Use of large-mouth bass stomach polypeptide in preparation of antioxidant and / or immunoregulatory product

Abstract

The application discloses application of a large-mouth black bass stomach polypeptide in preparation of an antioxidant and / or immunoregulation product, relates to the field of bioactive peptides, and is characterized in that the large-mouth black bass stomach polypeptide is a polypeptide product obtained by alkaline protease enzymolysis of large-mouth black bass stomach, and components with molecular weights less than 3000 Da account for more than 98% of the total peptide amount. The polypeptide has significant DPPH free radical, hydroxyl free radical and ABTS cationic free radical scavenging capacity and reducing capacity, can promote macrophage proliferation, enhance phagocytosis, promote nitric oxide secretion, up-regulate immune-related cytokine expression, and play an immunoregulation role through activation of P38 and P44 MAPK signal pathways. The application realizes high-value utilization of large-mouth black bass processing by-products, has high product safety, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and in particular relates to the application of a largemouth bass stomach polypeptide in the preparation of antioxidant and / or immunomodulatory products. Background Technology

[0002] Bioactive peptides (BAPs) generally refer to short peptide chains composed of 2-20 amino acids that have positive physiological effects on the body. Numerous studies have shown that bioactive peptides possess various biological functions, including antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects. Given that synthetic drugs often come with varying degrees of adverse reactions, the academic and industrial communities are increasingly inclined to extract bioactive peptides with potential medicinal or health-promoting value from natural animal and plant proteins as an alternative. Aquatic products and their byproducts are rich in high-quality protein, making them ideal raw materials for extracting peptides and other bioactive substances. However, a large amount of byproducts generated during aquatic product processing (such as fish heads, bones, viscera, and skin) are usually discarded directly or used only as low-value feed, resulting in a huge waste of protein resources and serious environmental pollution. Therefore, how to achieve high-value utilization of aquatic product processing byproducts has become a critical issue that the industry urgently needs to address.

[0003] The immune system plays a crucial role in protecting organisms from harm by recognizing pathogens and clearing senescent and tumor cells. Therefore, enhancing the body's immunity using exogenous substances is of great significance. Currently, there is increasing attention on immunomodulators, with a wide variety of synthetic immunomodulators available, but these often come with side effects. Therefore, it is particularly important to develop a natural immunomodulator that is mild, safe, and effective. Recent studies have shown that immunomodulatory peptides derived from food proteins are easily absorbed and have higher safety profiles. Food-derived immunomodulatory peptides or protein hydrolysates can improve many chronic diseases and accelerate the body's recovery from injury. Aquaculture is the world's fastest-growing food production source, supporting the ever-increasing protein demand of humankind. Most fish processing byproducts are converted into low-value products such as animal feed and fishmeal. Therefore, enzymatic hydrolysis of aquaculture processing byproducts is an effective resource for preparing immunomodulatory bioactive peptides. Protein hydrolysates prepared from underutilized fish resources may provide a sustainable and safe alternative for therapeutic immunomodulation.

[0004] Meanwhile, oxidative stress is closely related to the occurrence and development of various diseases in the body. Excessive accumulation of reactive oxygen species (ROS) can lead to cell membrane lipid peroxidation, protein denaturation, and DNA damage. In recent years, food-derived antioxidant peptides have become a research hotspot as an alternative to synthetic antioxidants due to their advantages such as safe source, high absorption efficiency, and low toxicity. Currently, the methods for evaluating antioxidant activity mainly include two categories: chemical methods and cellular methods. Commonly used methods such as DPPH free radical scavenging, ABTS free radical scavenging, hydroxyl free radical scavenging, oxygen free radical uptake capacity (ORAC) method, and reducing power determination can analyze the antioxidant mechanism from multiple perspectives. Existing research on the extraction of antioxidant peptides from fish mainly focuses on fish meat or fish skin, with a lack of research and analysis on viscera. Furthermore, previous articles have focused more on introducing bioactive peptides, only briefly introducing their antioxidant properties, without in-depth summary and analysis of antioxidant peptides extracted from fish viscera.

[0005] In the study of immunomodulatory peptides, the presence of certain amino acids significantly influences their immunomodulatory function. These include hydrophobic amino acids (tryptophan, proline, and phenylalanine, etc.), branched-chain amino acids (leucine, isoleucine, and valine), and basic amino acids (lysine, arginine, and histidine, etc.). The immunomodulatory activity of peptides is closely related to hydrophobic and branched-chain amino acids. Hydrophobic amino acids can act as hydrogen donors, exerting antioxidant effects by scavenging free radicals. Oxidative stress is closely related to immune function; therefore, this antioxidant activity indirectly supports immunomodulatory function. Amino acid position is also an important factor affecting the activity of immunomodulatory peptides. When the N-terminus or C-terminus of an immunomodulatory peptide contains Arg, pSer, Gln, or Trp, its activity is often stronger. Relative molecular mass is closely related to the activity of fish immunomodulatory peptides. Smaller molecular weight peptides are better absorbed by organisms and therefore usually exhibit higher activity than larger molecular weight peptides.

[0006] Largemouth bass ( Micropterus salmoidesLargemouth bass, commonly known as California bass, is considered the fifth largest freshwater aquaculture species in China and is one of the most important freshwater aquaculture species in the country. The protein content of largemouth bass ranges from 17.97% to 20.15%, fat from 0.81% to 6.41%, ash from 1.24% to 1.41%, and moisture from 72.12% to 79.98%. The total amino acid content of the muscle is 14.19% to 16.47%, with essential amino acids accounting for more than 44% of the total, indicating that largemouth bass muscle protein is a high-quality protein source and can be used as a raw material for extracting bioactive peptides. Early studies have indicated that hydrolysates of bass skin, bones, and flesh have effects such as promoting wound healing, antibacterial activity, antioxidant activity, anti-inflammatory activity, and immunomodulatory activity. Han Mengyao et al. used largemouth bass skin as raw material to obtain collagen peptides with high antioxidant activity and verified their ability to restore immune function and optimize intestinal flora structure in immunosuppressed mice. Li Peizhi investigated the mechanism of lactation-promoting effect of largemouth bass peptide compound powder. The results showed that the compound powder not only significantly promoted the proliferation of MCF-10A cells but also promoted the expression of proteins related to the milk protein synthesis pathway, such as mTOR and PI3K proteins. However, most existing research focuses on the breeding and resource conservation of largemouth bass, and fresh products still dominate the composition of largemouth bass consumer products, with low processing conversion rates. Meanwhile, globally, more than 20 million tons of fish tissue are discarded annually, accounting for approximately 70% of the total fish weight. As a type of fish, largemouth bass processing by-products are mostly processed at low cost for feed or discarded. Exploration of high-value-added development is still weak, especially research on the high-value utilization of largemouth bass processing by-products. The largemouth bass stomach, located between the esophagus and pyloric cecum, is easily identifiable and separable. Currently, most of it is processed into feed or discarded, resulting in low utilization. Therefore, it may be a high-quality raw material suitable for preparing bioactive peptides from the viscera.

