A yeast peptide-hydroxytyrosol complex with high ROS capture ability and its preparation method and application

Abstract

The present invention discloses a yeast peptide-hydroxytyrosol complex with high ROS capture ability and its preparation method and application. The yeast peptide-hydroxytyrosol complex contains hydroxytyrosol and yeast peptide, and the yeast peptide is a peptide with a molecular weight greater than or equal to 1kDa and less than 3kDa produced by hydrolysis of yeast protein; the preparation method of the yeast peptide-hydroxytyrosol complex with high ROS capture ability, the yeast peptide and hydroxytyrosol are dispersed in a low ionic strength PBS buffer respectively, and then the yeast peptide dispersion and the hydroxytyrosol dispersion are mixed; the application of the complex with high ROS capture ability in the manufacture of antioxidant drugs. The yeast peptide-hydroxytyrosol complex provided by the present invention has high ROS capture performance, has higher free radical and peroxide scavenging ability than using hydroxytyrosol or yeast peptide alone, and shows excellent application potential in the fields of food and medicine.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, in particular to a yeast peptide-hydroxytyrosol complex with high ROS capture capability and a preparation method and application thereof. Background Art

[0002] As the per capita protein demand and global protein consumption increase year by year, existing protein resources can no longer meet people's needs. Land restrictions and resource shortages make it difficult to significantly increase the supply of traditional proteins. Therefore, the development of sustainable alternative proteins has become a research hotspot. Yeast protein is rich in protein. Specifically, the weight of protein can account for 35% to 60% of the dry weight of yeast, and the amino acid composition of yeast protein is close to that of ideal protein. It is one of the most ideal new alternative proteins and has a wide range of uses in food processing.

[0003] Currently, carbon emissions from animal husbandry have become one of the main factors of environmental damage. Every kilogram of beef produced produces 32.49 kilograms of CO2 equivalent methane gas. In contrast, yeast protein is produced from food production waste through fermentation engineering, which is sustainable and environmentally friendly. The entire production process is not limited by agricultural technology and environmental factors, so yeast protein can partially replace animal and plant protein, reduce dependence on agriculture, and significantly reduce carbon emissions. It is a low-carbon, environmentally friendly and sustainable protein resource.

[0004] Yeast protein can be used to make bioactive peptides. Bioactive peptides have attracted widespread attention due to their non-toxic side effects and biological functions such as anti-oxidation, lowering blood pressure, lowering blood sugar, and improving immunity. They are widely used in the fields of functional foods, medicines, and cosmetics. In the structure of the parent protein, the polypeptide is in an unhydrolyzed state, and most of them do not have biological activity. Only after being released through processes such as enzymatic hydrolysis, digestion, or fermentation can the active peptides exert their physiological effects. Compared with ordinary proteins, bioactive peptides contain more hydrophobic residues, which can effectively avoid the hydrolysis of digestive enzymes. The functional activity of polypeptides involves almost all physiological functions. When preparing polypeptides by enzymatic hydrolysis, the activity of the peptides can be controlled by changing the type of enzyme.

[0005] ROS (Reactive Oxygen Species) are a class of highly reactive oxygen-containing molecules or ions that are usually produced during cellular metabolism, such as oxidative phosphorylation, phagocytosis, and drug metabolism. Excessive accumulation of ROS may lead to oxidative stress, a state that is harmful to cell structure and function. Therefore, in order to prevent oxidative stress, it is necessary to find ways to capture and remove ROS.

[0006] Currently, antioxidants are often used to capture and remove ROS. Hydroxytyrosol (HT) is a natural polyphenolic compound with strong antioxidant activity, which mainly exists in the form of esters in olive fruits and branches and leaves. However, the bioaffinity of HT needs to be improved. Summary of the invention

[0007] To this end, the technical problem to be solved by the present invention is to provide a yeast peptide-hydroxytyrosol complex with high ROS capture ability and a preparation method and application thereof. When the yeast peptide-hydroxytyrosol complex with high ROS capture ability is used for ROS capture, it can remove free radicals and peroxides more efficiently than using hydroxytyrosol or yeast peptide alone, and has excellent application potential in the fields of food, medicine and skin protection products.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A yeast peptide-hydroxytyrosol complex with high ROS capture ability, the yeast peptide-hydroxytyrosol complex contains hydroxytyrosol and yeast peptide, the yeast peptide is a peptide with a molecular weight greater than or equal to 1kDa and less than 3kDa produced by yeast protein hydrolysis, and the enzyme used for yeast protein hydrolysis is a protease capable of hydrolyzing in the pH range of 7 to 8.

[0010] The yeast peptide-hydroxytyrosol complex with high ROS capture ability further contains phosphatidylcholine and / or a phospholipid membrane made of phosphatidylcholine, and the yeast peptide is a yeast peptide produced by hydrolyzing yeast protein with papain.

[0011] A method for preparing a yeast peptide-hydroxytyrosol complex with high ROS capture ability comprises the following steps:

[0012] S1. Add yeast protein powder into deionized water and stir to prepare yeast protein dispersion;

[0013] S2, adding protease to the yeast protein dispersion, hydrolyzing the yeast protein with the protease, then inactivating the enzyme, centrifuging and retaining the supernatant to obtain a yeast protein hydrolysate containing yeast peptides; the added protease is a protease that can exert a hydrolysis effect within the pH range of 7 to 8;

[0014] S3, separating yeast peptides having a molecular weight greater than or equal to 1 kDa and less than 3 kDa from the yeast protein hydrolysate;

[0015] S4, dispersing hydroxytyrosol and the yeast peptide obtained in step S3 in low ionic strength PBS buffer respectively, and then mixing the yeast peptide dispersion and the hydroxytyrosol dispersion, and sequentially performing ultrasonic treatment, high pressure homogenization treatment and freeze drying to obtain the above-mentioned yeast peptide-hydroxytyrosol complex with high ROS capture ability.

[0016] The method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability comprises the following steps: in step S1, stirring at 100-120 rpm for 3-5 min at 35-42° C.;

[0017] The protein content of the yeast protein powder is greater than or equal to 80wt%, and the mass concentration of the yeast protein powder in the yeast protein dispersion is 70-75g / L. The condition of 35-42°C can promote the swelling and dispersion of the yeast protein powder in water; when the mass fraction of the yeast protein powder is within the above range, it can be fully suspended and contacted with the enzyme after stirring at a speed of 100-120rpm for 3-5min.

