Yak milk casein antioxidant peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a yak milk casein antioxidant peptide as well as a preparation method and application thereof, and the preparation method comprises the following steps: step 1, dissolving yak milk casein in alkali liquor to obtain a casein solution; step 2, carrying out enzymolysis on the casein solution by using protease to obtain enzymatic hydrolysate; step 3, inactivating and centrifuging the enzymatic hydrolysate, taking supernate, and freeze-drying to obtain a crude polypeptide product; and step 4, purifying the polypeptide crude product through anion exchange column chromatography and gel filtration chromatography in sequence to obtain the antioxidant peptide component. The preparation method is efficient and clear in target, yak milk casein is subjected to enzymolysis through a single enzyme / complex enzyme combined strategy, a two-step purification process of DEAE-52 cellulose column chromatography and SephadexG-50 gel chromatography is combined, the high-activity polypeptide component is efficiently and repeatedly prepared, and the defect that traditional enzymolysis product components are complex is overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a yak milk casein antioxidant peptide, its preparation method, and its application. Background Technology

[0002] Liver disease is a serious global public health problem threatening human health. Oxidative stress is one of the key factors leading to hepatocellular damage, characterized by the excessive accumulation of intracellular reactive oxygen species (ROS), which triggers oxidative damage to lipids, proteins, and DNA, drives inflammatory responses, and ultimately leads to liver dysfunction. Therefore, finding effective and safe antioxidants to alleviate hepatocellular oxidative stress damage is of great significance for the prevention and adjuvant treatment of liver diseases.

[0003] Food-derived antioxidant peptides have become ideal candidates for the development of functional foods and drugs due to their high safety, easy absorption, and diverse bioactivities. Yak milk casein is a known high-quality protein source, and its enzymatic hydrolysates have been reported to possess in vitro antioxidant activity.

[0004] However:

[0005] The casein hydrolysates obtained by existing technologies are mostly complex mixtures of crude peptides with low activity. There is a lack of targeted purification processes, making it difficult to obtain specific peptides with high activity.

[0006] Its activity evaluation is mostly limited to chemical-level free radical scavenging experiments (such as DPPH and ABTS methods), lacking efficacy verification in cell models that are closer to the physiological environment, especially in hepatocyte oxidative stress injury models.

[0007] Therefore, existing technologies cannot confirm whether yak milk casein peptides can directly and effectively protect hepatocytes from oxidative stress damage, nor can they elucidate their specific mechanism of action. This severely limits their development and application in functional foods or drugs that target liver disease intervention.

[0008] The purpose of this invention is to overcome the above-mentioned defects of the prior art, provide a method for efficiently preparing a specific antioxidant peptide component from yak milk casein, and demonstrate the significant protective effect and mechanism of this component in a hepatocyte oxidative stress injury model. Summary of the Invention

[0009] To address the technical problems mentioned in the background section, this invention provides a yak milk casein antioxidant peptide, its preparation method, and its application.

[0010] This invention is achieved using the following technical solution: One of the objectives of this invention is to provide a method for preparing yak milk casein antioxidant peptides, comprising the following steps:

[0011] Step 1: Dissolve yak milk casein in alkaline solution to obtain casein solution;

[0012] Step 2: Enzymatically hydrolyze the casein solution using a protease to obtain an enzymatic hydrolysate;

[0013] Step 3: Inactivate the enzyme hydrolysate, centrifuge, collect the supernatant, and freeze-dry to obtain crude polypeptide;

[0014] Step 4: The crude polypeptide is purified sequentially by anion exchange column chromatography and gel filtration chromatography to obtain antioxidant peptide components;

[0015] Step 5: The purified antioxidant peptide components are screened for activity. The screening indicators include DPPH free radical scavenging rate, ABTS+ free radical scavenging rate and hydrogen peroxide-induced mouse AML12 hepatocyte survival rate. Based on this, target peptide components with high antioxidant activity are screened.

[0016] in:

[0017] In step 2, the protease is selected from one or more of neutral protease, papain, trypsin, pepsin, and alkaline protease;

[0018] In step 3, after the enzymatic hydrolysis reaction is completed, the sample is placed in a boiling water bath for 10 minutes to terminate enzyme activity. After cooling to room temperature, the sample is centrifuged to remove insoluble matter, thereby collecting the supernatant of the enzymatic hydrolysis product.

[0019] Furthermore, in step 2, the enzymatic hydrolysis method is single-enzyme hydrolysis, double-enzyme hydrolysis, or triple-enzyme hydrolysis;

[0020] When performing a single enzyme digestion, digest for 120 minutes under the optimal temperature and pH conditions for the corresponding enzyme.

[0021] When performing a double enzymatic hydrolysis, first add the first enzyme and hydrolyze for 60 minutes. After stopping the enzymatic hydrolysis, adjust the hydrolysate to the optimal temperature and pH of the second enzyme and then hydrolyze for another 60 minutes.

[0022] When performing a triple-enzyme hydrolysis, add the third enzyme sequentially according to the above method and continue hydrolysis for 60 minutes, based on the double-enzyme hydrolysis.

