A milk-derived bioactive peptide, DDLCDYWIR, with strong antioxidant and immunomodulatory properties, its preparation method, and its applications.

Abstract

This invention discloses a novel milk-derived bioactive peptide, DDLCDYWIR, with strong antioxidant and immunomodulatory properties, along with its preparation method and applications, belonging to the field of bioactive peptides. Using lactoferrin as a raw material, this invention involves enzymatic hydrolysis with protease and fermentation with *Lactobacillus paracasei*, from which a novel milk-derived bioactive peptide is screened. Experimental verification shows that this bioactive peptide exhibits excellent antioxidant and immunomodulatory activities: it not only enhances the body's immune function but also effectively combats oxidative stress, thus delaying aging. Furthermore, this bioactive peptide is derived from natural milk, without any added chemicals, and possesses good biocompatibility, making it widely applicable in the preparation of antioxidant, immunomodulatory, or dual-function products.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and more specifically, to a milk-derived bioactive peptide, DDLCDYWIR, which possesses strong antioxidant and immunomodulatory properties, as well as its preparation method and applications. Background Technology

[0002] In modern life, factors such as frequent late nights and high levels of stress are leading to increasingly common problems of oxidative stress imbalance and immune dysfunction in the human body. This has created an urgent market demand for functional food ingredients that are naturally sourced and possess both antioxidant and immune-regulating effects. Currently, most dairy products on the market are still primarily positioned as basic nutritional supplements (such as calcium and protein supplements), but there is a significant supply gap for dairy-derived bioactive peptide products that address core needs such as "antioxidation" and "immune regulation," making it difficult to meet market demand.

[0003] From the perspective of the actual market situation, most existing dairy products focus on basic nutritional supplementation (such as calcium supplementation and whey protein supplementation), and very few products use "milk-derived active peptides" as a core functional ingredient to specifically achieve targeted effects such as anti-oxidation or immune regulation. In the field of anti-oxidation, most dairy products that claim to be "antioxidant" either rely solely on the natural trace substances such as vitamin E and selenium contained in milk itself, without adding highly active milk-derived antioxidant peptides, or use chemically synthesized antioxidants such as BHA and BHT. These ingredients are far inferior to natural milk-derived active peptides in terms of safety and biocompatibility. Even if a few products are labeled as containing "milk-derived antioxidant peptides," their actual active peptide content is often less than 0.3%, and core activity indicators such as DPPH free radical scavenging rate and ABTS free radical inhibition rate do not reach the effective action threshold, thus failing to achieve a stable antioxidant effect. In the field of immune regulation, the supply gap is even more pronounced: most dairy products that claim to "enhance immunity" either achieve an indirect immune support effect by adding ordinary probiotics, without containing specific immune-regulating dairy bioactive peptides, or using non-dairy immune components such as plant polysaccharides and animal extracts—these components have low compatibility with the dairy matrix; at the same time, products generally lack actual verification data on "dairy bioactive peptides regulating the activity of immune cells such as macrophages and T cells," and some products even vaguely replace the "immune regulation" function with the concept of "supplementing protein," making it difficult to meet consumers' demand for "precise immune support."

[0004] The core reason for the aforementioned supply gap lies in the technological bottlenecks in the preparation and research of milk-derived bioactive peptides. On the one hand, traditional preparation processes struggle to selectively obtain highly active single-function milk-derived peptides, and subsequent chromatographic separation and purification are required, increasing costs by 6-8 times compared to ordinary milk protein processing. Many companies have abandoned related research due to cost pressures. While microbial fermentation is a preferred technological path for preparing milk-derived bioactive peptides, the industry currently lacks sufficient screening of specific strains for "high-yield antioxidant milk-derived peptides" or "high-yield immunomodulatory milk-derived peptides." Existing strains (such as conventional lactic acid bacteria) mostly produce only trace amounts of bioactive peptides, insufficient to support commercial production needs. On the other hand, the industry's research focus is misaligned. Existing milk-related research is largely concentrated on optimizing basic nutritional components, with insufficient depth in research on the amino acid sequence analysis and activity mechanisms of "antioxidant milk-derived bioactive peptides," as well as the interaction targets of "immunomodulatory milk-derived bioactive peptides" with immune cells. This lack of feasible industrialization solutions further exacerbates the supply gap for targeted products in the market.

[0005] Therefore, developing a natural, highly active, and industrially producible "antioxidant" and / or "immunomodulatory" milk-derived bioactive peptide has become an urgent problem for the industry.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a milk-derived bioactive peptide DDLCDYWIR with strong antioxidant and immunomodulatory properties, its preparation method, and its applications.

