Artemia antioxidant peptide fragments, antioxidant peptides, preparation methods and applications

Abstract

The application provides an Artemia antioxidant peptide peptide segment, an antioxidant peptide, a preparation method and application, belongs to the technical field of biology, and can solve the problem that the deep development of Artemia proteins and the preparation research of antioxidant active ingredients are still in the blank stage at present. The application provides an Artemia antioxidant peptide peptide segment, and the amino acid sequence of the Artemia antioxidant peptide peptide segment is one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. The Artemia antioxidant peptide peptide segment is applied to the preparation of antioxidant related drugs, the Artemia antioxidant peptide includes polypeptides with different molecular weights, can be applied to different scenes such as the fields of food, medicine and cosmetics, and widens the utilization way of Artemia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to an anti-oxidative peptide fragment from Artemia salina, the anti-oxidative peptide, its preparation method, and its application. Background Technology

[0002] Peptides are polymers formed by two or more amino acids covalently linked by peptide bonds, falling between amino acids and proteins. Peptide compounds with specific physiological functions are collectively called bioactive peptides. Studies have shown that bioactive peptides possess functions such as antioxidation and immunomodulation. Among them, peptides with significant antioxidant properties are called antioxidant peptides, a class of natural bioactive peptides that have been extensively studied in recent years. Antioxidant peptides are short-chain compounds formed by amino acids linked by peptide bonds. As natural antioxidants, antioxidant peptides have relatively simple structures, are easily absorbed, have good stability, and are non-immunoreactive. They not only possess strong antioxidant activity but also have other specific effects such as lowering blood pressure and anti-cancer properties, attracting increasing attention in the food and healthcare product industries. Antioxidant peptides are mostly derived from the enzymatic hydrolysis products of natural proteins, with common sources including animal proteins, plant proteins, and fermentation products.

[0003] Artemia, an arthropod belonging to the genus Artemia in the family Artemiaceae, order Anapiformes, is also known as brine shrimp. It inhabits extreme environments such as high-salinity salt pans and brackish lakes, exhibiting remarkable salt tolerance. Artemia contains abundant protein, amino acids, unsaturated fatty acids, and inorganic elements. However, due to their tiny size, the actual collection, processing, and handling of artemia present numerous technical challenges and cost issues, significantly limiting the comprehensive development and effective utilization of artemia resources. Furthermore, current research on artemia largely focuses on the development and utilization of its dormant eggs or its use as aquaculture feed. Research on the in-depth development of its proteins and the preparation of antioxidant active ingredients remains incomplete, and the preparation and application of antioxidant peptides from artemia are not reported. Summary of the Invention

[0004] This invention addresses the current lack of research on the in-depth development of Artemia proteins and the preparation of antioxidant active ingredients, particularly the unreported preparation and application of antioxidant peptides from Artemia. It proposes an Artemia antioxidant peptide fragment, preparation method, and application. By employing ceramic membrane fractionation and ultrafiltration purification techniques, Artemia antioxidant peptides are prepared and separated, yielding multiple polypeptides with different molecular weight ranges. These peptides can be applied in various scenarios, including food, pharmaceuticals, and cosmetics, broadening the utilization pathways of biological resources from extreme environments. Furthermore, mass spectrometry identification and screening yield structurally clear Artemia antioxidant peptide fragments, which can be used in the preparation of antioxidant-related drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: an Artemia antioxidant peptide segment, wherein the amino acid sequence of the Artemia antioxidant peptide segment is one of the sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0006] In another aspect, the present invention provides an artesian antioxidant peptide, comprising the aforementioned artesian antioxidant peptide fragment.

[0007] Another aspect of the present invention provides a method for preparing the aforementioned Artemia antioxidant peptides, comprising: homogenizing an Artemia sample, enzymatically hydrolyzing it, and then sequentially separating it by ultrafiltration and gel chromatography to obtain the Artemia antioxidant peptides. In this technical solution, the homogenization method is grinding or homogenizing using a homogenizer.

[0008] In one embodiment, the enzymatic hydrolysis step includes: adding flavor protease at a material-to-liquid ratio of 0.5-2:1 and an enzyme dosage of 600 U / g to obtain an artesian hydrolysate, wherein the hydrolysis pH is 4-7, the hydrolysis temperature is 30-60℃, and the hydrolysis time is 1-5 h.

[0009] In one embodiment, the material-to-liquid ratio is 1:1, the enzymatic hydrolysis pH is 6, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis time is 2 hours, the degree of hydrolysis of the artichoke hydrolysate is 13.80%, and the DPPH free radical scavenging rate is 94.02%.

