Puffer fish polypeptide with anti-fatigue activity as well as preparation method and application of puffer fish polypeptide

Abstract

The invention relates to a puffer fish polypeptide with anti-fatigue activity and a preparation method and application thereof. The amino acid sequence of the puffer fish polypeptide is DWHFSK. The preparation method comprises the following steps: S1, hydrolyzing testis of puffer fish with papain, deactivating enzyme after enzymolysis, centrifuging, collecting supernate, filtering, collecting polypeptide with relative molecular weight less than 1kDa, and freeze-drying for later use; s2, performing mass spectrometry and analysis on the extracted polypeptide to obtain a plurality of non-repetitive polypeptide sequences; s3, carrying out bioinformatics predictive analysis on the screened polypeptide sequences, screening out polypeptide sequences with strong bioactivity, then carrying out molecular docking, and screening out polypeptide sequences with top docking score ranks; and S4, performing solid-phase synthesis on the screened polypeptide sequence to obtain the puffer fish polypeptide with anti-fatigue activity. The amino acid sequence of the puffer fish polypeptide with anti-fatigue activity is obtained by taking the puffer fish as a raw material, and a new thought and a more economic source are provided for developing food-borne polypeptide with an anti-fatigue effect.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a pufferfish polypeptide with anti-fatigue activity, its preparation method, and its application. Background Technology

[0002] Fatigue is a complex physiological and psychological syndrome that occurs after prolonged physical or mental activity, and it is a self-protective mechanism evolved in animals. With the accelerated pace of modern life, chronic fatigue syndrome caused by prolonged physical or mental labor has become a significant problem affecting human health. Long-term fatigue not only reduces work efficiency and quality of life but may also induce chronic diseases such as cardiovascular disease and metabolic syndrome. Therefore, screening safe and effective anti-fatigue active ingredients from natural biological resources has become a research hotspot in the fields of food science and medicine.

[0003] Numerous studies have demonstrated that various marine-derived bioactive peptides, such as sea cucumber peptides and turtle peptides, can effectively alleviate fatigue by increasing energy reserves, clearing metabolic waste products, and reducing oxidative stress. Small molecule peptides with anti-fatigue activity are widely available, highly safe, and have well-defined mechanisms of action, holding significant theoretical value and industrialization potential in the development of functional foods and adjuvant therapeutic drugs. Furthermore, the search for broader, more economical, and stable peptide sources remains a continuous pursuit within the industry.

[0004] Pufferfish, also known as river pufferfish, belongs to the class Osteichthyes, order Tetraodontiformes, and family Tetraodontidae. With the iterative optimization of modern aquaculture technology, especially breakthroughs in key technologies for controlling pufferfish toxicity, the tetrodotoxin content in their bodies has been significantly controlled, successfully achieving safe industrial-scale farming. Among them, the pufferfish 'Chrysanthemum' is one of the most economically valuable species currently farmed in Fujian Province. Pufferfish meat is characterized by high protein and low fat, and is rich in minerals and essential amino acids, making it highly sought after in the market. However, during pufferfish processing, the testes, as one of the core byproducts, are non-toxic and, although they account for up to 20% of the total weight of the pufferfish, their utilization rate is extremely low due to their distinctive odor. Summary of the Invention

[0005] The purpose of this invention is to provide a pufferfish polypeptide with anti-fatigue activity, its preparation method and application. Using pufferfish as raw material, the amino acid sequence of the pufferfish polypeptide with anti-fatigue activity is obtained, providing a new idea and a more economical source for developing food-derived polypeptides with anti-fatigue effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention discloses a pufferfish polypeptide with anti-fatigue activity, the amino acid sequence of which is: DWHFSK.

[0007] This invention also discloses a method for preparing the above-mentioned pufferfish polypeptide, which includes the following steps: S1, polypeptide extraction The testes of pufferfish were hydrolyzed using papain. After hydrolysis, the enzyme was inactivated. After centrifugation, the supernatant was collected and filtered. Peptides with a relative molecular weight of less than 1 kDa were collected and freeze-dried for later use. S2, Sequence Identification The peptides extracted in step S1 were subjected to mass spectrometry. The results of the mass spectrometry were analyzed using mass spectrometry analysis software to obtain multiple non-repetitive peptide sequences. S3, Virtual Filtering The peptide sequences selected in step S2 are subjected to bioinformatics prediction analysis using software to identify peptide sequences with strong biological activity. Then, the peptide sequences selected by bioinformatics prediction analysis are subjected to molecular docking using software to identify peptide sequences with high docking scores. S4, polypeptide synthesis The polypeptide sequences screened in step S3 were synthesized in a solid phase to obtain the pufferfish polypeptide with anti-fatigue activity.

[0008] Preferably, in step S1, the testes of pufferfish are homogenized with ultrapure water at a material-to-liquid ratio of 15:1 for 2 minutes. Then, papain is added at a concentration of 10,000 U / g, the pH is adjusted to 6.5, and the mixture is stirred at 55°C for 4 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the hydrolysate is placed in a boiling water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, it is centrifuged at 8,000 r / min for 15 minutes at 4°C. The supernatant is then collected to obtain the crude peptide solution. The crude peptide solution is first filtered through a ceramic membrane to remove impurities, and then passed through a 1 kDa ultrafiltration membrane. The peptides that have passed through the 1 kDa ultrafiltration membrane are collected and then freeze-dried under vacuum for later use.

[0009] Preferably, the pufferfish is the yellow pufferfish.

[0010] Preferably, in step S2, the peptide extracted in step S1 is subjected to mass spectrometry analysis by LC-MS / MS equipped with an online nanospray ion source. The chromatographic column is a C18 column, and the sample is separated by a gradient of 60 min. The column flow rate is controlled at 300 nL / min, the column temperature is 40℃, the electrospray voltage is 2 kV, the gradient starts from 4% B phase, increases to 39% in a non-linear gradient at 53 min, increases to 50% within 40 s, increases to 95% within another 40 s, and is maintained for 340 s. The mass spectrum is analyzed by mass spectrometry analysis software, and the peptide card value is set to -10lgP≥20. The peptide sequence is exported after searching the library by mass spectrometry analysis software.

[0011] Preferably, in step S3, the bioinformatics prediction analysis includes bioactivity prediction analysis, solubility prediction analysis, toxicity prediction analysis, molecular weight and isoelectric point prediction analysis, sensitization prediction analysis, and prediction analysis of human intestinal absorption, blood-brain barrier, CYP metabolism, and acute oral toxicity; molecular docking involves molecularly docking the peptide sequences screened by the bioinformatics prediction analysis with the receptor proteins Keap1, LDH, and CK, respectively, wherein the crystal structure of the receptor protein Keap1 is 4IQK, the crystal structure of the receptor protein LDH is 7EPM, and the crystal structure of the receptor protein CK is 3B6R.

[0012] Furthermore, this invention also discloses the application of the above-mentioned pufferfish polypeptide in the preparation of anti-fatigue products.

[0013] Preferably, the anti-fatigue product is a functional food, health product, or pharmaceutical preparation.