[0008] This invention is implemented as follows:

[0009] In a first aspect, embodiments of the present invention provide a polypeptide whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0010] Secondly, embodiments of the present invention provide a biological material selected from: (a) an isolated nucleic acid molecule encoding the polypeptide described in the foregoing embodiments; (b) a carrier containing the nucleic acid molecule; and (c) a cell containing the carrier.

[0011] Thirdly, embodiments of the present invention provide a method for preparing milk-derived bioactive peptides, which includes the following steps: artificially synthesizing the polypeptides described in the foregoing embodiments or culturing the cells described in the foregoing embodiments.

[0012] Fourthly, embodiments of the present invention provide a method for preparing milk-derived bioactive peptides, comprising the following steps: mixing lactoferrin solution with protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate; mixing the enzymatic hydrolysate, Lactobacillus paracasei, and lactose for fermentation to obtain a fermentation broth; inactivating the fermentation broth and centrifuging it to obtain a supernatant; and ultrafiltration the supernatant to obtain milk-derived bioactive peptides.

[0013] Fifthly, embodiments of the present invention provide a composition whose active ingredient includes the polypeptide described in the foregoing embodiments, the biomaterial described in the foregoing embodiments, or the product prepared by the preparation method described in the foregoing embodiments.

[0014] Sixthly, embodiments of the present invention provide the use of the polypeptides, biomaterials, products prepared by the preparation methods described in the foregoing embodiments, or compositions described in the foregoing embodiments in the preparation of antioxidant and / or immunomodulatory products.

[0015] The present invention has the following beneficial effects:

[0016] This invention uses lactoferrin as a raw material, and after enzymatic hydrolysis with protease and fermentation with Lactobacillus paracasei, a novel milk-derived bioactive peptide is screened from the product. Experimental verification shows that this bioactive peptide exhibits excellent antioxidant and immunomodulatory effects: it not only enhances the body's immune function but also effectively combats oxidative stress, thus delaying aging. Furthermore, this bioactive peptide is derived from natural milk, without any added chemicals, and possesses good biocompatibility, making it widely applicable in the preparation of antioxidant, immunomodulatory, or dual-function products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The mass spectrum of the fragment with a mass-to-charge ratio of 628.2802 is shown.

[0019] Figure 2 The secondary mass spectrum of the fragment with a mass-to-charge ratio of 628.2802 and the fragmentation of peptides az and by are shown.

[0020] Figure 3 The spatial structure predicted for the bioactive peptide (SEQ ID NO:1);

[0021] Figure 4The images show the survival curves of nematodes under oxidative stress; Blank is the blank control, with only deionized water added; Sample is a 1 mg / ml active peptide sample, with deionized water as the solvent.

[0022] Figure 5 The images show the immune survival curves of nematodes; Blank is the blank control, with only deionized water added; Sample is a 1 mg / ml active peptide sample, with deionized water as the solvent. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] Definition of noun

[0025] The term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. Amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0026] The term "pharmaceutical acceptable" means that the form of the compound must meet requirements for safety, stability, and suitability for formulation.

[0027] On one hand, embodiments of the present invention provide a polypeptide whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:1 (DDLCDYWIR).

[0028] DDLCDYWIR, specifically Asp-Asp-Leu-Cys-Asp-Tyr-Trp-Ile-Arg.

[0029] In some possible implementations, the at least 80% includes: 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0030] On the other hand, embodiments of the present invention provide a biomaterial selected from:

[0031] (a) An isolated nucleic acid molecule encoding the polypeptide described in any of the foregoing embodiments;

[0032] (b) A carrier containing the nucleic acid molecule;

[0033] (c) Cells containing the carrier.

[0034] In some implementations, the vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells via transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0035] In some possible implementations, the cells include prokaryotic cells or eukaryotic cells; the prokaryotic cells include, but are not limited to, bacterial cells, such as Escherichia coli; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, or animal cells; the yeast cells may be, but are not limited to, Pichia pastoris or Saccharomyces cerevisiae cells; the animal cells may be, but are not limited to, CHO cells, COS cells, NSO cells, 293T cells, HT-1080 cells, BHK (young hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, or PERC.6 (human retinal cells).

[0036] On the other hand, embodiments of the present invention provide a method for preparing milk-derived bioactive peptides, which includes the following steps: artificially synthesizing the polypeptides described in any of the foregoing embodiments or culturing the cells described in any of the foregoing embodiments.