[0010] In one embodiment, the ultrafiltration separation step includes: clarifying the enzymatic hydrolysate of Artemia salina obtained by enzymatic hydrolysis through a ceramic membrane, and then performing ultrafiltration using ultrafiltration tubes with molecular weights of 1 kDa, 3 kDa, and 5 kDa, respectively, to obtain a first component with a molecular weight range of <1 kDa, a second component with a molecular weight range of 1 kDa to 3 kDa, and a third component with a molecular weight range of 3 kDa to 5 kDa. The optimal component is screened using DPPH free radical scavenging rate, ABTS cation free radical scavenging rate, and total reducing power as indicators. In this technical solution, the ceramic membrane pore size is preferably 50 nm. Through pore size classification, the ceramic membrane can separate the artesian enzymatic hydrolysate according to particle size, efficiently separating large molecular impurities such as un-enzymatically hydrolyzed proteins and polysaccharides from target peptides with a molecular weight <5 kDa, reducing the loss of target peptides in subsequent processing. The ceramic membrane can recover more than 95% of soluble components. Ultrafiltration further concentrates the target peptides while removing small molecular impurities such as salts and free amino acids, significantly increasing peptide concentration. The continuous operation mode of the ceramic membrane and ultrafiltration tube in this invention reduces intermediate transfer steps, minimizing the loss caused by target peptide adsorption on the container or filter material surface. This effectively reduces the risk of cross-reactions or microbial contamination caused by impurities and shortens the production cycle. The ultrafiltration separation step of this invention operates at low pressure and at room temperature, avoiding peptide bond breakage problems caused by high temperatures or strong acid / alkali conditions. Furthermore, the ultrafiltration separation process is carried out in a closed system, reducing contact with air and lowering the oxidation risk of sensitive amino acids such as tryptophan and tyrosine in the peptides.

[0011] In one embodiment, the gel chromatography separation step includes: performing gel chromatography separation on the polypeptide solution obtained by ultrafiltration using Sephadex G-25, measuring the absorbance at a wavelength of 280 nm, collecting the filtrate at room temperature using an automatic collector, and then lyophilizing the mixture according to the chromatographic peaks to obtain purified artichoke antioxidant peptides. Specifically, the polypeptide solution is prepared with ultrapure water to a concentration of 10 mg / mL, then eluted with deionized water at a flow rate of 0.7 mL / min, collecting the filtrate every 5 min, measuring the absorbance at a wavelength of 280 nm, collecting the filtrate at room temperature using an automatic collector, and then lyophilizing the mixture according to the chromatographic peaks. The artichoke antioxidant peptides are then screened based on DPPH free radical scavenging rate, ABTS cation free radical scavenging rate, and total reducing power to obtain the peak component with the strongest antioxidant activity.

[0012] In one embodiment, a mass spectrometry identification step is also included, comprising: analyzing and identifying the Artemia antioxidant peptide using nano-HPLC-MS / MS. The mass spectrometry identification step specifically includes setting chromatographic conditions and setting mass spectrometry conditions. The chromatographic conditions setting step includes: separation using a nano-HPLC system; liquid phase A being 0.1% formic acid-water solution and liquid phase B being 0.1% formic acid-acetonitrile solution; using a 75μm×2cm PepMap100 C18 column, equilibrated with 100% liquid phase A; elution flow rate of 750 nL / min; elution 0–20 min, 5% B; 20–22 min, 38% B; 22–30 min, 95% B. The mass spectrometry conditions setting step includes: primary mass spectrometry scan resolution of 60,000, scan range of 350–1500 m / z, maximum injection time of 118 ms; DDA cycle time of 2 s; maximum ion injection time of secondary mass spectrometry of 22 ms; collision chamber energy set to 30%, applicable to all precursor ions; and dynamic exclusion set to 35 s.

[0013] In another aspect, the present invention also provides the application of the said Artemia antioxidant peptide fragment, wherein the Artemia antioxidant peptide fragment is used in the preparation of antioxidant-related drugs.

[0014] In another aspect, the present invention also provides the application of the said Artemia antioxidant peptide, which is used in the fields of food, medicine and cosmetics.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: the antioxidant peptides of the present invention have clear peptide structures and high purity, and can be applied to the preparation of antioxidant-related drugs; the antioxidant peptides of the present invention include multiple polypeptides with different molecular weight segments, which can be applied to different scenarios such as food, medicine and cosmetics, thus broadening the utilization pathways of biological resources in extreme environments; the antioxidant peptides of the present invention use readily available and low-cost artichokes as raw materials, and are prepared and separated by ceramic membrane fractionation and ultrafiltration membrane purification technology, which significantly improves the yield and bioactivity stability of the antioxidant peptides of artichokes. Attached Figure Description

[0016] Figure 1 The effects of different proteases on the degree of hydrolysis and DPPH free radical scavenging rate provided in the embodiments of the present invention are shown. Different uppercase letters indicate that there are significant differences in the degree of hydrolysis among different treatment groups (P<0.05), and different lowercase letters indicate that there are significant differences in the DPPH free radical scavenging rate among different treatment groups (P<0.05).

[0017] Figure 2The figures represent the DPPH radical scavenging rate and degree of hydrolysis of the Artemia hydrolysate under different feed-to-liquid ratios provided in this embodiment of the invention. Different uppercase letters indicate significant differences in the degree of hydrolysis between different treatment groups (P<0.05), and different lowercase letters indicate significant differences in the DPPH radical scavenging rate between different treatment groups (P<0.05).