[0007] In this study, the inventors successfully prepared a polypeptide (MsGP) with significant antioxidant and immunomodulatory activities from the stomach of largemouth bass for the first time through extensive experiments. Its physicochemical properties, molecular weight distribution, peptide composition, amino acid composition, and biological activities were systematically characterized. More than 98% of the polypeptide's molecular weight is less than 3000 Da, it contains 19 specific peptide segments, and is rich in functional amino acids related to antioxidant and immunomodulatory activities. In vitro experiments confirmed its highly efficient scavenging ability against DPPH free radicals, hydroxyl radicals, and ABTS cation radicals, and it can promote macrophage proliferation, phagocytosis, and cytokine secretion by activating the P38 and P44 subunits of the MAPK signaling pathway. However, there are currently no reports on the use of this largemouth bass stomach polypeptide in the preparation of antioxidant and / or immunomodulatory products. Therefore, developing new uses for largemouth bass stomach polypeptide in the preparation of antioxidant and / or immunomodulatory products has significant practical implications and broad application prospects. Summary of the Invention

[0008] This invention aims to provide a novel use for largemouth bass stomach peptides, specifically their application in the preparation of antioxidant and / or immunomodulatory products. These peptides are derived from the stomach, a byproduct of largemouth bass processing, and are prepared through a specific enzymatic hydrolysis process. They exhibit a well-defined low molecular weight distribution and a specific peptide composition, demonstrating significant antioxidant and immunomodulatory activities. Therefore, they can be used as an effective ingredient in functional foods, health supplements, or cosmetics.

[0009] To achieve the above objectives, the present invention provides an application of largemouth bass stomach peptides in the preparation of antioxidant and / or immunomodulatory products. The largemouth bass stomach peptides are polypeptide products obtained by alkaline protease hydrolysis of largemouth bass stomach, wherein the components with a molecular weight of less than 3000 Da account for more than 98% of the total peptide weight, and the polypeptide product contains one or more peptides selected from the following: DAKELEVM, IDWEYPGSR, LDQEHSL, DSGDGVTH, GPPGPPGPPGQP, VEPLDPPE.

[0010] Furthermore, the components of the largemouth bass gastric polypeptide with a molecular weight of less than 3000 Da account for 98.48% of the total peptide weight, and the components with a molecular weight of less than 1000 Da account for more than 90% of the total peptide weight.

[0011] Furthermore, the largemouth bass stomach polypeptide contains 19 peptide segments with a molecular weight distribution range of 786.31 Da to 2959.17 Da, of which short peptides with a length of 11 amino acids or less account for 42.11% of the total number of peptide segments.

[0012] Furthermore, the Fourier transform infrared spectrum of the largemouth bass gastric polypeptide was observed in the amide I band at 1621.82 cm⁻¹. -1 Amide II band 1582.49 cm -1 CH2 stretching vibration peak 2924.44 cm⁻¹ -1 The C=O stretching vibration peak is 1742.81 cm⁻¹. -1 It has a characteristic absorption peak.

[0013] Further, the preparation method of the largemouth bass stomach polypeptide includes the following steps: adding the pretreated largemouth bass stomach to phosphate buffer, adjusting the pH to 8.0-9.0, adding alkaline protease at an enzyme amount of 7000-10000 U / g, enzymatically hydrolyzing at 50-60℃ for 4-6 hours, inactivating the enzyme, centrifuging, and freeze-drying to obtain the polypeptide.

[0014] Furthermore, in the preparation method of the largemouth bass gastric polypeptide, the enzymatic hydrolysis conditions are: pH 8.5, temperature 55.4℃, time 5.0 hours, and enzyme dosage 9000 U / g.

[0015] Furthermore, the antioxidant product is used to scavenge at least one of DPPH free radicals, hydroxyl free radicals, and ABTS cationic free radicals, and / or to provide reducing power; the largemouth bass stomach peptide has a scavenging rate of 12.03% to 78.03% for DPPH free radicals at a concentration of 2 to 10 mg / mL; and / or, the largemouth bass stomach peptide has a scavenging rate of 95.53% for hydroxyl free radicals at a concentration of 5 mg / mL; and / or, the largemouth bass stomach peptide has a scavenging rate of 99.99% for ABTS cationic free radicals at a concentration of 5 mg / mL.

[0016] Furthermore, the immunomodulatory product is used to promote macrophage proliferation, enhance macrophage phagocytic capacity, promote macrophage secretion of nitric oxide, and upregulate the expression levels of immune-related cytokine genes at at least one of the following: largemouth bass gastric peptide can significantly promote RAW264.7 macrophage proliferation at a concentration range of 10–1250 μg / mL; and / or, the largemouth bass gastric peptide can significantly enhance the phagocytic capacity of RAW264.7 macrophages for neutral red at a concentration range of 10–6250 μg / mL; and / or, the largemouth bass gastric peptide at a concentration of 1250 μg / mL can increase the nitric oxide secretion of RAW264.7 macrophages to 5.37 μM.

[0017] Furthermore, the immune-related cytokines are selected from one or more of IL-1β, IL-6, iNOS, and TNF-α; the largemouth bass gastric polypeptide can significantly upregulate the mRNA expression level of the cytokines in RAW264.7 macrophages at concentrations of 250 μg / mL and 1250 μg / mL.

[0018] Furthermore, the largemouth bass gastric polypeptide at a concentration of 1250 μg / mL significantly increased the expression levels of p-P38 and p-P44 proteins in RAW264.7 macrophages.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects: The largemouth bass gastric polypeptide provided by this invention exhibits significant technical effects in both antioxidant and immunomodulatory aspects. In terms of antioxidant activity, this polypeptide can efficiently scavenge DPPH free radicals, hydroxyl radicals, and ABTS cationic free radicals, and possesses stable reducing power. Furthermore, these activities are concentration-dependent, indicating that it can serve as a highly efficient electron or hydrogen atom donor, making it suitable for preparing antioxidant products that scavenge free radicals and provide reducing power. In terms of immunomodulation, this polypeptide can significantly promote macrophage proliferation, enhance their phagocytic capacity, promote nitric oxide secretion, and upregulate the gene expression levels of immune-related cytokines such as IL-1β, IL-6, iNOS, and TNF-α. Mechanistic studies show that this polypeptide mainly exerts its immunomodulatory effect by activating the P38 and P44 (ERK1 / 2) subunits in the MAPK signaling pathway. Therefore, this polypeptide can enhance macrophage immune function at multiple levels, including cell proliferation, phagocytosis, cytokine secretion, and signaling pathways, making it suitable for preparing immunomodulatory products. Due to its dual activities, this polypeptide can also be used to prepare products with both antioxidant and immunomodulatory functions. The largemouth bass stomach polypeptide used in this invention is derived from aquatic product processing byproducts. The preparation process is green and environmentally friendly, highly safe, and has no toxic side effects, showing promising application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The graph shows the effect of different proteases on the degree of hydrolysis.

[0022] Figure 2 The figure shows the results of a single-factor experiment. A: Effect of enzyme concentration on the degree of hydrolysis; B: Effect of hydrolysis temperature on the degree of hydrolysis; C: Effect of hydrolysis time on the degree of hydrolysis; D: Effect of hydrolysis pH on the degree of hydrolysis.

[0023] Figure 3 The response surface and contour plots show the effects of enzymatic hydrolysis pH and temperature on the degree of hydrolysis of MsGP.

[0024] Figure 4 The response surface and contour plots show the effects of enzymatic hydrolysis time and temperature on the degree of hydrolysis of MsGP.

[0025] Figure 5 The response surface and contour plots show the effects of enzymatic hydrolysis pH and enzymatic hydrolysis time on the degree of hydrolysis of MsGP.

[0026] Figure 6This is a distribution diagram of the length of MsGP peptide fragments in the stomach of largemouth bass.

[0027] Figure 7 A statistical chart of GO function classification of gastric polypeptide MsGP from largemouth bass.