[0037] On the other hand, embodiments of the present invention provide a method for preparing milk-derived bioactive peptides, which includes the following steps: mixing lactoferrin solution with protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate; mixing the enzymatic hydrolysate, Lactobacillus paracasei, and lactose for fermentation to obtain a fermentation broth; inactivating the fermentation broth and centrifuging it to obtain a supernatant; and ultrafiltration the supernatant to obtain milk-derived bioactive peptides.

[0038] This preparation method maximizes the peptide production capacity and bioactivity of the target strain, while ensuring bioactivity and reducing production costs.

[0039] In some embodiments, the protein concentration of the lactoferrin solution is 1% to 10% (wt / wt), and the protein concentration can be any one or any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

[0040] In some embodiments, the preparation of the lactoferrin solution includes: mixing lactoferrin with water and adjusting the pH to 5.0-8.0. This pH can be any one or a range between any two of the following: 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0.

[0041] In some implementations, the protease includes a neutral protease.

[0042] In some embodiments, the enzymatic hydrolysis conditions include a temperature of 30–70°C and a time of 4–6 hours. The enzymatic hydrolysis temperature can be any one or any combination of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70°C. The specific time can be any one or any combination of 4 hours, 4 hours 10 minutes, 4 hours 20 minutes, 4 hours 30 minutes, 4 hours 40 minutes, 4 hours 50 minutes, 5 hours, 5 hours 10 minutes, 5 hours 20 minutes, 5 hours 30 minutes, 5 hours 40 minutes, 5 hours 60 minutes, and 6 hours.

[0043] In some embodiments, the amount of protease added is 0.1% to 2.0% of the substrate lactoferrin. This amount can be any one or a range between any two of the following: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2.0%.

[0044] In some possible implementations, 10 mg of enzyme solution is added per 1 mL of enzymatic hydrolysis solution. 4 ~10 6 CFU of Lactobacillus paracasei and 0.01~1.00g lactose; the amount of Lactobacillus paracasei added can be 10. 4 10 5 10 6 The amount of lactose added can be any one or any two of the following CFUs: 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 and 1.00g.

[0045] In some possible implementations, the *Lactobacillus paracasei* includes *Lactobacillus paracasei* ET-22, with accession number CGMCC No. 15077, which has been disclosed in the invention titled "A *Lactobacillus paracasei* ET-22 with function of relieving intestinal inflammation" and patent number CN110893195A.

[0046] In some possible implementations, the fermentation conditions include: a temperature of 30-45°C and a fermentation time of 2-10 hours. Specifically, the fermentation temperature can be any one or any two of the following: 30, 32, 34, 36, 38, 40, 42, 44, and 45°C. Specifically, the fermentation time can be any one or any two of the following: 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 hours.

[0047] In some embodiments, the centrifugation conditions are: 6000~10000 RCF (×g), 0~10℃, 1~30 min. The relative centrifugal force can be any one or any two of 6000, 7000, 7500, 8000, 8500, 9000, and 10000 RCF. The temperature can be any one or any two of 0, 2, 4, 6, 8, and 10℃. The time can be any one or any two of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 min.

[0048] In some possible implementations, the ultrafiltration membrane has a molecular weight cutoff of ≤10kDa, specifically any one or any two of 5, 6, 7, 8, 9 and 10kDa.

[0049] In some embodiments, the preparation method further includes sterilizing, concentrating and / or drying the ultrafiltration product.

[0050] In some implementations, the drying includes vacuum freeze drying and / or low-temperature spray drying.

[0051] On the other hand, embodiments of the present invention provide a composition whose active ingredients include the polypeptide or its derivatives described in any of the foregoing embodiments, the biomaterials described in any of the foregoing embodiments, or the products prepared by the preparation methods described in any of the foregoing embodiments.

[0052] In some embodiments, the polypeptide derivative includes polypeptide derivatives obtained by modifying the amino acid side chain groups, amino terminus, or carboxyl terminus of the polypeptide DDLCDYWIR with hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation.

[0053] In some possible implementations, the composition further includes excipients.

[0054] In some possible implementations, the excipients include any one or more of the following: carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent, and colorant.

[0055] In optional embodiments, the carrier includes at least one of a food-grade carrier and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is, but is not limited to, any pharmaceutically acceptable excipient, carrier, adjuvant, additive, surfactant, desiccant, or diluent. A pharmaceutically acceptable carrier can be solid, semi-solid, or liquid.