[0018] Figure 3 The figures represent the DPPH radical scavenging rate and degree of hydrolysis of the Artemia hydrolysate under different pH conditions provided in this embodiment of the invention. Different uppercase letters indicate significant differences in the degree of hydrolysis between different treatment groups (P<0.05), and different lowercase letters indicate significant differences in the DPPH radical scavenging rate between different treatment groups (P<0.05).

[0019] Figure 4 The figures represent the DPPH radical scavenging rate and degree of hydrolysis of the Artemia hydrolysate at different enzymatic hydrolysis temperatures provided in this embodiment of the invention. Different uppercase letters indicate significant differences in the degree of hydrolysis between different treatment groups (P<0.05), and different lowercase letters indicate significant differences in the DPPH radical scavenging rate between different treatment groups (P<0.05).

[0020] Figure 5 The figures represent the DPPH radical scavenging rate and degree of hydrolysis of the Artemia hydrolysate at different enzymatic hydrolysis times provided in this embodiment of the invention. Different uppercase letters indicate significant differences in the degree of hydrolysis between different treatment groups (P<0.05), and different lowercase letters indicate significant differences in the DPPH radical scavenging rate between different treatment groups (P<0.05).

[0021] Figure 6 The effect of different molecular weight Artemia peptides on DPPH free radical scavenging rate provided in the embodiments of the present invention is shown. Different lowercase letters indicate that there are significant differences between different components at the same concentration (P<0.05).

[0022] Figure 7 The different molecular weight Artemia peptides for ABTS provided in the embodiments of the present invention + The effect of free radical scavenging rate: different lowercase letters indicate significant differences among different components at the same concentration (P<0.05).

[0023] Figure 8 The effect of different molecular weight Artemia polypeptides on reducing power provided in the embodiments of the present invention is shown. Different lowercase letters indicate that there are significant differences in different components at the same concentration (P<0.05).

[0024] Figure 9 The Sephadex G-15 gel column chromatography elution curve of the artesian peptide provided in the embodiments of the present invention;

[0025] Figure 10 This is a visualization of the docking of the peptide segment shown in SEQ ID NO.1 of this invention with SOD;

[0026] Figure 11 This is a visualization of the docking of the peptide segment shown in SEQ ID NO.2 of this invention with SOD;

[0027] Figure 12 This is a visualization of the docking of the peptide segment shown in SEQ ID NO.3 of this invention with SOD;

[0028] Figure 13 This is a visualization of the docking of the peptide segment shown in SEQ ID NO.4 of this invention with SOD;

[0029] Figure 14 This is a visualization of the docking between the peptide segment shown in SEQ ID NO.5 of this invention and SOD. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention provides an anti-oxidative peptide fragment from Artemia salina, an antioxidant peptide preparation method, and its applications. The anti-oxidative peptide from Artemia salina is prepared under mild reaction conditions by optimizing enzymatic hydrolysis process conditions and combining ceramic membrane fractionation and ultrafiltration purification technology. This preparation method has advantages such as being natural and green, easy to operate, and having mild reaction conditions. It effectively avoids the shortcomings of traditional chemical hydrolysis or high-temperature and high-pressure extraction methods, such as large loss of active ingredients, high energy consumption, and easy introduction of chemical pollution. It significantly improves the yield and bioactivity stability of Artemia salina antioxidant peptides, providing a sustainable technical solution for the high-value utilization of biological resources in extreme environments. The Artemia salina antioxidant peptides can be applied to the preparation of high-end health products or functional foods, serving as a core active ingredient in antioxidant capsules and oral liquids; applied to the preparation of sports nutrition foods; and also applied to the preparation of high-end daily chemical products, added to anti-aging skincare products such as serums and eye creams, and post-medical aesthetic repair products. Through high activity, it scavenge skin free radicals and reduces skin damage caused by ultraviolet radiation and environmental stress. The artichoke antioxidant peptides obtained through mass spectrometry identification and screening have clear and traceable peptide structures, can be directly synthesized, and have high purity. They can be used in the preparation of antioxidant-related drugs and can also be used as standards for cell experiments, animal experiments, and other studies on the mechanism of action of antioxidant peptides. This clarifies the antioxidant pathway and target binding mode of a single peptide segment, providing theoretical support for subsequent product optimization.

[0032] Example 1

[0033] The preparation method of the artesian antioxidant peptide of the present invention includes:

[0034] S1. The brine shrimp sample was homogenized using a homogenizer. Distilled water was added to adjust the material-to-liquid ratio to 1:1. The amount of flavor protease added was 600 U / g, and the pH was adjusted to 6.0. Enzymatic hydrolysis was carried out at 50℃ for 2 hours, followed by enzyme inactivation in a boiling water bath for 5 minutes. After cooling, the sample was centrifuged at 8000 r / min for 10 minutes. The supernatant was collected to obtain the brine shrimp enzymatic hydrolysate. The degree of hydrolysis of the brine shrimp enzymatic hydrolysate was 13.80%, and the DPPH free radical scavenging rate was 94.02%.