[0028] Figure 8 A statistical chart of KOG function classification of MsGP, a gastric polypeptide from largemouth bass.

[0029] Figure 9 A statistical chart showing the KEGG functional classification of MsGP, a gastric polypeptide from largemouth bass.

[0030] Figure 10 This is a diagram showing the structural domains of MsGP, a stomach polypeptide from largemouth bass.

[0031] Figure 11 Fourier transform infrared spectrum of MsGP, a gastric polypeptide from largemouth bass.

[0032] Figure 12 The graph shows the DPPH free radical scavenging capacity of the gastric polypeptide MsGP from largemouth bass.

[0033] Figure 13 This is a graph showing the hydroxyl radical scavenging capacity of MsGP, a gastric polypeptide from largemouth bass.

[0034] Figure 14 This is a graph showing the oxygen free radical scavenging capacity of MsGP, a gastric polypeptide from largemouth bass.

[0035] Figure 15 The graph shows the ABTS free radical scavenging capacity of MsGP, a gastric polypeptide from largemouth bass.

[0036] Figure 16 The image shows the results of the reducing power test. A represents the reducing power of glutathione; B represents the reducing power of MsGP, a gastric polypeptide from largemouth bass.

[0037] Figure 17 The figure shows the effect of MsGP on the proliferation rate of RAW264.7 cells.

[0038] Figure 18 The figure shows the effect of MsGP on the phagocytic ability of RAW264.7 cells.

[0039] Figure 19 The figure shows the effect of MsGP on NO secretion in RAW264.7 cells.

[0040] Figure 20 The figure shows the effect of MsGP on the expression level of IL-1β mRNA in RAW264.7 macrophages.

[0041] Figure 21The figure shows the effect of MsGP on the expression level of RAW264.7 IL-6 mRNA gene in macrophages.

[0042] Figure 22 The figure shows the effect of MsGP on the expression level of iNOS mRNA gene in RAW264.7 macrophages.

[0043] Figure 23 The figure shows the effect of MsGP on the expression level of TNF-α mRNA in RAW264.7 macrophages.

[0044] Figure 24 The figures show the Western Blot results for proteins related to the MAPK signaling pathway. A: Gray-scale analysis of the relevant target proteins in the Western Blot; B: Effect of MsGP on p-P38 expression in RAW264.7 macrophages; C: Effect of MsGP on p-P44 expression in RAW264.7 macrophages; D: Effect of MsGP on p-JNK expression in RAW264.7 macrophages. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0047] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0048] Example 1: Preparation and process optimization of gastric polypeptide (MsGP) from largemouth bass 1.1 Raw material pretreatment After thawing fresh or frozen largemouth bass stomachs (from Huzhou Xincheng Aquatic Products Company), cut open the stomachs with scissors, remove visible fatty tissue, rinse repeatedly with pure water until the water is clear, drain the water, put the stomachs into a food processor and grind them thoroughly, then package them and freeze them at -20℃ for later use.

[0049] 1.2 Screening of optimal enzyme species Alkaline protease (AP), neutral protease (NP), papain (PP), and trypsin (TP) were used for enzymatic hydrolysis experiments. A suitable amount of pretreated largemouth bass stomach was weighed and added to PBS at a mass-to-volume ratio of 1:20 (w / v). The pH of the system was adjusted to the optimal conditions for each protease using 1 mol / L NaOH and HCl (alkaline protease: pH 8.0, 50℃; neutral protease: pH 7.0, 50℃; papain: pH 7.0, 50℃; trypsin: pH 8.0, 50℃). Then, 1 mL of pre-prepared protease solution (enzyme activity 8000 U / g) was added, and the mixture was thoroughly shaken and incubated in a constant-temperature shaker for 4 h for hydrolysis. After the enzymatic hydrolysis reaction was completed, the sample was immediately placed in a boiling water bath for 10 min to inactivate the enzymes and terminate the reaction. The sample was then centrifuged at 4000 r / min for 10 min, and the supernatant was collected to calculate the degree of hydrolysis.

[0050] Degree of hydrolysis determination: The degree of hydrolysis (DH) was calculated according to formula (1) by measuring the content of amino acid nitrogen in the enzymatic hydrolysate and the total nitrogen content in the raw material. The amino acid nitrogen was determined by formaldehyde titration: 5 mL of enzymatic hydrolysate was accurately pipetted into a centrifuge tube and titrated with 0.1 M NaOH solution to a pH of 8.2. Then 10 mL of neutral formaldehyde solution was added, and the solution was titrated with 0.1 M NaOH to a pH of 9.2. The volume of titrant consumed was recorded, and deionized water was used as a control. The total nitrogen content of the sample was determined by the fully automatic Kjeldahl nitrogen analyzer method, referring to the "National Food Safety Standard for Determination of Protein in Food" (GB 5009.5-2016). The main steps were to first digest the stomach of the largemouth bass, then process it with a fully automatic Kjeldahl nitrogen analyzer and titrate it with hydrochloric acid.

[0051] The formula for calculating the degree of hydrolysis is: Degree of hydrolysis (DH) = N1 / N2 × 100% (1) Wherein, N1 is the amino acid nitrogen content in the enzymatic hydrolysate; N2 is the total nitrogen content in the raw material.

[0052] The results are as follows Figure 1 As shown, alkaline protease exhibited the best enzymatic hydrolysis effect, with a degree of hydrolysis reaching 17.8%, significantly higher than neutral protease, papain, and trypsin (P<0.01). Therefore, alkaline protease was selected as the optimal enzyme for the preparation of MsGP.

[0053] 1.3 Optimization of enzymatic digestion conditions through single-factor experiments Single-factor experiments were conducted with enzyme concentration, hydrolysis pH, hydrolysis temperature, and hydrolysis time as independent variables and degree of hydrolysis as the dependent variable. Each experiment was repeated three times.

[0054] (1) Effect of enzyme concentration: With pH 8.5, temperature 50℃ and time 4 h fixed, enzyme concentrations were set to 6000, 7000, 8000, 9000 and 10000 U / g.

[0055] The results are as follows Figure 2 As shown in Figure A, the degree of hydrolysis increases with increasing enzyme concentration. In the range of 6000 U / g to 9000 U / g, the degree of hydrolysis increases with the amount of enzyme added. However, in the range of 9000 U / g to 10000 U / g, the increasing trend of the degree of hydrolysis becomes stable with further increases in enzyme concentration. This is because when the enzyme concentration exceeds the maximum amount of substrate that can bind, further increasing the enzyme concentration does not significantly improve the degree of hydrolysis. In fact, high enzyme concentrations may even lead to intermolecular interactions or feedback inhibition of enzyme activity by the hydrolysis products. Considering production costs, 9000 U / g was chosen as the optimal enzyme concentration.

[0056] (2) Effect of enzymatic hydrolysis temperature: The enzyme concentration was fixed at 8000 U / g, pH 8.5, and time was 4 h. The enzymatic hydrolysis temperatures were set at 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ respectively.

[0057] The results are as follows Figure 2 As shown in Figure B, the degree of hydrolysis reaches its maximum value of 16.23% at an enzymatic hydrolysis temperature of 55 ℃, which is the optimal enzymatic hydrolysis temperature for this enzyme. When the enzymatic hydrolysis temperature is between 40 and 55 ℃, the degree of hydrolysis increases from 11.46% to 16.23%. When the temperature is further increased to 60 ℃, the degree of hydrolysis decreases to 14.6%. As the temperature increases, the degree of hydrolysis decreases instead of increasing. This is because temperature has a significant impact on enzyme activity. Appropriate heating can increase the probability of collision between the enzyme and the substrate and increase the reaction rate. However, after exceeding the enzyme's temperature tolerance threshold, the spatial structure of the alkaline protease gradually dissociates and becomes inactive, resulting in a decrease in catalytic efficiency, which manifests as a decrease in the degree of hydrolysis.