[0056] In optional embodiments, the composition is in the form of powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0057] In an optional embodiment, the composition is obtained by spray drying, for example, electrostatic spray drying.

[0058] In optional embodiments, the composition is an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0059] In optional embodiments, the composition may be applied to humans (e.g., infants, children, adolescents, the elderly, etc.) or animals (e.g., mammals).

[0060] In some embodiments, the composition is a health product, food, cosmetic, or pharmaceutical. In some embodiments, the composition has antioxidant and / or immunomodulatory effects.

[0061] In some possible implementations, the food is a functional food, such as a novel dietary supplement or infant formula. For example, adding the polypeptides described in any of the foregoing embodiments to infant formula can enhance the infant's immunity, protect them from oxidative stress damage, and promote the healthy growth of the infant; using the polypeptides as a health supplement or adding them to the formula can help regulate the body's immune function, delay aging, and prevent and improve chronic diseases related to immunity and oxidation.

[0062] Furthermore, embodiments of the present invention also provide the use of the polypeptides, biomaterials, products prepared by the preparation methods described in any of the foregoing embodiments, or compositions described in any of the foregoing embodiments in the preparation of antioxidant and / or immunomodulatory products.

[0063] In some possible implementations, the immune regulation includes: enhancing or inhibiting the function of the body's immune system to maintain immune balance, regulating the activity of immune cells (such as macrophages), reducing the excessive release of inflammation-related cytokines (such as IL-6) by immune cells under external stimuli (such as LPS), enhancing the organism's tolerance or resistance to pathogens (such as PA14) to maintain survival functions (such as prolonging lifespan), preventing or mitigating abnormal states caused by immune dysfunction, promoting the recovery of immune function from an unbalanced state to a balanced state, or a combination of the above.

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Experimental reagents used in the examples:

[0066] Lactoferrin powder; Lactobacillus paracasei ET-22; neutral protease (enzyme activity 1.1×10⁻⁶) 6 U / g (enzyme activity was determined by spectrophotometry) - Xiasheng (Shanghai) Biotechnology Co., Ltd.; Lactose - Ningxia Xiasheng Industrial Group Co., Ltd.; Sodium chloride - Shanghai Lingfeng Chemical Reagent Co., Ltd.; Peptone - BBI Life Sciences Co., Ltd.; DPPH assay kit - purchased from Shanghai Beyotime Biotechnology Co., Ltd.; PBS - Nanjing Kaiji Biotechnology Co., Ltd.; DMEM incomplete high-glucose culture medium - Nanjing Kaiji Biotechnology Co., Ltd.; Fetal bovine serum - GIBCO Ltd.; Macrophages RAW264.7 - Shanghai Institute of Cell Biology, Chinese Academy of Sciences; Lipopolysaccharide (LPS, E. coli O111:B4) - Sigma Ltd.; Mouse interleukin-6 (IL-6) ELISA kit - Sangon Biotech (Shanghai) Co., Ltd.

[0067] Table 1: Reagent Preparation

[0068]

[0069] Table 2: Culture medium preparation

[0070]

[0071] The equipment involved in the embodiments:

[0072] Electronic balance, Sartorius, Germany; Constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; 3 kda ultrafiltration tubes, Millipore; DR-200Bc microplate reader, Changsha Deco Instrument Equipment Co., Ltd.; JB-VS-1300U clean bench, Shanghai Yiming Purification Equipment Co., Ltd.; LRH-250F biochemical incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.; GI36T autoclave, Xiamen Zhiwei Instrument Co., Ltd.; Froma 700 low-temperature freezer, Thermo Fisher Scientific (China) Co., Ltd.; RO15 pure water system, Likang Biomedical Technology Holdings Co., Ltd.; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd. Centrifuge 5414 D small high-speed centrifuge, Eppendorf GmbH; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.; Sartorius electronic balance, Germany; constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; DR-200Bc microplate reader, Changsha Deco Instrument Equipment Co., Ltd.; JB-VS-1300U ultra-clean workbench, Shanghai Yiming Purification Equipment Co., Ltd.

[0073] Example 1: Isolation of the active peptide DDLCDYWIR

[0074] 1. Preparation of lactoferrin solution

[0075] Lactoferrin was dissolved in an aqueous solution, the pH was adjusted to 7.0, sterilized, and cooled to obtain a lactoferrin solution with a concentration of 5% (wt / wt).