[0018] In the above-mentioned method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability, in step S2, the added protease is papain;

[0019] The amount of papain and yeast protein dispersion is calculated as follows: first, the total protein content in the yeast protein dispersion is measured, then the casein mass in the yeast protein dispersion is calculated based on the casein content accounting for 86 wt % of the total protein content, and a hydrolysis reaction system is prepared based on the papain activity / casein mass of 9000-11000 U / g and hydrolysis is performed;

[0020] In step S2, the reaction conditions during hydrolysis are: temperature of 50-55°C, pH of the hydrolysis reaction system of 7-8, and hydrolysis time of 3.5-4.5h; when inactivating the enzyme, incubate in a boiling water bath for 10min, the speed during centrifugation is 8000-9000rpm, and the centrifugation time is 20-30min. The temperature and pH during hydrolysis are determined according to the properties of papain. The mass ratio of papain to substrate and the hydrolysis time will affect the amount of yeast peptides, and will also affect the structure of yeast peptides to a certain extent. If the enzyme dosage is too much or the hydrolysis time is too long, the yeast protein hydrolysis degree is too high, which will affect the activity of the obtained yeast peptides.

[0021] In the method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability, in step S3, an ultrafiltration membrane is used to centrifuge and separate yeast peptides with a molecular weight greater than or equal to 1 kDa and less than 3 kDa from yeast protease hydrolysate; the temperature during centrifugation is 4°C, the centrifugal force is 5000-8600g, and the centrifugation time is 15-20 minutes. When the centrifugal force is too high and the centrifugation time is too long, the ultrafiltration membrane may fail to retain the yeast peptide, and the yeast peptide with the target molecular weight cannot be separated.

[0022] In the method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability, step S4 comprises the following steps:

[0023] S4-A, dispersing the yeast peptide separated in step S3 in a low ionic strength PBS buffer to prepare a yeast peptide dispersion; in the yeast peptide dispersion, the mass concentration of the yeast peptide is 0.5-1.5 mg / mL; the low ionic strength PBS buffer helps to maintain the stability of the yeast peptide dispersion, while reducing ion interference and improving the subsequent binding efficiency with hydroxytyrosol;

[0024] S4-B, dispersing hydroxytyrosol in a low ionic strength PBS buffer to prepare a hydroxytyrosol dispersion; in the hydroxytyrosol dispersion, the mass concentration of hydroxytyrosol is 0.5-1.5 mg / mL, and the mass concentration of hydroxytyrosol in the hydroxytyrosol dispersion is equal to the mass concentration of yeast peptide in the yeast peptide dispersion; the concentration of the hydroxytyrosol dispersion is the same as that of the yeast peptide dispersion, ensuring equal mass reactions of the two components in the subsequent mixing process, promoting uniform formation of the complex, and improving the stability and functionality of the complex;

[0025] S4-C, mixing the yeast peptide dispersion and the hydroxytyrosol dispersion in equal quantities and stirring evenly to obtain a precursor solution; the stirring temperature is 20 to 30° C., the rotation speed is 80 to 120 rpm, and the stirring time is 0.5 to 1.5 h;

[0026] S4-D, heating the precursor solution to the preheating temperature, and then subjecting the precursor solution to ultrasonic treatment; after the start of ultrasonic treatment, first keep the precursor solution at the preheating temperature for 5 to 8 minutes, then cool it to 25°C at a cooling rate of 8 to 10°C / min, and keep it warm until the end of ultrasonic treatment; the preheating temperature is 40 to 45°C, the ultrasonic treatment time is 11 to 20 minutes, the ultrasonic power is 300 to 400W, the ultrasonic frequency is 40kHz, and the precursor solution is continuously stirred at a speed of 100 to 120rpm during the ultrasonic process; ultrasonic treatment can promote the mixing of yeast peptide and hydroxytyrosol. In addition, the ultrasonic process is first carried out at a higher temperature, which can accelerate the molecular thermal motion in the solution, promote the collision and contact of hydroxytyrosol and yeast peptide in the precursor solution, and is conducive to the full combination of the two; gradually cooling during the ultrasonic treatment process can improve the stability of the formed complex;

[0027] S4-E, subjecting the precursor solution after ultrasonic treatment to high-pressure homogenization treatment; the pressure of the high-pressure homogenization treatment is 280-320 MPa, and the number of cycles is 3-5 times; the high-pressure homogenization treatment effectively improves the stability and uniformity of the composite, ensuring that the composite has good and stable antioxidant properties;

[0028] S4-F, freeze-drying the precursor solution after high-pressure homogenization to obtain a yeast peptide-hydroxytyrosol complex.

[0029] The method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability is as follows: in step S4-C, after a precursor solution is prepared, a phospholipid membrane made of phosphatidylcholine is added to the precursor solution; the phospholipid membrane is prepared by dissolving phosphatidylcholine in chloroform and then evaporating the chloroform under reduced pressure; the amount of the phospholipid membrane added is calculated based on phosphatidylcholine, and the amount / volume ratio of phosphatidylcholine to the precursor solution is 0.1 to 0.5 mmol / L.

[0030] The phosphatidylcholine in the phospholipid membrane has a bilayer structure, with its hydrophobic tail located on the inside and the hydrophilic head located on the outside. Yeast peptide and hydroxytyrosol contain more hydrophilic groups (such as hydroxyl groups), which can bind to the head of phosphatidylcholine, so that the yeast peptide and hydroxytyrosol form a relatively stable connection with the help of phospholipids. At the same time, some hydrophobic groups in yeast peptides can also bind to the tails of some phospholipids, and the heads of these phospholipids bind to the hydrophilic hydroxytyrosol, which increases the number of hydroxytyrosol that yeast peptides can bind. In addition, phospholipids mainly bind to the alcoholic hydroxyl group of hydroxytyrosol, and have less interference with the phenolic hydroxyl group of hydroxytyrosol, which has an antioxidant effect. And phospholipids can improve the biocompatibility of yeast peptide-hydroxytyrosol complexes to a certain extent. Under the temperature conditions of 40-45°C at the initial stage of ultrasonic treatment, the phospholipid membrane swells and disperses rapidly, allowing it to better bind to yeast peptide and hydroxytyrosol.

