Preparation method of soybean polypeptide with antioxidant and antagonistic lead damage effects

Abstract

The present application relates to a kind of soybean polypeptide preparation method with antioxidant and antagonistic lead damage effect.The soybean polypeptide prepared in the present application has good DPPH free radical clearance rate, ABTS free radical clearance rate and Fe3+ reduction capacity in vitro;It can also alleviate the case that the body weight of mouse is slowly increased due to lead exposure, improve the learning and memory ability of lead exposure mouse, alleviate the organ damage of mouse caused by lead exposure, enhance the level of reduced glutathione, superoxide dismutase and catalase of organism, and alleviate the abnormal increase of malondialdehyde caused by lead exposure.The soybean polypeptide prepared in the present application has good in vitro antioxidant capacity, and has antagonistic effect on oxidative stress caused by lead exposure mouse, and can be used as non-toxic and harmless natural antioxidant.For the development of natural antioxidant food, it has far-reaching application prospect, and also provides a new feasible idea for the combined application of bioinformatics technology and traditional enzymatic technology.

Description

Technical Field

[0001] This invention relates to a method for preparing soybean polypeptides with antioxidant and lead-damage-antagonistic effects, belonging to the field of biotechnology. Background Technology

[0002] Oxidation is a crucial metabolic process in humans. Exposure to exogenous pollutants often leads to the formation of free radicals, further causing oxidative stress. Oxidative stress is associated with various human diseases such as cancer, diabetes, Alzheimer's disease, and Parkinson's disease. Exogenous antioxidants can scavenge free radicals and inhibit peroxidation, thereby reducing oxidative stress in the body.

[0003] Lead is a heavy metal toxicant widely present in the environment. Both acute and chronic lead exposure pose potential hazards to the physiological, biochemical, and behavioral functions of humans and animals. Lead exposure can reduce the activity of antioxidant enzymes such as superoxide dismutase and catalase, as well as the content of antioxidant substances such as glutathione in body tissues, causing oxidative damage. Therefore, developing novel natural antioxidants is particularly important to mitigate the damage caused by lead exposure.

[0004] Soybean peptides are hydrolyzed products of soybean protein, typically composed of 3-6 amino acids, along with small amounts of free amino acids, sugars, water, and inorganic salts. Soybean peptides obtained through soybean protein hydrolysis have small molecular weights and are rich in various bioactive peptides, offering numerous potential health benefits. These include good DPPH free radical scavenging activity and in vitro reducing capacity, lowering blood pressure, regulating immunity, and combating fatigue. Therefore, developing and using soybean peptides with high safety and antioxidant activity is currently a hot research topic.

[0005] However, the discovery and development of bioactive peptides is time-consuming, labor-intensive, and dependent on various factors. The application of computer simulations can quickly and effectively predict the bioactivity of peptides, enabling precise definition of the functions associated with bioactive peptides. Based on the vast amounts of data in databases, bioinformatics technology accelerates the process of predicting the bioactivity of peptides, simplifies the screening process, improves the efficiency of peptide preparation, and reduces production costs. For example, the BIOPEP database not only links protein sequences and bioactive peptides but also has a built-in program that can predict protein hydrolysates. The UniProtKB and NCBI databases contain various protein sequences that can be used to analyze the amino acid profiles of precursor proteins. BIOPEP can be used to predict peptide-related activities, and PeptideCutter can obtain theoretically cleaved peptides by selecting specific enzymes. PeptideRanker assesses the probability of activity of a peptide using a score from 0 to 1. Therefore, employing computer simulation technology can overcome the limitations of traditional methods and improve the antioxidant activity of soybean peptides.

[0006] This invention investigates the preparation of soybean peptides by screening proteases through computer simulation and combining different enzymatic hydrolysis strategies. The obtained soybean peptides have antioxidant and anti-lead-induced oxidative stress effects. Summary of the Invention

[0007] In order to obtain a non-toxic and harmless natural antioxidant, this invention provides a method for preparing soybean polypeptides with antioxidant and lead-damage antagonistic effects.

[0008] The technical solution adopted in this invention is:

[0009] A method for preparing soybean polypeptides, comprising the following steps:

[0010] (1) Preparation of soybean protein hydrolysate: Weigh 5-20g of soybean protein and dissolve it in 100-300ml of HCl-Tris buffer. After boiling water treatment, adjust the pH and add protease stepwise for hydrolysis. After hydrolysis, inactivate the enzyme, centrifuge, and collect the supernatant for later use. The specific steps of enzymatic hydrolysis are as follows: First, use pepsin for 1-3 hours; second, use chymotrypsin and bromelain simultaneously for 2-6 hours.