[0014] Preferably, the functional food is a solid beverage, meal replacement powder, or baked goods; the health product is a tablet or oral liquid; and the pharmaceutical preparation is a capsule or granule.

[0015] Preferably, the amount of pufferfish polypeptide added in functional foods is 0.2-6 wt%; the amount of pufferfish polypeptide added in health products is 2-12 wt%; and the amount of pufferfish polypeptide added in pharmaceutical preparations is 8-35 wt%.

[0016] The present invention has the following beneficial effects: 1. This invention uses the testes of pufferfish as raw material to obtain the amino acid sequence of pufferfish polypeptide with anti-fatigue activity, providing a new idea and a more economical source for developing food-derived polypeptides with anti-fatigue effects, and also making use of waste.

[0017] 2. The pufferfish polypeptide of this invention has the characteristics of being non-toxic, non-allergenic, and highly water-soluble. Although the initial intestinal absorption rate needs to be optimized, it can be improved through mature formulation technology. The overall safety and bioavailability are excellent, making it suitable for wide application in the food, health product, and pharmaceutical fields.

[0018] 3. Molecular docking experiments confirmed that the pufferfish polypeptide of this invention can exert anti-fatigue effects through multiple pathways by targeting Keap1, LDH, and CK target proteins. The mechanism of action is clear, providing solid theoretical support for subsequent in vivo functional verification and product development. Attached Figure Description

[0019] Figure 1 The effect of different proteases on the degree of hydrolysis of the testes of the pufferfish.

[0020] Figure 2 The effect of different enzymatic hydrolysis times on the degree of hydrolysis.

[0021] Figure 3 The effect of different enzymatic hydrolysis times on the degree of hydrolysis.

[0022] Figure 4 The effect of different enzyme dosages on the degree of hydrolysis.

[0023] Figure 5 The effect of different liquid-to-solid ratios on the degree of hydrolysis.

[0024] Figure 6 The effect of different pH values ​​on the degree of hydrolysis.

[0025] Figure 7 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between enzymatic hydrolysis temperature and time.

[0026] Figure 8 The diagram shows the three-dimensional response surface and corresponding contour plot of the interaction between enzyme dosage and enzymatic hydrolysis time.

[0027] Figure 9 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and the enzymatic hydrolysis time.

[0028] Figure 10 The diagram shows the three-dimensional response surface and corresponding contour plot of the interaction between enzyme dosage and enzymatic hydrolysis temperature.

[0029] Figure 11 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and the enzymatic hydrolysis temperature.

[0030] Figure 12 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and the amount of enzyme added.

[0031] Figure 13 This serves as a comparison of the DPPH free radical scavenging rates of different concentrations of TFPPs.

[0032] Figure 14 This serves as a comparison of the hydroxyl radical scavenging rates of different concentrations of TFPPs.

[0033] Figure 15 This serves as a comparison of the scavenging rates of superoxide anion free radicals by different concentrations of TFPPs.

[0034] Figure 16 3D and 2D schematic diagrams of the interaction between DWHFSK and the active site of Keap1 (PDB ID: 4IQK).

[0035] Figure 17 3D and 2D model diagrams of the interaction between DWHFSK and LDH (PDB ID:7EPM).

[0036] Figure 183D and 2D model diagrams of the interaction between DWHFSK and CK (PDB ID: 3B6R). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] This invention discloses a pufferfish polypeptide with anti-fatigue activity, the amino acid sequence of which is DWHFSK, as shown in the sequence listing SEQ ID NO: 1.

[0039] The above-mentioned method for preparing pufferfish polypeptide includes the following steps: S1, polypeptide extraction The testes of *Pueraria lobata* were hydrolyzed using protease. After hydrolysis, the enzyme was inactivated, and the supernatant was collected by centrifugation and filtered. Peptides with a relative molecular weight less than 1 kDa were collected and freeze-dried for later use. In this case, the *Pueraria lobata* testes were purchased from Fujian Hongpu Aquaculture Co., Ltd.

[0040] Before enzymatic hydrolysis, the testes of *Pueraria lobata* were pretreated as follows: the purchased testes were transported back to the laboratory and stored at -20°C for later use. Before the experiment, the frozen testes were removed, thawed by rinsing under running water at room temperature, and after complete thawing, the surface connective tissue and impurities were removed. The testes were then rinsed three times with distilled water, drained, and cut into 1cm × 1cm pieces for later use.

[0041] The general steps of enzymatic hydrolysis are as follows: Ultrapure water and pretreated pufferfish testes are homogenized for 2 min at a specific liquid-to-solid ratio (V / W, mL / g) to obtain a homogenate. A certain amount of protease is added and thoroughly mixed. The pH is adjusted with 1 mol / L NaOH or HCl, and the solution is placed in a constant-temperature water bath at the optimal temperature for protease hydrolysis for a certain period of time, with continuous stirring. The hydrolysate is then inactivated by boiling in a water bath (100℃) for 10 min. After cooling to room temperature, the solution is centrifuged at 4℃ and 8000 r / min for 15 min, and the supernatant is collected to obtain the crude peptide solution (TFPPs).

[0042] To screen for the optimal enzymatic hydrolysis conditions, the following experiments were conducted: 1. Screening of proteases Pretreated *Pueraria lobata* testes were weighed and homogenized with ultrapure water at a liquid-to-solid ratio of 20:1. After reaching the optimal enzymatic hydrolysis temperature for each protease, the pH was adjusted to the optimal value for each protease using 1 mol / L NaOH or HCl (see Table 1). 5000 U of protease was added per gram of testes, and enzymatic hydrolysis was carried out in a constant temperature water bath for 5 h. Afterward, the enzymes were inactivated by boiling in a water bath for 10 min, cooled, and centrifuged at 4℃ and 8000 r / min for 15 min. The supernatant was then freeze-dried. The degree of hydrolysis was used as the evaluation index to compare the effects of five proteases on the degree of hydrolysis of peptides derived from *Pueraria lobata* testes.

[0043] Table 1. Process parameters for enzymatic hydrolysis of pufferfish testes with different proteases

[0044] The results are as follows Figure 1 As shown, from Figure 1 It can be seen that different proteases have significant differences in their hydrolytic ability on the testes of *Pueraria lobata*. P <0.05, which may be closely related to the substrate specificity of each protease. The degree of hydrolysis, from high to low, is trypsin > papain > pepsin > alkaline protease > neutral protease. Among them, trypsin showed the highest degree of hydrolysis, reaching 37.36%, with no significant difference between it and papain and pepsin. P Papain (>0.05%) showed good hydrolysis efficiency, but significantly higher than neutral and alkaline proteases. This is mainly attributed to the fact that papain is a thiol protease with a broad amino acid substrate range and abundant cleavage sites, resulting in high hydrolysis efficiency. Furthermore, papain's optimal operating environment is neutral conditions, and its cost is relatively low in industrial production. Therefore, considering both hydrolysis efficiency and economic benefits, papain was chosen as the enzyme for subsequent experiments.