[0023] Furthermore, the amount of protease added is 5000 U / g casein, and the enzymatic hydrolysis conditions are as follows:

[0024] Trypsin: pH 8.0, temperature 37℃;

[0025] Neutral protease: pH 7.0, temperature 40℃;

[0026] Pepsin: pH 3.0, temperature 40℃;

[0027] Papain: pH 7.0, temperature 50℃;

[0028] Alkaline protease: pH 10.0, temperature 40℃.

[0029] Furthermore, in step 4, the anion exchange column chromatography is a DEAE-52 cellulose column, eluted with a linear gradient of 0–0.3 mol / L NaCl, and the eluted fraction with absorbance at 280 nm is collected.

[0030] Furthermore, in step 4, the gel filtration chromatography is performed using a Sephadex G-50 gel column, eluted with deionized water, and the eluted fraction with absorbance at 280 nm is collected.

[0031] The second objective of this invention is to provide a yak milk casein antioxidant peptide composition, prepared by the above method, which contains one or more of the antioxidant peptide components Z1-C, Z3-B, YM3-B, YZW1-B, and YZW3-B.

[0032] A third objective of this invention is to propose the application of an antioxidant peptide composition in the preparation of functional foods or pharmaceuticals for protecting hepatocytes from oxidative stress damage.

[0033] Furthermore, the hepatocytes are AML12 hepatocytes, and the oxidative stress damage is induced by H2O2.

[0034] Furthermore, the antioxidant peptide composition can reduce the levels of ROS, MDA, NO, and the expression of inflammatory factors in hepatocytes.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The preparation method of this invention is highly efficient and has a clear objective. It uses a specific single-enzyme / complex-enzyme combination strategy (such as neutral protease, trypsin-papain, trypsin-neutral protease-pepsin) to enzymatically hydrolyze yak milk casein, and combines a two-step purification process of DEAE-52 cellulose column chromatography and Sephadex G-50 gel chromatography to efficiently and reproducibly prepare specific antioxidant peptide components (Z1-C, Z3-B, YM3-B, YZW1-B, YZW3-B), overcoming the shortcomings of traditional enzymatic hydrolysis products that are complex in composition and have low activity.

[0037] The specific antioxidant peptide components prepared by this invention exhibit superior free radical scavenging capabilities at the chemical level. For example, the DPPH free radical scavenging rate of the pancreaticogastric trienzyme hydrolysate can reach 42.50%, and the ABTS free radical scavenging rate of the neutral protease hydrolysate can reach [missing information]. + The free radical scavenging rate can reach 87.09%, which is significantly better than many single enzymatic hydrolysis products.

[0038] Cellular experiments have confirmed that the specific antioxidant peptide components obtained in this invention can significantly improve cell survival in an H2O2-induced AML12 hepatocyte oxidative stress injury model. The purified active components (such as YZW3-B) can increase cell survival to 82.19%, demonstrating a significant effect and clarifying its ability to directly protect hepatocytes, which has not been revealed in existing technologies.

[0039] The mechanism of action of this invention is clear. Through systems biology evaluation, it has been confirmed that the antioxidant peptides of this invention can not only reduce the level of reactive oxygen species (ROS) in hepatocytes, but also significantly inhibit the production of oxidative damage markers malondialdehyde (MDA) and nitric oxide (NO), and effectively downregulate the expression of key inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This reveals the mechanism by which it exerts a synergistic hepatocyte protective effect through a dual "antioxidant-anti-inflammatory" pathway, providing a solid theoretical basis for product development.

[0040] This invention provides a clear technical route and experimental support for developing functional foods or drugs with yak milk casein antioxidant peptides as the core ingredient, which can be used to prevent or assist in the treatment of oxidative stress-related liver damage, thereby enhancing the added value of yak milk resources. Attached Figure Description

[0041] Figure 1 This is a comparative analysis of the effects of peptides on H2O2-induced AML12 cell survival.

[0042] Figure 2 Comparative diagram showing the protective effect of peptides separated by DEAE-52 cellulose column chromatography against H2O2-induced oxidative stress damage in AML12 cells;

[0043] Figure 2 A, Figure 2 B Figure 2 C represents the elution curve of DEAE-52 cellulose column chromatography; where: Figure 2 A is a neutral single-enzyme hydrolysate polypeptide; Figure 2 B is a pancreatic-wood double-enzyme hydrolysate polypeptide; Figure 2 C contains polypeptides hydrolyzed by three enzymes in the pancreas and stomach.

[0044] Figure 2 D is a comparison of the results of AML12 cytotoxicity experiments using neutral single-enzyme hydrolysis of peptides;

[0045] Figure 2 E is a comparison of the results of cytotoxicity experiments using pancreatic xylase and pancreatic stomach three-enzyme hydrolysis of polypeptide AML12;

[0046] Figure 2 F is a comparison of the effects of each polypeptide component on the survival rate of AML12 cells under oxidative stress.

[0047] Figure 3 Comparison of results showing the protective effect of peptides separated by Sephadex G-50 gel chromatography against H2O2-induced oxidative stress damage in AML12 cells;

[0048] Figure 3 A- Figure 3 G represents the elution curve of Sephadex G-50 gel chromatography; where: Figure 3 A is component Z1; Figure 3 B is component Z2; Figure 3 C is component Z3; Figure 3 D is component Z4; Figure 3 E is component YM3; Figure 3 F represents component YZW1; Figure 3 G is the YZW3 component;

[0049] Figure 3 H is a comparison of the results of the neutral single enzyme AML12 cytotoxicity assay;

[0050] Figure 3 Figure I shows a comparison of the results of the pancreatic-xylene dual-enzyme AML12 cytotoxicity assay.