[0011] (2) Ultrafiltration separation: The enzymatic hydrolysis product is separated by ultrafiltration membrane and divided into three components with different molecular weight ranges. The component with a molecular weight of less than 3 kDa is freeze-dried to obtain soybean antioxidant peptides.

[0012] Further, (1) Preparation of soybean protein hydrolysate: Weigh 10g of soybean protein and dissolve it in 200ml of HCl-Tris buffer. After boiling water treatment, adjust the pH and add the protease step by step for hydrolysis. After hydrolysis, inactivate the enzyme, centrifuge, and take the supernatant for later use. The specific steps of enzymatic hydrolysis are: first step: use pepsin for 2h hydrolysis, second step: use chymotrypsin and bromelain for 4h hydrolysis.

[0013] (2) Ultrafiltration separation: The enzymatic hydrolysis products were separated by ultrafiltration using 30KDa and 3KDa ultrafiltration membranes to separate them into three components with different molecular weight ranges. The components with molecular weight less than 3KDa were freeze-dried to obtain soybean antioxidant peptides.

[0014] Furthermore, computer simulations of enzymatic hydrolysis were used to determine the types of proteases, soybean protein sequences were identified through databases, and the antioxidant activity of polypeptide sequences after enzymatic hydrolysis was simulated and predicted, thereby determining the proteases required for subsequent processing.

[0015] The specific steps of computer-simulated enzymatic hydrolysis are as follows: soybean protein sequence P0DO16 is obtained by screening through UniProtKB and NCBI databases; enzymatic hydrolysis is simulated based on the Biopep database; and pepsin, chymotrypsin and bromelain are obtained by predicting the antioxidant activity of the polypeptide sequence after enzymatic hydrolysis.

[0016] The enzymatic hydrolysis conditions are as follows: pepsin, pH 1-2.5, temperature 20-50℃, enzyme-substrate ratio 1000-3000 U / g, hydrolysis time 1-3 h; chymotrypsin and bromelain, pH 6-8, temperature 40-60℃, enzyme-substrate ratio of 1000-3000 U / g for both enzymes, hydrolysis time 2-5 h.

[0017] The enzymatic hydrolysis conditions were as follows: pepsin, pH 1.8, temperature 37℃, enzyme-substrate ratio 1000 U / g, hydrolysis time 2 h; chymotrypsin and bromelain, pH 7.5, temperature 50℃, enzyme-substrate ratio 2000 U / g for both enzymes, hydrolysis time 4 h.

[0018] This invention also relates to soybean peptides prepared by the above-mentioned method, and the application of these soybean peptides in alleviating lead exposure-induced slow weight gain in mice, alleviating lead exposure-induced organ damage in mice, and improving the learning and memory abilities of lead-exposed mice. Furthermore, the invention relates to the application of these soybean peptides in enhancing the levels of reduced glutathione, superoxide dismutase, and catalase in the body, alleviating the abnormal increase in malondialdehyde caused by lead exposure, and antagonizing lead-induced oxidative stress.

[0019] Animal experiments were conducted to verify the effects of lead exposure on mice. A Pb exposure model was established by using Kunming mice via drinking water. Soybean antioxidant peptides were administered to protect the mice and to investigate their antagonistic effect and antioxidant capacity in vivo.

[0020] This invention aims to find a natural and highly effective antioxidant peptide agent. Starting with soybean protein, it uses computer simulation to determine the use of protease through enzymatic hydrolysis. By specifically cleaving the enzyme, peptides with more antioxidant activity fragments are obtained, thereby preparing soybean peptides with high antioxidant activity and antagonistic effect against lead-induced oxidative stress.

[0021] The advantages and positive effects of this invention are as follows:

[0022] 1. This invention is based on computer-simulated enzymatic hydrolysis to prepare natural and highly efficient antioxidant peptides. The combined use of bioinformatics and bioenzymology technologies changes the existing extraction methods of antioxidant peptides, improves the bioactivity of peptides, and greatly shortens the research time and cost, making it suitable for widespread use.

[0023] 2. The combination of specific proteases in this invention has achieved unexpected technical effects in the extraction of soybean polypeptides. In particular, polypeptides prepared by the simultaneous use of chymotrypsin and bromelain have better antioxidant activity than those prepared by using one enzyme alone or by sequential enzymatic hydrolysis of the two enzymes. This has a synergistic effect and is the applicant's first discovery.