[0045] 2. Single-factor experimental design Based on the selection of the optimal protease, the effects of five factors on the enzymatic hydrolysis efficiency of pufferfish spermatophore peptides were investigated, using the degree of hydrolysis as the evaluation index. These factors included different hydrolysis temperatures (30, 40, 50, 60, 70℃), different pH values ​​(4.5, 5.5, 6.5, 7.5, 8.5), different enzyme dosages (2500, 5000, 7500, 10000, 12500 U / g), different liquid-to-solid ratios (4:1, 8:1, 12:1, 16:1, 20:1), and different hydrolysis times (2, 3, 4, 5, 6 h). When investigating one factor, the other conditions were fixed as follows: temperature 50℃, pH 7, liquid-to-solid ratio 20:1 (mL / g), enzyme dosage 5000 U / g, and hydrolysis time 5 h.

[0046] 2.1 Effect of enzymatic hydrolysis time on the degree of hydrolysis Enzymatic hydrolysis time is one of the key factors affecting the degree of hydrolysis of pufferfish testis protein. Figure 2 It can be seen that, within a certain range, the degree of hydrolysis increases with the extension of enzymatic hydrolysis time. The degree of hydrolysis reaches its highest value of 33.30% when the enzymatic hydrolysis time is 5 hours. Further extending the enzymatic hydrolysis time does not significantly increase the degree of hydrolysis. P The concentration of phospholipids (>0.05) decreased slightly and then leveled off. This is because the enzymatic hydrolysis reaction was relatively complete at 5 hours, and the binding of the substrate and enzyme, as well as the catalytic reaction, reached a relative equilibrium. However, excessively long hydrolysis times could lead to non-specific degradation of some active peptides and increase time and energy consumption during production. Considering both the hydrolysis effect and production economics, 5 hours was chosen as the optimal enzymatic hydrolysis time.

[0047] 2.2 Effect of enzymatic hydrolysis temperature on the degree of hydrolysis Enzymatic hydrolysis temperature, as one of the core environmental factors affecting the catalytic activity of proteases, plays a crucial regulatory role in the hydrolysis process of pufferfish testis protein. Figure 3 It can be seen that within the experimentally set temperature range of 30–70℃, the degree of hydrolysis first increases and then decreases with increasing enzymatic hydrolysis temperature. The degree of hydrolysis reaches its highest value of 39.20% when the enzymatic hydrolysis temperature reaches 50℃. As the temperature continues to rise, the degree of hydrolysis decreases significantly. P The value <0.05 indicates that excessively high temperatures inhibit protease activity, hindering efficient hydrolysis and significantly reducing the enzymatic hydrolysis effect. Therefore, 50℃ was determined to be the optimal enzymatic hydrolysis temperature.

[0048] 2.3 Effect of enzyme dosage on degree of hydrolysis The amount of enzyme added is a crucial factor affecting the hydrolysis of testicular protein in pufferfish. From... Figure 4 It can be seen that the degree of hydrolysis gradually increases as the enzyme dosage increases from 2500 U / g to 12500 U / g. The degree of hydrolysis reaches its highest value of 46.77% when the enzyme dosage is 12500 U / g; the degree of hydrolysis is 45.11% when the enzyme dosage is 10000 U / g, which is not significantly different from the degree of hydrolysis at 12500 U / g. P >0.05). This is because, within a certain range of enzyme dosage, an increase in the number of enzyme molecules provides more opportunities for substrate-enzyme binding, thereby promoting the hydrolysis reaction. However, when the enzyme dosage exceeds a certain threshold, the enzyme active sites that the substrate can bind to tend to saturate. Excess enzyme cannot fully bind with the substrate, which not only wastes enzyme resources but also increases production costs. Considering both hydrolysis efficiency and production economy, 10,000 U / g was selected as the optimal enzyme dosage for papain.

[0049] 2.4 Effect of liquid-to-solid ratio on degree of hydrolysis The liquid-to-solid ratio is also one of the key factors affecting the enzymatic hydrolysis process of *Pueraria lobata* testes. Figure 5 It was found that within the experimentally set liquid-to-solid ratio range of 4:1 to 20:1 (mL / g), the degree of hydrolysis initially increased and then slowly decreased with increasing liquid-to-solid ratio. The degree of hydrolysis reached its highest value (38.46%) when the liquid-to-solid ratio increased to 16:1. At a liquid-to-solid ratio of 12:1, the degree of hydrolysis was 35.69%, slightly lower than that at 16:1. When the liquid-to-solid ratio further increased to 20:1, the degree of hydrolysis showed a slow decreasing trend. This is because, within a certain liquid-to-solid ratio range, increasing the liquid-to-solid ratio provides a more favorable mass transfer environment for sufficient contact between the enzyme and the substrate, accelerating the catalytic reaction rate of the protease and the diffusion rate of product molecules, which is beneficial to the enzymatic hydrolysis reaction. However, when the liquid-to-solid ratio increases to a certain extent, the relative concentration of the protease in the system will decrease due to the excessive increase in solvent volume, thereby reducing the efficiency of the enzymatic hydrolysis reaction. Considering both the hydrolysis effect and the feasibility of subsequent separation and purification processes, 16:1 was selected as the optimal liquid-to-solid ratio.

[0050] 2.5 Effect of pH on the degree of hydrolysis pH is one of the key environmental factors affecting the catalytic activity of proteases. Figure 6 It can be seen that within the pH range of 4.5 to 8.5, the degree of hydrolysis of the testes of *Pueraria lobata* by papain showed a slight increasing trend followed by a slight decreasing trend, but there was no significant difference in the degree of hydrolysis under different pH conditions. P >0.05). This is partly due to the inherent characteristics of papain, which has a relatively wide optimal pH range, giving it the flexibility to maintain a certain level of catalytic activity across a broad pH spectrum. Furthermore, the testes of the pufferfish (Tetraodon chrysanthum) are rich in nucleic acids and arginine, which form a natural buffer system that neutralizes and buffers pH changes during the enzymatic hydrolysis process, thus reducing the impact of pH fluctuations on enzyme activity. Based on these results, pH was no longer considered as a separate optimization factor in subsequent experiments.

[0051] 3. Response surface optimization experimental design Based on the results of the single-factor experiments, a Box-Behnken design with four factors and three levels was used to conduct a three-level experiment. The factors that significantly affected the enzymatic hydrolysis of the testes of *Pueraria lobata* were: hydrolysis time (A), hydrolysis temperature (B), enzyme dosage (C), and liquid-to-solid ratio (D) as independent variables, with the degree of hydrolysis as the response value. The levels of the response surface methodology factors are shown in Table 2.