[0051] Figure 3 J is a comparative graph showing the effects of each polypeptide component on the survival rate of AML12 cells under oxidative stress.

[0052] Figure 4 The effect of casein antioxidant peptides on the relative ROS content induced by H2O2 in AML12 cells;

[0053] in: Figure 4 A represents the blank control group; Figure 4 Group B represents the oxidative damage group; Figure 4 C represents the glutathione group; Figure 4 D represents the Z1-C drug administration group; Figure 4 E represents the Z3-B treatment group; Figure 4 F represents the YM3-B treatment group; Figure 4 G represents the YZW1-B treatment group; Figure 4 H represents the YZW3-B treatment group; Figure 4 The bottom left corner shows a comparison of DCF fluorescence intensity.

[0054] Figure 5 A- Figure 5 D is a comparative graph showing the effects of casein antioxidant peptides on the levels of MDA, NO, TNF-α, and IL-1β in AML12 cells.

[0055] Figure 6 This is a flowchart of the preparation method of yak milk casein antioxidant peptides proposed in this invention. Detailed Implementation

[0056] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0057] The present invention will be further explained below with reference to specific embodiments.

[0058] Regarding materials and reagents in this plan:

[0059] Casein was provided by Gansu Hualing Dairy Co., Ltd.; neutral protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; papain, alkaline protease, pepsin, and trypsin were purchased from Solarbio; DPPH, ABTS, and L-ascorbic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; thiazolyl blue tetrazolium bromide (MTT) was purchased from Shanghai Aladdin Co., Ltd.; and AML12 complete culture medium and AML12 cell line were purchased from Wuhan Pronosei Life Science Co., Ltd.

[0060] Regarding instruments and equipment in this plan:

[0061] FA2004 electronic analytical balance, Shanghai Liangping Instrument Co., Ltd.; centrifuge, Hunan Xiangyi; multi-functional microplate reader, Meigu Molecular; IX51 inverted microscope, Olympus, Japan; HealForce HF160W CO2 incubator, Shanghai Lishen; 85-2 digital display constant temperature magnetic stirrer, Changzhou Yuexin Instrument Manufacturing Co., Ltd.; R-1001VN rotary evaporator, Zhengzhou Great Wall Industry & Trade Co., Ltd.

[0062] Example 1:

[0063] This embodiment presents a method for preparing yak milk casein antioxidant peptides, the specific process of which is as follows:

[0064] Weigh 1g of yak milk casein and add 50mL of 0.1mol / L NaOH solution to dissolve it completely. Perform enzymatic hydrolysis according to the conditions shown in the table below.

[0065] Table 1: Enzyme dosage, pH value, and temperature during casein hydrolysis

[0066] name Dosage pH value temperature trypsin 5000U / g 8.0 37℃ neutral protease 5000U / g 7.0 40℃ pepsin 5000U / g 3.0 40℃ Papain 5000U / g 7.0 50℃ alkaline protease 5000U / g 10.0 40℃

[0067] Preparation of single-enzyme hydrolysed peptides: Referring to Table 1, a protease was added at the corresponding temperature and pH, and the mixture was hydrolyzed for 120 min. After hydrolysis, the reaction was terminated by boiling in a water bath for 10 min, cooled, and centrifuged at 4000 r / min for 20 min. The supernatant was collected, freeze-dried, and five single-enzyme hydrolysed peptide samples were obtained.

[0068] Preparation of peptides by double enzymatic hydrolysis: 1g of casein was dissolved, and the first enzyme (e.g., trypsin) was added for 60 min of enzymatic hydrolysis. After inactivation by boiling water bath, the temperature and pH of the system were adjusted to the optimal conditions for the second enzyme (e.g., papain), and enzymatic hydrolysis was continued for another 60 min. The peptides were then inactivated, centrifuged, and the supernatant was collected and lyophilized to obtain 6 peptide samples by double enzymatic hydrolysis.

[0069] Preparation of peptides by triple-enzyme hydrolysis: Based on double-enzyme hydrolysis, after inactivation and adjustment of conditions, a third enzyme was added and hydrolysis continued for 60 min. This process was repeated to obtain four peptide samples with triple-enzyme hydrolysis.

[0070] The 15 enzymatically hydrolyzed polypeptide samples obtained above were diluted 20-fold with water, and their DPPH free radical scavenging rate and ABTS⁺ free radical scavenging rate were measured. The results are shown in Table 2. Among them, the neutral protease hydrolyzed polypeptide, the trypsin-papain dual-enzyme hydrolyzed polypeptide (pancreatic-papain dual-enzyme), and the trypsin-neutral protease-pepsin tri-enzyme hydrolyzed polypeptide (pancreatic-pepsin tri-enzyme) showed high free radical scavenging activity.