[0031] The method for preparing the yeast peptide-hydroxytyrosol complex with high ROS capture ability is as follows: in step S1, stirring at 120 rpm for 5 min at 40° C.; the protein content in the yeast protein powder is 82 wt %, and the mass concentration of the yeast protein powder in the yeast protein dispersion is 70.9 g / L;

[0032] In step S2, a hydrolysis reaction system is prepared according to the papain activity / casein quality of 10000U / g; the temperature during hydrolysis is 50°C, the pH of the hydrolysis reaction system is 7, and the hydrolysis time is 4h; the speed during centrifugation is 8000rpm, and the centrifugation time is 30min;

[0033] In step S3, the centrifugal force during the ultrafiltration membrane centrifugal separation is 5000g, and the centrifugation time is 15min;

[0034] In step S4-A, the mass concentration of yeast peptide in the yeast peptide dispersion is 1 mg / mL;

[0035] In step S4-B, the mass concentration of hydroxytyrosol in the hydroxytyrosol dispersion is 1 mg / mL;

[0036] In step S4-C, the stirring temperature is 25° C., the rotation speed is 100 rpm, and the stirring time is 1 h. The amount of phospholipid membrane added to the precursor solution is calculated based on phosphatidylcholine, and the amount / volume ratio of phosphatidylcholine to the precursor solution is 0.1 mmol / L.

[0037] In step S4-D, the preheating temperature is 45°C, the precursor solution is kept at 45°C for 5 minutes, then cooled to 25°C at a cooling rate of 10°C / min, and kept warm until the ultrasonic treatment is completed; the ultrasonic treatment time is 15 minutes, the ultrasonic power is 400W, and the precursor solution is continuously stirred at a speed of 100rpm during the ultrasonic treatment;

[0038] In step S4-E, the pressure of the high-pressure homogenization treatment is 300 MPa, and the number of cycles is 3 times.

[0039] The yeast peptide-hydroxytyrosol complex with high ROS capture ability is used in any of the following applications:

[0040] As food antioxidant additive;

[0041] Used to manufacture foods with antioxidant function;

[0042] Used in the manufacture of antioxidant drugs;

[0043] Used in the manufacture of UV protection products for skin;

[0044] Antioxidant stress protectant for cell culture in vitro;

[0045] Used as an indicator of cellular oxidative stress processes;

[0046] Used in the manufacture of plant protection agents.

[0047] The technical solution of the present invention achieves the following beneficial technical effects:

[0048] 1. The present invention combines the strong antioxidant properties of hydroxytyrosol with the antioxidant properties and bioaffinity properties of yeast peptides, and forms a stable composite structure through efficient ultrasonic treatment and high-pressure homogenization. This composite has high antioxidant properties and more efficient free radical scavenging ability than using hydroxytyrosol or yeast peptide alone, showing excellent application potential in the fields of food, medicine and skin protection products.

[0049] 2. The present invention provides a method for preparing a yeast peptide-hydroxytyrosol complex. Under suitable conditions, papain is used to hydrolyze the protein in yeast protein powder, and ultrafiltration is performed to obtain a yeast peptide with a molecular weight greater than or equal to 1 kDa and less than 3 kDa. The yeast peptide itself has a strong ·OH capture ability and biological affinity. Further, the yeast peptide is compounded with hydroxytyrosol having SOD activity and ·OH capture ability in equal amounts to prepare a yeast peptide-hydroxytyrosol complex, and a phospholipid membrane made of phosphatidylcholine is added during the preparation process. The unique bilayer structure of the phospholipid membrane enhances the stability of the yeast peptide and hydroxytyrosol, and enables the hydrophobic groups in the yeast peptide to combine with the alcoholic hydroxyl groups of the relatively hydrophilic hydroxytyrosol with the help of phosphatidylcholine, thereby increasing the binding amount of the yeast peptide and hydroxytyrosol. Phosphatidylcholine also increases the biological affinity of the complex. The invention discloses a novel method for preparing a novel compound of yeast peptide-hydroxytyrosol, which is characterized by high molecular weight, high molecular weight and low molecular weight. The method has the advantages of good stability, good chemical stability, good chemical stability, and good chemical resistance, and will not affect the active groups such as phenolic hydroxyl groups with antioxidant capacity in the complex. In the process of preparing the complex, the cavitation effect of ultrasound is used to break the molecular agglomeration, and the temperature condition of 40-45℃ is used to promote the swelling of the phospholipid membrane in the precursor solution, so that phosphatidylcholine, yeast peptide and hydroxytyrosol can fully collide and combine through molecular thermal motion. Rapid cooling and continuous ultrasonic treatment after 5-8min can make the binding between phosphatidylcholine, yeast peptide and hydroxytyrosol more firm. In the ultrasonic treatment after cooling, ultrasound only breaks the agglomeration between the molecules of the yeast peptide-hydroxytyrosol complex, but does not damage the structure of the complex itself. The preparation process is carried out in a low ionic strength PBS buffer, which can not only protect the activity of the yeast peptide and the complex, but also prevent charged metal ions from interfering with the complex between the yeast peptide and hydroxytyrosol. The yeast peptide-hydroxytyrosol complex finally obtained has a strong ROS capture ability and good biocompatibility. The complex can play a more effective role in the body and has good biocompatibility, which is suitable for a variety of biomedical applications.

[0050] 3. The yeast peptide-hydroxytyrosol complex provided by the present invention has efficient ROS capture performance and can be used as an indicator of ROS for the study of cellular oxidative stress processes. In addition, the complex can also provide cells with anti-oxidative stress protection, help study the mechanism of action of ROS in cells and organisms, and promote scientific research and technological development in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The experimental results of the methyl violet fading spectrophotometric experiment of the yeast peptide prepared in Example 1 of the present invention;

[0052] Figure 2 ESR spectra of the yeast peptide and hydroxytyrosol prepared in Example 1 of the present invention;

[0053] Figure 3 Comparison of the experimental results of the yeast peptide-hydroxytyrosol complex prepared in Example 1 of the present invention, yeast peptide wood 3 and hydroxytyrosol methyl violet fading spectrophotometry experiment;

[0054] Figure 4 Photos of the yeast peptide-hydroxytyrosol complex prepared in Example 1 of the present invention, yeast peptide wood 3 and samples to be tested in the hydroxytyrosol methyl violet fading spectrophotometry experiment;

[0055] Figure 5 XDR spectrum of the yeast peptide prepared in Example 1 of the present invention;

[0056] Figure 6 The SOD activity test results of the yeast peptide-hydroxytyrosol complex, yeast peptide 3 and hydroxytyrosol prepared in Example 1 of the present invention;

[0057] Figure 7 Biocompatibility test results of the yeast peptide-hydroxytyrosol complex prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0058] Example 1

[0059] The yeast protein powder used in this embodiment is commercially available yeast protein powder, which is prepared using Saccharomyces cerevisiae as a raw material. The method for preparing yeast peptide-hydroxytyrosol using yeast protein powder is further described in detail below.