[0024] 3. The soybean antioxidant peptides prepared in this invention possess both highly efficient in vitro antioxidant capacity and good in vivo antioxidant capacity. They can enhance the levels of reduced glutathione, peroxidase, and total superoxide dismutase in the brain tissue of lead-exposed mice, reduce malondialdehyde levels, and enhance the body's antioxidant capacity. The soybean peptides prepared in this invention have high antioxidant activity and can be used in the preparation of drugs to antagonize lead damage, which is of great significance for maintaining health and preventing disease. Attached Figure Description

[0025] Figure 1 This invention illustrates the effects of different proteases on the simulated enzymatic hydrolysis results and antioxidant activity of soybean protein sequences.

[0026] Figure 2 The effects of different enzymatic hydrolysis methods on the degree of hydrolysis, DPPH free radical scavenging rate, ABTS free radical scavenging rate, and Fe2+ in this invention. 3+ The effect diagram of reducing power;

[0027] Figure 3 The effects of different components after ultrafiltration on DPPH radical scavenging rate, ABTS radical scavenging rate and Fe2+ radical scavenging rate in this invention are described. 3+ The effect diagram of reducing power;

[0028] Figure 4 This is a graph showing the effect of soybean antioxidant peptides on lead-induced changes in mouse body weight in this invention.

[0029] Figure 5 The effect of soybean antioxidant peptides on lead-induced organ indices in mice in this invention;

[0030] Figure 6 This is a graph showing the effect of soybean antioxidant peptides on the average escape latency, number of platform crossings, and target time in lead-induced mice.

[0031] Figure 7 The figure shows the effect of soybean antioxidant peptides in this invention on the levels of reduced glutathione, catalase activity, superoxide dismutase activity and malondialdehyde content in lead-induced mouse brain tissue. Detailed Implementation

[0032] The preparation method is as follows:

[0033] Computer-simulated enzymatic hydrolysis: The soybean protein sequence P0DO16 was obtained by screening the UniProtKB and NCBI databases. The Biopep database was used to simulate enzymatic hydrolysis of this protein sequence, yielding the hydrolyzed soybean polypeptide sequences. Based on the Biopep database, the antioxidant activity of the hydrolyzed sequences was predicted, and the protease to be used for subsequent hydrolysis was determined according to the prediction results.

[0034] Preparation of soybean protein hydrolysate: Weigh 10g of soybean protein and dissolve it in 200ml of HCl-Tris buffer. Boil in water for 5min, cool to a certain temperature, and then bring the volume to the original volume. Adjust the pH to the optimal value for the enzyme using 1M HCl or 1M NaOH. Place in a constant temperature water bath and adjust to the optimal temperature for the enzyme. Add protease to initiate the hydrolysis reaction. After hydrolysis, inactivate the enzyme at 100℃ for 10min. After cooling, centrifuge at 4℃ and 5000r / min for 15min, and collect the supernatant for later use.

[0035] Determination of degree of hydrolysis: Take 10 mL of sample solution, add 30 mL of distilled water (CO2-free), adjust the pH to 8.20, add 10 mL of formaldehyde neutralized to pH 8.20, and then titrate the pH to 9.20 with 0.05 mol / L standard NaOH. Record the volume V1. Use distilled water instead of the sample and follow the same procedure to determine the blank volume V0. The formula for calculating the degree of hydrolysis (DH) is as follows:

[0036]

[0037] C: NaOH concentration, mol / L;

[0038] M: Sample mass, g;

[0039] W: Protein mass fraction in the sample;

[0040] U: Free amino nitrogen content per gram of soy protein, which is 0.33 mmol / g for soy protein;

[0041] V: Total volume of hydrolysate, mL;

[0042] K: The number of peptide chain equivalents per gram of protein; for soy protein, it is 7.8 mmol / g.