[0035] S1.1 Optimal protease screening

[0036] Six enzymes—flavor protease, animal protease, papain, pepsin, trypsin, and alkaline protease—were selected for screening. Enzymes were added at a material-to-liquid ratio of 1:1 and an enzyme dosage of 600 U / g. Enzymatic hydrolysis was performed for 3 hours at the optimal temperature and pH for each enzyme. The optimal protease was selected as the flavor protease based on DPPH free radical scavenging rate and degree of hydrolysis. The optimal temperatures and pH values ​​for each protease are shown in Table 1. The effects of different proteases on the degree of hydrolysis and DPPH free radical scavenging rate are shown in Table 2. Figure 1 As shown. The presence of large hydrophobic amino acids at the C-terminus of peptides often endows them with strong antioxidant activity. Therefore, utilizing the different cleavage sites of various proteases, screening for suitable proteases is crucial for enhancing the antioxidant activity of peptides. Figure 1 It was found that different proteases could hydrolyze Artemia to varying degrees. Among them, flavor protease showed the best hydrolysis effect, with a degree of hydrolysis of 17.93%, significantly higher than other proteases (P<0.05). Trypsin group was the second best at 15.14%, while pepsin showed a poor hydrolysis effect on Artemia, with only 8.76%. In terms of DPPH free radical scavenging rate, the flavor protease group had the highest scavenging rate at 94.68%, significantly higher than other proteases (P<0.05), followed by animal protease group at 83.13%. Trypsin and alkaline protease groups had the lowest scavenging rates, at 50.09% and 48.07%, respectively, with no significant difference between them (P>0.05). Considering both the degree of hydrolysis and DPPH free radical scavenging rate, the flavor protease group showed the best hydrolysis effect and the highest DPPH free radical scavenging rate. Therefore, flavor protease was selected for subsequent experiments.

[0037] Table 1. Optimal temperature and pH of proteases

[0038]

[0039] S1.2, Single-factor enzymatic digestion test

[0040] Using DPPH free radical scavenging rate and degree of hydrolysis as indicators, a single-factor enzymatic hydrolysis experiment was conducted using flavor protease to investigate the effects of four factors: material-liquid ratio, hydrolysis pH, hydrolysis temperature, and hydrolysis time.

[0041] S1.2.1 The screening steps for the material-liquid ratio specifically include: homogenizing the brine shrimp samples, adding distilled water to adjust the material-liquid ratio to 0.5:1, 1:1, 1.5:1, and 2:1 respectively, adding 600 U / g of flavor protease, adjusting the pH to 6.0, enzymatically hydrolyzing at 50℃ for 2 hours, inactivating the enzyme in a boiling water bath for 5 minutes, cooling, centrifuging at 8000 r / min for 10 minutes, collecting the supernatant to obtain the brine shrimp enzymatic hydrolysate, and determining the DPPH free radical scavenging rate and degree of hydrolysis of the brine shrimp enzymatic hydrolysate under different material-liquid ratios. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that when the material-liquid ratio is 1:1, the degree of hydrolysis and DPPH free radical scavenging rate are the highest, with the degree of hydrolysis being 15.93% and the DPPH free radical scavenging rate being 95.32%.

[0042] S1.2.2, The screening steps for enzymatic hydrolysis pH specifically include: homogenizing the artichoke sample, adding distilled water to adjust the material-to-liquid ratio to 1:1, adding flavor protease at 600 U / g, adjusting the pH to 4.0, 5.0, 6.0, and 7.0 respectively, enzymatically hydrolyzing at 50℃ for 2 h, inactivating the enzyme in a boiling water bath for 5 min, cooling, centrifuging at 8000 r / min for 10 min, collecting the supernatant to obtain the artichoke enzymatic hydrolysate, and determining the DPPH free radical scavenging rate and degree of hydrolysis of the artichoke enzymatic hydrolysate under different pH conditions. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the degree of hydrolysis and DPPH free radical scavenging rate are highest when the pH is 6.0, with the degree of hydrolysis being 12.35% and the DPPH free radical scavenging rate being 89.19%.

[0043] S1.2.3. The screening steps for enzymatic hydrolysis temperature specifically include: homogenizing the brine shrimp sample, adding distilled water to adjust the material-to-liquid ratio to 1:1, adding 600 U / g of flavor protease, adjusting the pH to 6.0, and enzymatically hydrolyzing at 30℃, 40℃, 50℃, and 60℃ for 2 h respectively, followed by enzyme inactivation in a boiling water bath for 5 min, cooling, centrifuging at 8000 r / min for 10 min, collecting the supernatant to obtain the brine shrimp enzymatic hydrolysate, and determining the DPPH free radical scavenging rate and degree of hydrolysis of the brine shrimp enzymatic hydrolysate at different enzymatic hydrolysis temperatures. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the degree of hydrolysis and DPPH free radical scavenging rate are highest when the enzymatic hydrolysis temperature is 50℃, with a degree of hydrolysis of 13.98% and a DPPH free radical scavenging rate of 93.31%.