[0076] 2. Preparation of enzymatic hydrolysate

[0077] Add 1% of the amount of neutral protease to the prepared lactoferrin solution, mix well, and then enzymatically hydrolyze at 37°C for 4 hours to obtain the lactoferrin hydrolysate solution.

[0078] 3. Preparation of fermentation broth

[0079] Add 10 to each milliliter of lactoferrin hydrolysate solution 4 CFU of Lactobacillus paracasei ET-22 lyophilized powder and lactose were mixed and then fermented in an incubator at 40℃ for 4 hours to obtain the fermentation broth.

[0080] 4. Extraction of polypeptides

[0081] The fermentation broth was inactivated by heating in a 95°C water bath for 5 min, cooled in an ice-water bath, and then centrifuged at low temperature under the following conditions: 8000 RCF, 4°C, for 10 min. The bottom precipitate was discarded, and the supernatant was collected. The supernatant was then transferred to the inner tube of an ultrafiltration tube for ultrafiltration using a 10 kDa molecular weight cutoff membrane. The ultrafiltration centrifugation conditions were: 4800 RCF, 4°C, for 30 min. The bottom ultrafiltrate was collected, and the resulting peptide solution was stored at 4°C.

[0082] 5. Preparation of polypeptide powder

[0083] The polypeptide solution obtained in step 5 was spray-dried to obtain the milk concentrated protein peptide powder. The spray-drying conditions were: inlet air temperature 180℃, outlet air temperature 90℃, and flow rate 25mL / min.

[0084] 6. Screening of bioactive peptide DDLCDYWIR

[0085] (1) UPLC analysis

[0086] The UPLC conditions are:

[0087] Instruments: Waters ACQUITY UPLC (ultra-high performance liquid chromatography, electrospray ionization, quadrupole, time-of-flight mass spectrometer); Column specifications: BEH C18 column; Flow rate: 0.4 mL / min; Temperature: 50℃; UV detection wavelength: 210 nm; Injection volume: 2 μL; Gradient conditions: Solution A: water containing 0.1% (v / v) formic acid, Solution B: acetonitrile containing 0.1% (v / v) formic acid.

[0088] Table 3: Gradient elution procedure

[0089]

[0090] (2) Mass spectrometry analysis

[0091] Mass spectrometry conditions are:

[0092] Ion mode: ES+; Mass range (m / z): 100, 1000; Capillary voltage (kV): 3.0; Sampling cone (V): 35.0; Ion source temperature (°C): 115; Desolvent temperature (°C): 350; Desolvent gas flow (L / hr): 700.0; Collision energy (eV): 4.0; Scan time (sec): 0.25; Intra-scan time (sec): 0.02.

[0093] Based on the above analytical methods, ultra-high performance liquid chromatography (UHPLC), electrospray ionization (ESI), quadrupole chromatography (QPC), and time-of-flight mass spectrometry (TOF-MS) were used to perform chromatographic and mass spectrometric analyses on milk concentrate peptides, obtaining the amino acid sequences of all peptides in the fermentation broth. The same amino acid sequences from the parallel control group were selected, and amino acid sequences with lower bioactivity potential scores in the peptide ranker and previously published amino acid sequences were excluded to obtain the sequence of the bioactive peptide DDLCDYWIR.