[0052] Table 2 Factors and levels in response surface methodology

[0053] The four-factor, three-level response surface methodology constructed in this experiment, along with the corresponding hydrolysis degree measurement results, is shown in Table 3. Through multiple quadratic regression fitting analysis of the experimental data, a quadratic regression equation was established between the degree of hydrolysis (Y) and enzymatic hydrolysis time (A), enzymatic hydrolysis temperature (B), enzyme dosage (C), and liquid-to-solid ratio (D). The specific expression is as follows: Y=39.76+1.70A+1.12B+0.7182C+4.98D-1.88AB+0.2570AC+0.7709AD+0.0856BC+3.14BD+0.6786CD-5.34.A2-2.74.B2-6.42.C2-5.04.D2 This regression equation can be used to quantitatively describe the influence of various process parameters and their interactions on the degree of hydrolysis of TFPPs, providing mathematical model support for subsequent significance testing of process parameters, interaction analysis, and solution of optimal process conditions.

[0054] Table 3 Response Surface Experimental Design and Results

[0055] The above quadratic regression equation quantitatively reflects the nonlinear relationship between four factors—hydrolysis time (A), hydrolysis temperature (B), enzyme dosage (C), and liquid-to-solid ratio (D)—and the degree of hydrolysis (response value Y). To further verify the reliability of the model and the influence of each factor, an analysis of variance was performed on the regression equation. The specific results are shown in Table 4. The F-value of this quadratic regression model is 41.32. P <0.0001 indicates that the model is highly significant and can effectively describe the relationship between each factor and the degree of hydrolysis; the p-values ​​of each significance level in the model are all less than 0.05, further confirming the statistical significance of the model.

[0056] Table 4. Analysis of Variance of Regression Model

[0057] By comparing the F-values ​​(or standardized regression coefficients) of each factor, the influence of the four factors on the degree of hydrolysis, from strongest to weakest, was determined to be D (liquid-to-solid ratio) > A (enzymatic hydrolysis time) > B (enzymatic hydrolysis temperature) > C (enzyme dosage). Among these, the liquid-to-solid ratio is the most critical influencing factor, and its regulatory effect requires close attention. Meanwhile, the interactions between enzymatic hydrolysis time and temperature (AB), the interaction between enzymatic hydrolysis temperature and liquid-to-solid ratio (BD), and the quadratic terms of enzymatic hydrolysis time (A2), enzymatic hydrolysis temperature (B2), enzyme dosage (C2), and liquid-to-solid ratio (D2) of the three individual factors (A, B, and D) all had highly significant effects on the response surface methodology. P <0.01, the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature (AB) has a significant effect on the response surface value. P<0.05); C and other interactions (AC, BC, CD) had no significant effect on the degree of hydrolysis. P >0.05), which is consistent with the results of the previous single-factor experiment where the degree of hydrolysis increased only to a limited extent after the amount of enzyme added exceeded the threshold.

[0058] Furthermore, the model lack of fit term is not significant ( P The value >0.05 indicates that the model does not exhibit significant misfit; the model's coefficient of determination R² = 0.9764 indicates that the model can explain 97.64% of the response value variation, demonstrating a good fit of the equation and a high correlation between predicted and measured values, thus possessing good theoretical predictive ability. In summary, the Box-Behnken response surface methodology used is scientifically sound, and the constructed quadratic regression model is reliable and effective. It can be used to optimize the enzymatic hydrolysis process of pufferfish spermatophore-derived peptides, providing a solid statistical and mathematical basis for improving the degree of hydrolysis of TFPPs by regulating key process parameters.

[0059] To intuitively reveal the influence of the interaction between various factors on the degree of hydrolysis, based on the constructed quadratic regression model, and with the other two factors fixed as the central level, three-dimensional response surface plots and corresponding contour plots were drawn for each pair of factors. The interaction effect between factors was quantitatively analyzed through the surface morphology and contour features. The results are shown in Figure 7-12. Figure 7 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between enzymatic hydrolysis temperature and time. Figure 8 The diagram shows the three-dimensional response surface and corresponding contour plot of the interaction between enzyme dosage and enzymatic hydrolysis time. Figure 9 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and enzymatic hydrolysis time. Figure 10 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between enzyme dosage and enzymatic hydrolysis temperature. Figure 11 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and the enzymatic hydrolysis temperature. Figure 12 This is a three-dimensional response surface plot and corresponding contour plot showing the interaction between the feed-to-liquid ratio and the amount of enzyme added.

[0060] Depend on Figure 7 The 3D response surface plot shows that the surface is a parabolic shape with the opening facing downwards, indicating that there is an interaction point within the experimental range that maximizes the degree of hydrolysis. In the contour plot, the rate of change of the color gradient (blue → red) along the enzymatic hydrolysis time (A) is significantly faster than that along the enzymatic hydrolysis temperature (B), and the slope of the response surface is steeper in the enzymatic hydrolysis time dimension. This phenomenon indicates that, in the interaction between the two, the enzymatic hydrolysis time has a stronger influence on the degree of hydrolysis, which is consistent with the conclusion in the analysis of variance that "the influence of enzymatic hydrolysis time is stronger than that of enzymatic hydrolysis temperature". Figure 8The response surface also exhibits a downward opening characteristic, with more pronounced color transitions in the contour lines along the enzymatic hydrolysis time direction and a greater slope in this direction; while the color change along the enzyme dosage direction is gradual, and the surface fluctuations are smaller. This indicates that the effect of enzymatic hydrolysis time on the degree of hydrolysis is significantly stronger than that of the enzyme dosage, further verifying the rule that the contribution of the enzyme dosage to the increase in degree of hydrolysis is limited after exceeding the threshold. Figure 9 The contour lines are nearly circular, indicating that the interaction between enzymatic hydrolysis time and liquid-to-solid ratio is not significant. P >0.05), but judging from the surface slope and color gradient, the surface in the direction of liquid-to-material ratio is steeper and the color change rate is faster, indicating that the independent influence of liquid-to-material ratio on the degree of hydrolysis is significantly stronger than that of enzymatic hydrolysis time, which is consistent with the conclusion that "liquid-to-material ratio is the most critical influencing factor". Figure 10 The response surface opens downwards, and the contour lines are narrow ellipses with the enzymatic hydrolysis temperature as the major axis. The color gradient along the enzymatic hydrolysis temperature direction is more significant, and the surface inclination is greater. With the synergistic increase of enzyme dosage and enzymatic hydrolysis temperature, the degree of hydrolysis first increases and then decreases, and the change in enzymatic hydrolysis temperature has a more prominent effect on the peak position of the degree of hydrolysis. This indicates that in the interaction between the two, the regulatory effect of enzymatic hydrolysis temperature is dominant, while the effect of enzyme dosage is relatively weak. Figure 11 In the middle, the contour lines are typically elliptical, combined with the results of the analysis of variance ( P <0.05), confirming a significant interaction between enzymatic hydrolysis temperature and liquid-to-solid ratio; the rate of change of the color gradient along the liquid-to-solid ratio direction is much faster than that along the enzymatic hydrolysis temperature direction, and the slope of the response surface is steeper in the liquid-to-solid ratio dimension, indicating that the liquid-to-solid ratio has a stronger influence on the degree of hydrolysis in the interaction between the two, and is the core regulatory factor in this interactive system. Figure 12 The contour plots are close to circular, indicating that the interaction between the amount of enzyme added and the liquid content is not significant; however, the fluctuation of the response surface in the direction of the liquid-to-solid ratio is significantly greater than that in the direction of the amount of enzyme added, and the color gradient changes are more obvious, which further confirms that the independent influence of the liquid-to-solid ratio on the degree of hydrolysis is much stronger than that of the amount of enzyme added.