[0044] S1.2.4 The screening steps for enzymatic hydrolysis time specifically include: homogenizing the brine shrimp sample, adding distilled water to adjust the material-to-liquid ratio to 1:1, adding 600 U / g of flavor protease, adjusting the pH to 6.0, and enzymatically hydrolyzing at 50℃ for 1h, 2h, 3h, 4h, and 5h respectively. The enzyme is then inactivated by boiling in a water bath for 5min, cooled, and centrifuged at 8000r / min for 10min. The supernatant is collected to obtain the brine shrimp enzymatic hydrolysate. The DPPH free radical scavenging rate and degree of hydrolysis of the brine shrimp enzymatic hydrolysate at different enzymatic hydrolysis times are measured. The results are as follows: Figure 5 As shown. By Figure 5 It can be seen that the degree of hydrolysis and DPPH free radical scavenging rate are highest when the enzymatic hydrolysis time is 2 hours, with the degree of hydrolysis being 13.37% and the DPPH free radical scavenging rate being 78.95%.

[0045] S2. After the brine shrimp enzymatic hydrolysate obtained in the example was clarified by a ceramic membrane, it was ultrafiltered using ultrafiltration tubes with molecular weights of 1 kDa, 3 kDa, and 5 kDa, respectively, to obtain a first component (LCT-1) with a molecular weight range of <1 kDa, a second component (LCT-2) with a molecular weight range of 1 kDa to 3 kDa, and a third component (LCT-3) with a molecular weight range of 3 kDa to 5 kDa. The optimal component was screened using DPPH free radical scavenging rate, ABTS cation free radical scavenging rate, and total reducing power as indicators.

[0046] As can be seen from the above, the optimal enzymatic hydrolysis conditions are: a material-to-liquid ratio of 1:1, a hydrolysis pH of 6, a hydrolysis temperature of 50℃, and a hydrolysis time of 2 hours.

[0047] Because the enzymatic hydrolysate contains polysaccharides, free amino acids, inorganic salts, peptides of different molecular weights, and proteins, all of which can affect the bioactivity of polypeptides, the hydrolysate needs to be separated and purified. This example uses DPPH and ABTS... + The antioxidant activity of Artemia peptides with different molecular weights was studied using free radical scavenging rate and reducing power as indicators.

[0048] S2.1 Scavenging effect of Artemia peptides of different molecular weights on DPPH free radicals

[0049] LCT-1, LCT-2, and LCT-3 were prepared into solutions of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively. The antioxidant activity of artichoke peptides with different molecular weights was studied using DPPH free radical scavenging rate as an indicator. The results are as follows: Figure 6 As shown in Table 2, the regression equation analysis of the DPPH free radical scavenging rate of Artemia peptides with different molecular weights is presented.

[0050] Table 2. Regression equation analysis of DPPH free radical scavenging rate by artichoke peptides of different molecular weights

[0051]

[0052] Depend on Figure 6 As shown in Table 2, all three artesian peptides with different molecular weights exhibited strong scavenging activity against DPPH free radicals, and the scavenging rate increased with increasing sample concentration. At the same concentration, LCT-1 showed superior scavenging ability compared to LCT-2 and LCT-3, and there were significant differences in the DPPH free radical scavenging rates among the three artesian peptides with different molecular weights (P<0.05). The half-maximal inhibitory concentration (IC50) was... 50 In terms of values, the DPPH free radical scavenging effects of different samples, from high to low, are LCT-1 (IC50, 10 ... 50 0.39 mg / mL), LCT-2 (IC50) 50 0.44 mg / mL) and LCT-3 (IC50) 50 The concentration of 0.58 mg / mL indicates that different molecular weight Artemia peptides have different DPPH free radical scavenging abilities. The LCT-1 group, with the lowest molecular weight, has the strongest scavenging ability. This is because peptides with smaller molecular weights have less steric hindrance and can therefore react with free radicals better.

[0053] S2.2, Effects of Artemia peptides of different molecular weights on ABTS + Free radical scavenging effect

[0054] LCT-1, LCT-2, and LCT-3 were prepared into solutions with concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL, respectively, and then analyzed using ABTS. + Free radical scavenging rate was used as an indicator to study the antioxidant activity of artichoke peptides with different molecular weights. The results are as follows: Figure 7 As shown, different molecular weight Artemia peptides affect ABTS + The regression equation analysis of the free radical scavenging rate is shown in Table 3.