[0058] (3) Effect of enzymatic hydrolysis time: The enzyme concentration was fixed at 8000 U / g, pH 8.5, and temperature at 50℃, and the enzymatic hydrolysis time was set to 2, 3, 4, 5, 6 and 7 h respectively.

[0059] The results are as follows Figure 2 As shown in Figure C, the degree of hydrolysis increased from 11.1% to 19.47% as the hydrolysis time increased from 4 h to 5 h. The degree of hydrolysis peaked at 5 h. With further increases in hydrolysis time, the degree of hydrolysis began to decrease, dropping to 15.53% at 7 h. In the early stages of hydrolysis, sufficient substrate allows for rapid binding between the substrate and enzyme, leading to an increased reaction rate and degree of hydrolysis. In the later stages, insufficient substrate concentration causes enzyme-substrate binding to become saturated, resulting in a decreased reaction rate and consequently, a lower degree of hydrolysis.

[0060] (4) Effect of pH on enzymatic hydrolysis: The enzyme concentration was fixed at 8000 U / g, the temperature was 50℃, and the time was 4 h. The pH values ​​were set to 8.0, 8.5, 9.0, 9.5 and 10.0.

[0061] The results are as follows Figure 2 As shown in Figure D, the degree of hydrolysis initially increases and then decreases with increasing pH, reaching a maximum of 18.27% at pH 8.5. pH affects the active conformation of enzyme molecules, the charge state and solubility of substrate proteins, and the stability of enzyme-substrate complexes. Changes in pH directly influence the binding and catalysis of enzymes and substrates, thus affecting the enzyme's hydrolysis efficiency. Deviations from the optimal pH can lead to enzyme denaturation or difficulty in binding the substrate complex, thereby reducing the degree of hydrolysis.

[0062] The optimal conditions determined by the single-factor experiment were: enzyme concentration of 9000 U / g, enzymatic hydrolysis temperature of 55℃, enzymatic hydrolysis time of 5 h, and enzymatic hydrolysis pH of 8.5.

[0063] 1.4 Optimization of Enzymatic Hydrolysis Process Using Response Surface Methodology Based on the single-factor experiments, a three-factor, three-level response surface methodology was conducted using the degree of hydrolysis as the response value (Y). Three factors were selected: enzymatic hydrolysis temperature (A), enzymatic hydrolysis time (B), and enzymatic hydrolysis pH (C). The Box-Behnken design was used. The factor levels are shown in Table 1, and the experimental scheme and results are shown in Table 2. A total of 17 experiments were conducted, including 5 centerpoint replicates.

[0064] Table 1 Factors and Levels in Response Surface Analysis Table 2 Response Surface Experimental Design and Results The experimental data were subjected to regression analysis using Design-Expert 13 software, and the regression equation model was obtained as follows: Y=18.76+0.3375A+0.425B-0.0625C-0.425AB+0.25AC-0.075BC-1.08A²-0.555B²-0.68C².

[0065] The results of the analysis of variance are shown in Table 3. The model F-value was 66.69, P < 0.0001, indicating that the model was highly significant; the lack-of-fit term P = 0.2114 > 0.05, indicating that the model fits the experimental data well; the coefficient of determination R... 2=0.9885, indicating that the model can explain 98.85% of the response value variation. The order of influence of each factor on the degree of hydrolysis is: hydrolysis time (B) > hydrolysis temperature (A) > hydrolysis pH (C). Among the interactions, the interaction term between hydrolysis temperature and hydrolysis time (AB) is extremely significant (P=0.0006), the interaction term between hydrolysis temperature and hydrolysis pH (AC) is significant (P=0.0103), while the interaction term between hydrolysis time and hydrolysis pH (BC) is not significant (P=0.3312).

[0066] Table 3 Response Surface Experimental Variance Analysis Response surface 3D plot and contour plot, such as Figure 3 (Enzymatic hydrolysis pH and temperature) Figure 4 (Enzymatic hydrolysis time and temperature) Figure 5 (Enzymatic hydrolysis time and pH) are shown. Figure 3 The contour lines are elliptical, indicating an interaction between temperature and pH. Figure 4 The contour lines are elliptical, indicating a significant interaction between temperature and time. Figure 5 The contour lines are nearly circular, indicating a weak interaction between time and pH. All 3D response surface plots are downward-opening parabolic surfaces, suggesting the existence of an optimal combination of parameters that maximizes the degree of hydrolysis.

[0067] The optimal enzymatic hydrolysis conditions, predicted by response surface methodology, were: hydrolysis temperature 55.405℃, hydrolysis time 5.356 h, and pH 8.474, with a predicted degree of hydrolysis of 18.851%. For practical application, the conditions were modified to: hydrolysis temperature 55.4℃, hydrolysis time 5.0 h, and pH 8.5. Three replicate validation experiments were conducted under these conditions, yielding an average degree of hydrolysis of 18.35%, close to the predicted value, indicating the model's reliability.

[0068] 1.5 Preparation of freeze-dried powder of gastric polypeptide (MsGP) from largemouth bass The preparation was carried out on a scale-up basis according to the above-mentioned optimal process: Pretreated largemouth bass stomachs were taken, and PBS was added at a material-to-liquid ratio of 1:20 (w / v). The pH was adjusted to 8.5, and alkaline protease was added until the enzyme activity reached 9000 U / g. Enzymatic hydrolysis was performed at 55.4℃ with constant shaking for 5.0 h. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min, and centrifuged at 4000 r / min for 10 min. The supernatant was collected. The supernatant was pre-frozen and then dried into powder in a vacuum freeze dryer to obtain largemouth bass stomach peptide lyophilized powder, named MsGP. The obtained peptide powder was sealed and packaged, and stored at -20℃ or -80℃ for later use. The MsGP prepared in this example was used for various tests in the following examples.

[0069] Example 2: Detection of the physicochemical properties of gastric polypeptide (MsGP) from largemouth bass 2.1 Basic Component Detection The basic components of MsGP were tested according to national standard methods: Fat content: Tested according to Method II of GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food".

[0070] Protein content: Tested according to Method I of GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food".

[0071] Moisture content: Tested according to Method I of GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food".

[0072] Ash content: Tested according to Method I of GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food".

[0073] Table 4. Basic components of MsGP, a gastric polypeptide from largemouth bass. The test results are shown in Table 4: MsGP contained 45.6 g / 100g of protein, 3.0 g / 100g of fat, 2.19 g / 100g of moisture, and 46.6 g / 100g of ash. The low fat and moisture content and high ash content are presumably due to the use of phosphate buffer and sodium hydroxide to adjust the pH during the enzymatic hydrolysis process.

[0074] 2.2 Peptidomics Analysis The peptide composition of MsGP was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). After desalting and lyophilizing the samples using a C18 Cartridge, they were reconstituted with 0.1% formic acid (FA) solution and separated using a nano-flow HPLC system. Buffer A was a 0.1% formic acid aqueous solution, and buffer B was a 0.08% formic acid-80% acetonitrile aqueous solution. The elution gradient parameters are shown in Table 5. The separated samples were then analyzed by mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer.