[0043] Determination of DPPH free radical scavenging capacity: Take 1 mL of sample solution in a test tube, add 1 mL of 0.15 mmol / L DPPH·ethanol solution, mix well, and let stand in the dark for 30 min. Measure its absorbance (Ax) at 517 nm. Alternatively, add DPPH solution with ethanol instead of the sample and measure its absorbance (A0). Mix the sample with an equal volume of anhydrous ethanol (without adding other reagents) and measure its absorbance (Ax0). Perform the test three times for each sample and take the average of the three tests. The calculation formula is as follows:

[0044]

[0045] Fe 3+Determination of reducing power: Take 1 mL of sample solution into a test tube, add 2.5 mL of phosphate buffer (pH 6.6), shake well, then add 2.5 mL of 1% potassium ferricyanide solution, mix thoroughly, and react at 50℃ for 20 min. Next, add 2.5 mL of 10% trichloroacetic acid solution, centrifuge at 3000 r / min for 10 min, take 5 mL of the supernatant, add 5 mL of distilled water and 1 mL of 0.1% ferric chloride solution, react for 10 min, and measure the absorbance (A) at 700 nm. Use distilled water as a reference solution to measure its absorbance (A0). Perform the test three times for each sample, and take the average of the three tests. The calculation formula is as follows:

[0046] Fe 3+ Reducing power (%) = (A - A0) × 100% Determination of ABTS free radical scavenging ability: Mix 7 mmol / L ABTS with 2.45 mmol / L potassium persulfate at a volume ratio of 1:1 and react in the dark for 12-16 h to prepare an ABTS stock solution. Dilute the ABTS solution with 0.2 M phosphate buffer (pH 7.4) to an absorbance level of 0.70 ± 0.02 at 734 nm to prepare ABTS working solution (prepare fresh before use). Mix 10 ml of sample with 990 ml of diluted ABTS solution, react for 5 min, and measure the absorbance (A) at 734 nm. Measure the blank (A0) using distilled water instead of the sample.

[0047]

[0048] Ultrafiltration separation: The enzymatic hydrolysis products were separated by ultrafiltration using 30 kDa and 3 kDa ultrafiltration membranes, resulting in three fractions with different molecular weight ranges: fractions with molecular weights greater than 30 kDa, fractions with molecular weights between 30 kDa and 3 kDa, and fractions with molecular weights less than 3 kDa. The antioxidant capacity of each fraction was measured, and the resulting soybean antioxidant peptides were obtained by freeze-drying.

[0049] The present invention will be further illustrated below with specific examples of different enzymatic hydrolysis schemes; the following examples are illustrative and not limiting, and should not be used to limit the scope of protection of the present invention.

[0050] Example 1

[0051] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, chymotrypsin (50℃, pH 7.5, 2000 U / g) was added for the second step of hydrolysis for 4 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 4℃ and 5000 rpm for 15 minutes, and the supernatant was collected for later use.

[0052] Example 2

[0053] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, bromelain (50℃, pH 7.5, 2000 U / g) was added for the second step of hydrolysis, and the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected for later use.

[0054] Example 3

[0055] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, chymotrypsin (50℃, pH 7.5, 2000 U / g) and bromelain (50℃, pH 7.5, 2000 U / g) were added simultaneously for the second step of hydrolysis, and the enzyme was inactivated at 100℃ for 10 minutes after hydrolysis. After cooling, the sample was centrifuged at 4℃ and 5000 rpm for 15 minutes, and the supernatant was collected for later use.

[0056] Example 4

[0057] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, bromelain (50℃, pH 7.5, 2000 U / g) was added for the second step of hydrolysis, and the enzyme was inactivated at 100℃ for 10 minutes. After cooling, chymotrypsin (50℃, pH 7.5, 2000 U / g) was added for the third step of hydrolysis, and the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected for later use.

[0058] Example 5

[0059] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, chymotrypsin (50℃, pH 7.5, 2000 U / g) was added for the second step by hydrolysis for 4 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, bromelain (50℃, pH 7.5, 2000 U / g) was added for the third step by hydrolysis for 4 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected for later use.

[0060] Example 6

[0061] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, chymotrypsin (50℃, pH 7.5, 4000 U / g) was added for the second step of hydrolysis for 4 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected for later use.

[0062] Example 7

[0063] The soybean protein sample was hydrolyzed for the first step by adding pepsin (37℃, pH 1.8, 1000 U / g) for 2 hours. After hydrolysis, the enzyme was inactivated at 100℃ for 10 minutes. After cooling, bromelain (50℃, pH 7.5, 4000 U / g) was added for the second step of hydrolysis, and the enzyme was inactivated at 100℃ for 10 minutes. After cooling, the sample was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected for later use.