[0055] Table 3. Effects of Artemia peptides of different molecular weights on ABTS + Regression equation analysis of free radical scavenging rate

[0056]

[0057] Depend on Figure 7 As shown in Table 3, the three different molecular weight Artemia peptides affect ABTS. + All free radicals exhibited strong scavenging activity, and the scavenging rate increased with increasing sample concentration. At concentrations below 8 mg / mL, LCT-1 showed superior scavenging ability compared to LCT-2 and LCT-3. The three artesian peptides with different molecular weights showed similar scavenging activity against ABTS. +The free radical scavenging rates showed significant differences (P<0.05). At a concentration of 10 mg / mL, the ABTS values ​​of the three different molecular weight Artemia peptides were significantly different. + The free radical scavenging rate was 100%. (From the half-inhibitory concentration IC50) 50 In terms of values, different samples affect ABTS + The free radical scavenging activity, from highest to lowest, is LCT-1 (IC). 50 1.88 mg / mL), LCT-2 (IC50) 50 2.18 mg / mL) and LCT-3 (IC) 50 2.76 mg / mL), indicating that different molecular weight peptides affect ABTS. + The free radical scavenging abilities differ, with the LCT-1 group, having the lowest molecular weight, exhibiting the strongest scavenging ability. This is because smaller molecular weight peptides have better hydrophilicity, while ABTS... + Free radicals are hydrophilic, making them more prone to reaction with each other; therefore, ABTS... + It has a higher free radical scavenging ability.

[0058] S2.3, Reducing power analysis of Artemia peptides with different molecular weights

[0059] LCT-1, LCT-2, and LCT-3 were prepared into solutions with concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL, respectively, to treat Fe. 3+ Reduced to Fe 2+ Using the antioxidant capacity as an indicator, the antioxidant activity of Artemia peptides with different molecular weights was studied, and the results are as follows: Figure 8 As shown in Table 4, the regression equation analysis of the reducing power of Artemia peptides with different molecular weights is presented.

[0060] Table 4. Regression equation analysis of reducing power of Artemia peptides with different molecular weights

[0061]

[0062] Depend on Figure 8 As shown in Table 4, all three artesian peptides with different molecular weights possessed reducing power, and the reducing power increased with increasing sample concentration. LCT-1 exhibited superior reducing power compared to LCT-2 and LCT-3, and significant differences existed among the three artesian peptides with different molecular weights (P<0.05). At a concentration of 10 mg / mL, LCT-1 showed the highest reducing power (0.75), while LCT-2 and LCT-3 had reduction values ​​of 0.58 and 0.42, respectively. The IC50 values ​​were calculated from the half-maximum inhibitory concentration (IC50). 50 In terms of values, the reducing power of different samples from high to low is as follows: LCT-1 IC50 50 =5.90 mg / mL; IC50 of LCT-250 =8.13 mg / mL; IC50 of LCT-3 50 =12.70 mg / mL. This indicates that peptides of different molecular weights have different reducing powers, with the LCT-1 group having the lowest molecular weight and the strongest reducing power. This is because the shorter the peptide segment, the more fully the groups with more electrons are exposed, thus resulting in the strongest reducing ability.

[0063] In summary, the LCT-1 group with the lowest molecular weight exhibited the best DPPH radical scavenging rate and the best ABTS scavenging rate. + The free radical scavenging rate and reducing power were the strongest, both superior to the LCT-2 and LCT-3 groups. The LCT-1 group was the component with the strongest antioxidant activity.

[0064] S3. Using dextran gel electrophoresis to separate peptides based on molecular weight offers advantages such as mild conditions and minimal impact on sample activity. This example uses Sephadex G-15 for separation and purification, including: preparing the LCT-1 fraction with the strongest antioxidant activity after ultrafiltration to a concentration of 10 mg / mL with ultrapure water, eluting with deionized water, adjusting the flow rate to 0.7 mL / min, collecting every 5 min, measuring the absorbance at 280 nm, collecting the filtrate at room temperature using an automatic collector, mixing according to the chromatographic peaks, and freeze-drying. The results are as follows: Figure 9 As shown.

[0065] Depend on Figure 9 It was found that the LCT-1 group of Artemia antioxidant peptides with a molecular weight less than 1 kDa was separated into three fractions by Sephadex G-15 gel column chromatography, labeled as F1, F2, and F3, respectively. The DPPH and ABTS of F1, F2, and F3 were compared. + Free radical scavenging rate and reducing power: at a concentration of 1 mg / mL, the scavenging rate of component F1 against DPPH free radicals and its reducing power against ABTS free radicals... + The free radical scavenging rate and reducing power of F1 were higher than those of F2 and F3, and the DPPH of F1 component was 96.10%, while the ABTS of F1 component was lower. + The free radical scavenging rate was 71.62%, and the reducing power of the F1 component was 0.54.

[0066] S4. The peptide structure of the F1 fraction was identified using a liquid chromatography-mass spectrometry (LC-MS) system consisting of an Easy-nLC 1200 ultra-high performance liquid chromatography-tandem Orbitrap Fusion Lumos DDA high-resolution mass spectrometer.

[0067] Chromatographic conditions: Separation was performed using a nano-HPLC system. Solution A was 0.1% formic acid-water solution, and solution B was 0.1% formic acid-acetonitrile solution. The chromatographic column (PepMap 100 C18, 75 μm × 2 cm) was equilibrated with 100% solution A. The elution flow rate was 750 nL / min, and the elution conditions were: 0–20 min, 5% B; 20–22 min, 38% B; 22–30 min, 95%.