[0060] 1. Preparation of Yeast Protein Dispersion

[0061] The yeast protein powder was added into deionized water and stirred at 120 rpm for 5 min at 40° C. to prepare a yeast protein dispersion.

[0062] 2. Determination of protein content of yeast protein powder

[0063] 2.1 Preparation of reagents

[0064] Preparation of biuret reagent: Weigh 1.5g copper sulfate (CuSO4·5H2O) and 6.0g potassium sodium tartrate (KNaC4H4O6·4H2O), dissolve them in 500mL deionized water, add 300mL of 10wt% NaOH solution under stirring, make up to 1L with deionized water, and set aside.

[0065] Preparation of standard protein solution: Accurately weigh 0.05g of standard bovine serum albumin (BSA), dissolve it and add it to a 10mL volumetric flask, and make up to volume with distilled water. The standard protein solution contains 5mg / mL BSA.

[0066] 2.2 Drawing the standard curve

[0067] Take 6 test tubes and divide them into two groups, and prepare the standard solution according to the formula in Table 1. After the volume is fixed, mix thoroughly and let it stand at room temperature for 30 minutes, then measure the A540 absorbance.

[0068] Table 1

[0069] serial number 1 2 3 4 5 6 5mg / mL standard protein solution (mL) 0.0 0.2 0.4 0.6 0.8 1.0 Deionized water (mL) 1.0 0.8 0.6 0.4 0.2 0.0 Biuret reagent (mL) 4.0 4.0 4.0 4.0 4.0 4.0 Protein concentration (mg / mL) 0.0 0.2 0.4 0.6 0.8 1.0

[0070] Take the average value of the A540 values ​​measured in the two groups, that is, the A540 absorbance value is the horizontal axis and the protein concentration is the vertical axis, and draw a standard curve.

[0071] 2.3 Sample determination

[0072] Weigh 25mg yeast protein powder in a triangular flask, add 5mL of 0.05M NaOH and 20mL of biuret reagent to the flask, vibrate for 15s, let stand for 30min, filter the filtrate and add a cuvette, measure A540, and compare with the standard curve to obtain the protein content in the yeast protein powder. Take the average value of the two groups of measurements, and finally determine that the protein content in the yeast protein powder used in this embodiment is 82wt% (in the yeast protein powder used to prepare yeast peptides, the protein content should be greater than or equal to 80wt%).

[0073] Calculate the casein content in yeast protein powder according to the following formula:

[0074] Casein content in yeast protein powder (%) = (y×N) / c×100%×r

[0075] y is the protein concentration obtained from the standard curve (mg / mL)

[0076] N is the total volume (25mL)

[0077] c is the weight of the yeast protein powder weighed

[0078] r is the casein mass fraction in yeast protein (calculated as 86 wt%)

[0079] 3. Yeast proteolysis

[0080] 3.1 Enzyme activity assay

[0081] When enzymatic hydrolysis of yeast protein is performed to prepare yeast peptides, the activity of the enzyme will affect the amount of yeast peptides obtained. In this section, the ability of alkaline protease, flavor protease and papain to hydrolyze proteins is tested.

[0082] Prepare L-tyrosine standard solution with reference to Table 2, take 1 mL of standard solution in each test tube, add 5 mL of 0.4 M sodium carbonate and 1 mL of Folin-phenol reagent (i.e. Folin-phenol reagent, also known as Folin-phenol reagent B, a commercial product, concentration 1 mol / L), respectively, and place in a 40°C water bath for 20 min. Take tube 1 without L-tyrosine as a control, and measure the absorbance value of each tube solution at 660 nm. Draw a standard curve with absorbance as the vertical axis and tyrosine concentration as the horizontal axis.

[0083] Table 2

[0084]

[0085] Take 0.01g of alkaline protease, 0.01g of flavor protease and 0.01g of papain respectively, add these three enzymes to 10mL of BR buffer (Britton-Robinson buffer solution) respectively, make them dispersed evenly, and then use BR buffer to supplement the volume to 50mL respectively to obtain the enzyme solution to be tested. For alkaline protease, the pH of the BR buffer used is 8, and for flavor protease and papain, the pH of the BR buffer used is 7.

[0086] For each enzyme, 4 portions of 1 mL each of the enzyme solution to be tested were taken, 3 of which were used as 3 parallel replicates of the experimental group, and the other was used as a blank control.

[0087] First, preheat the enzyme solution to be tested at 40°C for 2 minutes. For the enzyme solution to be tested in the experimental group, add 1 mL of casein dispersion preheated at 40°C and shake well; for the enzyme solution in the blank control group, add 2 mL of trichloroacetic acid and shake well. Heat the experimental and control groups in a water bath at 40°C for 10 minutes.

[0088] After the water bath heating was completed, for the blank control group, 1 mL of casein was added, shaken and allowed to stand for 10 min; for the experimental group, 2 mL of trichloroacetic acid was added to each test sample, shaken and allowed to stand for 10 min.

[0089] After standing, for the samples of the experimental group and the control group, 0.5 mL of filtrate was filtered out with a 0.22 μm microporous filter membrane. Finally, according to the standard determination method, 2.5 mL of 0.4 M sodium carbonate solution was added to each 0.5 mL of filtrate and mixed, and the mixture was allowed to stand at 23°C for 10 minutes. Then, 0.5 mL of Folin phenol reagent was added and quickly shaken, and the mixture was allowed to stand at 23°C for 30 minutes. 1 mL of water was used as a blank control, and the absorbance was measured at 680 nm. By comparing the absorbance with the standard curve, the tyrosine concentration in the enzymatic hydrolysis product of the sample to be tested can be obtained, and the activity of the enzyme can be calculated by combining the volume of the enzymatic reaction system, the reaction time, and the mass of the added enzyme.

[0090] The final calculation results showed that the enzyme activity of alkaline protease was 200,000 U / g, the enzyme activity of flavor protease was 50,000 U / g, and the enzyme activity of papain was 250,000 U / g.

[0091] 3.2 Preparation of Yeast Protein Hydrolysate

[0092] With reference to the casein content in the yeast protein powder calculated in 2.3, the yeast protein powder is calculated and accurately weighed, and according to the method in "1. Preparation of Yeast Protein Dispersion", a yeast protein dispersion with a casein mass concentration of 50 mg / mL (the mass concentration of yeast protein powder in the yeast protein dispersion is 70.9 g / L) is prepared, and alkaline protease, flavor protease and papain are added to the yeast protein dispersion, respectively, and the mass of the added enzyme is adjusted to ensure that the final enzyme activity / substrate mass (E / S) is 10000 U / g. Wherein, the mass of the substrate refers to the mass of casein. The hydrolysis of yeast protein is carried out according to the system and procedure in Table 3.