[0075] Table 5 Elution gradient parameters for liquid chromatography The analysis results are shown in Table 6. A total of 19 peptides were identified, with molecular weights ranging from 786.31 Da to 2959.17 Da. Experiments demonstrated that peptides with molecular weights less than 3 kDa possess immunomodulatory activity. Based on the start and end positions, the peptide lengths ranged from 8 to 24 amino acid residues.

[0076] DAKELEVM (933.45 Da) contains glutamic acid (E) and methionine (M), with the methionine residue providing a sulfur atom to scavenge free radicals. IDWEYPGSR (1121.51 Da) has an arginine (R) C-terminus and a hydrophobic isoleucine (I) N-terminus, consistent with the structural characteristics of an antioxidant peptide. Peptides such as LDQEHSL (969.44 Da) and DSGDGVTH (786.31 Da) contain histidine (H), which can exert antioxidant effects by chelating metal ions. The N-terminal and C-terminal amino acids have a significant impact on the antioxidant activity of MsGP; hydrophobic amino acids at the N-terminus, such as valine, leucine, and glycine, can increase the antioxidant activity of the peptide.

[0077] Table 6. Basic Information on MsGP Peptide Fragments from the Stomach of Largemouth Bass peptide length distribution as follows Figure 6 As shown: there are 8 short peptides with a length of 11 amino acids or less (accounting for 42.11%), 5 medium-length peptides with a length of 11-20 amino acids, and 6 long peptides with a length of more than 20 amino acids. The higher proportion of short peptides is beneficial for intestinal absorption and bioavailability.

[0078] 2.3 GO Functionality Analysis Gene Ontology (GO) functional annotation was performed on MsGP-derived proteins, and the results are as follows: Figure 7 As shown, the functional distribution of peptide MsGP-related proteins is presented from three aspects: biological processes (BP), cellular components (CC), and molecular functions (MF). In the biological processes category, cellular processes are the most abundant, involving 6 proteins, indicating that MsGP plays a core role in basic cellular life activities; immune system processes involve 1 protein, indicating that MsGP has immune functions. In the cellular components (CC) category, cell structure-related proteins account for a relatively high proportion. In the molecular functions (MF) category, binding function is the most prevalent functional type, involving 11 proteins, suggesting that MsGP may have potential for metal ion chelation, lipid binding, or receptor interaction, which are important characteristics of antioxidant peptides.

[0079] 2.4 KOG Functional Analysis KOG (EuKaryotic Orthologous Groups) functional classification results are as follows: Figure 8As shown, among the MsGP-derived proteins, extracellular structures (W) and the cytoskeleton (Z) accounted for the highest proportions, indicating that MsGP peptides are mainly derived from myosin and actin, suggesting good antioxidant activity. Secondly, signal transduction (T) components accounted for a relatively high proportion, suggesting that MsGP may have functions in cell communication regulation and immune regulation.

[0080] 2.5 KEGG Pathway Analysis The results of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis are as follows: Figure 9 As shown, the two most enriched pathways are cytoskeleton in muscle cells and regulation of actin cytoskeleton, involving six proteins, which corroborates the high abundance of cytoskeleton proteins in the KOG analysis. Furthermore, the enrichment of pathways such as focal adhesion, Salmonella infection, phagosomes, endocytosis, and the intestinal immune network for IgA production suggests that MsGP may be involved in innate immune defense and mucosal immune responses, possessing immunomodulatory activity.

[0081] 2.6 Domain Analysis The results of the domain analysis are as follows Figure 10As shown, the kinesin motor domain superfamily is the most abundant domain type (approximately 2.5 proteins), with a wide range of integrin-related domains including the integrin domain superfamily, the integrin alpha (N-terminal), the integrin alpha chain (C-terminal cytoplasmic region), integrin alpha-2, the integrin alpha beta-propellor, and the integrin alpha chain. Integrins are key molecules in cell adhesion and signal transduction, corresponding to the focal adhesion pathway in KEGG analysis. The presence of collagen triplehelix repeat domains suggests that MsGP contains collagen components.

[0082] 2.7 Fourier Transform Infrared Spectroscopy Analysis Take an appropriate amount of MsGP lyophilized powder and potassium bromide, mix thoroughly, grind, and compress into tablets. Use a Fourier transform infrared spectrometer to perform full-band scanning detection, and use Origin software to process and analyze the obtained spectral data.

[0083] The results are as follows Figure 11 As shown, the MsGP spectrum exhibits typical protein / peptide characteristic absorption peaks, with the amide I band at 1621.82 cm⁻¹. -1 ) and amide II band (1582.49 cm) -1 The presence of ) confirms the secondary structure of the polypeptide, in which the position of the amide I band indicates that β-sheets and random coils are the main conformations; 2924.44 cm -1 The CH2 stretching vibration peak at 1742.81 cm⁻¹ is consistent with previously identified collagen peptides rich in Pro and Gly (such as GPPGPPGPPGQP) (Table 3-5); -1 The C=O stretching vibration may originate from the ester group or the carboxylic acid group.

[0084] 2.8 Relative Molecular Mass Distribution Analysis The relative molecular mass distribution of MsGP was determined using high performance size exclusion chromatography. The detection was performed according to GB 31645-2018 "National Food Safety Standard for Collagen Peptides" at a UV wavelength of 220 nm, and the molecular weight distribution was calculated based on the relative molecular mass calibration curve.

[0085] Table 7. Relative molecular mass distribution of gastric polypeptide MsGP from largemouth bass. The results are shown in Table 7. The relative molecular mass distribution of MsGP is as follows: peptides <189 Da account for 42.17%, 189–500 Da account for 32.48%, 500–1000 Da account for 16.97%, 1000–2000 Da account for 5.33%, 2000–3000 Da account for 1.53%, 3000–5000 Da account for 0.97%, 5000–10000 Da account for 0.48%, and >10000 Da account for 0.07%. Among them, peptides with a molecular weight less than 3000 Da account for 98.48%, indicating that MsGP is mainly composed of small molecule peptides and has good antioxidant and immunomodulatory potential.

[0086] 2.9 Amino acid composition analysis The amino acid composition of MsGP was determined using an automatic amino acid analyzer, in accordance with GB / T 18246-2019 "Determination of Amino Acids in Feed" and GB 5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food".

[0087] Table 8. Amino acid composition of gastric polypeptide MsGP from largemouth bass. The results are shown in Table 8. MsGP contained 18 amino acids, including 8 essential amino acids for adults (threonine, valine, isoleucine, leucine, phenylalanine, lysine, methionine, and tryptophan), with essential amino acids accounting for 30.6% of the total amino acids. It also contained histidine, which is needed by infants and young children. MsGP was rich in acidic amino acids (aspartic acid 3.64%, glutamic acid 5.21%), aromatic amino acids (tyrosine 1.02%, phenylalanine 1.52%, tryptophan 0.23%), and hydrophobic amino acids (alanine 3.08%, valine 1.71%, leucine 2.37%, proline 3.03%, etc.). The presence of these amino acids is an important structural basis for the strong antioxidant activity of MsGP.

[0088] Example 3: Validation of the application of largemouth bass stomach peptide (MsGP) in the preparation of antioxidant products This embodiment is used to verify the antioxidant activity of largemouth bass stomach peptide (MsGP) to demonstrate that it can be used to prepare antioxidant products.