[0068] Mass spectrometry conditions: Level 1 mass spectrometry scan resolution is 60000, scan range is 350-1500 m / z, maximum injection time is 118 ms; DDA cycle time is 2 s; Level 2 mass spectrometry maximum ion injection time is 22 ms; collision chamber energy is set to 30%, applicable to all precursor ions; dynamic exclusion is set to 35 s.

[0069] This invention uses LC-MS / MS to analyze and identify the F1 component, obtaining a total of 195 peptides. Five peptides with local confidence scores higher than 99 were analyzed, and their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The results are shown in Table 5. Local confidence score refers to the confidence assessment of the correctness of the matching of local amino acid residues in a peptide, used to judge the reliability of the identification of a single or several consecutive amino acids in the sequencing results. The higher the value, the more accurate the sequence matching in that region.

[0070] Table 5 Peptide Sequences

[0071]

[0072] Table 5 shows that the molecular weights of the five selected peptides range from 578.61 to 1262.49 Da, containing 7 to 13 amino acids. The antioxidant activity of the peptides shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 was verified. At a concentration of 1 mg / mL, the peptide shown in SEQ ID NO.1 showed a 56.44% scavenging rate against DPPH free radicals, while the peptide shown in SEQ ID NO.4 showed a 56.44% scavenging rate against ABTS. + The free radical scavenging rate can reach 67.72%.

[0073] The five peptides obtained in this invention have clear structures and can be used as standards for studying the mechanism of action of antioxidant peptides (such as cell experiments and animal experiments). They clarify the antioxidant pathway and target binding mode of a single peptide, providing theoretical support for subsequent product optimization. They can also be used as candidate compounds for the early screening of antioxidant-related drugs, and their clear structures facilitate drug modification and activity optimization.

[0074] Molecular docking

[0075] Superoxide dismutase (SOD) is a metalloproteinase widely found in animals, plants, and microorganisms. It is closely related to the scavenging of reactive oxygen species and plays a crucial role in the body's oxidation-antioxidant balance. SOD can effectively remove free radicals that damage health and cause disease, breaking them down into harmless oxygen and water molecules for excretion. This invention selected SOD as the receptor protein and performed docking simulations with Artemia antioxidant peptides. The docking binding energies are shown in Table 6, and the docking visualization is shown in [Figure number missing]. Figures 10-14 As shown.

[0076] Table 6. Binding energy between antioxidant peptides and SOD

[0077]

[0078] A negative binding energy indicates the possibility of binding; generally, the smaller the value, the greater the likelihood of binding. Table 6 shows that all five purified peptides bound well to the active site of the target protein, with binding energies all less than -5. The peptide represented by SEQ ID NO.2 had the lowest binding energy, -7.369.

[0079] Depend on Figure 10 It is known that the peptide shown in SEQ ID NO.1 mainly relies on hydrophobic interactions and hydrogen bonds to bind in the SOD binding pocket. Among them, HIS-30, TRP-161, HIS-163, and TYR-166 provide significant hydrophobic effects, while HIS-30 (bond length: 2.5 Å), ASN-142 (bond length: 2.5 Å / 3.3 Å), and HIS-163 (bond length: 2.9 Å / 2.4 Å) further stabilize the binding conformation through hydrogen bonds. The overall binding mode is dominated by hydrophobic interactions and assisted by hydrogen bonds, ensuring the chimerism effect of the ligands in the pocket.

[0080] Depend on Figure 11As can be seen, the peptide represented by SEQ ID NO.2 exhibits strong interaction characteristics within the SOD binding pocket, and the synergistic effect of multiple interactions ensures the high stability of the ligand within the binding pocket. Among these, hydrophobic interactions mainly involve LYS-1, ALA-32, VAL-40, and LEU-4; these residues form a stable hydrophobic environment, which helps enhance the spatial adaptability between the ligand and receptor. Regarding hydrogen bonding, the ligand establishes hydrogen bond interactions with HIS-27 (bond length: 3.0 Å) and GLU-43 (bond length: 2.5 Å / 2.8 Å), further improving the directionality and stability of the binding. In addition, the ligand forms an electrostatic interaction with GLU-43 (bond length: 5.3 Å), playing a crucial role in maintaining a stable binding mode.

[0081] Depend on Figure 12 It is known that the peptide shown in SEQ ID NO.3 exhibits strong stability in the binding pocket of the SOD protein. It forms hydrophobic interactions with PRO-83 and HIS-71, enhancing the chimerism effect in the hydrophobic environment. At the same time, GLN-147 (bond length: 2.6 Å), ASN-185 (bond length: 2.1 Å / 2.4 Å), ASN-188 (bond length: 2.5 Å), ARG-192 (bond length: 2.2 Å / 2.0 Å / 2.7 Å), THR-79 (bond length: 2.3 Å), and PRO-154 (bond length: 3.4 Å) form hydrogen bonds with the ligands. Compared with simple hydrophobic interactions, these polar interactions provide the ligands with higher binding specificity. In addition, GLU-187 (bond length: 3.3 Å) also participates in electrostatic interactions, which overall enhances the spatial adaptability of the ligands in the binding pocket.