[0061] Based on the results of the three-dimensional response surface and contour line analysis, the order of influence of each factor on the degree of hydrolysis is: liquid-to-solid ratio (D) > hydrolysis time (A) > hydrolysis temperature (B) > enzyme dosage (C), which is consistent with the F-value ranking results in the analysis of variance. Regarding factor interactions, the interaction effect between hydrolysis temperature (B) and liquid-to-solid ratio (D) is the most significant, while the interactions between other factors are weak or insignificant. This conclusion is consistent with the finding in the analysis of variance that "the BD term is highly significant (…)". P The results (<0.01) are highly consistent. The above analysis not only intuitively quantifies the interaction law between factors, but also further verifies the effectiveness and reliability of the response surface model, providing an intuitive basis for determining the optimal process parameters.

[0062] To verify the predictive reliability of the response surface regression model and the practical feasibility of the optimal process parameters, based on the quadratic regression model between the degree of hydrolysis (Y) and each factor, Design-Expert 8.0.6 software was used to predict and analyze four key parameters: enzymatic hydrolysis time (A), enzymatic hydrolysis temperature (B), enzyme dosage (C), and liquid-to-solid ratio (D). The results showed that the theoretically optimal enzymatic hydrolysis process parameters were: hydrolysis time 4.11 h, hydrolysis temperature 55.62℃, enzyme dosage 10224.09 U / g, and liquid-to-solid ratio 14.74:1 (mL:g). Under this parameter combination, the theoretically predicted degree of hydrolysis was 41.91%. Considering the accuracy of instruments and ease of operation in actual production, the theoretical parameters needed to be adjusted for engineering purposes. The final practically operable process parameters were: hydrolysis time 4 h, hydrolysis temperature 55℃, enzyme dosage 10000 U / g, and liquid-to-solid ratio 15:1 (mL:g). To verify the effectiveness of the optimized process parameters and the model fit, three parallel enzymatic hydrolysis experiments were conducted under the aforementioned actual parameter conditions, and the mean degree of hydrolysis was measured to be (42.31±0.63)%. A one-way ANOVA was performed between the "model predicted value (41.91%)" and the "actual measured mean (42.31%)", and the results showed no significant difference between the two. P The value >0.05 indicates that the constructed response surface regression model has excellent fitting effect and prediction accuracy. The adjusted process parameters can accurately reflect the theoretical optimal conditions and have practical application value.

[0063] 4. Ultrafiltration membrane fractionation The enzymatic hydrolysate prepared under optimal enzymatic hydrolysis conditions was first separated using a ceramic membrane (5 mesh) to remove small molecules such as free amino acids and inorganic salts. Then, it was fractionated sequentially using ultrafiltration membranes with different molecular weight cutoffs (3 kDa and 1 kDa), collecting three fractions: TFPPs1 (<1 kDa), TFPPs2 (1-3 kDa), and TFPPs3 (3-10 kDa). These three fractions were then concentrated by nanofiltration (molecular weight cutoff of 150 Da) and freeze-dried to obtain TFPPs of different molecular weights. Subsequently, the in vitro antioxidant activity of the crude peptide and each ultrafiltration fraction was systematically evaluated using DPPH free radical scavenging, hydroxyl free radical scavenging, and superoxide anion free radical scavenging experiments. The specific steps are as follows.

[0064] 4.1 DPPH free radical scavenging rate The DPPH free radical scavenging capacity assay kit (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) was tested according to the instructions. The specific method is as follows: Take 80 μL of TFPPs solutions with concentrations of 1, 5, 10, 15, 20, and 25 mg / mL, respectively, add 120 μL of reagent one, mix thoroughly, and incubate at 25°C in the dark for 30 min. After the reaction, run at 8000 r / min at room temperature for 5 min. Measure the absorbance (A) of the supernatant at 515 nm. The DPPH free radical scavenging rate is calculated using the following formula:

[0065] 4.2 Hydroxyl radical scavenging rate The hydroxyl radical scavenging ability assay kit (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) was tested according to the instructions, as follows: 100 μL of Reagent 1, 5 μL of Reagent 2, and 100 μL of Reagent 3 were mixed to prepare working solutions. The stock solution of Reagent 4 was diluted with distilled water at a ratio of 1:9 to prepare Reagent 4. For each test tube, 50 μL of TFPPs solution at concentrations of 1, 5, 10, 15, 20, and 25 mg / mL were added, followed by 250 μL of working solution, and then 50 μL of Reagent 4, and mixed thoroughly. For the control tube, 250 μL of working solution, 50 μL of distilled water, and Reagent 4 were added, and mixed thoroughly. For the blank tube, 250 μL of working solution and 100 μL of distilled water were added, and mixed thoroughly. All solutions were incubated at 37℃ for 60 min. After the reaction, the solution was centrifuged at 8000 r / min at 25℃ for 5 min. 200 μL of the supernatant was taken and the absorbance value (A) was measured at a wavelength of 536 nm. The hydroxyl radical scavenging rate can be calculated using the following formula:

[0066] 4.3 Superoxide anion free radical scavenging rate The superoxide anion scavenging capacity assay kit (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) was tested according to the instructions. The specific method is as follows: Dissolve Reagent II powder in 6 mL of distilled water. For each test tube, add 10 μL of Reagent I and 40 μL of Reagent II, mix thoroughly, and react at 25°C for 1 min. Then add 25 μL of TFPPs solutions at concentrations of 1, 5, 10, 15, 20, and 25 mg / mL, and 50 μL of Reagent III, respectively. Mix thoroughly and react at 37°C for 30 min. Finally, add 50 μL of Reagent IV and Reagent V, respectively, and mix thoroughly. For the control tube, add 10 μL of Reagent I, 40 μL of Reagent II, and 25 μL of distilled water, mix thoroughly, and react at 25°C for 1 min. Then add 50 μL of Reagent III, mix thoroughly, and react at 37°C for 30 min. Finally, add 50 μL of Reagent IV and Reagent V, and mix thoroughly. Develop color at 37°C for 20 min, and measure the absorbance at 530 nm. Record these values ​​as Test Tube A and Control Tube A, respectively. The superoxide anion radical scavenging rate can be calculated using the following formula:

[0067] The results are as follows Figure 13-15 As shown, where, Figure 13 This serves as a control for the DPPH free radical scavenging rate of different concentrations of TFPPs. Figure 14 This serves as a control for the scavenging rate of hydroxyl radicals by different concentrations of TFPPs. Figure 15 This serves as a comparison of the scavenging rates of superoxide anion free radicals by different concentrations of TFPPs. Figure 13-15 It can be seen that all tested components exhibited certain antioxidant activity, and the scavenging rates of DPPH free radicals, hydroxyl free radicals, and superoxide anion free radicals by each TFPP component increased significantly with increasing sample concentration, showing a good dose-response positive correlation. P <0.05).

