant activity. This embodiment designs and verifies lipophilic antioxidant peptides based on the relationship between the lipophilic short peptide structure and antioxidant activity obtained in Example 3, as detailed below: 1. Rational Design: Based on the relationship between the lipophilic short peptide structure and antioxidant activity obtained in Example 3, the tripeptide WYF was designed. R1 is Trp (aromatic, hydrophobic), R2 is Tyr (aromatic, hydrophobic, strong hydrogen bond donor), and R3 is Phe (aromatic, hydrophobic, moderately sized). After optimizing the WYF structure model, it was input into the 3D-QSAR model of Example 2, which predicted that its primary oxidation product inhibition rate was much higher than that of the natural antioxidant VE. Replacing Tyr in R1 with Glu (acidic, hydrophilic, hydrogen bond acceptor / donor) was used to test the importance of N-terminal hydrogen bond donor / hydrophobicity. The model predicted that its activity would be significantly lower than that of WYF. A total of 15 novel lipophilic short peptides were designed, covering various situations from strictly following to partially changing the rules.

[0070] 2. Synthesis and Activity Assay of Novel Lipid-Soluble Short Peptides: The 15 designed peptides described above were synthesized. The actual antioxidant properties of each novel lipid-soluble short peptide were tested strictly following the same sample preparation, accelerated oxidation, and activity assay methods as described in Example 1.

[0071] 3. Results Analysis and Method Validation: Peptides predicted to be highly active (such as WYF) showed measured inhibition rates of primary and secondary oxidation products that were comparable to or higher than those of the natural antioxidant vitamin E, consistent with the model's predicted trend. Peptides predicted to be low-active (such as EWF) indeed exhibited lower measured antioxidant activity. Among the 15 designed peptides, the measured activity rankings of 12 peptides highly matched the model's predicted rankings, and the deviations between the absolute activity values ​​and the predicted values ​​were within acceptable limits, achieving a design success rate of 80%. This result strongly demonstrates that rational design based on the structure-activity relationship rules resolved by the 3D-QSAR model of this invention can efficiently and accurately create novel highly active antioxidant peptides.

[0072] Table 2. Antioxidant activity data of peptides

[0073] Example 6: Targeted Enzymatic Hydrolysis Method for Peptides Based on Rational Design To bridge the gap between rational design and large-scale production, this embodiment provides a method for the targeted enzymatic hydrolysis preparation of a designed highly active peptide, as detailed below: 1. Precursor protein selection and enzymatic digestion strategy design: (1) Screening of natural precursor proteins: Based on the amino acid sequence of the target peptide obtained through rational design (such as WYF in Example 4), natural precursor proteins are screened through bioinformatics analysis. Zeat gliadin, rice protein, or soybean protein rich in Tyr, Trp, and Phe and with a high frequency of occurrence of the target peptide sequence are selected as enzymatic hydrolysis substrates.

[0074] (2) Design and screen proteases: Analyze the cleavage sites of the target peptide and select proteases with specific recognition sites, such as chymotrypsin (which cleaves the carboxyl terminus of aromatic amino acids) or complex proteases (such as a combination of alkaline protease and flavor protease) to maximize the release of the target peptide or a peptide that is highly similar to its core active fragment.

[0075] 2. Optimization of enzymatic hydrolysis process: The selected protein substrate was prepared into a solution or suspension with a certain mass concentration (1%~4%). Response surface methodology was used to optimize key enzymatic hydrolysis parameters: within a set temperature range (45-60℃) and pH range (set according to the optimal pH of the protease), the effects of the enzyme-to-substrate ratio (E / S) and reaction time on the degree of hydrolysis were investigated.

[0076] Optimized enzymatic hydrolysis process: Rice protein substrate concentration: 2%, hydrolysis temperature: 50℃, pH: 7.5, chymotrypsin to substrate mass ratio (E / S): 1:12, hydrolysis time: 180 min.

[0077] The results showed that the degree of protein hydrolysis reached 15%, the inhibition rate of primary oxidation products reached 31%, and the inhibition rate of secondary oxidation products reached 27%.

[0078] 3. Isolation and enrichment of enzymatic hydrolysis products: Immediately after the enzymatic hydrolysis reaction is complete, heat the solution to inactivate the enzyme. Centrifuge the hydrolysate to remove insoluble matter.