[0071] Table 2: Free radical scavenging rate of some enzymatic hydrolysis products

[0072] Enzymatic hydrolysis products DPPH removal rate (%) ABTS+ clearance rate (%) Neutral protease hydrolysates peptides 36.87±4.19 87.09±5.14 Pancreatic wood dual-enzyme hydrolysis polypeptide 23.21±0.60 76.78±3.62 Pancreatic-gastric three-enzyme hydrolysis of polypeptides 40.97±1.01 69.10±2.77 Positive control VC 97.67±0.65 98.54±0.30

[0073] Table 3: Scavenging rates of DPPH and ABTS+ free radicals of 15 enzymatic hydrolysates

[0074] Different enzymatic hydrolysis products DPPH free radical scavenging rate (%) DPPH scavenging rate (%) <![CDATA[ABTS + Free radical scavenging rate (%) ABTS + scavengingrate (%) Neutral protease hydrolysates peptides <![CDATA[36.87±4.19 b ]]> <![CDATA[87.09±5.14 ab ]]> Papain hydrolysate polypeptides <![CDATA[29.23±1.08 bcde ]]> <![CDATA[53.82±2.19 fgh ]]> alkaline proteolytic peptides <![CDATA[26.17±4.81 cf ]]> <![CDATA[62.01±6.13 dg ]]> Pepsin hydrolysate polypeptides <![CDATA[27.03±1.44 cf ]]> <![CDATA[44.77±4.53 hi ]]> Trypsin hydrolysis of polypeptides <![CDATA[23.88±0.41 cf ]]> <![CDATA[66.03±0.91 def ]]> Zhongmu double-enzyme hydrolysate polypeptide <![CDATA[31.81±3.47 bc ]]> <![CDATA[59.50±4.83 eg ]]> Wood stomach double-enzyme hydrolysate polypeptide <![CDATA[21.30±1.31 f ]]> <![CDATA[42.96±4.00 hi ]]> Mid-gastric dual-enzyme hydrolysis of polypeptides <![CDATA[21.97±0.60 ef ]]> <![CDATA[38.94±5.13 i ]]> Pancreatic and gastric dual-enzyme hydrolysis of polypeptides <![CDATA[24.26±0.41 cf ]]> <![CDATA[44.12±4.87 hi ]]> Pancreatic wood dual-enzyme hydrolysis polypeptide <![CDATA[23.21±0.60 df ]]> <![CDATA[76.78±3.62 bd ]]> Double-enzyme hydrolysis of polypeptides in the pancreas <![CDATA[23.97±0.44 df ]]> <![CDATA[70.43±6.08 cde ]]> Pancreatic-gastrointestinal three-enzyme hydrolysis of polypeptides <![CDATA[42.50±2.48 bcd ]]> <![CDATA[84.62±5.99 abc ]]> Zhongmu stomach three-enzyme hydrolysate polypeptide <![CDATA[36.10±1.58 b ]]> <![CDATA[50.30±1.68 gi ]]> Pancreatic-gastric three-enzyme hydrolysis of polypeptides <![CDATA[40.97±1.01 b ]]> <![CDATA[69.10±2.77 cdef ]]> Pancreatic polysaccharide hydrolysis <![CDATA[38.97±1.47 bcd ]]> <![CDATA[69.52±7.36 cdef ]]> Positive control VC <![CDATA[97.67±0.65 a ]]> <![CDATA[98.54±0.30 a ]]>

[0075] More specifically, in this embodiment, the method for determining the free radical scavenging rate of the polypeptide solution is as follows:

[0076] Weigh 4 mg of DPPH powder, dissolve it in 95% ethanol, and bring the volume to 10 mL to obtain a 0.1 mmol / L DPPH working solution. DPPH is photoinstable, so the experiment must be conducted in the dark; the solution should be prepared fresh before use. Mix 100 µL of the sample solution to be tested with 100 µL of DPPH solution, mix thoroughly, and react at room temperature in the dark for 30 min. Measure the absorbance value A0 of the mixture at 517 nm. Replace the DPPH solution with an equal volume of 95% ethanol and measure the absorbance value A1 of the sample and ethanol mixture; replace the sample solution with an equal volume of 95% ethanol and measure the absorbance value A2 of the ethanol and DPPH mixture.

[0077] Accurately weigh 40.602 mg of ABTS powder, dissolve it in 95% ethanol, and dilute to 10 mL to prepare a 7.4 mmol / L ABTS stock solution. Separately weigh 7.028 mg of potassium persulfate (K₂S₂O₈), and similarly dilute to 10 mL with 95% ethanol. Mix the two solutions in a 1:1 (v / v) ratio and allow to stand in the dark for 12 h. Dilute with 95% ethanol; the absorbance at 734 nm is 0.70 ± 0.02. Add 200 µL of ABTS solution to 10 µL of the sample solution, mix well, and react at room temperature in the dark for 30 min. Measure the absorbance A0 of the mixed solution at 734 nm. Replace the ABTS solution with an equal volume of 95% ethanol and measure the absorbance A1 of the 95% ethanol and sample solution mixture. Replace the sample solution with an equal volume of 95% ethanol and measure the absorbance A2 of the 95% ethanol and ABTS solution mixture.

[0078] The scavenging rates of DPPH and ABTS+ radicals are calculated using the following formula:

[0079] .