[0064] Effect verification experiment

[0065] 1. Grouping of experimental animals and administration of test substances

[0066] Male Kunming mice, 20±2g, SPF grade, were purchased from Beijing Spaford Laboratory Animal Co., Ltd. After acclimatization, they were randomly divided into 4 groups of 10 mice each, for a total of 40 mice. The control group mice received 0.01mol / L NaAc aqueous solution (equivalent to 2g / L pb). 2+ As a control group); except for the blank control group, the mice in the other groups drank (CH3COO)2Pb aqueous solution (2g / L pb). 2+ A lead-exposed mouse model was established. Mice in the polypeptide protection group were given 400 mg / (kg·bw) of soybean antioxidant polypeptide daily; mice in the vitamin C control group were given 100 mg / (kg·bw) of vitamin C daily; mice in the blank control group and the lead model group were given the same volume of physiological saline by gavage daily, and the surgical procedures were kept consistent. After 30 days of continuous administration, the mice were fasted for 24 hours before dissection, and the brain, liver, and kidneys were harvested. Serum was separated at 1500 rpm for 10 minutes.

[0067] 1. Changes in mouse body weight

[0068] The mice were weighed and their weight was recorded every six days during the experiment.

[0069] 2. Morris water maze experiment

[0070] In the final week of the experiment, each group of mice underwent the Morris water maze test, which included a navigation and spatial exploration test. The escape latency, swimming trajectory, number of times the mouse crossed the platform within 60 seconds, and time spent in the target quadrant were recorded.

[0071] 3. Determination of organ indices in mice

[0072] After separating the brain, liver, and kidneys, the surface blood was rinsed off with saline solution, and the surface moisture was absorbed with filter paper. The weight of the brain, liver, and kidneys was then measured and calculated using the formula: Organ Index (%) = Organ weight / 10 × Body weight.

[0073] 4. Determination of indicators of mouse oxidative defense system

[0074] The levels of reduced glutathione, catalase activity, superoxide dismutase activity, and malondialdehyde content in mouse brain tissue were detected using a kit from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0075] The relevant test results regarding the antioxidant and lead-induced oxidative stress antagonistic effects of the soybean polypeptides prepared in this invention are as follows:

[0076] 1. The soybean protein sequence (P0DO16) was simulated using the Biopep database. The antioxidant activity of the resulting polypeptide sequences was predicted, and the three most active proteases were selected for subsequent experiments. Results are as follows: Figure 1 As shown, the predicted antioxidant activities of the peptides after hydrolysis by pepsin, chymotrypsin, and bromelain were 0.0083, 0.0066, and 0.005, respectively, and the theoretical degrees of hydrolysis were 44.5364%, 46.5232%, and 70.1987%, respectively. This indicates that the soybean peptides obtained by hydrolysis by pepsin, chymotrypsin, and bromelain have higher antioxidant activities.

[0077] 2. The degree of hydrolysis, DPPH radical scavenging rate, ABTS radical scavenging rate, and Fe were measured in Examples 1-5. 3+ Reducing capacity is used to determine the optimal enzymatic hydrolysis method among different enzymatic hydrolysis strategies. For example... Figure 2 As shown, the DPPH radical scavenging rate and ABTS radical scavenging rate of Examples 3-5 were high and showed no significant difference; the Fe radical scavenging rate of Example 3 was... 3+ The reducing power was 30.57%, the highest compared to other examples; indicating that the soybean peptide prepared in Example 3 has higher antioxidant activity.

[0078] 3. The enzymatic hydrolysis products of Example 3 were separated by ultrafiltration using 30 kDa and 3 kDa ultrafiltration membranes to obtain components with molecular weights greater than 30 kDa, components with molecular weights between 30 kDa and 3 kDa, and components with molecular weights less than 3 kDa. These three components, along with the unseparated component, were freeze-dried to prepare a 5 mg / mL sample solution. The DPPH radical scavenging rate, ABTS radical scavenging rate, and Fe were then measured. 3+ Restorative ability. Results as follows: Figure 3 As shown, the DPPH radical scavenging rate for components with a free radical scavenging capacity of less than 3 kDa was 90.70%, the ABTS radical scavenging rate was 85.15%, and the Fe... 3+ The reducing power of 32.20% is higher than that of other components. This demonstrates that the soybean peptides prepared in this invention have high antioxidant activity.