[0079] An ultrafiltration membrane system (molecular weight cutoff <3 kDa) was used to perform preliminary separation of the enzymatic hydrolysate, and the permeate or retentate rich in the target short peptide was collected.

[0080] Further, preparative reversed-phase high-performance liquid chromatography or macroporous adsorption resin column chromatography can be used to finely purify and enrich the target peptide components.

[0081] 4. Product identification and verification: Mass spectrometry analysis (LC-ESI-MS / MS) of the purified product confirmed that the main peptide sequences were consistent with the design target.

[0082] Finally, following the method described in Example 1, the antioxidant activity of the enzymatically prepared peptide product was determined, verifying that the prepared peptide maintained the high activity expected by the rational design.

[0083] Example 7: Validation of the antioxidant activity of novel lipophilic short peptides This embodiment tests the antioxidant activity of WYF, as detailed below: 1. Sample preparation: Select the highly active designed peptide WYF, which was successfully verified in Example 5, as well as VE and blank oil sample as controls.

[0084] 2. ESR test: The sample to be tested (sunflower seed oil with added WYF or VE, concentration of 200 mg / kg) was mixed with a toluene solution of free radical scavenger PBN. The mixture was then placed in a quartz capillary tube and placed in the heating chamber of the electron spin resonance spectrometer.

[0085] 3. Free radical generation monitoring: The temperature was rapidly increased to 150℃, and ESR signals were captured at this temperature at 5, 10, 15, and 20 min after heating. The amount of free radicals generated in different samples was quantitatively compared by measuring the characteristic signal intensity of the PBN-free radical adduct.

[0086] 4. Results Analysis: Compared with the blank group and the control group, the oil sample with added WYF showed a significant decrease in ESR signal intensity throughout the entire heating process, and the decrease was greater than that of the positive control VE group. This directly proves that the highly active peptide WYF designed in this invention can effectively remove free radicals generated during the high-temperature oxidation of oils, thereby exerting an antioxidant effect by interrupting the free radical chain reaction.

[0087] Table 3

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A lipophilic short peptide, or a pharmaceutically or food-industrially acceptable salt thereof, characterized in that, The lipophilic short peptide has an amino acid sequence as shown in Formula I: Xaa1-Xaa2-Xaa3-Xaa4 (Formula I). Xaa1 is selected from phenylalanine F, tyrosine Y, tryptophan W, serine S, threonine T, methionine M, valine V, leucine L, proline P, and isoleucine I. Xaa2 is selected from tyrosine Y, phenylalanine F, tryptophan W, histidine H, serine S, valine V, glycine G, leucine L, proline P, arginine R, isoleucine I, and alanine A. The Xaa3 is selected from proline P, tryptophan W, tyrosine Y, phenylalanine F, histidine H, valine V, leucine L, aspartic acid D, histidine H, glutamine Q, isoleucine I, and alanine A. Xaa4 is either absent or selected from tyrosine Y, histidine H, glutamine Q, threonine T, aspartic acid D, tryptophan W, proline P, and isoleucine I.

2. The lipophilic short peptide or its salt according to claim 1, characterized in that, The short peptides are selected from the group consisting of: tripeptides: FIH, FIL, FLA, FLY, FYV, HFY, IAV, ILF, IYP, LFA, LFQ, MFQ, PVY, TVL, VFH, VFY, VPF, WFH, WFQ, WYW, YFP, YIH, YPF, YPI, YVH, FYP, FYW, MFY, WYF, YSW; tetrapeptides: FFLK, FHLP, IIVS, IPFG, KIGI, LPFP, LPVP, LRVF, LVIF, TIYP, TLPY, TLVP, TVPY, TVYL, TVYP, TWYW, VTIV, FYLT, FWHD, SWYQ, TGYQ, THYQ, TWYH; Preferably, the N-terminus of the short peptide is acetylated, and / or its C-terminus is amidated.

3. The use of the lipophilic short peptide according to claim 1 or 2 in products for inhibiting or delaying the oxidative deterioration of oils or oil-containing compositions, characterized in that, The oil or oil-containing composition is selected from food, health products, pharmaceuticals, cosmetics, feed, or industrial products.

