Bullfrog protein peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and provides bullfrog protein peptide and a preparation method and application thereof, and the method comprises the following steps: decoloring pretreated bullfrog leftovers to obtain decolored leftovers; after degreasing treatment, stirring at normal temperature and washing to obtain crude protein; adding a reaction medium and protease for enzymolysis, separating soluble peptide after inactivation, and freeze-drying to obtain solid protein peptide. The bullfrog whole leftovers are subjected to decoloration, degreasing and enzymolysis for preparation, the method is simple and efficient, protein resource utilization is improved, and industrial application is facilitated; when the additive is applied to a perch feed, the oxidation resistance and the growth performance of perch can be improved, when 1% of the additive is added, the CAT activity and the SOD activity of perch are improved by 40.72% and 51.94% respectively compared with those of a control group, when 0.5% of the additive is added, the weight gain rate and the specific growth rate are improved by 11.46% and 4.41% respectively, and the additive has a good application prospect.

Description

[0001] This application is a divisional application of the invention patent application filed on November 20, 2025, entitled "Bullfrog protein peptides with antioxidant activity and their preparation method and application", with application number 202511708070.7. Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to a bullfrog protein peptide, its preparation method, and its application. Background Technology

[0003] Sea bass (Lateolabrax japonicus), an important freshwater and marine aquaculture species, is highly favored by consumers for its tender meat, rich nutrition, and lack of fishy smell, making it a highly valuable economic product. In recent years, with the expansion of aquaculture scale, the sea bass industry has faced problems such as high-temperature stress (e.g., summer water temperatures often exceed 28°C, increasing sea bass metabolic rate and oxygen consumption, leading to increased reactive oxygen species) and frequent diseases, resulting in slower growth rates and lower survival rates, thus affecting the development of sea bass aquaculture. Research shows that improving the antioxidant capacity of sea bass is a key way to alleviate these problems; therefore, developing efficient, safe, and low-cost antioxidant feed additives is an urgent need in the sea bass aquaculture sector. Bullfrog (Lithobates cates beiana) is also an important characteristic economic aquatic product, with its aquaculture scale expanding year by year and widely used in the catering industry. However, the processing of bullfrogs for consumption generates a large amount of by-products (such as heads, feet, skin, bones, and internal organs), which are usually discarded directly, resulting in a waste of protein resources.

[0004] Protein peptides are small molecule peptides prepared through enzymatic hydrolysis and fermentation. They possess characteristics such as small molecular weight, easy absorption, high bioavailability, and diverse functional activities (e.g., antioxidant and immunomodulatory), making them a research hotspot in feed additives. Among these, enzymatic hydrolysis is widely used in protein peptide preparation due to its mild reaction conditions, high specificity, high safety, and ease of control. Currently, existing enzymatic hydrolysis processes for all components of bullfrog by-products are not yet mature, and most studies focus on a single hypoglycemic function, failing to effectively combine the growth and antioxidant needs of aquaculture animals. For example, Chinese invention patent application CN119285752A discloses a bullfrog skin peptide with DPP-IV inhibitory activity, its purification method, and its application. This method involves enzymatic hydrolysis to extract polypeptides, followed by column chromatography purification to extract components containing peptide fragments. Finally, the components are evaluated and screened to obtain purified bullfrog skin peptides with DPP-IV inhibitory activity, which are then formulated into hypoglycemic drugs. However, this method only uses bullfrog skin. In addition to the directly peeled bullfrog skin, the bullfrog by-products also include unpeeled bullfrog skin. The above method requires screening and peeling of bullfrog skin. The processing method also requires column chromatography for purification. The whole processing method is relatively complicated and the actual operation process is time-consuming, making it difficult to prepare on a large scale. Summary of the Invention

[0005] In view of this, as one aspect of the present invention, an embodiment of the present invention provides a method for preparing bullfrog protein peptides, the method comprising: The pre-treated bullfrog by-products were decolorized to obtain decolorized bullfrog by-products. The decolorized bullfrog by-products were subjected to defatting treatment to obtain bullfrog crude protein. A reaction medium and a protease were added to the crude bullfrog protein, and enzymatic hydrolysis was performed according to a preset hydrolysis time to obtain the hydrolysate. After inactivating the protease, soluble bullfrog protein peptides were separated from the enzymatic hydrolysate, and then freeze-dried to obtain solid bullfrog protein peptides.

[0006] As a second aspect of the present invention, embodiments of the present invention provide a bullfrog protein peptide, prepared by the method described in any of the preceding claims, wherein the amino acid sequence of the protein peptide is one of Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr, Leu-Leu-Pro-Tyr-Asp-Gln-Tyr, and Trp-Arg-Pro-Pro-Asn-Trp.

[0007] As a third aspect of the present invention, embodiments of the present invention provide the application of bullfrog protein peptides as described above, characterized in that: the bullfrog protein peptides are used to prepare feed for aquatic species.

[0008] Beneficial effects: In summary, the embodiments of the present invention provide a bullfrog protein peptide, its preparation method, and its application. The method includes: decolorizing pretreated bullfrog by-products to obtain decolorized bullfrog by-products; defatting the decolorized bullfrog by-products to obtain crude bullfrog protein; adding a reaction medium and a protease to the crude bullfrog protein and performing enzymatic hydrolysis according to a preset time to obtain a hydrolysate; inactivating the protease and separating soluble bullfrog protein peptides from the hydrolysate; and freeze-drying the hydrolysate to obtain solid bullfrog protein peptides. This method uses bullfrog by-products as a substrate to prepare bullfrog protein peptides through decolorization, defatting, and enzymatic hydrolysis. The method is not only simple and efficient but also effectively utilizes all bullfrog by-products, improving the utilization of bullfrog protein resources and facilitating large-scale industrial application. When applied to sea bass feed, it enhances the antioxidant capacity and growth performance of sea bass. Adding 1% of the peptide increases the CAT and SOD activities of sea bass by 40.72% and 51.94% respectively compared to the control group. Adding 0.5% increases the weight gain rate and specific growth rate by 11.46% and 4.41% respectively, demonstrating promising application prospects. It addresses the problem of existing technologies that only utilize bullfrog skin as a single component for protein peptide preparation, resulting in the direct discarding of all by-products and wasting protein resources. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort, and these are all within the protection scope of the present invention.

[0010] Figure 1 Bar graph showing the peptide concentration and TCA-NSI data under different protease conditions.

[0011] Figure 2 Bar graphs showing the concentration of peptides and TCA-NSI under different protease combinations.

[0012] Figure 3 Bar graphs showing the concentrations of peptides and TCA-NSI under different ratios of alkaline protease and neutral protease.

[0013] Figure 4 This is a bar graph showing the peptide concentrations and TCA-NSI data under different enzyme addition conditions for alkaline and neutral proteases.

[0014] Figure 5 This is a bar graph showing the concentration of peptides and TCA-NSI data from enzymatic hydrolysis of alkaline and neutral proteases under different pH conditions.

[0015] Figure 6 This is a bar graph showing the peptide concentrations and TCA-NSI data from enzymatic hydrolysis of alkaline and neutral proteases under different temperature conditions.

[0016] Figure 7 This is a bar graph showing the peptide concentration and TCA-NSI data under different enzymatic hydrolysis time conditions for alkaline protease and neutral protease.

[0017] Figure 8 This is a response surface plot showing the effect of enzyme dosage and pH interaction on TCA-NSI.

[0018] Figure 9 This is a response surface plot showing the effect of temperature and pH interaction on TCA-NSI.

[0019] Figure 10 This is a response surface plot showing the effect of time and pH interaction on TCA-NSI.

[0020] Figure 11 This is a response surface plot showing the effect of the interaction between enzyme dosage and temperature on TCA-NSI.

[0021] Figure 12 This is a response surface plot showing the effect of time and temperature interaction on TCA-NSI.

[0022] Figure 13 This is a response surface plot of time and enzyme dosage interaction to TCA-NSI.

[0023] Figure 14 This is a line graph showing the scavenging rate of bullfrog protein peptides against hydroxyl radicals.

[0024] Figure 15 This is a line graph showing the measured data of the DPpH free radical scavenging rate of bullfrog protein peptides.

[0025] Figure 16 This is a line graph showing the measurement data of the ABTS+ free radical scavenging rate of bullfrog protein peptides.

[0026] Figure 17 This is a bar graph showing the measurement data of sea bass weight gain rate.