[0080] Example 2:

[0081] In this embodiment, the experimental method for enzymatic hydrolysis of peptides to combat hepatocyte oxidative stress damage is as follows:

[0082] An oxidative stress injury model of AML12 cells was constructed using H2O2, with a modeling time of 4 hours. AML12 cells were brought to 80% confluence, then digested with trypsin, centrifuged, and collected. The cells were resuspended and seeded into 96-well plates with 5000 cells per well, and cultured at 37°C in a 5% CO2 incubator for 24 hours. The following groups were set up: a blank group (containing culture medium but no cells), a control group (containing culture medium with cells), and an experimental group (containing culture medium with H2O2 concentrations of 100, 200, 300, 400, 500, 600, and 800 μmol / L, respectively). Each group was divided into 5 parallel groups. The inoculated cells were divided into groups and cultured for another 4 hours. 10 μL of 5 mg / mL MTT solution was added to each well. After incubation for 4 hours, the MTT solution was removed, and 100 μL of dimethyl sulfoxide was added. After thorough mixing, the H2O2 survival rate was calculated using a microplate reader at a detection wavelength of 490 nm according to the following formula, and an IC50 curve was fitted.

[0083]

[0084] Table 4; Experimental grouping of AML12 cells

[0085] Grouping Blank control group Oxidative damage group Positive control group peptide dosing group Dosage none <![CDATA[H2O2]]> <![CDATA[H2O2 + Glutathione]]> <![CDATA[H2O2 + polypeptide]]>

[0086] AML12 cells were seeded in 96-well plates and grouped as shown in Table 4. The blank control group received no drug, the H2O2-damaged group received H2O2, the positive control group received glutathione at a final concentration of 100 µg / mL, and the peptide-treated groups were divided into different groups according to different peptides. Each group received H2O2 and different concentrations of peptide, with 5 replicates per group. Cells were incubated in an incubator for 4 h, and cell viability was determined by the MTT assay to evaluate the protective effect of peptides against oxidative stress.

[0087] AML12 cells were seeded in T25 culture flasks and cultured to 80% confluence. Three groups were established: a blank control group, a glutathione group, and a peptide-treated group. Cell culture medium was added to the blank control group, cell culture medium containing 100 µg / ml glutathione was added to the glutathione group, and cell culture medium containing 100 µg / ml peptide sample was added to the peptide-treated group. The cells were incubated for another 24 hours, and the supernatant was discarded. Cells were collected by trypsin digestion and then disrupted using an ultrasonic cell disruptor. Intracellular MDA content was measured according to the kit (Solepro, BC5240) instructions.

[0088] AML12 cells were seeded into 6-well plates and cultured to 80% confluence. They were grouped as shown in Table 4, with three replicates per group. After 4 hours of culture, the culture medium was collected, centrifuged, and the supernatant was used. Culture medium containing 10 μmol / L DCFH-DA fluorescent probe was added to the 6-well plates, and the plates were reacted at 37°C in the dark for 20 minutes. The culture medium was then discarded, and the cells were washed twice with pre-cooled PBS. The supernatant was discarded, and the green fluorescence intensity was measured using a fluorescence microscope. The fluorescence intensity was analyzed using ImagJ software, and changes in ROS content in the cells were measured. The collected supernatant was used to determine the NO content using a kit (Beyotime Biotechnology Co., Ltd., S0021S), and the IL-1β and TNF-α levels were measured using ELISA kits (Jingmei Biotechnology Co., Ltd., JM-02323M2, JM-02415M2).

[0089] In this study, cell viability levels directly reflect the physiological state of cells. Therefore, by measuring the survival rate of AML12 hepatocytes under oxidative stress, the potential protective effect of the peptides can be evaluated. H2O2 can directly oxidize biomolecules (such as lipids and proteins), promoting the accumulation of free radicals, which in turn can lead to cell damage and even death.

[0090] Therefore, H2O2 was chosen to establish an AML12 cell oxidative stress injury model, such as... Figure 1As shown, the IC50 value of H2O2 oxidative damage to AML12 cells was 387 µmol / L. Using glutathione as a positive control at a concentration of 100 µg / mL, compared with the oxidative damage group, the cell viability in the glutathione group increased to 83.48%. The cell viability in neutral single-enzyme digestion, pancreaticogastric double-enzyme digestion, and pancreaticogastric triple-enzyme digestion polypeptide solutions (all diluted 20-fold) increased to 69.38%, 65.95%, and 63.32%, respectively.

[0091] Example 3:

[0092] The purification and activity verification methods for antioxidant peptides are as follows:

[0093] This embodiment utilizes DEAE-52 cellulose column chromatography and Sephadex G-50 gel chromatography to separate peptides.

[0094] 1. Separation of peptides by DEAE-52 cellulose column chromatography: Pretreated DEAE-52 cellulose packing material was equilibrated with deionized water (20 mm × 600 mm). A 50 mg / mL aqueous solution of casein-digested peptides was prepared, filtered through a 0.45 μm aqueous filter membrane, and loaded into a 4 mL volume. A linear gradient elution was performed using 0–0.3 mol / L NaCl solution. 5 mL of the eluent was collected per tube, and the absorbance was measured at 280 nm. Based on the absorbance curve, eluents at the same elution peak were collected and combined. The resulting separated fractions were freeze-dried for subsequent experiments.