[0093] Table 3

[0094] name Enzyme activity (10,000 U / g) Enzymatic hydrolysis temperature (℃) Enzymatic pH Enzymatic hydrolysis time (h) E / S(U / g) Alkaline protease 20 60 8 4 10000 Flavor protease 5 50 7 4 10000 Papain 25 50 7 4 10000

[0095] During the hydrolysis process, 1 mol / L NaOH or HCl is added to keep the pH constant. After the hydrolysis reaction is completed, the reaction solution is incubated in boiling water for 10 minutes to inactivate the enzyme, and then centrifuged at 8000 rpm for 30 minutes, and the supernatant is collected to obtain the yeast protein hydrolysate (the hydrolysate). If a large amount of hydrolysate is obtained at one time, it can be freeze-dried to obtain the yeast protein hydrolysate dry powder, which is stored at -20°C.

[0096] 4.Separation and purification of yeast protein hydrolysate

[0097] For the yeast protein hydrolysate obtained in 3.2, the polypeptides therein were retained using an ultrafiltration tube Amicon Ultra-15 equipped with 3kDa and 1kDa ultrafiltration membranes. During the retention, the yeast protein hydrolysate was transferred to an ultrafiltration tube equipped with an ultrafiltration membrane for centrifugation, the centrifuge temperature was 4°C, the centrifugal force was 5000g, and the centrifugation time was 15min. After centrifugation, two components with a molecular weight less than 3kDa and greater than or equal to 1kDa and less than 1kDa were retained. The filtered component (component less than 1kDa) and the retained component (component less than 3kDa and greater than or equal to 1kDa) were freeze-dried separately for performance characterization such as antioxidant capacity testing.

[0098] In some other embodiments, the centrifugation time and centrifugal force can also be adjusted according to demand, but the centrifugal force should not exceed 8600g, and the centrifugation time should not exceed 20 minutes.

[0099] 5. Characterization results of enzymatic hydrolysates

[0100] XRD analysis was performed on the above filtered components and retained components. The analysis results are as follows: Figure 5 As shown in the figure, "wood", "alkali" and "wind" represent papain treatment, alkaline protease treatment and flavor protease treatment respectively, "3" represents a component less than 3kDa and greater than or equal to 1kDa, and "1" represents a component less than 1kDa. Figure 5 It can be seen that different samples show different diffraction peaks at different 2θ angles, and the peak heights also vary greatly. This indicates that after yeast protein is treated with different enzymes, different types of polypeptides are obtained, and the contents of various polypeptides are also different.

[0101] In addition, the antioxidant capacity of the enzymatic hydrolysates after different enzyme treatments was tested. When testing the antioxidant capacity, the methyl violet fading spectrophotometry was used. This method can measure the content of hydroxyl radicals ·OH in the system by measuring the absorbance of the system containing methyl violet at 580nm. The basic principle is: F 2+ It reacts with H2O2 to generate ·OH through Fenton reaction, and ·OH reacts with the -C=C- group with high electron cloud density in methyl violet through electrophilic addition reaction, thereby fading methyl violet. In other words, assuming that the enzymatic hydrolysate (yeast peptide) in the above-mentioned filtered components and retained components has a high antioxidant capacity, the enzymatic hydrolysate will remove ·OH, making the methyl violet in the reaction system less likely to fade, the color of the system is darker, and the absorbance of the system at 580nm is higher.

[0102] like Figure 1The results of the antioxidant capacity test are shown in Figure MV, which indicates a methyl violet solution without ·OH, Blank, which indicates a water control, and the "wood", "alkali" and "wind" in brackets after "yeast peptide" respectively represent papain treatment, alkaline protease treatment and flavor protease treatment. "<3kDa" indicates a component less than 3kDa and greater than or equal to 1kDa, and "<1kDa" indicates a component less than 1kDa.

[0103] Comparing the absorbance measurement results of the methyl violet solution with the addition of 5 components, it can be seen that the "yeast peptide (wood) <3kDa" group has the highest absorbance, indicating that this component itself has a strong antioxidant capacity. Papain, alkaline protease and flavor protease can all be used to hydrolyze yeast protein and prepare yeast peptides with antioxidant capacity. The action pH of papain is 3.5-9, and the action pH of flavor protease is 5-9. The optimal pH of alkaline protease is greater than 9. Although it can hydrolyze protein under pH 8, the hydrolysis efficiency is low, and increasing the pH during protein hydrolysis may cause problems such as polypeptide inactivation. Taking into account the action pH of the enzyme and the antioxidant activity of the product yeast peptide, in this embodiment, "yeast peptide (wood) <3kDa" was finally selected to prepare the yeast peptide-hydroxytyrosol complex. In some other embodiments, the yeast peptide obtained by hydrolyzing yeast protein with flavor protease, alkaline protease or other proteases that can play a hydrolytic role in the pH range of 7-8 can also be selected to prepare the yeast peptide-hydroxytyrosol complex.

[0104] Furthermore, the ROS capture performance of hydroxytyrosol and yeast peptide (W) <3 kDa was tested by electron spin resonance (ESR). Figure 2 For test results. Figure 2 The ESR signals of the three samples in the magnetic field range of 3460G to 3540G are shown. In the figure, Blank represents the blank control, yeast peptide wood 3 represents "yeast peptide (wood) <3kDa", and the hydroxytyrosol group contains hydroxytyrosol of the same mass as "yeast peptide (wood) <3kDa". The three groups are all added with DMPO (5,5-dimethyl-1-pyrroline-N-oxide), Fe 2+ and H2O2(Fe 2+ When DMPO captures ·OH, a characteristic peak of 1:2:2:1 will be generated in the ESR graph.

[0105] Depend on Figure 2It can be seen that all three groups of samples showed characteristic peaks of 1:2:2:1, indicating that DMPO in all three groups captured ·OH. Yeast peptide wood 3 and hydroxytyrosol can compete with DMPO to capture ·OH, reducing the ·OH captured by DMPO, which is manifested as a decrease in the peak value of the ESR signal. The blank control does not contain any substance that can compete with DMPO to capture ·OH. In other words, if the peak value of a sample is significantly lower than that of the blank control, it means that the sample has a good ·OH capture ability (its ROS capture ability is also good).

[0106] Comparison of the ESR signal intensities of each sample showed that both hydroxytyrosol and yeast peptide 3 had good ·OH capture capabilities and were expected to be used to prepare yeast peptide-hydroxytyrosol complexes with good antioxidant properties.