4. The application according to claim 3, characterized in that, The food is selected from at least one of the following: edible oils, baked goods, fried foods, oily dairy products, meat products, confectionery products, infant formula, nuts and their products; The cosmetics are selected from at least one of the following: creams, lotions, serums, skin care oils, facial oils, massage oils, sunscreens, makeup products, hair care products, and cleansing products; The medicine or health product is selected from at least one of the following: oil-containing ointments, creams, drug delivery carriers, nutritional supplements containing unsaturated fatty acids, and vitamin preparations; The industrial product is selected from at least one of the following: lubricating oil, biodiesel, oil-based coatings, and leather fatliquoring agents; Preferably, the edible oils include soybean oil, peanut oil, olive oil, corn oil, sunflower oil, animal oil, margarine, shortening, blended oil, and salad dressing. Preferably, the drug delivery carrier comprises liposomes; Preferably, the nutritional supplement containing unsaturated fatty acids includes fish oil capsules and flaxseed oil capsules; Preferably, the vitamin preparation includes fat-soluble vitamins, such as vitamin A, D, and E soft capsules.

5. A method for inhibiting or delaying the oxidative deterioration of oils or oil-containing compositions, characterized in that, This includes adding the lipophilic short peptide described in claim 1 or 2 to oils or oil-containing compositions.

6. An antioxidant composition, characterized in that, It includes: (a) the lipophilic short peptide of claim 1 or 2 as an antioxidant active ingredient; and, (b) Oils and fats; Preferably, the composition further comprises at least one additive selected from emulsifiers, preservatives, fragrances, colorants, and other antioxidants; Preferably, the other antioxidants include tocopherol and BHT.

7. A method for preparing the lipophilic short peptide of claim 1 or 2, characterized in that, The method involves using natural precursor proteins as substrates and preparing lipophilic short peptides by enzymatic hydrolysis with proteases. The enzymatic hydrolysis method involves adding a protease to a natural precursor protein at a mass fraction of 1-4%, and hydrolyzing for 20-200 min at 20-80°C and pH 4.0-9.

0.

8. The method according to claim 7, characterized in that, The natural precursor protein includes zein, rice protein, or soybean protein; the protease includes chymotrypsin or a complex protease. Preferably, the complex protease includes an alkaline protease and / or a flavor protease.

9. A method for screening or designing lipophilic short peptides with antioxidant activity based on a 3D-QSAR model, characterized in that, Includes the following steps: (1) Provide the amino acid sequence of at least one candidate lipophilic short peptide; (2) Construct a minimum energy three-dimensional conformation for the candidate lipophilic short peptide and spatially superimpose it with the template molecule; (3) Calculate the three-dimensional structure descriptor for the superimposed candidate lipophilic short peptides; (4) Input the descriptor obtained in step (3) into the 3D-QSAR model to obtain its predicted antioxidant activity value; (5) Based on the predicted antioxidant activity value, determine whether the candidate lipophilic short peptide is a peptide with preset antioxidant activity.

10. The method according to claim 9, characterized in that, The method for constructing the 3D-QSAR model is as follows: (1) Obtain a training set, wherein the training set contains the amino acid sequences of multiple lipophilic short peptides and their corresponding measured antioxidant activity values; (2) Construct a minimum energy three-dimensional conformation for each lipophilic short peptide in the training set described in step (1); (3) Select a template molecule and, using the preset atoms of the template molecule as the superposition reference, spatially superimpose the three-dimensional conformation of each lipophilic short peptide obtained in step (2) with the template molecule; (4) Calculate a set of three-dimensional structure descriptors for each lipophilic short peptide after superposition in a preset molecular force field; (5) Using partial least squares regression, a mathematical relationship is established between the three-dimensional structure descriptor and the antioxidant activity value, thereby obtaining the 3D-QSAR model; Preferably, in step (1), the antioxidant activity values ​​are the inhibition rate of primary oxidation products and the inhibition rate of secondary oxidation products; Preferably, in step (3), the template molecule is a tripeptide WYF or YFP, and the superposition reference is the C of the peptide backbone. α atom; Preferably, the method further includes step (6), performing internal and / or external validation on the 3D-QSAR model, wherein the cross-validation correlation coefficient Q of the model is... 2 Greater than 0.5.

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