[0089] 3.1 DPPH free radical scavenging ability test The DPPH scavenging ability was measured using a kit (Edison Biotechnology Co., Ltd.). The visible spectrophotometer was preheated for at least 30 min, the wavelength was adjusted to 517 nm, and zeroed with anhydrous ethanol. Reagents were added sequentially to the EP tube (according to the kit instructions), mixed well, and incubated at room temperature (25°C) in the dark for 30 min. The tube was then centrifuged at 12000 rpm for 5 min at room temperature. 800 μL was transferred to a glass cuvette, and the absorbance was read at 517 nm.

[0090] The clearance rate is calculated according to formula (2): P (%) = [1 - (A determination - A control) / A blank] × 100% (2).

[0091] The results are as follows Figure 12 As shown in the figure, within the MsGP concentration range of 2–10 mg / mL, the DPPH radical scavenging rate increased in a concentration-dependent manner, gradually increasing from 12.03% to 78.03%, indicating that this peptide can effectively scavenge DPPH radicals by providing hydrogen atoms and blocking the free radical chain reaction. The positive control glutathione maintained a high scavenging rate of over 90% throughout the same concentration range, and the change with increasing concentration tended to be gradual, reaching near-saturation scavenging effect at low concentrations, demonstrating a stronger DPPH radical scavenging ability. The results indicate that MsGP has good DPPH radical scavenging ability and possesses antioxidant characteristics.

[0092] 3.2 Detection of hydroxyl radical scavenging ability Add 10 mmol / L salicylic acid-ethanol solution, MsGP solution, 10 mmol / L ferrous sulfate solution and distilled water to a test tube in sequence, and finally add 100 mmol / L hydrogen peroxide solution to start the Fenton reaction. After mixing, measure the absorbance at 510 nm.

[0093] The clearance rate is calculated according to formula (3): P (%) = [1-(A1-A2) / A3]×100% (3), where A1 is the absorbance value of the sample with added H2O2; A2 is the absorbance value of the sample without added H2O2; and A3 is the absorbance value of the blank control.

[0094] The results are as follows Figure 13 As shown, MsGP's ability to scavenge hydroxyl radicals increases rapidly with increasing concentration, showing a significant increasing trend in the range of 1–5 mg / mL. At 5 mg / mL, the scavenging rate reaches 95.53%, demonstrating excellent hydroxyl radical scavenging activity.

[0095] 3.3 Detection of oxygen free radical scavenging capacity Mix 4.5 mL of 50 mmol / L Tris-HCl buffer (pH 8.2) with 4.2 mL of distilled water, preheat at 25 °C for 20 min, add 1 mL of MsGP solution, and immediately add 0.3 mL of 30 mmol / L pyrogallic acid (prepared with 10 mmol / L HCl) that has been preheated in a 25 °C water bath. Mix quickly, react in a 25 °C water bath for 5 min, add hydrochloric acid to terminate the reaction, and measure the absorbance at 325 nm.

[0096] The clearance rate is calculated according to formula (4): P(%)=([A0-(A1-A2)) / A0×100% (4), where A0 is the absorbance value of the control group; A1 is the absorbance value of the experimental group; and A2 is the absorbance value of the blank group.

[0097] The results are as follows Figure 14 As shown, the oxygen free radical scavenging rate of MsGP showed a slow increasing trend in the range of 2–10 mg / mL, with a maximum scavenging rate of 35.43%. The positive control glutathione exhibited extremely strong oxygen free radical scavenging ability, with a scavenging rate approaching 100% at 4 mg / mL, and remaining saturated even with subsequent increases in concentration, demonstrating a significant oxygen free radical scavenging effect. MsGP possesses a certain oxygen free radical scavenging ability, but it is weaker than its ability to scavenge DPPH and hydroxyl radicals.

[0098] 3.4 ABTS Free Radical Scavenging Ability Test Preparation of ABTS stock solution: Weigh 0.0384 g of ABTS, dissolve and dilute to 10 mL; weigh 0.0134 g of potassium persulfate, dissolve and dilute to 10 mL; mix and let stand in the dark for 12–16 h. Dilute with PBS (pH 7.4) to obtain the ABTS assay solution. Take 0.4 mL of the assay solution, add 0.1 mL of MsGP solution, shake to mix, and react in the dark for 6 min. Read the absorbance B1 at 734 nm; use 0.1 mL of deionized water instead of the sample as a blank and measure the absorbance B0.

[0099] The clearance rate is calculated according to formula (5): P (%) = (1-(B2-B1) / B0)×100% (5), where B2 is the absorbance value of the experimental group; B1 is the absorbance value of the control group; and B0 is the absorbance value of the blank group.

[0100] The results are as follows Figure 15 As shown, MsGP's ability to scavenge ABTS free radicals increases with increasing concentration, reaching 87.37% at a concentration of 1 mg / mL and nearly 100% (99.99%) at 5 mg / mL, demonstrating highly efficient and stable ABTS free radical scavenging activity comparable to the positive control glutathione. It can exert a strong antioxidant effect even at low concentrations, showing potential for development as a natural antioxidant.

[0101] 3.5 Reduction Ability Test Take a certain concentration of MsGP solution, add 2.5 mL of 0.2 mol / L pH 6.6 PBS and 2.5 mL of 1% K3[Fe(CN)6] solution, mix well, preheat at 50℃ for 20 min, add 2.5 mL of 10% trichloroacetic acid, mix well, centrifuge at 3000 r / min for 10 min, take the supernatant, add distilled water and 0.1% FeCl3 in sequence, use PBS buffer instead of sample to zero the sample, and measure the absorbance at 700 nm.

[0102] The results are as follows Figure 16 As shown in B, within the MsGP concentration range of 2–10 mg / mL, the reducing power increased with increasing concentration, exhibiting a clear concentration dependence. Figure 16 A shows that the positive control glutathione exhibits strong reducing ability in the range of 0.02–0.1 mg / mL. Although MsGP is weaker than glutathione, it shows stable reducing ability, further confirming its in vitro antioxidant activity.

[0103] Based on the results of Example 3, MsGP exhibits scavenging and reducing abilities against DPPH, hydroxyl, oxygen, and ABTS radicals in a concentration-dependent manner, indicating that the largemouth bass stomach polypeptide has good antioxidant activity and can be used to prepare antioxidant products.

[0104] Example 4: Validation of the application of largemouth bass gastric polypeptide (MsGP) in the preparation of immunomodulatory products This embodiment is used to verify the immunomodulatory activity of largemouth bass stomach polypeptide (MsGP) to demonstrate its potential use in the preparation of immunomodulatory products.

[0105] 4.1 Cell Culture Mouse macrophages RAW264.7 were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and incubated at 37°C in a 5% CO2 incubator. Upon cell resuscitation, cryopreservation tubes were rapidly thawed in a 37°C water bath. After sterilization, cells were transferred to centrifuge tubes containing 1 mL of complete medium, centrifuged at 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 2 mL of complete medium. The cells were then transferred to T25 culture flasks, and 3 mL of complete medium was added. Cells were incubated daily. Cell growth was observed daily. When cell density reached 80%–90%, cells were passaged. The culture medium was discarded, 5 mL of fresh medium was added, and cells were gently detached by pipetting. Cells were transferred to centrifuge tubes, and medium was added to a final volume of 10–15 mL. After mixing, cells were aliquoted into new T25 flasks at a ratio of 1:2 or 1:3.