[0068] Further analysis of the differences in antioxidant capacity among the components revealed that the overall antioxidant activity of the TFPPs-1 component was significantly superior to the other two ultrafiltration components and the crude peptide (P < 0.05). Its half-maximal scavenging concentrations (IC50) for DPPH free radicals, hydroxyl free radicals, and superoxide anion free radicals were 8.94 mg / mL, 12.15 mg / mL, and 7.58 mg / mL, respectively. According to existing academic evaluation standards, when the IC50 of a substance's DPPH scavenging rate is... 50A concentration less than 10 mg / mL indicates strong in vitro antioxidant activity, demonstrating the excellent antioxidant potential of the TFPPs-1 component. This is likely because small peptides, with their simpler spatial structure and smaller molecular size, not only provide more transferable electrons to free radicals but also expose more active sites, making them more susceptible to redox reactions or complexation reactions with free radicals. This leads to the transformation of free radicals into more stable, inactive substances, ultimately exhibiting a stronger free radical scavenging ability. Based on these experimental results, the TFPPs-1 component was ultimately selected as the core research subject for subsequent experiments.

[0069] In summary, as a preferred implementation method, step S1 is as follows: First, transport the purchased pufferfish testes back to the laboratory and store them in a -20℃ freezer for later use. Before the experiment, take out the frozen pufferfish testes, rinse them under running water at room temperature to thaw them, and after they are completely thawed, remove the surface connective tissue and impurities, rinse them three times with distilled water, drain the water, and cut them into 1cm×1cm pieces for later use.

[0070] Add ultrapure water to the testicular fragments at a liquid-to-solid ratio of 14:1 (mL / g), homogenize for 2 minutes using a high-speed homogenizer (10000 r / min) to prepare a homogenate; add papain (enzyme activity 6.0 × 10⁻⁶). 6 The enzyme dosage was increased to 10000 U / g. The pH was adjusted to 6.5 with 1 mol / L HCl or NaOH. The mixture was then placed in a 54℃ constant temperature water bath and magnetically stirred for 4 hours for enzymatic hydrolysis.

[0071] After enzymatic hydrolysis, the hydrolysate was placed in a boiling water bath for 10 min to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 8000 r / min for 15 min at 4℃. The supernatant was then taken as the crude peptide solution TFPPs.

[0072] The crude peptide solution was first filtered through a 5-mesh ceramic membrane to remove insoluble impurities, and then passed through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa (operating pressure 0.12 MPa, temperature 30℃). The peptide component (TFPPs-1) that passed through the 1 kDa ultrafiltration membrane was collected and then freeze-dried under vacuum (-50℃, 0.1 Pa) to obtain TFPPs-1 powder.

[0073] S2, Sequence Identification The peptides (TFPPs-1) extracted in step S1 were subjected to mass spectrometry. The results of the mass spectrometry analysis were analyzed using mass spectrometry software to obtain multiple non-repeating peptide sequences. Specifically, the peptide sequences were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0074] The sample was analyzed by LC-MS / MS equipped with an online nano-spray ionization source. The entire system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC1200 in series. A total of 1 μL of sample was loaded (C18 column: 20 cm × 75 μm id, 1.9 μm particle size). The sample was separated using a gradient at 60 min, with the column flow rate controlled at 300 nL / min, column temperature at 40 °C, and electrospray voltage at 2 kV. The gradient started at 4% B phase, increased non-linearly to 39% at 53 min, increased to 50% within 40 s, increased to 95% within another 40 s, and held for 340 s. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0075] The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 100-1500; resolution: 120,000; normalized AGC target: 200%; maximum injection time: 100 ms; (2) HCD-MS / MS: resolution: 50,000; normalized AGC target: 200%; maximum injection time: 86 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s. The mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was set to uniprot-Takifugu flavidus (2025, 29296 entries) sequence, and the enzyme digestion was set to none. Search parameters: Fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 10 ppm, variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98, Hydroxylation (KP) 16. Protein card value: contains at least 1 unique peptide; peptide card value: -10lgP ≥ 20.

[0076] A total of 2085 specific peptide sequences were identified. Analysis of the peptide length distribution characteristics showed that they were mainly concentrated in the range of 3 to 10 amino acid residues. Peptides in this length range are generally more likely to cross biological membrane barriers and have a good basis for bioavailability. Amino acid composition analysis revealed that leucine (Leu), isoleucine (Ile), valine (Val), tyrosine (Tyr), glycine (Gly), and aspartic acid (Asp) were significantly more frequent than other amino acids in the identified peptide sequences. Leu, Ile, and Val, as branched-chain amino acids, are important substrates for energy metabolism and play a clear role in promoting muscle repair after exercise and alleviating exercise fatigue. Tyr, as a key component of antioxidant enzymes such as tyrosinase, participates in the scavenging of free radicals in the body. Gly and Asp can enhance the body's antioxidant capacity and delay oxidative stress-mediated aging by regulating intracellular redox balance and helping to increase glutathione (GSH) levels. These amino acid composition characteristics suggest that the peptide sequences in the TFPPs-1 component may possess both anti-fatigue and antioxidant potential functions.

[0077] S3, Virtual Filtering The peptide sequences selected in step S2 are subjected to bioinformatics prediction analysis using software to identify peptide sequences with strong biological activity. Then, the peptide sequences selected by bioinformatics prediction analysis are subjected to molecular docking using software to identify peptide sequences with high docking scores. Specifically, the bioactivity of pufferfish protamine from *Peptide Ranker* (http: / / distilldeep.ucd.ie / PeptideRanker / ) was predicted using the online software Peptide Ranker. The screening criterion was set at a score > 0.5 (this threshold is the industry-recognized critical value for screening potential bioactive peptides). Ten short peptide sequences that met the criteria were obtained, as detailed in Tables 5 and 6. The potential bioactive peptide sequences obtained above were imported into the online tool Innovagen Peptide Property Calculator (http: / / www.innovagen.com / proteomics-tools) to predict the peptides' solubility. The results showed that all target peptides had good water solubility, providing a feasible basis for subsequent in vitro activity validation and formulation development. The toxicity of the above peptides was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ), and the results showed that all peptides had no toxic risk. Sensitization analysis of the above peptides was performed using the Allergylab platform (https: / / sortaller.gzhmu.edu.cn / ), confirming that the target peptides were not sensitizing, thus preliminarily ensuring their safety as functional components. The molecular weight and isoelectric point (PI) of the peptides were predicted using Expasy ProtParam (http: / / web.expasy.org / compute_pi / ). The relevant physicochemical parameters are shown in Table 5, providing crucial reference for subsequent optimization of the separation and purification process.

[0078] The target peptide was converted into the Simplified Molecular-InputLine-Entry System (SMILES) format and uploaded to the AdmetSAR online system (http: / / lmmd.ecust.edu.cn / admetsar1 / predict / ). Its human intestinal absorption (HIA), blood-brain barrier (BBB) ​​penetration, cytochrome P450 (CYP) enzyme metabolism characteristics, and acute oral toxicity were predicted. The specific pharmacokinetic data are shown in Table 6.