[0094] Example 2: Artificial Synthesis of the Active Peptide DDLCDYWIR

[0095] I. Synthesis of Bioactive Peptides

[0096] 1. Weigh 3 g of RINK resin (degree of substitution 0.3 mmol / g) into a 150 mL reactor and soak it in 50 mL of dichloromethane (DCM); 2. After 2 hours, wash the resin with 3 times the volume of nitrogen-dimethylformamide (DMF), then dry it. Repeat this process four times. After drying the resin, set it aside for later use; 3. Add a certain amount of 20% piperidine (piperidine / DMF=1:4, v:v) to the reactor and shake it on a decolorizing shaker for 20 min to remove the Fmoc protecting groups on the resin. After deprotection, wash four times with 3 times the resin volume of DMF, then dry. 4. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, the deprotection is successful. 5. Weigh an appropriate amount of amino acid Lys and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake well. After the solution has clarified, add it to the reactor and then place the reactor in a shaker at 30°C to react; 6. After 2 hours, cap the solution with a certain amount of acetic anhydride (acetic anhydride:DIEA:DCM=1:1:2, v:v:v) for half an hour, then wash it four times with 3 times the volume of DMF and dry it for later use; 7. Add a certain amount of 20% piperidine (piperidine / DMF=1:4, v:v) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting groups on the resin. After deprotection, wash four times with DMF and then dry. 8. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, the deprotection is successful. 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake well for 1 min. After the solution becomes clear, add it to the reactor and then place the reactor in a shaker at 30℃ for reaction. 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin is colorless, the reaction is complete; if the resin is colored, the condensation is incomplete, and the reaction should continue. 11. After the reaction is complete, wash the resin four times with DMF, then dry it, add a certain amount of 20% piperidine (piperidine / DMF=1:4, v:v) to the reactor, and shake it on a decolorizing shaker for 20 min to remove the Fmoc protecting groups on the resin.After deprotection, wash four times with DMF, then dry the resin to check if the protection has been removed; 12. Following steps 9-11, sequentially add amino acids Ile, Trp, Tyr, Asp, Cys, Leu, Asp, and Asp; 13. After adding the last amino acid, deprotect the resin, wash four times with DMF, and then dry the resin with methanol. Then, use 95% cutting buffer (trifluoroacetic acid: 1,2-ethylenedithiol: 3, isopropylsilane: water = 95:2:2:1, v:v:v) to cut the bioactive peptide from the resin (10 ml of cutting buffer per gram of resin), and centrifuge four times with ice-cold diethyl ether (cutting buffer: ether = 1:9, v:v); thus, the bioactive peptide DDLCDYWIR has been artificially synthesized.

[0097] II. Identification of Bioactive Peptides

[0098] (1) UPLC analysis

[0099] The UPLC conditions are:

[0100] Instruments: Waters ACQUITY UPLC (ultra-high performance liquid chromatography, electrospray ionization, quadrupole, time-of-flight mass spectrometer); Column specifications: BEH C18 column; Flow rate: 0.4 mL / min; Temperature: 50℃; UV detection wavelength: 210 nm; Injection volume: 2 μL; Gradient conditions: Solution A: water containing 0.1% formic acid (v / v), Solution B: acetonitrile containing 0.1% formic acid (v / v); Gradient elution program is the same as in Table 3.

[0101] (2) Mass spectrometry analysis

[0102] The mass spectrometry conditions are as follows:

[0103] Ion mode: ES+; Mass range (m / z): 100, 1000; Capillary voltage (kV): 3.0; Sampling cone (V): 35.0; Ion source temperature (°C): 115; Desolvent temperature (°C): 350; Desolvent gas flow (L / hr): 700.0; Collision energy (eV): 4.0; Scan time (sec): 0.25; Intra-scan time (sec): 0.02.

[0104] Based on the above analytical methods, ultra-high performance liquid chromatography (UHPLC), electrospray ionization (ESI), quadrupole chromatography (QPC), and time-of-flight mass spectrometry (TOF-MS) were used to perform chromatographic and mass spectrometric analyses on the bioactive peptide DDLCDYWIR. The primary mass spectrum of the bioactive peptide DDLCDYWIR is shown below. Figure 1 As shown, the secondary mass spectrum of the extracted peaks and the fragmentation of az and by are as follows. Figure 2 As shown, the bioactive peptide mass-to-charge ratio of this peak is 628.2802, and the retention time is 45.63 min.

[0105] (3) Results

[0106] Depend on Figure 2 Based on the breakage of az and by, and after analysis and calculation using Mascot software, the sequence of the fragment with a mass-to-charge ratio of 628.2802 is Asp-Asp-Leu-Cys-Asp-Tyr-Trp-Ile-Arg (DDLCDYWIR), denoted as SEQ ID NO: 1. Its molecular structure is shown below. Figure 3 This fragment corresponds to residues 94-102 of the Lipocalin 2 protein, whose Uniprot number is E1B6Z6.

[0107] Example 3: Antioxidant Activity Experiment of Milk-Derived Bioactive Peptides

[0108] I. Determination of DPPH free radical scavenging rate of milk-derived bioactive peptide DDLCDYWIR

[0109] 1. Experimental Methods

[0110] Dissolve 9.858 mg of DPPH in anhydrous ethanol and dilute to a final volume in a 25 mL brown volumetric flask to prepare a 1 mmol / L solution. Store in the refrigerator. When needed, dilute 10 times with anhydrous ethanol to prepare a 0.1 mmol / L solution.