[0027] Figure 18 This is a bar graph showing the measured growth rate data of sea bass.

[0028] Figure 19 This is a bar graph showing the catalase activity data from sea bass livers.

[0029] Figure 20 This is a bar graph showing the superoxide dismutase activity assay data of sea bass liver.

[0030] Figure 21This is a flowchart of a method for preparing bullfrog protein peptides according to an embodiment of the present invention. Detailed Implementation

[0031] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only configured to explain the present invention and are not configured to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the invention.

[0032] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0033] Example 1 See Figure 21 This invention mainly relates to a bullfrog protein peptide, its preparation method, and its application, wherein the method includes the following steps: S1: Decolorize the pre-treated bullfrog by-products to obtain decolorized bullfrog by-products; S2: Degrease the decolorized bullfrog by-products to obtain bullfrog crude protein; S3: Add reaction medium and protease to the bullfrog crude protein, and perform enzymatic hydrolysis according to the preset hydrolysis time to obtain hydrolysate; S4: After inactivating the protease, soluble bullfrog protein peptides are separated from the enzymatic hydrolysate, and then freeze-dried to obtain solid bullfrog protein peptides.

[0034] In step S1, before processing the washed bullfrog by-products, the by-products are cut into small pieces of 1 to 2 centimeters to increase their specific surface area. This allows the by-products to fully contact the decolorizing agent, degreasing agent, and protease, avoiding uneven reactions caused by a small contact area due to direct processing. It should be noted that some pigments have strong adsorption properties, which can coat protein particles or bind to the active sites of proteases, hindering the contact between the protease and the bullfrog by-products, thus affecting the enzymatic hydrolysis efficiency. The decolorizing agent is mainly used to remove melanin, carotenoids, and other pigments from the skin and internal organs of the bullfrog by-products to prevent pigment residues from adsorbing protein particles or binding to the active sites of proteases.

[0035] In step S2, because bullfrog by-products contain a large amount of fat, the fat will coat the protein particles, hindering the contact between the protease and the bullfrog by-products; at the same time, fat oxidation products will affect the antioxidant activity of peptides. A defatting agent is used to remove lipid impurities. After defatting, the relative protein content in the bullfrog by-products can be increased, interference from the enzymatic hydrolysis system can be reduced, and the enzymatic hydrolysis can be made more efficient.

[0036] In step S3, the catalytic activity of the protease needs to be realized in a reaction medium. The reaction medium can be a protein molecule that expands and exposes its active site, while promoting the dissolution and dispersion of the protein and ensuring that the protease and bullfrog protein are in full contact.

[0037] In step S4, inactivating the protease avoids excessive hydrolysis that could lead to overly short peptides, loss of activity, or bitterness. After separating the soluble bullfrog protein peptides, freeze-drying at low temperatures removes moisture, preventing peptide oxidation or degradation and preserving their antioxidant activity.

[0038] In one embodiment, the decolorization treatment of the pretreated bullfrog by-products to obtain decolorized bullfrog by-products includes: S11: The bullfrog offal is cleaned and cut to a preset size, wherein the preset size is between 1 and 2 centimeters; The preset size refers to the range of material geometry dimensions set for subsequent mass transfer and reaction uniformity. This step involves cleaning to remove soluble impurities such as mud and blood from the surface, and cutting the bullfrog scraps into blocks or strips of one to two centimeters. The purpose is to increase the specific surface area and pore openness, allowing the subsequent decolorizing agent to penetrate the tissue more easily and act uniformly, avoiding the situation where large pieces of material cause excessive reaction on the outer layer and insufficient reaction on the inner layer. This is achieved by repeatedly rinsing with running water or diluted weak alkaline water until clear, and then quickly cutting the material to the specified size using clean knives or shearing tools, maintaining a consistent thickness as much as possible, so that the material can be fully spread and submerged in the container. This results in more stable mass transfer conditions and a more controllable reaction start point, reducing batch variations and shortening the decolorization time.

[0039] S12: According to the first preset mass ratio, a decolorizing agent is added to the sheared bullfrog scraps, wherein the decolorizing agent is a sodium hydroxide solution containing 1% hydrogen peroxide, and the first preset mass ratio is the mass ratio of bullfrog scraps to sodium hydroxide solution of 1:3 to 1:5. The decolorizing agent is a sodium hydroxide solution containing one mass percentage of hydrogen peroxide, with a first preset mass ratio of material to solution of 1:3 to 1:5. The purpose of this step is to induce the generation of active hydroxyl radicals from hydrogen peroxide in an alkaline environment, thereby breaking the conjugated structure of pigment molecules. Simultaneously, sufficient liquid phase ensures complete immersion and adequate convection, efficiently removing oxidized soluble small molecules. During implementation, a fresh solution is prepared according to the target mass ratio. After adding the sheared material, it is gently stirred to ensure full immersion and prevent agglomeration, maintaining a stable margin of liquid level above the material surface in the container. The beneficial effects of this step are a balance between oxidation efficiency and protein protection. The one mass percentage of hydrogen peroxide prevents over-oxidation of amino acid side chains, and the 1:3 to 1:5 liquid-to-solid ratio ensures uniform distribution of alkalinity and active species, thus improving decolorization uniformity and reducing the risk of subsequent enzymatic hydrolysis failure.

[0040] S13: Replace the decolorizing agent according to a preset time interval until decolorized bullfrog by-products are obtained, wherein the preset time interval is 20 to 40 minutes.

[0041] Specifically, the replacement strategy with a preset time interval of 20 to 40 minutes refers to promptly replacing the consumed and contaminated decolorizing solution after a period of time in a single oxidation reaction to maintain the system's effective oxidation capacity and mass transfer driving force. The aim is to avoid reverse adsorption phenomena that occur after the pigment concentration in the solution increases and to inhibit the efficiency decline caused by the self-decomposition of active species. To implement this, a timer is set, and at the end of each time window, the old solution is quickly poured out or filtered, and new solution of the same formulation is immediately added while resuming gentle stirring. If necessary, a short rinse is performed at the moment of replacement to remove free pigments from the surface. This maintains a stable reaction potential and a clean boundary layer, reduces localized residual color spots, and makes the decolorization endpoint more predictable.

[0042] In this embodiment, hydrogen peroxide can generate hydroxyl radicals under alkaline conditions, which can attack the conjugated double bonds in the pigment molecules and oxidize them into colorless small molecules. The 1% low concentration of hydrogen peroxide avoids excessive oxidation that affects the oxidative inactivation of amino acid residues in bullfrog crude protein. The 1% concentration can effectively decolorize while reducing damage to the protein structure.

[0043] In the above embodiments, sodium hydroxide provides alkaline conditions, which can promote the decomposition of hydrogen peroxide; at the same time, sodium hydroxide can dissolve some water-soluble pigments and mucopolysaccharide impurities, reduce the adsorption of pigments on the protein surface, and make tissues such as skin and internal organs swell and loosen, so that the pigments can fully contact the decolorizing agent.

[0044] In the above embodiments, as the decolorization time is extended, the concentration of dissolved pigment molecules in the solution increases, and some or all of them are re-adsorbed back onto the material surface. Replacing the decolorizing agent according to a preset time interval can reduce the pigment concentration in the solution and reduce the reverse adsorption phenomenon.

[0045] In the above embodiments, the preset time interval can be set according to the composition ratio of bullfrog by-products. For example, when the skin proportion in the bullfrog by-products is high, the pigment content is relatively high, such as skin melanin. The preset time interval can be set to 20 minutes, that is, the decolorizing agent is replaced every 20 minutes. 20 minutes can quickly reduce the pigment load and avoid local pigment residue. When the internal organs of the bullfrog by-products are high, the preset time can be set to 40 minutes, which can reduce the number of operations and reduce reagent consumption and labor costs. It should be noted that the preset time intervals can also be set in combination. For example, when the decolorizing agent is added for the first time, it can be replaced after 20 minutes of decolorization, and then replaced again after 30 minutes of decolorization.

[0046] In one embodiment, the mass ratio of the bullfrog by-products to the sodium hydroxide solution is 1:3 to 1:5.

[0047] In this embodiment, since the bullfrog by-products are dense and contain a large amount of connective tissue, a sufficient amount of liquid is required to achieve complete immersion. The mass ratio of bullfrog by-products to sodium hydroxide solution is 1:3. The sodium hydroxide solution can completely immerse the bullfrog by-products and has enough sodium hydroxide to penetrate into the interior of the material, dissolving and carrying out the fat-soluble pigments.