[0107] 6. Preparation of Yeast Peptide-Hydroxytyrosol Complex

[0108] Using "yeast peptide (wood) <3 kDa" as a raw material, the yeast peptide-hydroxytyrosol complex was prepared according to the following method:

[0109] (1) Disperse "yeast peptide (wood) <3 kDa" in a low ionic strength PBS buffer to prepare a yeast peptide dispersion. In the yeast peptide dispersion, the mass concentration of the yeast peptide is 1 mg / mL.

[0110] (2) Dispersing hydroxytyrosol in a low ionic strength PBS buffer to prepare a hydroxytyrosol dispersion with a hydroxytyrosol mass concentration of 1 mg / mL.

[0111] (3) According to the mass ratio of the yeast peptide dispersion to the hydroxytyrosol dispersion of 1:1, the two dispersions were mixed and stirred evenly to obtain a precursor solution; during stirring, the stirring was continued at a speed of 100 rpm at 25° C. for 1 hour.

[0112] (4) Dissolve phosphatidylcholine in chloroform and evaporate the chloroform under reduced pressure until the chloroform evaporates to obtain a phospholipid membrane. Add the phospholipid membrane to the precursor solution. The amount of the phospholipid membrane added is calculated based on phosphatidylcholine, and the amount / volume ratio of phosphatidylcholine to the precursor solution is 0.1 mmol / L.

[0113] (5) The precursor solution was preheated to 45°C, and then the precursor solution was ultrasonically treated for 15 min at 40 kHz and 400 W ultrasonic power. During the ultrasonic process, the precursor solution was continuously stirred at a speed of 100 rpm, and the precursor solution was first kept at 45°C for 5 min. After 5 min, the precursor solution was cooled to 25°C at a cooling rate of 10°C / min, and maintained at 25°C until the ultrasonic treatment was completed.

[0114] (6) Use a high-pressure homogenizer to perform high-pressure homogenization on the precursor solution. Set the pressure to 300 MPa and cycle 3 times.

[0115] (7) The precursor solution after high pressure homogenization is freeze-dried to obtain the yeast peptide-hydroxytyrosol complex. The freeze-drying can be carried out according to the general polypeptide freeze-drying method.

[0116] In this embodiment, the preparation method of the low ionic strength PBS buffer used is: weigh 1.37g NaCl, 0.027g KCl, 0.144g anhydrous Na2HPO4 and 0.018g KH2PO4, dissolve in 800mL distilled water, stir evenly, adjust the pH to 7.2, and then adjust the volume to 1L. The use of low ionic strength PBS buffer can not only protect the biological activity of yeast peptides, but also prevent the excessive ion concentration in the buffer from interfering with the binding between yeast peptides and hydroxytyrosol. In some other embodiments, low ionic strength PBS buffers of other formulations can also be used.

[0117] After phosphatidylcholine is made into a phospholipid membrane, phosphatidylcholine will form a bilayer structure, with the hydrophobic tail located on the inside and the hydrophilic head located on the outside. Yeast peptide and hydroxytyrosol contain more hydrophilic groups (such as hydroxyl groups), which can bind to the head of phosphatidylcholine, so that yeast peptide and hydroxytyrosol form a relatively stable connection with the help of phospholipids. At the same time, some hydrophobic groups in the polypeptide can also bind to the tails of some phosphatidylcholines, and the heads of these phosphatidylcholines bind to the hydrophilic hydroxytyrosol, which increases the number of hydroxytyrosol that yeast peptide can bind. In addition, phosphatidylcholine (phospholipid membrane) mainly binds to the alcohol hydroxyl group of hydroxytyrosol, and has less interference with the phenolic hydroxyl group of hydroxytyrosol, which has an antioxidant effect. And the phospholipid membrane can improve the biocompatibility of the yeast peptide-hydroxytyrosol complex to a certain extent. Providing a heating temperature of 45°C at the beginning of ultrasonic treatment is conducive to accelerating the swelling and dispersion of the phospholipid membrane, so that it can better bind to yeast peptide and hydroxytyrosol.

[0118] In some other embodiments, step (4) can be omitted, and the yeast peptide-hydroxytyrosol complex can be obtained while the other conditions remain unchanged. Alternatively, the amount of phospholipid membrane added to the precursor solution can be appropriately increased or phosphatidylcholine can be directly added, but the amount added (calculated as phosphatidylcholine) should not exceed 0.5 mmol / L to prevent the phospholipid membrane from interfering with the contact and capture of ROS by the yeast peptide-hydroxytyrosol complex.

[0119] 7. Characterization of ROS Capture Performance of Yeast Peptide-Hydroxytyrosol Complex

[0120] ROS include free radicals and non-radical compounds, the former including hydroxyl radicals (·OH) and the latter including hydrogen peroxide (H2O2), which can be neutralized by the antioxidant superoxide dismutase (SOD). In this part, the ·OH-capturing ability and superoxide dismutase activity of the yeast peptide-hydroxytyrosol complex were characterized.

[0121] like Figure 3 These are the test results of the ·OH capture ability of yeast peptide-hydroxytyrosol complex, hydroxytyrosol and yeast peptide wood 3, which were tested using methyl violet fading spectrophotometry. Figure 4 The following are the actual photos of each group. Figure 3 and Figure 4 It can be seen that for the yeast peptide-hydroxytyrosol complex group, the absorbance of the characteristic peak of methyl violet at 580nm did not decrease significantly, indicating that it has a good antioxidant capacity, that is, a high ability to capture ·OH. The ability to capture ·OH is ranked as follows: yeast peptide-hydroxytyrosol complex>hydroxytyrosol>yeast peptide wood 3.

[0122] like Figure 6 The figure shows the SOD activity comparison of yeast peptide-hydroxytyrosol complex, hydroxytyrosol and yeast peptide. CeO2, which is used as the control group in the figure, is a ROS scavenger and has SOD activity. As can be seen from the figure, although yeast peptide itself has no SOD activity, the complex formed by it and hydroxytyrosol has a higher SOD activity, which is higher than the SOD activity of hydroxytyrosol itself.

[0123] 8. Biocompatibility test of yeast peptide-hydroxytyrosol complex

[0124] The biocompatibility of yeast peptide-hydroxytyrosol complex was detected by MTT method. During the test, cells were cultured with cell culture medium containing 12.5μg / mL, 25μg / mL, 50μg / mL and 100μg / mL of yeast peptide-hydroxytyrosol complex for 24h, and then the cell survival rate was detected. Figure 7 For the test results, Figure 7 It can be seen that the biocompatibility of the yeast peptide-hydroxytyrosol complex is good, as shown by the survival rates of the four groups of cells containing the yeast peptide-hydroxytyrosol complex in the culture medium being above 90%. In addition, the cell survival rates of some groups are greater than that of the control group, which may be because some cells proliferate.