[0111] Pipette 50 μL of peptide solutions with concentration gradients of 0, 0.1, 0.5, and 1.0 mg / mL into 96-well plates, setting up 5 replicates. Add 100 μL of DPPH ethanol solution (0.1 mmol / L) to each well, shake well, and incubate at room temperature in the dark for 30 min, measuring the absorbance A1 at 517 nm. Add 100 μL of anhydrous ethanol solution to each well, shake well, and incubate at room temperature in the dark for 30 min, measuring the absorbance A2 at 517 nm. Use 1.0 mL of deionized water as a blank control instead of the sample, add 2.0 mL of anhydrous ethanol, and measure its absorbance A0. Calculate the results according to the following formula.

[0112] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100%

[0113] 2. Experimental Results and Analysis

[0114] Table 4. Determination of DPPH free radical scavenging rate by milk-derived bioactive peptide DDLCDYWIR

[0115]

[0116] Note: **, compared with the negative control group, there was a highly significant difference ( P <0.01).

[0117] The experimental results are shown in Table 4. The total antioxidant activity of the peptide DDLCDYWIR in vitro was determined by measuring the DPPH free radical scavenging rate. It was found that the absorbance of the experimental group with added milk-derived bioactive peptide DDLCDYWIR was lower than that of the blank group, indicating a better ability to reduce oxidizing substances. Table 4 shows that the total antioxidant capacity of the milk-derived bioactive peptide DDLCDYWIR increased with increasing peptide concentration, and the optimal total antioxidant level was achieved at a concentration of 1 mg / mL. Therefore, it can be concluded that the invented milk-derived bioactive peptide DDLCDYWIR has significant antioxidant capacity.

[0118] II. Determination of the oxidative survival curve of lactoferrin bioactive peptide DDLCDYWIR in nematodes

[0119] 1. Experimental Methods

[0120] N2 nematodes were washed off NGM medium with M9 buffer and transferred to 15 mL centrifuge tubes. After standing for 2 minutes to allow the nematodes to settle naturally to the bottom, the supernatant was discarded. Nematode lysis buffer was added to the centrifuge tubes, and the mixture was vortexed for 6 minutes until the nematodes were completely lysed. The lysis buffer was aliquoted into 1.5 mL centrifuge tubes and centrifuged at 4000 rpm for 3 minutes to allow the nematode eggs to settle completely; the supernatant was discarded. Then, 1 mL of M9 buffer was added to the centrifuge tubes, gently shaken to mix thoroughly, and centrifuged at 4000 rpm for 3 minutes. This process was repeated three times, and the collected nematode eggs were combined. The peptide sample was added to the middle of NGM medium containing E. coli OP50, and after drying, the nematode eggs were dropped into the area of ​​the medium containing the peptide sample and incubated at 20°C. After synchronization, the nematodes were cultured for 48-60 hours to the L4 stage. A 96-well plate was prepared, and 200 μL of 60 mmol / L paraquat solution was added to each well. Fifteen nematodes were picked from each well, and the counts were performed every 2 hours. Each group had 3-5 replicates.

[0121] 3. Experimental Results and Analysis

[0122] Further verification was conducted through animal experiments, and the experimental results are as follows: Figure 4 As shown, at a concentration of 1 mg / mL, the lactoferrin polypeptide DDLCDYWIR effectively improved the survival rate of nematodes under oxidative stress. Therefore, it can be concluded that the invented lactoferrin polypeptide DDLCDYWIR has significant antioxidant capacity.

[0123] Example 4: Immunomodulatory Activity Experiment of Milk-Derived Bioactive Peptides

[0124] I. Determination of the effect of milk-derived bioactive peptide DDDCYWIR on the release of cytokines from macrophages after LPS stimulation

[0125] 1. Experimental Methods

[0126] 1.1 Macrophage Culture

[0127] Prepare DMEM incomplete medium containing 10% fetal bovine serum (containing 80 U / mL penicillin and 0.08 g / L streptomycin), and use this medium to equilibrate RAW264.7 cells to a density of 2 × 10⁶ cells / mL. 5 At a concentration of / mL, the cell suspension was seeded in 25cm... 2 In disposable culture flasks or 96-well plates, culture in a saturated water vapor / carbon dioxide incubator at 37°C and 5% CO2 concentration. Change the culture medium after 24 hours. When the cells have essentially reached the bottom of the flask using an inverted microscope, passage them. For passage, carefully aspirate the old culture medium, add 1.5 mL of trypsin digestion solution, incubate at 37°C for 2 minutes, and then add 2 mL of culture medium to stop the reaction. Gently pipette the cells repeatedly to disperse any adherent cells at the bottom. Centrifuge at 800 g for 2 minutes, collect the cells at the bottom of the tube, and resuspend in culture medium to a final volume of 2 × 10⁻⁶. 5 Cells / L were cultured.