[0048] In this embodiment, a mass ratio of 1:3 to 1:5 can ensure the stability of the system pH. If the mass ratio of bullfrog by-products to sodium hydroxide solution is 1:5, it will lead to dilution of sodium hydroxide concentration and affect enzymatic hydrolysis efficiency.

[0049] In one embodiment, the degreasing agent is any one of petroleum ether, polysorbate, or lipase.

[0050] In this embodiment, the nonpolar molecules of petroleum ether can penetrate into adipose tissue, forming intermolecular forces with fat molecules, causing fat to dissolve from proteins and forming a mixed solution of oil and ether. The petroleum ether is then removed by static separation or rotary evaporation, achieving the purpose of fat removal. The hydrophobic groups in polysorbate molecules can insert into fat particles, while the hydrophilic groups are exposed to the aqueous phase, forming a stable emulsion that allows fat to detach from the protein surface. Crude bullfrog protein is retained by filtration. Lipase acts on the ester bonds of fat molecules, hydrolyzing them into glycerol and free fatty acids. The products are soluble in water or neutralized by a weak alkali to form soap salts. This process only hydrolyzes fat and does not affect proteins; it may include active peptide precursors from bullfrog by-products.

[0051] It should be noted that, in the above embodiments, petroleum ether is preferably used as the degreasing agent.

[0052] In one embodiment, the defatting treatment of the decolorized bullfrog by-products to obtain bullfrog crude protein includes: S21: According to the second preset mass ratio, a degreasing agent is added to the decolorized bullfrog by-products, wherein the degreasing agent is one of petroleum ether, polysorbate or lipase, and the second preset mass ratio is the mass ratio of the decolorized bullfrog by-products to the degreasing agent is 1:3 to 1:5. Specifically, the second preset mass ratio refers to a mass ratio of 1:3 to 1:5 between the decolorized material and the degreasing agent. The degreasing agent is one of petroleum ether, polysorbate, or lipase. Its purpose is to provide a sufficient continuous phase and a stable mass transfer channel, allowing lipids to migrate from the tissue to the outside and be carried out, while avoiding excessive dilution that could cause protein immersion damage and increased residue. During implementation, the degreasing agent is added to the decolorized material according to the stated mass ratio, ensuring complete immersion and maintaining gentle convection. If petroleum ether is selected, operation is preferably carried out under closed and ventilated conditions. If polysorbate is selected, it is diluted with aqueous phase to the working concentration to form a stable emulsion. If lipase is selected, the initial pH is recorded for subsequent gentle washing. This allows for efficient degreasing while ensuring safety and controllability, reducing the risk of re-adsorption of lipid-soluble inhibitors and pigments, and improving the consistency of the starting point for subsequent enzymatic hydrolysis.

[0053] S22: Under a preset temperature condition, the bullfrog offal and degreasing agent are stirred according to a preset stirring time of 1 to 3 hours; The preset temperature conditions refer to the safe and effective operating temperature range that matches the selected degreasing agent. Petroleum ether is best used at a slightly lower temperature to reduce volatility and safety hazards, polysorbate is best used at room temperature to obtain a stable emulsion system, and lipase is best used at a mild temperature range to maintain catalytic activity. The preset stirring time is one to three hours, the purpose of which is to improve the interface renewal frequency and diffusion rate through a moderate flow field, while avoiding excessive shear that could lead to protein structure damage and the release of soluble impurities. During implementation, constant-speed stirring is used to keep the system in uniform suspension without forming obvious eddies. If necessary, short-term stops are made to facilitate lipid phase separation or emulsion stabilization. Once the preset time is reached, the solid-liquid separation process begins. This control balances degreasing efficiency with protein integrity, reducing the subsequent washing load and protein loss.

[0054] S23: After stirring, the bullfrog by-products are washed to obtain bullfrog crude protein. After stirring, washing is performed to obtain bullfrog crude protein. The purpose is to remove residual defatting agents, carried-out lipids, and soluble impurities, restoring the system to a neutral or near-neutral environment suitable for subsequent processes. The process involves first performing solid-liquid separation, followed by batch washing with appropriate amounts of clean water or weakly alkaline water until the effluent is clear and free of a noticeable oil film. If petroleum ether was used in the previous step, an additional standing separation or short-term low-temperature evaporation is added to reduce residual solubility. If polysorbate was used, a short wash with warm water is added to break the emulsion and remove surface-adsorbed lipids. If lipase was used, washing is performed under mild conditions to avoid further hydrolysis. This step yields bullfrog crude protein with significantly reduced lipid content and well-preserved structure, providing a cleaner and more stable substrate for subsequent enzymatic reactions.

[0055] The mass ratio of the decolorized bullfrog by-products to the degreasing agent is 1:3 to 1:5; the preset stirring time is 1 to 3 hours.

[0056] In this embodiment, a mass ratio of bullfrog by-products to defatting agent of 1:3 to 1:5 ensures the defatting rate of the bullfrog by-products. A stirring time of 1 to 3 hours avoids over-stirring that could damage the protein structure.

[0057] In one embodiment, the step of adding a reaction medium and a protease to the bullfrog crude protein, and performing enzymatic hydrolysis according to a preset hydrolysis time to obtain the hydrolysate includes: S31: Add protease to the liquid according to the preset enzyme dosage, wherein the preset enzyme dosage is 100 to 800 U; Add a complex protease to the bullfrog crude protein according to the preset enzyme dosage, at a rate of 100 to 800 units per gram of substrate. The purpose of this step is to ensure sufficient catalytic sites cover the substrate surface and interior, achieving a controllable initial fragmentation rate and providing a basis for subsequent refinement. During implementation, first thoroughly disperse the bullfrog crude protein in the reaction medium, then add the alkaline protease and neutral protease in one go or in portions according to the target enzyme dosage, gently stirring to ensure uniform contact and avoid excessive hydrolysis caused by localized high concentrations. This ensures sufficient activity supply while reducing the risk of early runaway, allowing the reaction to enter a stable phase.

[0058] S32: Enzymatic hydrolysis is performed according to a preset hydrolysis time and a preset hydrolysis temperature to obtain the hydrolysate, wherein the protease includes alkaline protease and neutral protease; the preset hydrolysis time is 5 to 50 minutes.

[0059] The reaction is carried out under preset enzymatic hydrolysis temperature and time conditions. The proteases include alkaline and neutral proteases, and the hydrolysis time is five to fifty minutes to obtain the hydrolysate. The purpose of this step is to complete the controllable conversion of macromolecules into small and medium-sized peptides in a mild aqueous phase, while maintaining enzyme activity and accumulating target peptides. Water is used as the reaction medium to stabilize the three-dimensional conformation of the enzyme and avoid denaturation by organic solvents. The reaction is carried out at a set temperature until the preset time is reached, after which the enzyme inactivation and separation process begins. This results in higher peptide concentrations and better TCA-NSI, while reducing the process complexity caused by solvent treatment.

[0060] In the above embodiments, the preset enzymatic hydrolysis time is 5 to 50 minutes, which can avoid insufficient or excessive hydrolysis of bullfrog crude protein.

[0061] In one embodiment, the mass ratio of bullfrog crude protein to water is 1:4 to 1:6; the mass ratio of alkaline protease to neutral protease is 3:1 to 1:3.

[0062] In this embodiment, a bullfrog crude protein to water mass ratio of 1:4 ensures complete dispersion of the bullfrog crude protein, allowing the protease to penetrate the substrate and thus guaranteeing enzymatic hydrolysis efficiency. A bullfrog crude protein to water mass ratio of 1:6 avoids excessive water leading to dilution of the protease concentration, thereby ensuring enzymatic hydrolysis efficiency.

[0063] In the above embodiments, the mass ratio of alkaline protease to neutral protease is set from 3:1 to 1:3 according to the component proportion of bullfrog by-products. If the skin proportion is high, the proportion of neutral protease is increased to hydrolyze neutral amino acid peptide bonds. If the viscera proportion is high, the proportion of alkaline protein is increased to efficiently break alkaline amino acid peptide bonds. When the proportion of alkaline protease is relatively high, the crude bullfrog protein can be hydrolyzed into intermediate peptides, providing more action sites for neutral protease. When the proportion of neutral protease is high, the intermediate peptides can be further hydrolyzed into small molecule peptides, thereby enhancing the antioxidant activity of the peptides.