[0095] like Figure 2 As shown, the neutral single-enzyme hydrolysate obtained by elution with NaCl solutions of different concentrations consisted of four components, named Z1, Z2, Z3 and Z4 in elution order; the pancreatic-to-gastric double-enzyme hydrolysate consisted of three components, named YM1, YM2 and YM3 in elution order; and the pancreatic-to-gastric triple-enzyme hydrolysate consisted of three components, named YZW1, YZW2 and YZW3 in elution order.

[0096] The 10 isolated polypeptide fractions were then desalted by dialysis, freeze-dried, and prepared into aqueous solutions of different concentrations. The MTT assay was used to detect their cytotoxic effects on AML12 cells. Figure 2 As shown, 50-200 µg / ml polypeptide aqueous solutions were found to have no significant toxicity to cells. Therefore, 200 µg / ml was selected for AML12 cell oxidative stress injury experiments, as shown... Figure 2As shown in Figure F, compared with the oxidative damage group, the neutral single-enzyme hydrolysate peptides Z1, Z2, Z3, and Z4 fractions all exhibited significant anti-oxidative stress effects, with Z3 showing the strongest protective effect and a cell viability of 83.32%. The pancreatic-gastric dual-enzyme hydrolysate peptides YM1 and YM2 fractions showed no anti-oxidative stress effect on AML12 cells, while YM3 exhibited a significant anti-oxidative stress effect, with a cell viability of 72.31%. The pancreatic-gastric triple-enzyme hydrolysate peptides YZW1 and YZW3 fractions also showed significant anti-oxidative stress effects, with cell viability of 76.33% and 77.18%, respectively.

[0097] 2. Separation of peptides by Sephadex G-50 gel chromatography: Pretreated Sephadex G-50 gel was packed into a glass chromatography column (16mm × 800mm), and three column volumes were balanced. The peptide sample purified by DEAE-52 cellulose column chromatography was taken and a 50mg / mL aqueous solution was prepared. The solution was filtered through a 0.22μm aqueous filter membrane, and 4mL of sample was loaded. Deionized water was used as the eluent, and 5mL of eluent was collected per tube. The absorbance was measured at a wavelength of 280nm. The eluents at the same elution peak were collected and combined and freeze-dried for subsequent experiments.

[0098] like Figure 3 As shown, Z1 separated three components, named Z1-A, Z1-B, and Z1-C in elution order; Z2 separated two components, named Z2-A and Z2-B in elution order; Z3 separated two components, named Z3-A and Z3-B in elution order; Z4 separated two components, named Z4-A and Z4-B in elution order; YM3 separated two components, named YM3-A and YM3-B in elution order; YZW1 separated three components, named YZW1-A, YZW1-B, and YZW1-C in elution order; and YZW3 separated two components, named YZW3-A and YZW3-B in elution order.

[0099] The peptide fractions separated by Sephadex G-50 gel chromatography were freeze-dried and prepared into aqueous solutions of different concentrations. The MTT assay was used to detect the cytotoxic effects on AML12 cells. Figure 3 As shown, 25-100 µg / mL polypeptide aqueous solutions were found to have no significant toxicity to cells. Therefore, 100 µg / mL was selected for AML12 cell oxidative stress injury experiments, as shown... Figure 3 As shown, compared with the oxidative damage group, the five polypeptide components Z1-C, Z3-B, YM3-B, YZW1-B and YZW3-B showed significant anti-oxidative stress damage effects, with cell viability of 72.20%, 74.45%, 74.60%, 75.11% and 82.19%, respectively.

[0100] Example 4:

[0101] Experiments on the effects of antioxidant peptides

[0102] To elucidate the protective mechanism of antioxidant peptides, the five purified polypeptide components (Z1-C, Z3-B, YM3-B, YZW1-B, YZW3-B) obtained in Example 3 were subjected to the following tests.

[0103] 1. Intracellular ROS level detection: The DCFH-DA fluorescent probe was used for observation and quantification under a fluorescence microscope. Results are as follows: Figure 4 As shown, compared with the oxidative damage group (B), the green fluorescence intensity of each peptide-administered group (DH) was significantly reduced, indicating a significant decrease in intracellular ROS levels.

[0104] In detail, to elucidate whether the protective mechanism of casein-derived antioxidant peptides against H2O2-induced AML12 cell damage involves reactive oxygen species (ROS) regulation, this protocol quantitatively analyzed changes in intracellular ROS levels using DCFH-DA fluorescent labeling technology. When ROS accumulates excessively, the cell's antioxidant system cannot effectively regulate, thereby inducing oxidative stress and causing significant oxidative cell damage. Since DCFH-DA itself does not possess fluorescent properties, after being converted into DCFH by intracellular hydrolases, it can be oxidized by ROS into the fluorescent substance DCF, which then emits green fluorescence.

[0105] like Figure 4 As shown, the blank control group cells had very low ROS content. After the addition of H2O2, AML12 cells produced a large amount of ROS. After the addition of antioxidant peptides Z1-C, Z3-B, YM3-B, YZW1-B, and YZW3-B, the intensity of green fluorescence in the cells decreased to varying degrees, indicating that these peptide components can significantly reduce the ROS content in the cells.