[0079] Table 5. Sensitization, toxicity, and physicochemical properties of peptides in TFPPs-1

[0080] Table 6. ADMET of peptides in TFPPs-1

[0081] The 3D structure of the peptide was drawn using ChemDraw 20.0 software, and energy optimization was performed before exporting it to Mol2 format. After optimization, it was imported into AutoDocktools v1.5.6 for hydrogenation, charge calculation, charge distribution, and setting of rotatable bonds. The crystal structures of the receptor molecules Keap1 (PDB ID: 4IQK), LDH (PDB ID: 7EPM), and CK (PDB ID: 3B6R) were downloaded from the RCSB protein database (https: / / www.rcsb.org / ). The receptor proteins were preprocessed using PyMol software, including removing ligands and water molecules from the receptors. AutoDock software (V1.5.6) was then used for hydrogenation, charge distribution calculation, and processing to specify the protein type. AutoDock was used to perform docking with Vina 1.1.2. The center coordinates of the Keap1 docking box were: x = -49.55, y = 0.425, z = -16.958, with a box size of 60 Å × 60 Å × 60 Å. The center coordinates of the LDH docking box were: x = -71.248, y = 38.106, z = -1.501, with a box size of 80 Å × 80 Å × 80 Å. The center coordinates of the CK docking box were: x = -18.504, y = 2.784, z = -18.102, with a box size of 60 Å × 60 Å × 80 Å. The results, obtained by selecting the highest affinity determined by Vina docking, were considered successful if the binding energy was ≤ -5 kJ / mol. Visualization, analysis, and processing were performed using Pymol and Discovery Studio 4.5.

[0082] In molecular docking studies, a larger absolute value of the binding energy (i.e., a smaller numerical value) indicates a higher spatial fit and interaction strength between the small molecule ligand and the target protein, and a stronger ability to form a stable complex. In this study, the potentially bioactive peptide DWHFSK was selected for molecular docking analysis with key target proteins related to antioxidation and anti-fatigue, including Kelch-like ECH-associated protein 1 (Keap1, a key protein in the antioxidant pathway), lactate dehydrogenase (LDH, an enzyme related to energy metabolism and fatigue), and creatine kinase (CK, a key enzyme in muscle energy metabolism). The docking results showed that the binding energy of the active peptide to Keap1 protein was between -7.0 and -6.2 kcal / mol; the binding energy to LDH protein was between -8.4 and -6.7 kcal / mol; and the binding energy to CK protein was between -8.0 and -7.4 kcal / mol, indicating that the peptide can form a strong and stable binding with the target protein and has potential targeted regulatory activity.

[0083] Antioxidant peptides can inhibit and regulate oxidative stress in the body through multiple mechanisms. Among them, the Keap1 / Nrf2 signaling pathway is a core defense pathway against oxidative damage. Under normal physiological conditions, Nrf2 binds to Keap1 and remains inactive. When the body is stimulated by oxidative stress, Keap1 undergoes a conformational change, and Nrf2 dissociates and enters the cell nucleus, initiating the expression of downstream antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, thereby enhancing the body's antioxidant capacity. Therefore, the binding affinity and binding mode between bioactive peptides and Keap1 protein are key indicators for assessing their antioxidant potential. The molecular docking binding mode diagram of the bioactive peptide DWHFSK with Keap1 protein is shown below. Figure 16 .from Figure 16 It is known that the DWHFSK peptide can bind to Ser555, Ser508, Arg380, and Asn382 of the Keap1 active site via hydrogen bonds; bind to Gly433, Ser431, Asn414, Arg415, Tyr334, Asn387, Phe577, Tyr572, Ala556, Tyr525, and Phe D4 via hydrophobic interactions; and form additional stabilizing effects with Gly603, Ser363, and Gly509 via carbon-hydrogen bonds.

[0084] Furthermore, the surface binding diagram of Keap1 protein to active peptides ( Figure 16It was observed that the active peptide molecule could completely embed into the active pocket of the Keap1 protein, with a high degree of spatial fit and no obvious steric hindrance, further validating the rationality of the binding mode. Hydrophobic amino acids are the core source of the binding force maintaining the Keap1-Nrf2 complex, especially residues such as Tyr334, Arg380, Arg415, Asn382, Tyr525, and Tyr572 in the active pocket of the Keap1 protein, which directly participate in hydrophobic interactions and hydrogen bonding with ligand molecules. In this study, the DWHFSK active peptide could form specific interactions with the above key residues, further suggesting its potential to exert antioxidant activity in vivo by regulating the Keap1 / Nrf2 signaling pathway.

[0085] Lactate dehydrogenase, a key rate-limiting enzyme in the glycolytic metabolic pathway, has the core function of catalyzing the reduction of pyruvate to lactate, accompanied by the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD. + When the body is under high-intensity exercise or hypoxia, increased LDH activity leads to a large accumulation of lactic acid in muscle tissue, causing a decrease in muscle pH and reduced contraction efficiency, ultimately resulting in physical exhaustion. Therefore, inhibiting LDH activity is an important target for screening anti-fatigue active molecules.

[0086] This study analyzed the binding mode of the active peptide DWHFSK to LDH protein, demonstrating its precise targeting of the active pocket region of LDH protein and the formation of a stable complex through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and C-H bonds. The binding mode of the DWHFSK active peptide to CK protein is as follows: Figure 17 As shown, from Figure 17 It can be seen that the DWHFSK peptide can form hydrogen bond interactions with amino acid residues Ser238, Arg170, Asp259, Asn165, and Met258 at the active site of LDH protein; it can also form hydrophobic interactions with lys43, Tyr173, Ala169, and Leu255, further consolidating the complex structure through van der Waals forces of the hydrophobic amino acid side chains. Notably, the DWHFSK active peptide forms specific interactions with key residues of LDH protein, such as Arg170, Asn165, and Ala169. These residues are the core sites for maintaining the conformation of the LDH enzyme active site and binding the substrate (pyruvate) and coenzyme (NADH). By binding to these residues, the active peptide can competitively inhibit the binding of the substrate to LDH or induce conformational changes in the enzyme active site, thereby reducing LDH catalytic activity and decreasing lactate production.

[0087] The binding energy between the active peptide and LDH molecules is between -8.4 and -6.7 kcal / mol, indicating a strong binding ability. Based on the analysis of the binding mode in this section, it can be seen that the DWHFSK peptide sequence can exert an anti-fatigue effect by targeting and regulating LDH activity, and has the potential application value of realizing anti-fatigue function in vivo.