[0064] In one embodiment, step S3 further includes: S031: Preprocess the historical experimental data to obtain the target dataset. The historical experimental data includes multiple experimental records. Each experimental record contains independent variables and corresponding experimental results. The independent variables include pH, temperature, enzyme dosage, and time. The experimental results include TCA-NSI and peptide concentration. Specifically, historical experimental data refers to records of multiple completed enzymatic hydrolysis batches, each record containing independent variables and outcome indicators. Independent variables include pH, temperature, enzyme dosage, and hydrolysis time; outcome indicators include TCA-NSI (the proportion of non-precipitable nitrogen in the TCA system, used to characterize soluble peptide levels and the degree of hydrolysis) and peptide concentration. The purpose of this step is to standardize data from diverse sources and with varying definitions, reducing the interference of noise and bias on subsequent conclusions. The resulting dataset exhibits better structural integrity, comparability, and stability, supporting more reliable parameters and significance conclusions from subsequent regression and response surface methodology.

[0065] S032: Based on the target dataset, with TCA-NSI as the response variable and the independent variable and its two-factor interaction term as the explanatory variables, least squares fitting is performed to obtain a linear regression model; The response variable is the explained indicator, and TCA-NSI is selected here. The explanatory variables are the factors used to explain the response, in this case, pH, temperature, enzyme dosage, and enzymatic hydrolysis time, including pairwise interaction terms to characterize the combined effect. The purpose of this step is to quickly identify the direction and strength of the main effects with minimal modeling cost, initially screening out potentially significant interactions, and providing a basis for subsequently narrowing down the optimization space. In implementation, a linear model is fitted to the target dataset using the least squares method, outputting the coefficients of the main effects and interaction terms, standard errors, p-values, and goodness-of-fit indices. If necessary, robustness checks are performed on high-impact, high-leverage samples, and the model is compared with a baseline model without interactions to confirm the gain and rationality of the interaction terms.

[0066] S033: Perform significance testing and effect size assessment on the linear regression model to obtain the order of primary and secondary independent variables and a list of two-factor interaction combinations; Significance testing is used to determine whether the coefficients are statistically significant, while effect size is used to measure the strength and engineering significance of the effect. The purpose of this step is to identify the key factors that need to be prioritized and the interaction combinations that must be included in the constraints, avoiding wasting experimental resources on secondary factors. In practice, the main effects are ranked by influence using indicators such as p-values, confidence intervals, and standardized regression coefficients, and statistically significant two-factor interactions are selected to form a "list of dominant factors" and a "list of interaction constraints." This approach focuses subsequent optimization efforts on high-contribution factors and their key interactions, reducing disturbances from invalid variables and improving the efficiency and interpretability of the experiment and parameter design.

[0067] S034: Based on the order of primary and secondary independent variables and the list of two-factor interaction combinations, a quadratic polynomial fitting is performed with TCA-NSI as the target response to obtain the response surface model. The response surface model employs a quadratic polynomial form, including first-order terms, second-order terms, and retained significant interaction terms, to describe the curvature and optimal neighborhood of the multi-factor-response relationship. This step aims to supplement the nonlinear information that linear models cannot provide, giving the shape, boundaries, and predicted optimal points of the high-response region, thus facilitating parameter setting within a safer and more effective range. In implementation, based on the priority order and interaction list from the previous step, secondary modeling is performed around the key factors. Accumulated data can be combined with newly added center point and axis point data for fitting, outputting significance results, contour plots, and predicted optimal intervals. Robustness is checked through leave-out validation or cross-validation. This yields a "spatial description of the high-response region," providing both direction and boundaries in parameter setting, reducing repeated trials in inefficient or unstable regions.

[0068] S035: Based on the current raw material information of bullfrog by-products, and combining the linear regression model and the response surface model, obtain the target parameter set corresponding to the current raw material.

[0069] Raw material information is an objective attribute of the current batch, including at least tissue composition, feed composition parameters, and initial pH, reflecting differences in cleavable sites, diffusion conditions, and initial pH. The purpose of this step is to transform model conclusions into executable batch-specific parameters, achieving adaptive adaptation to batch differences. In implementation, the acid-base shift and mass transfer differences in the system are first determined based on the raw material information. Then, linear regression is used to provide directional corrections for each factor. Within the high-response region provided by the response surface, an initial landing point satisfying interaction constraints and process safety boundaries is selected. If necessary, the initial ratio is determined by incorporating disclosed compound enzyme ratio rules. The final output target parameter set includes initial pH, initial temperature, initial enzyme dosage, initial hydrolysis time, and the initial compound enzyme ratio. This combined approach accelerates the rate at which TCA-NSI and peptide concentrations reach the target, reduces the risk of over-hydrolysis or deviation from the optimal region, and improves batch-to-batch consistency and first-pass yield.

[0070] In one embodiment, step S035 includes: S0351: Obtain the model parameters of the linear regression model and the contour information of the response surface model. The model parameters include the significance test results of the main effect coefficient and the interaction term coefficient, and the goodness-of-fit index. The contour information includes the boundary of the target high-response region. The model parameters here refer to the main effect coefficients and interaction term coefficients output by the linear regression model, as well as the significance test results and goodness-of-fit indices for each coefficient; "contour information" refers to the boundary trajectory of the response surface model at the same response level and its closed region within the multi-factor space. The purpose of this step is to simultaneously grasp the direction and magnitude of influence (from linear regression) and the spatial boundary and curvature (from the response surface), providing both quantitative weights and geometric constraints for subsequent parameter selection. In implementation, the model file or modeling report is read and organized into unified tables and graphical elements, such as a list of coefficients, a list of p-values, and the coordinates of contour line boundaries or inversely calculable interval expressions.

[0071] S0352: Based on the significance test results, sort the main effects of the independent variables and identify significant two-factor interaction terms to obtain a list of dominant factors and a list of interaction constraints; Main effects ranking involves ranking independent variables according to the strength of their effect on the response; "significant interaction terms" are statistically significant two-factor joint effects. The purpose of this step is to identify the key factors to be prioritized and to recognize two-factor constraints that must be considered concurrently during parameter setting. This is implemented by ranking based on significance as a threshold, combined with effect sizes (such as standardized coefficients); for interaction terms, their signs and relative strengths are recorded, provided they pass the significance threshold, for subsequent amplitude limiting and peak shifting settings. This concentrates attention on a few key combinations that genuinely have interaction, narrowing the subsequent search domain.

[0072] S0353: Extract the target high-response region based on the contour information and convert it into the candidate range of each variable; The target high-response region is the high-value band and its boundaries predicted by the response surface model; the "candidate range" is the projection interval of this high-value band onto each individual variable axis. The purpose of this step is to transcribe the preferred region on the multidimensional surface into a one-dimensional interval selectable by each factor, facilitating subsequent interval cross-cutting and amplitude limiting. In implementation, closed regions reaching the target threshold are selected from the contour lines or isosurfaces of the response surface, projected one by one along the variable dimension, and their upper and lower limits are recorded. Simultaneously, coupling annotations with other variables are retained, resulting in an intuitive and executable set of primary "candidate intervals" that both incorporates the optimal information from the response surface and facilitates merging with other information sources.

[0073] S0354: Based on the raw material information of the current bullfrog by-products, feature extraction is performed to obtain raw material feature data, which includes at least tissue composition, liquid composition parameters and initial pH value; Specifically, tissue composition refers to the proportions of skin, muscle, and viscera, which directly affect cleavable sites and mass transfer pathways; feed composition parameters refer to indicators such as moisture, lipids, ash, or salt content, which affect enzyme activity, diffusion, and pH buffering; initial pH value is the acidity or alkalinity of the system before adjustment after feeding. The purpose of this step is to identify the objective differences in this batch of raw materials, providing a basis for subsequent directional corrections and boundary adjustments. This information is obtained through raw material receiving slips, rapid physicochemical testing, or empirical grading, and recorded as raw material characteristic data after standardization, thereby making batch differences explicit and avoiding deviations caused by directly applying previous parameters.