[0125] Example 2

[0126] In this example, the yeast peptide-hydroxytyrosol complex prepared in Example 1 was used to test its ability to prevent cells from generating oxidative stress due to ultraviolet irradiation.

[0127] Experimental materials include:

[0128] (1) Cell line: human skin fibroblasts (HSF);

[0129] (2) yeast peptide-hydroxytyrosol complex;

[0130] (3) DMEM medium containing 10 wt% fetal bovine serum (FBS);

[0131] (4) pH 7.5 phosphate buffered saline (PBS);

[0132] Human skin fibroblasts were seeded in 6-well plates at 1×10 5 The cells were cultured in a 37°C, 5% CO2 incubator using DMEM medium (containing 10wt% FBS) for 24 hours.

[0133] The cells were randomly divided into the following three groups, with 3 replicate wells in each group:

[0134] Blank control group: no treatment, no UV irradiation.

[0135] UV irradiation group: without any protection, direct UV irradiation.

[0136] Yeast peptide-hydroxytyrosol complex group: UV irradiation after complex treatment.

[0137] 30 minutes before UV irradiation, the original culture medium was removed and 3.5 mL of fresh culture medium containing yeast peptide-hydroxytyrosol complex (the mass concentration of yeast peptide-hydroxytyrosol complex contained was 25 μg / mL) was added for pre-culture.

[0138] After the pre-culture, the culture medium was removed, the cells were washed with PBS, fresh culture medium without yeast peptide-hydroxytyrosol complex was added, and then the cells were irradiated with UV light. The irradiation dose and time were: UV-B: 50mJ / cm 2 , lasting 6h.

[0139] After UV irradiation, the cells were cultured for 24 hours. The cells were then digested with trypsin and the apoptosis rate was detected using a TUNEL cell apoptosis detection kit according to the instructions. The results are shown in Table 4.

[0140] Table 4

[0141]

[0142] The above results show that the apoptosis rate of cells with the addition of yeast peptide-hydroxytyrosol complex was significantly lower than that of the UV irradiation group, which indicates that the yeast peptide-hydroxytyrosol complex can effectively capture ROS in the cell culture system and has the ability to prevent cells from producing oxidative stress due to ultraviolet irradiation.

[0143] Example 3

[0144] Since the yeast peptide-hydroxytyrosol complex has good ROS capture ability, it can be used as an indicator in biochemical research. For example, studies have shown that a high sugar environment may cause endothelial cell apoptosis. When it is necessary to explore whether high sugar induces endothelial cell apoptosis by inducing oxidative stress in endothelial cells, the yeast peptide-hydroxytyrosol complex can be used as an "indicator" to make a preliminary judgment based on the survival of the cells.

[0145] It is known that a high-glucose environment can indeed induce apoptosis of vascular endothelial cells by inducing oxidative stress in vascular endothelial cells. In this example, a confirmation experiment was carried out using the yeast peptide-hydroxytyrosol complex prepared in Example 1.

[0146] Experimental Materials:

[0147] (1) Human umbilical vein endothelial cells (HUVECs).

[0148] (2) DMEM culture medium (containing 10 wt % fetal bovine serum).

[0149] (3) Yeast peptide-hydroxytyrosol complex.

[0150] (4) Cell apoptosis detection kit (Annexin V / PI double staining kit).

[0151] Endothelial cells were cultured under standard conditions (37°C, 5% CO2) until the logarithmic growth phase. The cells were digested with trypsin and the cells were counted. The cells were seeded into 6-well plates at 1x10 cells per well. 5 cells for subsequent experiments.

[0152] Experimental groups:

[0153] Control group: cells were cultured using DMEM medium containing normal sugar concentration (4.5 mM glucose).

[0154] High glucose group: cells were cultured using high glucose (30 mM glucose) DMEM medium.

[0155] High glucose + yeast peptide-hydroxytyrosol complex group: yeast peptide-hydroxytyrosol complex (dispersed in DMEM medium containing 30 mM glucose, yeast peptide-hydroxytyrosol complex mass concentration of 25 μg / mL) was added to high glucose medium to culture cells.

[0156] The cells were treated according to the above grouping, and after culturing the cells for 24 hours, the cell apoptosis rate was detected using the Annexin V / PI double staining kit. The results are shown in Table 5.

[0157] Table 5

[0158] Control group High sugar group High sugar + yeast peptide-hydroxytyrosol complex group Apoptosis rate (%) 3.8 16.8 10.1

[0159] The above results indicate that the addition of yeast peptide-hydroxytyrosol complex can alleviate the apoptosis of human umbilical vein endothelial cells caused by high glucose, that is, high glucose may induce apoptosis of human umbilical vein endothelial cells by inducing oxidative stress in human umbilical vein endothelial cells.

[0160] In addition, yeast peptide-hydroxytyrosol complex has other uses, including:

[0161] Food additives: Add this complex to foods such as juice, yogurt, and nutrition bars as a natural antioxidant to enhance the antioxidant properties of food and extend its shelf life.

[0162] Radiotherapy / chemotherapy adjuvant therapy: During cancer radiotherapy or chemotherapy, a large amount of ROS will be generated in the patient's body, causing cell damage. This complex can be used as an adjuvant therapy to reduce the damage of ROS to normal cells.

[0163] Sports Supplements: After strenuous exercise, athletes' ROS levels will increase, affecting their body's recovery. Adding this complex to sports drinks or supplements can help athletes recover faster.

[0164] Plant protectants: During plant growth, the accumulation of ROS can cause damage to the plants. The compound can be made into a plant protectant and sprayed on plants to help them resist the damage of ROS and improve their stress resistance.

[0165] Anti-aging skin care products: used in skin care products such as creams, essences, and masks to protect the skin from free radical damage and delay skin aging.