[0088] Creatine kinase (CK), a core enzyme in the muscle energy metabolism pathway, catalyzes the high-energy phosphate bond transfer between creatine and adenosine triphosphate (ATP) to generate phosphocreatine (PCr) and adenosine diphosphate (ADP). This reaction is reversible and maintains dynamic equilibrium—in the resting state of muscles, CK catalyzes the conversion of ATP to PCr for energy storage; during exercise, PCr is rapidly converted to ATP via CK catalysis to provide energy for muscle contraction. When CK activity is abnormal or its function is limited, muscle energy supply efficiency decreases, easily leading to fatigue accumulation. Therefore, regulating CK activity is an important direction for developing anti-fatigue active molecules. This study systematically analyzed the binding mode of the active peptide DWHFSK to the CK protein. This active peptide can precisely target the active pocket region of the CK protein, forming a stable complex through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and carbon-hydrogen bonds. The binding mode of the DWHFSK active peptide to the CK protein is as follows: Figure 18 As shown, from Figure 18 It can be seen that the DWHFSK peptide can form hydrogen bond interactions with amino acid residues Arg130, Arg132, Glu232, His191, Arg236, Arg341, Arg320, and Asn286 at the active site of the CK protein, thereby enhancing the binding affinity between the ligand and the protein through the strong polarity of hydrogen bonds. It can also form a hydrophobic interaction with Pro197, maintaining the conformational stability of the complex through the hydrophobic stacking effect of the amino acid side chains. In addition, the C-H bond formed with Arg292 further supplements the binding force and enhances the overall binding stability.

[0089] DWHFSK active peptides form specific interactions with key residues of CK protein, including His191, Arg130, Arg132, Arg236, Arg320, and Glu232. These residues are core components of the CK protein's active site. His191 directly participates in the proton transfer process during enzyme catalysis, while basic amino acid residues such as Arg130, Arg132, and Arg236 are responsible for binding polar groups of the substrate (creatine / ATP). Glu232 maintains the conformation of the active site, ensuring enzyme catalytic efficiency. By binding to these key residues, DWHFSK active peptides can competitively inhibit the binding of substrates to CK or induce conformational changes in the enzyme's active site, thereby regulating CK catalytic activity, optimizing muscle energy metabolism efficiency, and reducing fatigue accumulation.

[0090] The binding energy between the active peptide and the CK molecule is between -8.0 and -7.4 kcal / mol, indicating a strong ligand-protein binding ability. Based on the analysis of the binding mode in this section, the DWHFSK peptide sequence can participate in the regulation of muscle energy metabolism by targeting and regulating CK activity, and has the potential application value of exerting anti-fatigue function in vivo.

[0091] S4, polypeptide synthesis The peptide sequence DWHFSK selected in step S3 was synthesized in a solid phase to obtain the pufferfish peptide with anti-fatigue activity.

[0092] Based on the above research, this invention also discloses the application of the aforementioned pufferfish polypeptide in the preparation of anti-fatigue products. The anti-fatigue products can be functional foods, health supplements, or pharmaceutical preparations. More specifically, functional foods can be solid beverages, meal replacement powders, or baked goods; health supplements can be tablets or oral liquids; and pharmaceutical preparations can be capsules or granules.

[0093] Regarding the amount added, the preferred amount of pufferfish polypeptide in functional foods is 0.2-6 wt%, the preferred amount of pufferfish polypeptide in health products is 2-12 wt%, and the preferred amount of pufferfish polypeptide in pharmaceutical preparations is 8-35 wt%.

[0094] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A pufferfish polypeptide with anti-fatigue activity, characterized in that, Its amino acid sequence is: DWHFSK.

2. The method for preparing pufferfish polypeptide as described in claim 1, characterized in that, Includes the following steps: S1, polypeptide extraction The testes of pufferfish were hydrolyzed using papain. After hydrolysis, the enzyme was inactivated. After centrifugation, the supernatant was collected and filtered. Peptides with a relative molecular weight of less than 1 kDa were collected and freeze-dried for later use. S2, Sequence Identification The peptides extracted in step S1 were subjected to mass spectrometry. The results of the mass spectrometry were analyzed using mass spectrometry analysis software to obtain multiple non-repetitive peptide sequences. S3, Virtual Filtering The peptide sequences selected in step S2 are subjected to bioinformatics prediction analysis using software to identify peptide sequences with strong biological activity. Then, the peptide sequences selected by bioinformatics prediction analysis are subjected to molecular docking using software to identify peptide sequences with high docking scores. S4, polypeptide synthesis The polypeptide sequences screened in step S3 were synthesized in a solid phase to obtain the pufferfish polypeptide with anti-fatigue activity.

3. The preparation method according to claim 2, characterized in that: In step S1, the testes of pufferfish were homogenized with ultrapure water at a material-to-liquid ratio of 15:1 for 2 minutes. Papain was then added at a concentration of 10,000 U / g, and the pH was adjusted to 6.

5. The mixture was then stirred at 55°C for 4 hours for enzymatic hydrolysis. After hydrolysis, the hydrolysate was placed in a boiling water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, the solution was centrifuged at 8,000 r / min for 15 minutes at 4°C. The supernatant was collected to obtain the crude peptide solution. The crude peptide solution was first filtered through a ceramic membrane to remove impurities, and then passed through a 1 kDa ultrafiltration membrane. The peptides that passed through the 1 kDa ultrafiltration membrane were collected and then freeze-dried under vacuum for later use.

4. The preparation method according to claim 2, characterized in that: The pufferfish mentioned is the yellow pufferfish.

5. The preparation method according to claim 2, characterized in that: In step S2, the peptide extracted in step S1 was analyzed by LC-MS / MS equipped with an online nanospray ion source. A C18 column was used, and the sample was separated by a gradient of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40℃, and the electrospray voltage was 2 kV. The gradient started from 4% B phase, increased to 39% non-linearly in 53 min, increased to 50% within 40 s, increased to 95% within another 40 s, and maintained for 340 s. The mass spectrum was analyzed by mass spectrometry analysis software, and the peptide alpha value was set to -10lgP≥20. The peptide sequence was exported after searching the library using the mass spectrometry analysis software.

6. The preparation method according to claim 2, characterized in that: In step S3, the bioinformatics prediction analysis includes bioactivity prediction analysis, solubility prediction analysis, toxicity prediction analysis, molecular weight and isoelectric point prediction analysis, sensitization prediction analysis, and prediction analysis of human intestinal absorption, blood-brain barrier, CYP metabolism, and acute oral toxicity. Molecular docking involves molecularly docking the peptide sequences screened by the bioinformatics prediction analysis with the receptor proteins Keap1, LDH, and CK, respectively. The crystal structure of the receptor protein Keap1 is 4IQK, the crystal structure of the receptor protein LDH is 7EPM, and the crystal structure of the receptor protein CK is 3B6R.

7. The application of the pufferfish polypeptide as described in claim 1 in the preparation of anti-fatigue products.

8. The application as described in claim 7, characterized in that: The anti-fatigue products mentioned are functional foods, health products, or pharmaceutical preparations.

9. The application as described in claim 8, characterized in that: The functional foods are solid beverages, meal replacement powders, or baked goods; the health products are tablets or oral liquids; and the pharmaceutical preparations are capsules or granules.

10. The application as described in claim 8, characterized in that: The amount of pufferfish polypeptide added in functional foods is 0.2-6 wt%; the amount of pufferfish polypeptide added in health products is 2-12 wt%; and the amount of pufferfish polypeptide added in pharmaceutical preparations is 8-35 wt%.

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