[0074] S0355: Based on the raw material characteristic data, generate a process label, wherein the process label includes a compound enzyme ratio direction label, a composition level label, and a pH deviation label; The compound enzyme ratio directional label indicates the direction in which this batch tends to increase the proportion of alkaline or neutral proteases; the composition level label indicates the complexity or load level of the feed solution (e.g., high lipids, high salts); and the pH deviation label indicates the direction and degree of the initial pH deviation relative to the target pH. The purpose of this step is to extract complex, continuous characteristics into a few clear decision signals, providing clear guidance for subsequent screening and parameter setting. In implementation, simple thresholds or grading rules are set to map the original measurements of S0354 to directional or level labels, thereby reducing cognitive burden and improving consistency. Parameter setting for the same batch is clear and verifiable.

[0075] S0356: Based on the list of dominant factors and the candidate range, and in conjunction with the interaction constraints, the value ranges of each independent variable are cross-filtered to obtain the initial range of each independent variable; Cross-screening refers to finding the intersection of the candidate ranges of each factor. For factor pairs with significant interactions, the boundaries or staggered intervals are narrowed according to the direction and intensity of the interaction to generate a more robust "initial range," ensuring that the selected interval is both in the high-response zone and avoids known unfavorable linkage combinations. In implementation, the intersection of the candidate ranges of each factor is first calculated, and then amplitude limits or hierarchical combinations are applied to significant interaction terms according to the sign and magnitude of the interaction coefficient (for example, limiting the upper limit of a factor to only take effect when another factor is near its midpoint). This yields an executable and risk-controllable initial range, laying the foundation for determining the initial values.

[0076] S0357: Determine the initial values ​​of each variable based on the initial range and the pH deviation label; The initial values ​​determine the initial range of each factor as the specific setpoint. The pH deviation label determines whether the pH is fine-tuned upwards or downwards. This step provides the starting point for executable parameters in a single run, taking into account both the response surface curvature and batch pH deviation. During implementation, values ​​are preferentially taken within the steady-state zone or the gently sloping contour area of ​​the initial range. The pH is adjusted according to the deviation direction to a near-target position within the feasible region. Temperature, enzyme dosage, and enzymatic hydrolysis time can be selected using a centering or centroidal principle, while considering significant interactions to avoid falling on steep slope boundaries. This forms a stable initial parameter setting, increasing the probability of achieving the target on the first attempt and leaving room for necessary fine-tuning.

[0077] S0358: Based on the compound enzyme ratio direction label and the composition level label, set the initial ratio of alkaline protease and neutral protease in the compound protease to obtain the initial ratio of the compound enzyme. The initial ratio of the complex enzyme is a preliminary proportion of alkaline protease to neutral protease. The purpose of this step is to ensure that the ratio is determined synchronously with the raw material structure characteristics and feed grade, rather than through post-hoc adjustments based on experience. In practice, the bias is determined by the complex enzyme ratio direction label, increasing the neutral proportion when the skin / muscle content is high or the alkaline proportion when the visceral content is high. Simultaneously, it is constrained by the composition grade label to avoid choosing directions prone to inactivation or inhibition at high-salt or high-fat levels. This ensures that the ratio and process factors are linked in a homologous manner, reducing the risk of excessive hydrolysis or activity inhibition.

[0078] S0359: Based on the target high-response region, verify the compliance of the initial values ​​of each independent variable with the initial ratio of the compound enzyme, and obtain the target parameter set corresponding to the current raw materials.

[0079] Compliance verification refers to reviewing the safety and feasibility of initial values ​​and proportions within the process, equipment, and quality boundaries. This includes the upper temperature limit, pH safety window, economic and residual boundaries of enzyme dosage, and the impact of time on subsequent enzyme inactivation and separation load. The target parameter set is the set of parameters ultimately used for execution in this batch. The purpose of this step is to ensure that the selected parameters and proportions meet the high response range without violating safety and quality red lines.

[0080] Specifically, the results of S0357 and S0358 can be compared with the predetermined boundaries item by item. If necessary, the results can be returned to the second-best high-response zone according to priority or the minor factors can be finely adjusted before verification. After verification, the target parameter set containing pH, temperature, enzyme dosage, enzyme digestion time and the initial ratio of the compound enzyme can be output.

[0081] The overall beneficial effects of this embodiment are reflected in three aspects. First, the process settings are more batch-appropriate. While determining pH, temperature, enzyme dosage, and hydrolysis time, the current raw material composition, feed solution composition parameters, and initial pH are taken into consideration. The strength of influence and key interactions are identified first, and then a steady-state landing point is selected within the high-response region. Therefore, the initial parameters are closer to the effective range, reducing the dual risks of insufficient or excessive hydrolysis, making it easier to achieve TCA-NSI and peptide concentrations in one go. Second, the ratio of the complex enzyme is no longer fine-tuned afterward, but is set synchronously with process factors and constrained by interactions and safety boundaries. This allows for smoother cascade hydrolysis along a "first break down large segments, then refine" path based on the raw material's structural characteristics, thereby accumulating more small-molecule active peptides and avoiding further cleavage into free amino acids that would lead to decreased activity. Third, the process is more stable and economical. By selecting parameters that avoid steep curvature regions and unfavorable linkage combinations, and by combining compliance and safety margin checks, the pressure of overheating, overalkaliing, excessive enzyme addition, and residual treatment can be reduced, the number of test rounds and rework can be reduced, batch-to-batch consistency and scale-up controllability can be improved, and ultimately more stable antioxidant activity output can be obtained with lower trial and error costs.

[0082] In one embodiment, the step of inactivating the protease, separating soluble bullfrog protein peptides from the enzymatic hydrolysate, and then freeze-drying to obtain solid bullfrog protein peptides includes: The enzymatic hydrolysate was inactivated in a boiling water bath for 5 to 15 minutes and then cooled to room temperature. The cooled enzymatic hydrolysate was centrifuged at 4000-6000 rpm for 5 to 15 minutes to separate the soluble bullfrog protein peptides from the precipitate. The supernatant was then filtered to obtain the soluble bullfrog protein peptides. The soluble bullfrog protein peptides were then frozen and dried to obtain solid bullfrog protein peptides.

[0083] In this embodiment, the activity of the protease is terminated by a high temperature of 5 to 15 minutes in a water bath. This ensures that the enzymatic hydrolysis system reaches 100°C and is maintained for a sufficient time, while avoiding the continuous enzymatic hydrolysis reaction that could lead to excessive hydrolysis or structural damage of peptides.

[0084] This invention also provides a bullfrog protein peptide, prepared using the method described in any of the above embodiments, wherein the amino acid sequence of the protein peptide is one of Ile-Ala-Ser-Glu-Ser-Pro-Ser-Ala-Phe, Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr, Leu-Leu-Pro-Tyr-Asp-Gln-Tyr, and Trp-Arg-Pro-Pro-Asn-Trp.

[0085] This invention provides the application of bullfrog protein peptides as described above, wherein the bullfrog protein peptides are used to prepare feed for aquaculture species.

[0086] For ease of understanding, the following will provide a detailed explanation using specific examples: Alkaline protease: refers to an enzyme that can hydrolyze the peptide bonds of proteins under alkaline conditions, with an optimal pH range of 9-11. The alkaline protease used in this experiment is a proteolytic enzyme produced by deep fermentation, extraction, and purification of Bacillus licheniformis 2709, which was selected through bacterial protoplast mutagenesis. Its main enzyme component is Bacillus licheniformis protease, a serine-type endopeptide that can hydrolyze protein molecules into peptide chains to produce polypeptides or amino acids, and has a strong ability to decompose proteins.

[0087] Neutral proteases are a class of proteases that can function in neutral, weakly acidic, or weakly alkaline environments, with their optimal pH range being 6.0–7.5. The neutral proteases used in this study were microbial metabolites obtained through fermentation using Bacillus subtilis as the production strain.

[0088] The alkaline protease and neutral protease used in this invention were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The trichloroacetoc acid-nitrogen solubility index (TCA-NSI) accurately reflects the enzymatic hydrolysis of proteins and is a key indicator for detecting the degree of protein hydrolysis. Specifically, it refers to the percentage of soluble nitrogen soluble in 10% trichloroacetic acid in the hydrolysis products relative to the total nitrogen. A higher TCA-NSI value indicates a higher proportion of protein hydrolyzed into small peptides.

[0089] Example 1: Preparation of bullfrog protein peptides S1: Cut bullfrog offal into small pieces of 1 to 2 cm, wash them, and add a sodium hydroxide solution containing 1% hydrogen peroxide at a mass ratio of 0.15 mol / L to the bullfrog offal for decolorization. Change the sodium hydroxide solution every 30 minutes until the pigment is completely removed, and obtain decolorized bullfrog offal. The mass ratio of bullfrog offal to sodium hydroxide solution is 1:4.