[0128] RAW264.7 cells were cultured in DMEM medium containing the milk-derived bioactive peptide DDLCDYWIR at concentrations of 0 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 1.0 mg / mL for 24 h. At the 24-h mark, LPS solution with a final concentration of 100 μg / L was added to each culture flask. After culturing for another 2 h, the concentration of cytokine IL-6 in the supernatant of the culture medium was measured using an ELISA kit.

[0129] 2. Experimental Results and Analysis

[0130] Table 5. Measurement of IL-6 release from macrophages after LPS stimulation by milk-derived bioactive peptide DDLCDYWIR

[0131]

[0132] Note: **, compared with the negative control group, there was a highly significant difference ( P <0.01);*, compared with the negative control group, there was a significant difference ( P <0.05).

[0133] When the body is stimulated by LPS, macrophages can reach the local tissue where the pathogen is located through chemotaxis, rapidly engulf foreign substances, and generate a large number of inflammatory factors, such as IL-6. The experimental results are shown in Table 5. The in vitro immunomodulatory activity of the peptide DDLCDYWIR was measured by measuring the release of IL-6 from macrophages after LPS stimulation. It was found that the concentration of IL-6 in the cell culture supernatant of the experimental group with added milk-derived bioactive peptide DDLCDYWIR was lower than that of the control group, indicating a reduction in the release level of pro-inflammatory factors from macrophages, thus reducing the damage caused by inflammation to the body. Table 5 shows that the immunomodulatory capacity of the milk-derived bioactive peptide DDLCDYWIR increases with increasing peptide concentration, and the immunomodulatory capacity of DDLCDYWIR is optimal at a concentration of 1 mg / mL. Therefore, it can be concluded that the invented milk-derived bioactive peptide DDLCDYWIR has significant immunomodulatory capacity.

[0134] II. Assay of the nematode immune survival curve of lactoferrin bioactive peptide DDLCDYWIR

[0135] 1. Experimental Methods

[0136] N2 nematodes were washed off NGM medium with M9 buffer and transferred to 15 mL centrifuge tubes. After standing for 2 minutes to allow the nematodes to settle naturally to the bottom, the supernatant was discarded. Nematode lysis buffer was added to the centrifuge tubes, and the mixture was vortexed for 6 minutes until the nematodes were completely lysed. The lysis buffer was aliquoted into 1.5 mL centrifuge tubes and centrifuged at 4000 rpm for 3 minutes to allow the nematode eggs to settle completely; the supernatant was discarded. Then, 1 mL of M9 buffer was added to the centrifuge tubes, gently shaken to mix thoroughly, and centrifuged at 4000 rpm for 3 minutes. This process was repeated three times, and the collected nematode eggs were combined. The peptide sample was added to the middle of NGM medium containing E. coli OP50, and after drying, the nematode eggs were dropped into the area of ​​the medium containing the peptide sample and incubated at 20°C. After synchronization, nematodes were cultured for 48-60 h to the L4 stage. The nematodes were then washed off NGM medium with M9 buffer and transferred to 15 mL centrifuge tubes. After standing for 2 min to allow the nematodes to settle naturally to the bottom of the tube, the supernatant was discarded. The remaining nematodes were then added dropwise to PA14-coated medium, 30-60 nematodes per medium, and cultured at 25°C. Counting was performed every 12 h until all nematodes died.

[0137] 2. Experimental Results and Analysis

[0138] Further verification was conducted through animal experiments, and the experimental results are as follows: Figure 5As shown, at a concentration of 1 mg / mL, the lactoferrin peptide DDLCDYWIR effectively enhances the immunomodulatory capacity of nematodes, thereby prolonging their lifespan. Therefore, it can be concluded that the lactoferrin peptide DDLCDYWIR possesses significant immunomodulatory capabilities.

[0139] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of peptides in the preparation of antioxidant and / or immunomodulatory products, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The application of biomaterials in the preparation of antioxidant and / or immunomodulatory products, characterized in that, The biomaterial is an isolated nucleic acid molecule that encodes the polypeptide described in claim 1.

3. The use of the composition in the preparation of antioxidant and / or immunomodulatory foods, characterized in that, The active ingredient of the composition is the polypeptide described in claim 1.

4. Application of the composition in the preparation of health products with antioxidant and / or immunomodulatory properties.

5. The application according to claim 3 or 4, characterized in that, The composition also includes excipients.

6. The application according to claim 5, characterized in that, The excipients include any one or more of the following: carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent, and colorant.

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