[0106] 4.2 Effect of MsGP on the proliferation rate of RAW264.7 cells RAW264.7 cells in logarithmic growth phase were harvested, adjusted to a suitable density, and seeded into 96-well plates (100 μL / well) and cultured at 37℃ and 5% CO2 for 24 h. The original culture supernatant was discarded, and the cells were treated according to the following groups: the MsGP treatment group had 6 doses (10, 50, 250, 500, 1250, and 6250 μg / mL); the negative control group (NC) received an equal volume of fresh culture medium; and the positive control group received 1 μg / mL LPS. Each group was divided into 3 replicates, with PBS added to the edge wells to prevent edge effects. After culturing for another 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0107] Cell viability is calculated using the formula: Cell viability (%) = [A(drug-added) - A(blank)] / [A(0-drug-added) - A(blank)] × 100%, where A(drug-added): absorbance of wells containing cells, CCK-8 solution, and drug solution; A(blank): absorbance of wells containing culture medium and CCK-8 solution but no cells; A(0-drug-added): absorbance of wells containing cells and CCK-8 solution but no drug solution.

[0108] The results are as follows Figure 17 As shown in the figure. Compared with the NC group (100%), the LPS group significantly promoted cell proliferation (P<0.01). MsGP at concentrations of 10, 50, 250, 500, and 1250 μg / mL all significantly increased cell proliferation rate (P<0.01) in a dose-dependent manner, with the strongest promoting effect at 1250 μg / mL. At 6250 μg / mL, cell viability significantly decreased to approximately 65% ​​(P<0.01), suggesting that this concentration was cytotoxic.

[0109] 4.3 Effect of MsGP on the phagocytic capacity of RAW264.7 cells for neutral red Cell seeding and drug administration were the same as in Example 4.2. After culturing for 24 h, the supernatant was discarded, and the cells were washed 1–2 times with PBS. 100 μL of cell culture medium and 20 μL of neutral red staining solution were added to each well, and the cells were incubated for 2 h. The culture medium containing neutral red staining solution was discarded, and the cells were washed 1–2 times with PBS. 200 μL of neutral red detection lysis buffer was added, and the cells were lysed on a shaker at room temperature for 10 min. The absorbance at 450 nm was measured using a microplate reader.

[0110] The results are as follows Figure 18As shown in the figure. Compared with the NC group, the LPS group significantly promoted the phagocytosis of neutral red by cells (P<0.01). The phagocytic capacity of each concentration of MsGP (10–6250 μg / mL) was significantly higher than that of the NC group (P<0.05), with the best effect achieved at 50 μg / mL (absorbance value 1.35), which was close to the level of the LPS group.

[0111] 4.4 Effect of MsGP on NO secretion in RAW264.7 cells Cells were seeded in 6-well plates and cultured for 24 h. Then, the cells were added according to the groupings in Example 4.2 (MsGP concentrations: 10, 50, 250, 500, 1250, 6250 μg / mL), and cultured for another 24 h. Cell supernatants were collected, and NO content was detected using the Griess reagent method. Griess Reagent I and II were brought to room temperature. Standards (1–100 μM) were diluted with the solution used for the test samples. Standards and samples were added at 50 μL / well in 96-well plates, followed by 50 μL each of room temperature Griess Reagent I and II. The absorbance at 540 nm was measured using a microplate reader.

[0112] The results are as follows Figure 19 As shown in the figure, compared with the NC group, the LPS group significantly promoted NO secretion (P<0.01). The MsGP treatment group showed a clear dose-dependent increase in NO production: at low concentrations (10–500 μg / mL), NO production gradually increased from approximately 0.8 μM to 1.85 μM; at 1250 μg / mL, NO production significantly increased to 5.37 μM; and at 6250 μg / mL, it further increased to 7.4 μM. These results indicate that MsGP can effectively activate macrophages and promote NO secretion, and the activation effect gradually increases with increasing concentration. However, even at the highest concentration, its ability to induce NO production is still lower than that of LPS, suggesting that MsGP may activate macrophages in a mild manner rather than causing a strong inflammatory response. This is of great significance for the development of functional foods or drugs with immunomodulatory functions.

[0113] 4.5 Effects of MsGP on the expression levels of secreted cytokine genes in RAW264.7 cells 4.5.1 Total RNA Extraction Cells were seeded in 6-well plates and cultured for 24 h with MsGP (concentrations: 10, 50, 250, 1250 μg / mL). Cells were washed twice with pre-chilled PBS, and lysed with 1 mL of Trizol reagent. 200 μL of chloroform was added, and the mixture was vigorously inverted for 30 seconds. The mixture was incubated on ice for 15 min, then centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected. 400 μL of pre-chilled isopropanol was added, and the mixture was incubated at room temperature for 10 min, then centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was discarded, and the precipitate was washed with 1 mL of 75% ethanol (prepared with DEPC-treated water). The precipitate was centrifuged at 7500 rpm for 15 min at 4°C, and this process was repeated once. The ethanol was discarded, and the precipitate was air-dried at room temperature until translucent. 50 μL of DEPC-treated water was added to dissolve the RNA. The extracted RNA had OD260 / OD280 values ​​between 1.9 and 2.0, and concentrations between 800 and 1400 μg / μL, indicating good RNA integrity.

[0114] 4.5.2 Reverse transcription and qRT-PCR cDNA was synthesized by reverse transcription according to the FastKing cDNA First-Strand Synthesis Kit instructions. qRT-PCR was performed using SuperReal Premixed PCR kit. The reaction mixture (20 μL) consisted of 10 μL of 2×SuperRealPreMix Plus, 1 μL each of forward and reverse primers (10 μM), an appropriate amount of cDNA template, and RNase-free ddH2O to a final volume of 20 μL. The reaction program was: 95℃ pre-denaturation for 15 min; 40 cycles: 95℃ denaturation for 10 sec, 60℃ annealing / extension for 32 sec (fluorescence signal acquisition). Primer sequences are shown in Table 9, with β-actin as an internal reference gene. The relative expression level of the target gene was calculated using the 2^-ΔΔCt method.

[0115] Table 9 Primers for Real-Time Quantitative PCR 4.5.3 Results IL-1β gene expression: such as Figure 20 As shown, the expression level of IL-1β mRNA in the LPS group was approximately 1200-fold higher than that in the NC group (P<0.01). Low concentrations of MsGP (10, 50 μg / mL) had no significant effect; the expression level increased to approximately 8-fold at 250 μg / mL (P<0.01); and further increased to approximately 30-fold at 1250 μg / mL (P<0.01), showing a dose-dependent effect.

[0116] IL-6 gene expression: such as Figure 21As shown, the expression level of IL-6 mRNA in the LPS group was approximately 5000 times that in the NC group (P<0.01). After MsGP treatment, IL-6 expression was upregulated in a dose-dependent manner with increasing concentration, showing a significant increase at concentrations of 250 μg / mL and above, and reaching a peak at 1250 μg / mL.

[0117] iNOS gene expression: such as Figure 22 As shown, the expression level of iNOS mRNA in the LPS group was significantly increased (P<0.01). The expression of MsGP was significantly increased at concentrations of 50 μg / mL and above, reaching the highest value at 1250 μg / mL, showing a dose-dependent effect.

[0118] TNF-α gene expression: such as Figure 23 As shown, TNF-α mRNA expression was significantly increased in the LPS group (P<0.01). MsGP expression was significantly increased at 250 μg / mL and extremely significantly increased at 1250 μg / mL, showing a dose-dependent effect.

[0119] The above results indicate that MsGP can effectively activate RAW264.7 cells at the transcriptional level and upregulate the gene expression of immune-related cytokines such as IL-6, iNOS, IL-1β, and TNF-α.