[0090] S2: Take the decolorized bullfrog by-products from step S1, add petroleum ether to the decolorized bullfrog by-products for mixing and defatting, stir for 2 hours at room temperature, wash with water and homogenize to obtain bullfrog crude protein; wherein the mass ratio of decolorized bullfrog by-products to petroleum ether is 1:4.

[0091] S3: Take 10g of bullfrog crude protein, add distilled water to the 10g of bullfrog crude protein, the mass ratio of bullfrog crude protein to distilled water is 1:5, then add alkaline protease and neutral protease at a mass ratio of 1:1, the enzyme amount is 561U / g, the enzymatic hydrolysis time is 36min, the pH is 7.6, the temperature is 53.3℃, ​​and the enzymatic hydrolysate is obtained.

[0092] S4: Inactivate the protein in the enzymatic hydrolysate obtained in step S3 in a boiling water bath for 10 minutes, cool and shake well, centrifuge at 5000 r / min for 10 min, filter the enzymatic hydrolysate after centrifugation, collect the supernatant and separate the soluble bullfrog protein peptides from the enzymatic hydrolysate after centrifugation, finally freeze the supernatant and dry it to obtain solid bullfrog protein peptides.

[0093] Comparative Example 1: The only difference from Example 1 is that alkaline protease is added in step S3.

[0094] Comparative Example 2: The only difference from Example 1 is that a neutral protease is added in step S3.

[0095] Comparative Example 3: The only difference from Example 1 is that, in step S3, a flavor protease is added.

[0096] Comparative Example 4: The only difference from Example 1 is that papain was added in step S3.

[0097] Comparative Example 5: The only difference from Example 1 is that trypsin is added in step S3.

[0098] Comparative Example 6: The only difference from Example 1 is that in step S3, alkaline protease and papain are added in a mass ratio of 1:1.

[0099] Comparative Example 7: The only difference from Example 1 is that in step S3, papain and neutral protease are added in a mass ratio of 1:1.

[0100] Comparative Example 8: The only difference from Example 1 is that in step S3, alkaline protease and neutral protease are added at a mass ratio of 1:2.

[0101] Comparative Example 9: The only difference from Example 1 is that in step S3, alkaline protease and neutral protease are added at a mass ratio of 1:3.

[0102] Comparative Example 10: The only difference from Example 1 is that in step S3, alkaline protease and neutral protease are added at a mass ratio of 3:1.

[0103] Comparative Example 10: The only difference from Example 1 is that in step S3, alkaline protease and neutral protease are added at a mass ratio of 2:1.

[0104] Comparative Example 11: The only difference from Example 1 is that in step S3, the amount of enzyme added is 100 U / g.

[0105] Comparative Example 12: The only difference from Example 1 is that in step S3, the amount of enzyme added is 200 U / g.

[0106] Comparative Example 13: The only difference from Example 1 is that in step S3, the amount of enzyme added is 350 U / g.

[0107] Comparative Example 14: The only difference from Example 1 is that in step S3, the amount of enzyme added is 500 U / g.

[0108] Comparative Example 15: The only difference from Example 1 is that in step S3, the amount of enzyme added is 650 U / g.

[0109] Comparative Example 16: The only difference from Example 1 is that in step S3, the amount of enzyme added is 800 U / g.

[0110] Comparative Example 17: The only difference from Example 1 is that the pH is 5.5 in step S3.

[0111] Comparative Example 18: The only difference from Example 1 is that the pH is 6.5 in step S3.

[0112] Comparative Example 19: The only difference from Example 1 is that the pH is 7.5 in step S3.

[0113] Comparative Example 20: The only difference from Example 1 is that the pH is 8.5 in step S3.

[0114] Comparative Example 21: The only difference from Example 1 is that the pH is 9.5 in step S3.

[0115] Comparative Example 22: The only difference from Example 1 is that the pH is 10.5 in step S3.

[0116] Comparative Example 23: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 34°C.

[0117] Comparative Example 24: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 42°C.

[0118] Comparative Example 25: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 50°C.

[0119] Comparative Example 26: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 58°C.

[0120] Comparative Example 27: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 66°C.

[0121] Comparative Example 28: The only difference from Example 1 is that the enzymatic hydrolysis temperature in step S3 is 74°C.

[0122] Comparative Example 29: The only difference from Example 1 is that the enzymatic hydrolysis time in step S3 is 10 min.

[0123] Comparative Example 30: The only difference from Example 1 is that the enzymatic hydrolysis time in step S3 is 20 min.

[0124] Comparative Example 31: The only difference from Example 1 is that the enzymatic hydrolysis time in step S3 is 30 min.

[0125] Comparative Example 32: The only difference from Example 1 is that the enzymatic hydrolysis time in step S3 is 40 min.

[0126] Comparative Example 33: The only difference from Example 1 is that the enzymatic hydrolysis time in step S3 is 50 min.

[0127] This invention application uses the TCA-NSI assay method to test the bullfrog protein peptides prepared in Example 1 and Comparative Examples 1 to 33 above. The assay method is as follows: The enzymatic hydrolysate was precipitated with an equal volume of 15% TCA solution for 10 min, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. Protein solution and biuret reagent were added at a mass ratio of 1:4, and the reaction was allowed to proceed for 30 min. The absorbance was then measured at 540 nm. The soluble nitrogen content in the sample was calculated based on the protein standard curve. The TCA-NSI value was determined using the following formula: TCA-NSI = m0 / m1 × 100%; Wherein, m0 is the mass of soluble nitrogen in 15% TCA, and m1 is the mass of total nitrogen in the raw material; the mass of soluble nitrogen is determined by the biuret method, and the mass of total nitrogen is determined by the combustion method.

[0128] The method for determining peptide concentration is as follows: Take an equal volume of the sample solution to be tested, add 10% TCA solution to precipitate, mix well, let stand for 10 min, and then centrifuge (4000 r / min, 15 min). Take the supernatant and dilute it 10 times with 5% TCA. Add peptide solution and biuret reagent at a ratio of 3:2. After the reaction, centrifuge and take the supernatant to measure the absorbance value at 540 nm. Calculate the peptide concentration in the sample by referring to the standard curve (prepared with Gly-Gly-Tyr-Arg tetrapeptide as the standard reagent).

[0129] The test results are as follows: Please refer to Comparative Examples 1 through 5 and combine them. Figure 1Among the five proteases, alkaline protease had the highest peptide concentration and TCA-NSI value, while papain had the lowest. Therefore, alkaline protease showed the best hydrolysis effect, while papain showed the worst. This is because different proteases have different cleavage sites. Bullfrog by-products are rich in peptide bonds sensitive to alkaline protease, which can more effectively promote the hydrolysis of bullfrog proteins. Papain, on the other hand, has fewer peptide bonds sensitive to bullfrog by-products, resulting in a relatively lower degree of hydrolysis.

[0130] Please refer to Comparative Example 1 and Comparative Examples 6 and 7 in conjunction with them. Figure 2 Among the paired proteases, alkaline protease and neutral protease had the highest peptide concentrations and TCA-NSI values, at 10.99 mg / mL and 66.77%, respectively. Therefore, alkaline and neutral proteases showed the best enzymatic hydrolysis effect, and in the actual preparation of bullfrog protein peptides, the combination of alkaline and neutral protease was selected as the optimal protease for the hydrolysis of bullfrog by-products.

[0131] Please refer to Comparative Example 1 and Comparative Examples 8 to 10 and combine them. Figure 3 When the ratio of alkaline protease to neutral protease is 1:1, the peptide concentration and TCA-NSI value are highest, and the enzymatic hydrolysis effect is best. This is because when these two enzymes are combined in a 1:1 ratio, they can complement each other at different peptide bond sites, thereby improving the overall hydrolysis efficiency. Therefore, in the actual preparation of bullfrog protein peptides, the optimal ratio of alkaline protease to neutral protease is 1:1.

[0132] Please refer to Comparative Example 1 and Comparative Examples 11 to 16 in conjunction with them. Figure 4 As the amount of enzyme added increased, the peptide concentration and TCA-NSI value in bullfrog protein peptides showed a trend of first increasing and then decreasing, reaching the highest value at an enzyme addition of 500 U / g. This is because, when the bullfrog protein concentration is constant, increasing the amount of enzyme added can promote the enzymatic hydrolysis reaction, but excessive enzyme addition will lead to over-hydrolysis of the protein, producing free amino acids. Therefore, in the actual preparation of bullfrog protein peptides, the optimal enzyme addition amount is 500 U / g.