[0106] 2. Detection of oxidative damage markers and inflammatory factors: Cell culture supernatant and cell lysate were collected, and the levels of MDA, NO, TNF-α, and IL-1β were detected using appropriate kits. Results are as follows: Figure 5 As shown, each polypeptide component can significantly reduce the levels of these indicators, indicating that it has the effect of reducing lipid peroxidation and inhibiting inflammatory response.

[0107] In detail, MDA, as a hallmark product of lipid peroxidation, can damage the biological membrane system composed of phospholipid bilayers and membrane proteins when it accumulates excessively, ultimately leading to altered cell membrane permeability and loss of structural integrity. Therefore, detecting MDA levels can assess the degree of lipid peroxidation in the body, and thus indirectly reflect the degree of oxidative stress damage to cells.

[0108] In this protocol, antioxidant peptides Z1-C, Z3-B, YM3-B, YZW1-B, and YZW3-B were co-cultured with AML12 cells, and the cells were collected for lipid peroxide content determination. The results are as follows: Figure 5 As shown, compared with the blank control group, all five antioxidant peptides significantly reduced the lipid peroxide content in AML12 cells.

[0109] This protocol effectively assesses the degree of cellular inflammatory response by measuring the expression levels of pro-inflammatory mediators and inflammatory factors in cell culture supernatant. Figure 5 As shown, H2O2 treatment of AML12 cells resulted in a sharp increase in the levels of NO, TNF-α, and IL-1β in the culture medium, indicating that H2O2 induced an inflammatory response in AML12 cells. The addition of the peptide component significantly reduced the levels of NO, TNF-α, and IL-1β. These results demonstrate that casein antioxidant peptides can inhibit the expression of pro-inflammatory factors TNF-α and IL-1β, as well as the inflammatory marker NO, thereby improving the H2O2-induced inflammatory response in AML12 cells.

[0110] Example 5: Alternative Purification Methods This example provides two alternative purification techniques for antioxidant peptides, which can be used as supplements or alternatives to DEAE-52 cellulose column chromatography and Sephadex G-50 gel chromatography in Example 3.

[0111] (I) Ultrafiltration purification of peptides

[0112] Sample pretreatment: The crude peptide solution was first centrifuged (4000 r / min, 20 min) or filtered through a 0.45 μm filter membrane to remove insoluble impurities. If the solution concentration was too high (>50 mg / mL), it was appropriately diluted with phosphate buffer (PBS, pH 7.0) to avoid clogging the ultrafiltration membrane.

[0113] Ultrafiltration membrane selection and installation: Based on the target peptide molecular weight (usually <10 kDa), select an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, made of polyethersulfone or regenerated cellulose. Install the ultrafiltration membrane correctly into the ultrafiltration equipment (such as an Amicon ultrafiltration cup), ensuring a good seal.

[0114] Ultrafiltration separation operation: The pretreated sample is added to the ultrafiltration device and separated under a pressure of 0.1-0.3 MPa or a centrifugal force of 4000×g. Target small molecule peptides (molecular weight <10 kDa) will pass through the ultrafiltration membrane, while large protein molecules and peptides are retained.

[0115] Sample collection and processing; collection of filtrate (containing target small molecule peptides) or collection of retentate as needed.

[0116] The collected samples are concentrated using a rotary evaporator or directly freeze-dried to obtain refined peptide products.

[0117] Membrane cleaning and maintenance: After each use, immediately rinse the ultrafiltration membrane three times with distilled water. Disinfect the membrane by immersing it in 0.1 mol / L NaOH solution for 30 min, then rinse with distilled water until neutral (pH 7.0). For short-term storage, it can be immersed in 20% ethanol solution; for long-term storage, it must be dried and sealed.

[0118] (II) Purification of peptides by liquid chromatography

[0119] Sample pretreatment: The crude peptide solution was filtered through a 0.22-0.45 μm aqueous filter membrane to remove particulate impurities. If the sample salt concentration was high (NaCl > 0.1 mol / L), it was diluted with a mobile phase (5% acetonitrile-water, containing 0.1% trifluoroacetic acid) to a conductivity <10 mS / cm.

[0120] Chromatographic system preparation: Use a C18 reversed-phase column (column size: 4.6 mm × 250 mm, particle size: 5 μm). Wash the column with the initial mobile phase (95% aqueous phase + 5% organic phase, aqueous phase containing 0.1% trifluoroacetic acid, organic phase being acetonitrile). Equilibrate to a stable baseline (UV detection at 280 nm, drift <0.5 mAU / min). Sample loading and gradient elution: Inject 50-100 μL of sample (concentration 50 mg / mL). Use linear gradient elution: acetonitrile ratio increasing from 5% to 60%; gradient time: 60 min; flow rate: 1 mL / min; column temperature: 25℃; detection and component collection: real-time monitoring of the chromatographic peak at 280 nm.

[0121] Collect the target peak components based on the chromatogram (typically with a retention time of 15-45 min). Verify the purity of the target components (>90%) by HPLC analysis.

[0122] Post-processing and column maintenance: The collected components were removed by rotary evaporation to remove acetonitrile, and then freeze-dried to obtain purified peptides.