[0166] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing a yeast peptide-hydroxytyrosol complex with high ROS capture ability, characterized in that: The steps include: S1. Add yeast protein powder to deionized water and stir to prepare a yeast protein dispersion; the protein content of the yeast protein powder is greater than or equal to 80wt%, and the mass concentration of the yeast protein powder in the yeast protein dispersion is 70-75g / L; S2, adding protease to the yeast protein dispersion, hydrolyzing the yeast protein with the protease, then inactivating the enzyme, centrifuging and retaining the supernatant to obtain a yeast protein hydrolysate containing yeast peptides; the added protease is a protease that can exert a hydrolysis effect within the pH range of 7 to 8; S3, separating yeast peptides with a molecular weight greater than or equal to 1 kDa and less than 3 kDa from yeast protein hydrolysate; S4, dispersing hydroxytyrosol and the yeast peptide obtained in step S3 in a low ionic strength PBS buffer respectively, and then mixing the yeast peptide dispersion and the hydroxytyrosol dispersion, and sequentially performing ultrasonic treatment, high pressure homogenization treatment and freeze drying to obtain a yeast peptide-hydroxytyrosol complex with high ROS capture ability; In step S2, the added protease is papain; the amount of papain and yeast protein dispersion is calculated according to the following method: first measure the total protein content in the yeast protein dispersion, then calculate the casein mass in the yeast protein dispersion according to the casein content accounting for 86wt% of the total protein content, and prepare a hydrolysis reaction system according to the papain activity / casein mass of 9000-11000U / g and perform hydrolysis; In step S2, the reaction conditions during hydrolysis are: temperature of 50-55° C., pH of the hydrolysis reaction system of 7-8, and hydrolysis time of 3.5-4.5 h; when inactivating the enzyme, incubating in a boiling water bath for 10 min, the speed of centrifugation is 8000-9000 rpm, and the centrifugation time is 20-30 min; Step S4 includes the following steps: S4-A, dispersing the yeast peptide separated in step S3 in a low ionic strength PBS buffer to prepare a yeast peptide dispersion; In the yeast peptide dispersion, the mass concentration of the yeast peptide is 0.5 to 1.5 mg / mL; S4-B, dispersing hydroxytyrosol in a low ionic strength PBS buffer to prepare a hydroxytyrosol dispersion; in the hydroxytyrosol dispersion, the mass concentration of hydroxytyrosol is 0.5-1.5 mg / mL, and the mass concentration of hydroxytyrosol in the hydroxytyrosol dispersion is equal to the mass concentration of yeast peptide in the yeast peptide dispersion; S4-C, mixing equal masses of yeast peptide dispersion and hydroxytyrosol dispersion and stirring evenly to obtain a precursor solution; the stirring temperature is 20-30°C, the rotation speed is 80-120rpm, and the stirring time is 0.5-1.5h; adding a phospholipid membrane made of phosphatidylcholine to the precursor solution; the phospholipid membrane is prepared by dissolving phosphatidylcholine in chloroform and then evaporating the chloroform under reduced pressure; the amount of phospholipid membrane added is calculated based on phosphatidylcholine, and the amount / volume ratio of phosphatidylcholine to the precursor solution is 0.1-0.5mmol / L; S4-D, heating the precursor solution to the preheating temperature, and then subjecting the precursor solution to ultrasonic treatment; after the start of ultrasonic treatment, first keep the precursor solution at the preheating temperature for 5 to 8 minutes, then cool it to 25°C at a cooling rate of 8 to 10°C / min, and keep it warm until the end of ultrasonic treatment; the preheating temperature is 40 to 45°C, the ultrasonic treatment time is 11 to 20 minutes, the ultrasonic power is 300 to 400W, the ultrasonic frequency is 40kHz, and the precursor solution is continuously stirred at a speed of 100 to 120rpm during the ultrasonic process; S4-E, subjecting the precursor solution after ultrasonic treatment to high-pressure homogenization treatment; the pressure of the high-pressure homogenization treatment is 280-320 MPa, and the number of cycles is 3-5 times; S4-F, freeze-drying the precursor solution after high-pressure homogenization to obtain a yeast peptide-hydroxytyrosol complex.

2. The method for preparing a yeast peptide-hydroxytyrosol complex with high ROS capture ability according to claim 1, characterized in that: In step S1, stirring is performed at 35-42°C and 100-120 rpm for 3-5 min.

3. The method for preparing a yeast peptide-hydroxytyrosol complex with high ROS capture ability according to claim 2, characterized in that: In step S3, yeast peptides with a molecular weight greater than or equal to 1 kDa and less than 3 kDa are centrifuged from the yeast protease hydrolysate using an ultrafiltration membrane; the centrifugal temperature is 4° C., the centrifugal force is 5000 to 8600 g, and the centrifugal time is 15 to 20 min.

4. The method for preparing a yeast peptide-hydroxytyrosol complex with high ROS capture ability according to claim 3, characterized in that: In step S1, stirring was performed at 40° C. and 120 rpm for 5 min; the protein content of the yeast protein powder was 82 wt %, and the mass concentration of the yeast protein powder in the yeast protein dispersion was 70.9 g / L; In step S2, a hydrolysis reaction system is prepared according to the papain activity / casein quality of 10000U / g; the temperature during hydrolysis is 50°C, the pH of the hydrolysis reaction system is 7, and the hydrolysis time is 4h; the speed during centrifugation is 8000rpm, and the centrifugation time is 30min; In step S3, the centrifugal force during the ultrafiltration membrane centrifugal separation is 5000g, and the centrifugation time is 15min; In step S4-A, the mass concentration of yeast peptide in the yeast peptide dispersion is 1 mg / mL; In step S4-B, the mass concentration of hydroxytyrosol in the hydroxytyrosol dispersion is 1 mg / mL; In step S4-C, the stirring temperature is 25° C., the rotation speed is 100 rpm, and the stirring time is 1 h. The amount of phospholipid membrane added to the precursor solution is calculated based on phosphatidylcholine, and the amount / volume ratio of phosphatidylcholine to the precursor solution is 0.1 mmol / L. In step S4-D, the preheating temperature is 45°C, the precursor solution is kept at 45°C for 5 minutes, then cooled to 25°C at a cooling rate of 10°C / min, and kept warm until the ultrasonic treatment is completed; the ultrasonic treatment time is 15 minutes, the ultrasonic power is 400W, and the precursor solution is continuously stirred at a speed of 100rpm during the ultrasonic treatment; In step S4-E, the pressure of the high-pressure homogenization treatment is 300 MPa, and the number of cycles is 3 times.

5. The use of a yeast peptide-hydroxytyrosol complex having high ROS capture ability in any of the following applications, characterized in that: The yeast peptide-hydroxytyrosol complex with high ROS capture ability is prepared according to the preparation method of claim 1, and the application is any of the following: As food antioxidant additive; Used to manufacture foods with antioxidant function; Used in the manufacture of antioxidant drugs; Used in the manufacture of UV protection products for skin; Antioxidant stress protectant for cell culture in vitro; Used as an indicator of cellular oxidative stress processes; Used in the manufacture of plant protection agents.

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