[0120] 4.6 Effects of MsGP on the MAPK signaling pathway in RAW264.7 cells 4.6.1 Cell processing and protein extraction RAW264.7 cells in logarithmic growth phase were seeded into 10 cm culture dishes (12 mL / dish) and cultured for 12 h. Different concentrations of MsGP (10, 50, 250, 1250 μg / mL) were added to each dish. The positive control group received 1 μg / mL LPS, and the negative control group received an equal volume of culture medium. Cells were cultured for another 12 h. The supernatant was discarded, and the cells were washed with pre-chilled PBS. 500 μL of 1× lysis buffer (containing protease / phosphatase inhibitors) was added to each dish, and the cells were incubated on ice for 5 min. Cells were scraped, briefly sonicated, transferred to microcentrifuge tubes, centrifuged at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit (Shanghai Yuanye Biotechnology Co., Ltd.).

[0121] 4.6.2 Western Blot Detection Detection was performed using an automated Western blot quantitative analysis system (Abby). DTT, 5× Master Mix, Ladder, and samples were prepared and denatured at 95°C for 5 min, followed by cooling on ice for 5 min. Primary antibodies were diluted with Antibody Diluent II (p-P38, p-P44 / ERK1 / 2, p-JNK, and corresponding total protein antibodies). The chemiluminescent buffer was a mixture of equal volumes of Lumino-S and Peroxide. The prepared reagents were added to a dedicated plate, centrifuged at 1000 g for 5 min, and then analyzed using the system. Grayscale analysis was performed using Compass for SW 6.2.0 software.

[0122] 4.6.3 Results The results are as follows Figure 24 As shown. Figure 24 A is the grayscale analysis image of the relevant target protein in Western blotting.

[0123] p-P38 ( Figure 24 B): The expression levels of p-P38 protein in each MsGP treatment group were significantly higher than those in the blank control group (NC), showing a clear concentration-dependent upregulation trend, indicating that MsGP can effectively activate the P38 MAPK signaling pathway.

[0124] p-P44 / ERK1 / 2 ( Figure 24 C): When the MsGP concentration was 10, 50, 250, and 1250 μg / mL, the protein phosphorylation level of p-P44 was statistically different from that of the NC group (P<0.05), and the expression level gradually increased with the increase of MsGP concentration, indicating that MsGP can dose-dependently activate the P44 MAPK signaling pathway.

[0125] p-JNK ( Figure 24 D): After treatment with different concentrations of MsGP, the protein phosphorylation level of p-JNK was not significantly different from that of the NC group (P>0.05), suggesting that MsGP did not significantly activate the JNK MAPK signaling pathway.

[0126] In summary, MsGP mainly exerts its immunomodulatory effect by activating the P38 and P44 (ERK1 / 2) MAPK signaling pathways, exhibiting good immunomodulatory activity and can be used to prepare immunomodulatory products.

[0127] Example 5: Application of largemouth bass gastric peptides in the preparation of products with both antioxidant and immunomodulatory functions Based on the results of Examples 3 and 4, MsGP exhibits significant antioxidant activity (highly efficient scavenging and reducing capacity against DPPH, hydroxyl, and ABTS cation radicals) and immunomodulatory activity (promoting macrophage proliferation, phagocytosis, NO secretion, cytokine expression, and activation of the P38 / P44 MAPK signaling pathway). Therefore, MsGP can be used to prepare products requiring both antioxidant and immunomodulatory functions, such as health supplements or pharmaceuticals.

[0128] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and 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 defined by the claims of the present invention.

Claims

1. The application of largemouth bass gastric polypeptides in the preparation of antioxidant and / or immunomodulatory products, characterized in that, The largemouth bass stomach polypeptide is a polypeptide product obtained by alkaline protease hydrolysis of the largemouth bass stomach. The components with a molecular weight of less than 3000 Da account for more than 98% of the total peptides, and the polypeptide product contains one or more peptides selected from the following: DAKELEVM, IDWEYPGSR, LDQEHSL, DSGDGVTH, GPPGPPGPPGQP, VEPLDPPE.

2. The application according to claim 1, characterized in that, The components of the largemouth bass gastric polypeptide with a molecular weight less than 3000 Da account for 98.48% of the total peptide weight, and the components with a molecular weight less than 1000 Da account for more than 90% of the total peptide weight.

3. The application according to claim 1, characterized in that, The largemouth bass stomach polypeptide contains 19 peptide segments with a molecular weight distribution ranging from 786.31 Da to 2959.17 Da. Among them, short peptides with a length of 11 amino acids or less account for 42.11% of the total number of peptide segments.

4. The application according to claim 1, characterized in that, The Fourier transform infrared spectrum of the gastric polypeptide from the largemouth bass was at 1621.82 cm⁻¹ in the amide I band. -1 Amide II band 1582.49 cm -1 CH2 stretching vibration peak 2924.44 cm⁻¹ -1 The C=O stretching vibration peak is 1742.81 cm⁻¹. -1 It has a characteristic absorption peak.

5. The application according to claim 1, characterized in that, The preparation method of the largemouth bass stomach polypeptide includes the following steps: adding the pretreated largemouth bass stomach to phosphate buffer, adjusting the pH to 8.0-9.0, adding alkaline protease at a dosage of 7000-10000 U / g, enzymatically hydrolyzing at 50-60℃ for 4-6 hours, inactivating the enzyme, centrifuging, and freeze-drying to obtain the polypeptide.

6. The application according to claim 5, characterized in that, In the preparation method of the gastric polypeptide from largemouth bass, the enzymatic hydrolysis conditions are: pH 8.5, temperature 55.4℃, time 5.0 hours, and enzyme dosage 9000 U / g.

7. The application according to claim 1, characterized in that, The antioxidant product is used to scavenge at least one of DPPH free radicals, hydroxyl free radicals, and ABTS cationic free radicals, and / or to provide reducing power; the largemouth bass stomach peptide has a scavenging rate of 12.03% to 78.03% for DPPH free radicals at a concentration of 2 to 10 mg / mL; and / or, the largemouth bass stomach peptide has a scavenging rate of 95.53% for hydroxyl free radicals at a concentration of 5 mg / mL; and / or, the largemouth bass stomach peptide has a scavenging rate of 99.99% for ABTS cationic free radicals at a concentration of 5 mg / mL.

8. The application according to claim 1, characterized in that, The immunomodulatory product is used to promote macrophage proliferation, enhance macrophage phagocytic capacity, promote macrophage secretion of nitric oxide, and upregulate the expression levels of immune-related cytokine genes at at least one of the following: largemouth bass stomach peptide can significantly promote RAW264.7 macrophage proliferation at a concentration range of 10–1250 μg / mL; and / or, the largemouth bass stomach peptide can significantly enhance the phagocytic capacity of RAW264.7 macrophages for neutral red at a concentration range of 10–6250 μg / mL; and / or, the largemouth bass stomach peptide can increase the nitric oxide secretion of RAW264.7 macrophages to 5.37 μM at a concentration of 1250 μg / mL.

9. The application according to claim 8, characterized in that, The immune-related cytokines are selected from one or more of IL-1β, IL-6, iNOS, and TNF-α; the largemouth bass gastric polypeptide can significantly upregulate the mRNA expression level of the cytokines in RAW264.7 macrophages at concentrations of 250 μg / mL and 1250 μg / mL.

10. The application according to claim 9, characterized in that, The largemouth bass stomach peptide, at a concentration of 1250 μg / mL, significantly increased the expression levels of p-P38 and p-P44 proteins in RAW264.7 macrophages.

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