[0082] Depend on Figure 13 It is known that the peptide shown in SEQ ID NO.4 mainly relies on electrostatic interactions and hydrogen bonding to bind in the SOD binding pocket. Among them, HIS-71 (bond length: 3.2 Å), ARG-192 (bond length: 3.3 Å / 5.4 Å), GLU-187 (bond length: 4.2 Å), and GLU-191 (bond length: 3.1 Å) constitute the core region of electrostatic interaction, with HIS-71 (bond length: 3.5 Å) also forming electrostatic interactions with π-anions. At the same time, ASN-188 (bond length: 1.9 Å / 2.5 Å), SER-3 (bond length: 2.5 Å), and HIS-71 (bond length: 3.3 Å) form polar interactions with ligands through hydrogen bonds, improving the binding selectivity. In addition, PRO-5 and PRO-8 provide hydrophobic interactions, playing a key role in the fixation of the aromatic ligand backbone, so that LPDYN maintains a favorable orientation in the pocket.

[0083] Depend on Figure 14It is evident that the binding of the peptide represented by SEQ ID NO.5 to SOD is characterized by multiple interactions, ensuring the high stability of the ligand within the binding pocket. Specifically, the hydrophobic region formed by PRO-5 and ILE-76 provides initial stabilization; ASN-185 (bond length: 2.8 Å), ASN-188 (bond length: 2.4 Å / 2.3 Å), ARG-192 (bond length: 2.4 Å), HIS-71 (bond length: 3.4 Å), and SER-75 (bond length: 3.7 Å) form hydrogen bonds with the ligand, increasing the directionality and specificity of the binding; furthermore, GLU-191 (bond length: 5.1 Å) participates in electrostatic interactions, and HIS-71 (bond length: 3.2 Å / 4.0 Å) also forms electrostatic interactions with π-anions and salt bridges.

[0084] As can be seen from the above, the five Artemia antioxidant peptides of the present invention have high binding energies with SOD. The overall binding mode is dominated by hydrophobicity and hydrogen bonding. The synergistic effect of multiple interactions ensures the high stability of the ligand in the binding pocket, indicating that the Artemia antioxidant peptides can effectively bind to SOD and have good antioxidant activity.

[0085] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A composition of antioxidative peptides from artichokes, characterized in that, It includes the peptide shown in SEQ ID NO.1, the peptide shown in SEQ ID NO.2, the peptide shown in SEQ ID NO.3, the peptide shown in SEQ ID NO.4, and the peptide shown in SEQ ID NO.

5.

2. The method for preparing the Artemia antioxidant peptide composition according to claim 1, characterized in that, include: Enzymatic hydrolysis steps: After homogenizing the brine shrimp sample, distilled water was added to adjust the material-to-liquid ratio to 1:

1. The amount of flavor protease added was 600 U / g, the pH was adjusted to 6.0, and enzymatic hydrolysis was carried out at 50℃ for 2 hours. The enzyme was inactivated by boiling water bath for 5 minutes. After cooling, the sample was centrifuged at 8000 r / min for 10 minutes. The supernatant was collected to obtain the brine shrimp enzymatic hydrolysate. Ultrafiltration separation step: After the brine shrimp enzymatic hydrolysate is clarified through a ceramic membrane, it is ultrafiltered using an ultrafiltration tube with a molecular weight of 1 kDa to obtain the first component with a molecular weight range of <1 kDa. Gel chromatography separation steps: Gel chromatography separation was performed using Sephadex G-25. The first component was prepared with ultrapure water to a concentration of 10 mg / mL, eluted with deionized water, and the flow rate was adjusted to 0.7 mL / min. The sample was collected every 5 min, and the absorbance was measured at 280 nm. The filtrate was collected at room temperature using an automatic collector. After mixing according to the chromatographic peaks, the filtrate was freeze-dried to obtain three components: F1, F2, and F3. The component with the strongest antioxidant activity was selected as component F1, which is the Artemia antioxidant peptide composition, based on the DPPH free radical scavenging rate, ABTS cation free radical scavenging rate, and total reducing power.

3. The method for preparing the Artemia antioxidant peptide composition according to claim 2, characterized in that, In the enzymatic hydrolysis step, the degree of hydrolysis of the artichoke hydrolysate is 13.80%, and the DPPH free radical scavenging rate is 94.02%.

4. The method for preparing the Artemia antioxidant peptide composition according to claim 2, characterized in that, It also includes a mass spectrometry identification step, which involves analyzing and identifying the Artemia antioxidant peptide composition using nano-HPLC-MS / MS.

5. The application of the Artemia antioxidant peptide composition according to claim 1 in the food and cosmetic fields.

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