[0133] Please refer to Comparative Example 1 and Comparative Examples 17 to 22 and combine them. Figure 5 The peptide concentration and TCA-NSI value increased with increasing pH, reaching their maximum at pH 7.5, and then decreased. This is because changes in pH cause conformational changes in the enzyme, affecting its stability and activity, and consequently the reaction rate. Therefore, in practical preparation of bullfrog protein peptides, the optimal pH is 7.5.

[0134] Please refer to Comparative Example 1 and Comparative Examples 23 to 28 and combine them. Figure 6When the temperature is between 30 and 50 degrees Celsius, increasing the temperature can promote enzymatic hydrolysis efficiency. However, when the temperature exceeds 50 degrees Celsius, further heating will damage the active sites, leading to a decrease in enzymatic hydrolysis efficiency. Therefore, in the actual preparation of bullfrog protein peptides, the optimal enzymatic hydrolysis temperature is 50 degrees Celsius.

[0135] Please refer to Comparative Example 1 and Comparative Examples 29 to 33 and combine them. Figure 7 Within 30 minutes, both the peptide concentration and the TCA-NSI value increased with time. This is because in the initial stage of the reaction, there is ample bullfrog protein, leading to more opportunities for the protease to bind with the bullfrog protein, thus increasing the reaction rate. After 30 minutes, the bullfrog protein is essentially depleted, and the enzymatic hydrolysis reaches a stable state. Extending the reaction time then has no significant effect on the peptide concentration and TCA-NSI value. Therefore, in practical preparation of bullfrog protein peptides, the optimal enzymatic hydrolysis time is 30 minutes.

[0136] Based on the aforementioned single-factor experiments, and according to the principle of response surface methodology, this invention uses the TCA-NSI value as the corresponding Y value and selects four factors—pHA, temperature (B), enzyme dosage (C), and time (D)—to design a four-factor, three-level response surface experiment to optimize the process conditions for the enzymatic hydrolysis of bullfrog by-products. An example is shown below: Table 1 Response Surface Factor Level Design

[0137] After regression fitting of the experimental results, the optimal regression equation is obtained as: Y = 77.6373.36 + 0.9555A + 17B + 7.55C + 8.11D - 1.59AB - 5.3AC - 0.3296AD - 1.17BC + 2.98BD - 3.34CD - 7.01A 2 -18.47B 2 -14.6C 2 -9.32D 2 As can be seen from Table 2, the fitting model... P A value less than 0.0001 indicates that the model is extremely significant. Goodness of fit R0 2 =0.9626, adjust the goodness of fit R 2 Adj =0.9251, indicating that the model can explain 92.51% of the variation in response values. (Misfit term) P=0.0559>0.05, which is not significant, indicating that the model fits well and can predict TCA-NSI values. The F-value in the ANOVA can determine the influence of each factor and their interactions on TCA-NSI values; the larger the F-value, the more significant the influence. Table 2 shows that the order of influence of each factor on TCA-NSI is: enzymatic hydrolysis temperature > enzymatic hydrolysis time > enzyme dosage > pH. The interaction between pH and enzyme dosage has a significant impact on the enzymatic hydrolysis effect. P <0.05). Response surface plot as shown. Figures 8 to 13 As shown, the slope of the response surface reflects the degree of influence of each factor on the response value. If the slope of the surface is relatively steep, it indicates that these factors have a greater impact on the response value, and this is positively correlated with the results of the analysis of variance in the regression model.

[0138] Table 2. Analysis of variance of the response surface model

[0139] Where: P<0.01 indicates extremely significant (**); P<0.05 indicates significant (*); P>0.05 indicates not significant.

[0140] The optimal enzymatic hydrolysis conditions for bullfrog by-product protein peptides, predicted by Design-Expert software, were: time 36.202 min, pH 7.593, temperature 53.264°C, and enzyme dosage 561.031 U / g. The predicted TCA-NSI value was 78.63%. For ease of operation, the time, pH, temperature, and enzyme dosage were selected as 36 min, 7.6, 53.3°C, and 561 U / g for verification. Under these conditions, the TCA-NSI value was 77.89%, which was close to the predicted value (Table 3). The above results indicate that the enzymatic hydrolysis conditions obtained by this model have high reliability.

[0141] Table 3. Process determination and verification experiments for the enzymatic hydrolysis method for preparing protein peptides from bullfrog by-products.

[0142] Molecular weight distribution determination of bullfrog protein peptides prepared in Example 1: The determination was performed according to the national standard GB / T22729-2008. The results are as follows: Table 4. Molecular weight distribution of bullfrog protein peptides prepared in Example 1

[0143] Table 4 shows that the molecular weight of the bullfrog protein peptides prepared in Example 1 is mainly distributed in the range of 180~3000 Da, accounting for 83.57%, indicating that the combination of alkaline protease and neutral protease can effectively hydrolyze large molecular proteins in the by-products into small molecular peptides. The antioxidant activity of peptides is closely related to their molecular weight, and low molecular weight peptides exhibit better antioxidant activity than high molecular weight peptides.

[0144] The bullfrog protein peptide sequences prepared in Example 1 were analyzed by LC-MS / MS. The mass spectrometry parameters were as follows: reversed-phase column, aqueous phase of 0.1% formic acid, organic phase of a mixture of formic acid, acetonitrile, and water (acetonitrile being 80%), flow rate of 300 nl / min, and analysis time of 60 min. The following short peptides were obtained: Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr (IGAGGLDGY), Leu-Leu-Pro-Tyr-Asp-Gln-Tyr (LLPYDQY), and Trp-Arg-Pro-Pro-Asn-Trp (WRPPNW).

[0145] The method for testing the antioxidant activity of the bullfrog protein peptides prepared in Example 1 is as follows: Determination of DPpH free radical scavenging rate: Accurately weigh 3.94 mg of DPpH, dissolve it completely in anhydrous ethanol, and make up to volume to prepare a 0.1 mmol / L DPpH solution. Dilute bullfrog by-product protein peptide solution to different concentrations, add equal volumes of protein peptide solution and DPpH solution, mix well, and react in the dark for 30 min. Measure the absorbance at 517 nm. Record the absorbance value as A. b Anhydrous ethanol and water were used to replace the DPpH solution and protein peptide solution, respectively, as the control group and blank group. The absorbance was measured and recorded as A. c The above experiment was repeated with A0 and vitamin C as a control. The formula for calculating DPpH free radical scavenging rate is as follows: DPpH free radical scavenging rate (%) = (1 - (A0) / (A0) c -A b ) / A0)×100%.

[0146] ABTS + Determination of free radical scavenging rate: Weigh 40 mg ABTS and 6.88 mg potassium persulfate, dissolve them in distilled water, and bring the volume to 10 mL. Shake well and react at room temperature in the dark for 12–16 h to obtain ABTS. + Mother liquor. Dilute an appropriate amount of mother liquor with deionized water until the OD value is within 0.70±0.02. Add 900 μL of ABTS+ solution to 100 μL of the test solution, mix well, and react in the dark for 6 min. Measure the absorbance at 734 nm and record it as A1. Replace ABTS+ with an equal volume of deionized water. +The absorbance of the solution and the test solution is measured and recorded as A2 and A3, respectively, with vitamin C as a positive control. Calculate ABTS using the following formula. + Free radical scavenging rate: ABTS+ free radical scavenging rate (%) = (1-(A1-A2) / A3)×100%.

[0147] Determination of hydroxyl radical scavenging rate: 1.0 mL of 9.0 mmol / L FeSO4 solution, 1.0 mL of 9.0 mmol / L salicylic acid-ethanol, 1 mL of protein peptide solution, and 1 mL of 8.8 mmol / L H2O2 were mixed and heated in a water bath at 37°C for 30 min. The absorbance at 510 nm was measured and recorded as A1. Pure water was used to replace hydrogen peroxide and the protein peptide solution as the control and blank groups, respectively, and the absorbance was recorded as A2 and A0. Ascorbic acid was used as a control, and the above experiment was repeated. The hydroxyl radical scavenging rate was calculated as follows: Hydroxyl radical scavenging rate (%) = (1 - (A1 - A2) / A0) × 100.