[0123] After the experiment, the column was rinsed sequentially with the following solutions: 30% acetonitrile-water for 30 min; and pure methanol for 20 min. For long-term storage, the column should be stored in an 80% methanol-water solution.

[0124] Technical advantages:

[0125] Ultrafiltration is simple to operate, suitable for large-scale preliminary purification, and can quickly remove macromolecular impurities.

[0126] Liquid chromatography has high separation efficiency and can obtain high-purity single components, making it suitable for fine separation.

[0127] Both methods can be combined with the chromatography method in Example 3 to form a multi-stage purification process.

[0128] Purification schemes can be flexibly selected based on the purity requirements of the target product and the production scale.

[0129] The alternative purification method provided in this embodiment allows for flexible selection of appropriate purification processes based on different production needs and equipment conditions, ensuring the acquisition of high-quality antioxidant peptide products.

[0130] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing yak milk casein antioxidant peptides, characterized in that, Includes the following steps: Step 1: Dissolve yak milk casein in alkaline solution to obtain casein solution; Step 2: Enzymatically hydrolyze the casein solution using a protease to obtain an enzymatic hydrolysate; Step 3: Inactivate the enzyme hydrolysate, centrifuge, collect the supernatant, and freeze-dry to obtain crude polypeptide; Step 4: The crude polypeptide is purified sequentially by anion exchange column chromatography and gel filtration chromatography to obtain antioxidant peptide components; Step 5: Perform activity screening on the purified antioxidant peptide components. Screening indicators include DPPH free radical scavenging rate and ABTS. + Free radical scavenging rate and hydrogen peroxide-induced survival rate of mouse AML12 hepatocytes were used to screen for target peptide components with high antioxidant activity. in: In step 2, the protease is selected from one or more of neutral protease, papain, trypsin, pepsin, and alkaline protease; In step 3, after the enzymatic hydrolysis reaction is completed, the sample is placed in a boiling water bath for 10 minutes to terminate enzyme activity. After cooling to room temperature, the sample is centrifuged to remove insoluble matter, thereby collecting the supernatant of the enzymatic hydrolysis product.

2. The preparation method according to claim 1, characterized in that, In step 2, the enzymatic hydrolysis method is single-enzyme hydrolysis, double-enzyme hydrolysis, or triple-enzyme hydrolysis; When performing a single enzyme digestion, digest for 120 minutes under the optimal temperature and pH conditions for the corresponding enzyme. When performing a double enzymatic hydrolysis, first add the first enzyme and hydrolyze for 60 minutes. After stopping the enzymatic hydrolysis, adjust the hydrolysate to the optimal temperature and pH of the second enzyme and then hydrolyze for another 60 minutes. When performing a triple-enzyme hydrolysis, add the third enzyme sequentially according to the above method and continue hydrolysis for 60 minutes, based on the double-enzyme hydrolysis.

3. The preparation method according to claim 1, characterized in that, The amount of protease added was 5000 U / g casein, and the enzymatic hydrolysis conditions were as follows: Trypsin: pH 8.0, temperature 37℃; Neutral protease: pH 7.0, temperature 40℃; Pepsin: pH 3.0, temperature 40℃; Papain: pH 7.0, temperature 50℃; Alkaline protease: pH 10.0, temperature 40℃.

4. The preparation method according to claim 1, characterized in that, In step 4, the anion exchange column chromatography is a DEAE-52 cellulose column, eluted with a linear gradient of 0-0.3 mol / L NaCl, and the eluted fraction with absorbance at 280 nm is collected.

5. The preparation method according to claim 1, characterized in that, In step 4, the gel filtration chromatography is performed using a Sephadex G-50 gel column, eluted with deionized water, and the eluent fraction with absorbance at 280 nm is collected.

6. The preparation method according to claim 4 or 5, characterized in that, The purification step also includes purification using ultrafiltration or liquid chromatography; The ultrafiltration method includes: pretreating the peptide solution, separating it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and collecting the filtrate or retentate; the pretreatment of the ultrafiltration method includes centrifugation or filtration to remove insoluble impurities, and diluting with buffer to a concentration ≤50 mg / mL; The liquid chromatography method includes: using a C18 reversed-phase column, performing gradient elution with an acetonitrile-water system, and collecting the target peak components; the gradient elution of the liquid chromatography method involves linearly increasing the acetonitrile ratio from 5% to 60%, with an elution time of 60 min and a flow rate of 1 mL / min.

7. A composition of yak milk casein antioxidant peptides, characterized in that, Prepared by any one of claims 1-5, comprising one or more of the antioxidant peptide components Z1-C, Z3-B, YM3-B, YZW1-B, and YZW3-B.

8. The use of a yak milk casein antioxidant peptide composition prepared by any one of claims 1-5 in the preparation of functional foods, health products, sports nutrition products, food additives or cosmetics for anti-oxidation, protecting hepatocytes from oxidative stress damage, delaying aging or inhibiting lipid oxidation.

9. The application according to claim 8, characterized in that, In the protection of hepatocytes from oxidative stress damage, the hepatocytes are AML12 hepatocytes, and the oxidative stress damage is induced by H2O2.

10. The application according to claim 9, characterized in that, The antioxidant peptide composition can reduce the levels of ROS, MDA, NO, and inflammatory factors in hepatocytes. and The expression.

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