[0079] 4. The soybean antioxidant peptides obtained above were administered to lead-exposed mice to verify their efficacy in antagonizing lead-induced oxidative stress in vivo. To investigate the effect of the soybean antioxidant peptides on body weight changes in lead-exposed mice, body weight changes were recorded, such as... Figure 4 As shown in the figure, the body weight of mice in the lead model group was significantly lower than that of mice in the control group, indicating a slower growth rate. Compared with the model group, the protection of soybean antioxidant peptides effectively alleviated the decrease in body weight in mice, and there was no significant difference compared with the control group. This indicates that soybean antioxidant peptides can effectively protect mice from lead-induced weight loss.

[0080] 5. To investigate the effects of the soybean antioxidant peptides prepared in this invention on the organs of lead-exposed mice, we measured the organ coefficients of the mouse brain, liver, and kidneys. For example... Figure 5 As shown, compared with the control group, the brain and kidney indices of mice in the lead-exposed group decreased, while the liver indices increased, demonstrating that lead exposure causes certain damage to organs. Among these, the organ indices of the soybean polypeptide protection group and the vitamin C control group showed no significant difference from the control group, proving that the soybean antioxidant polypeptide prepared in this invention has a protective effect against organ damage caused by lead exposure in mice.

[0081] 6. Lead exposure can cause neurotoxicity, therefore we used the Morris water maze test to assess the cognitive level of mice. For example... Figure 6As shown, in the 5-day positioning and navigation experiment, with the increase of training days, the escape latency and the distance traveled to find the platform decreased for all mice, indicating that the mice formed spatial memory after acquired training. Lead exposure significantly increased the escape latency of mice, while the escape latency after protection with soybean antioxidant peptides and vitamin C showed no significant difference compared to the control group. In the spatial exploration experiment (60 s), lead exposure resulted in mice crossing platforms significantly fewer times and taking significantly less time to target than the control group, demonstrating the effect of lead on the spatial memory ability of mice. Compared with the lead-exposed group, after protection with soybean antioxidant peptides and vitamin C, the number of times mice crossed platforms increased by 164.29% and 171.43%, respectively, and the target time increased by 72.60% and 71.89%, respectively, with no significant difference compared to the control group. This proves that the soybean antioxidant peptides and vitamin C prepared in this invention effectively protect mice from lead exposure-induced damage to learning and memory function.

[0082] 7. Glutathione has antioxidant and detoxifying effects, participates in the body's redox processes, can scavenge excess free radicals, and reduce the degree of lipid peroxidation. For example... Figure 7 As shown, compared with the control group, the GSH content, SOD activity, and CAT activity in the brain tissue of mice exposed to lead decreased, while the MDA content increased. After administration of soybean antioxidant peptides, the GSH content in this group of mice increased by 79.76%, SOD activity increased by 31.72%, CAT activity increased by 81.19%, and MDA content decreased by 44.05% compared with the lead-exposed group, with no significant difference compared with the control group. This indicates that the soybean antioxidant peptides prepared in this invention can inhibit lead-induced oxidative stress in mice and have good in vivo antioxidant capacity.

[0083] In summary, the soybean antioxidant peptides prepared by this invention have both good in vitro antioxidant capacity and can effectively reduce lead-induced oxidative stress in mice, demonstrating good in vivo antioxidant capacity.

Claims

1. A method for preparing soybean polypeptides, characterized in that, The preparation steps are as follows: (1) Preparation of soybean protein hydrolysate: Weigh 5-20g of soybean protein and dissolve it in 100-300ml of HCl-Tris buffer. After boiling water treatment, adjust the pH and add protease step by step for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme, centrifuge, and take the supernatant for later use. The specific steps of enzymatic hydrolysis are as follows: First step: use pepsin for 2h, pH 1.8, temperature 37℃, enzyme-substrate ratio is 1000U / g. Second step: use chymotrypsin and bromelain for 4h simultaneously, pH 7.5, temperature 50℃, the ratio of both enzymes to substrate is 2000U / g. (2) Ultrafiltration separation: The enzymatic hydrolysis products were separated by ultrafiltration using 30 kDa and 3 kDa ultrafiltration membranes to separate them into three components with different molecular weight ranges. The components with molecular weight less than 3 kDa were freeze-dried to obtain soybean peptides.

2. Soybean polypeptide prepared by the method for preparing soybean polypeptide according to claim 1.

3. The use of the soybean polypeptide according to claim 2 in the preparation of pharmaceuticals that antagonize lead damage or enhance antioxidant capacity.

4. The use of the soybean polypeptide according to claim 2 in the preparation of a drug antagonizing lead-induced oxidative stress.

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