[0148] Depend on Figures 14 to 16 It can be seen that the scavenging rates of hydroxyl radicals, DPpH radicals and ABTS+ radicals of the bullfrog protein peptides prepared in Example 1 increased with the concentration of bullfrog by-product protein peptides, and their half-inhibitory concentrations (IC50) reached 8.90, 9.06 and 1.12 mg / mL, respectively, indicating good antioxidant activity.

[0149] The bullfrog protein peptides prepared in Example 1 were added to sea bass feed for aquaculture experiments to determine the growth performance and antioxidant capacity of the sea bass.

[0150] According to the feed formulation in Table 5, the bullfrog protein peptides prepared in Example 1 were added to sea bass feed at mass ratios of 0%, 0.25%, 0.5%, 1%, and 2%, respectively, labeled as groups CON, H0.25, H0.5, H1, and H2. 450 robust, uniformly sized juvenile sea bass (8.28 ± 0.03 g) were randomly assigned to 15 culture tanks in a high-temperature temperature-controlled system, with 30 fish per tank. The 15 tanks were randomly divided into 5 treatments, with 3 replicates per treatment. During the experiment, fish were fed twice daily (8:00 and 17:00), each time until their appetite was low and there was no uneaten food. During the rearing process, a 12-hour light-12-hour dark cycle was maintained daily, with a water temperature of 33°C and a pH of 6.8–7.5. The rearing period was 9 weeks, and the feeding amounts for the sea bass are shown in Table 6.

[0151] Table 5 Bullfrog Protein Peptide Feed Formula

[0152] Table 6 Total Feed Amount for Sea Bass

[0153] After the experiment, all experimental fish were starved for 24 hours to empty their gastrointestinal contents. Fifteen fish were randomly selected from each bucket, anesthetized with eugenol, weighed, and their body length measured. Three of these fish were preserved whole at -20°C for body composition analysis, while the remaining 12 fish were dissected, and their livers were separated. Samples were flash-frozen in liquid nitrogen and then stored at -80°C. The antioxidant indicators of superoxide dismutase (SOD) and catalase (CAT) in the sea bass liver were measured using kits from Nanjing Jiancheng Bioengineering Institute. The formula for calculating the weight gain rate of sea bass is as follows: Weight gain rate (%) = (W1 - W0) / W0 × 100; the formula for calculating the specific growth rate is as follows: Specific growth rate (% / day) = 100 × (lnW1 - lnW0) / t. Where W1 is the final body weight and W0 is the initial body weight. It should be noted that the specific growth rate is a core indicator for measuring the growth efficiency of an organism, and its calculation formula is: (ln final weight - ln initial weight) / "culture time". This indicator, by weakening the influence of the initial weight of the experimental animal, dynamically reflects the trend of weight change per unit time; the larger the value, the faster the weight gain. Compared with the weight gain rate, the specific growth rate incorporates the time dimension into the calculation system, and can more accurately assess growth efficiency.

[0154] Depend on Figures 17-18 It can be seen that after consuming bullfrog protein peptides containing the present invention, the weight gain rate and specific growth rate of sea bass are significantly improved. The weight gain rate and specific growth rate of the group consuming 0.5% bullfrog protein peptides (H0.5) are 11.46% and 4.41% higher than those of the group consuming no bullfrog protein peptides (CON), respectively, indicating that the bullfrog protein peptides of the present invention have the effect of promoting the growth of sea bass.

[0155] Depend on Figures 19 to 20 It is known that after consuming bullfrog protein peptides containing the present invention, the antioxidant activity of sea bass liver is significantly improved. The catalase activity (CAT) and superoxide dismutase (SOD) of the group consuming 1% bullfrog protein peptides (H1) are increased by 40.72% and 51.94% respectively compared with the group consuming no bullfrog protein peptides (CON), indicating that the bullfrog protein peptides of the present invention have the effect of improving the antioxidant capacity of sea bass liver.

[0156] The above description is merely a specific embodiment of the present invention. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, modules, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A bullfrog protein peptide, characterized in that, The amino acid sequence of the protein peptide is one of: Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr, Leu-Leu-Pro-Tyr-Asp-Gln-Tyr, and Trp-Arg-Pro-Pro-Asn-Trp.

2. A method for preparing bullfrog protein peptides, characterized in that, The method for preparing the bullfrog protein peptide as described in claim 1 comprises: The pre-treated bullfrog by-products were decolorized to obtain decolorized bullfrog by-products; According to the first preset mass ratio, a decolorizing agent is added to the sheared bullfrog scraps, wherein the decolorizing agent is a sodium hydroxide solution containing 1% hydrogen peroxide, and the first preset mass ratio is the mass ratio of bullfrog scraps to sodium hydroxide solution of 1:3 to 1:

5. The decolorizing agent is replaced at preset time intervals until decolorized bullfrog by-products are obtained, wherein the preset time interval is 20 to 40 minutes; The decolorized bullfrog by-products were subjected to defatting treatment to obtain bullfrog crude protein. A reaction medium and a protease were added to the crude bullfrog protein, and enzymatic hydrolysis was performed according to a preset hydrolysis time to obtain the hydrolysate. The protease includes alkaline protease and neutral protease, and the preset enzymatic hydrolysis time is 5 to 50 minutes. After inactivating the protease, soluble bullfrog protein peptides were separated from the enzymatic hydrolysate, and then freeze-dried to obtain solid bullfrog protein peptides.

3. The method for preparing bullfrog protein peptides according to claim 2, characterized in that, The step of decolorizing the pretreated bullfrog by-products to obtain decolorized bullfrog by-products includes: The bullfrog scraps are washed and cut to a preset size, wherein the preset size is between 1 and 2 centimeters.

4. The method for preparing bullfrog protein peptides according to claim 2, characterized in that, The degreasing treatment of the decolorized bullfrog by-products yields bullfrog crude protein, comprising: According to the second preset mass ratio, a degreasing agent is added to the decolorized bullfrog by-products, wherein the degreasing agent is one of petroleum ether, polysorbate or lipase, and the second preset mass ratio is the mass ratio of the decolorized bullfrog by-products to the degreasing agent is 1:3 to 1:

5. Under preset temperature conditions, the bullfrog by-products and degreasing agent are stirred according to a preset stirring time of 1 to 3 hours. After stirring, the bullfrog by-products are washed to obtain bullfrog crude protein.

5. The method for preparing bullfrog protein peptides according to claim 2, characterized in that, The process involves adding a reaction medium and protease to the crude bullfrog protein, followed by enzymatic hydrolysis according to a preset hydrolysis time, to obtain the hydrolysate, which includes: A reaction medium is added to the crude bullfrog protein to obtain a liquid, and the pH value of the liquid is adjusted to a preset pH value, wherein the reaction medium is water and the preset pH value is 5.5 to 10.5; According to the preset enzyme dosage, protease is added to the liquid, wherein the preset enzyme dosage is 100 to 800 U; The enzymatic hydrolysate is obtained by enzymatic hydrolysis according to a preset hydrolysis time and a preset hydrolysis temperature, wherein the protease includes alkaline protease and neutral protease; the preset hydrolysis time is 5 to 50 minutes.

6. The method for preparing bullfrog protein peptides according to claim 5, characterized in that, The mass ratio of bullfrog crude protein to water is 1:4 to 1:6; the mass ratio of alkaline protease to neutral protease is 3:1 to 1:

3.

7. The method for preparing bullfrog protein peptides according to claim 5, characterized in that, The step of adding a reaction medium and protease to the crude bullfrog protein, and performing enzymatic hydrolysis according to a preset hydrolysis time to obtain the hydrolysate, further includes: The target dataset is obtained by preprocessing historical experimental data. The historical experimental data includes multiple experimental records, each containing independent variables and corresponding experimental results. The independent variables include pH, temperature, enzyme dosage, and enzymatic hydrolysis time. The experimental results include TCA-NSI and peptide concentration. Based on the target dataset, a linear regression model is obtained by least squares fitting with TCA-NSI as the response variable and the independent variable and its two-factor interaction term as the explanatory variable. The linear regression model was subjected to significance testing and effect size assessment to obtain the order of primary and secondary independent variables and the list of two-factor interaction combinations. Based on the order of primary and secondary independent variables and the list of two-factor interaction combinations, a quadratic polynomial fitting was performed with TCA-NSI as the target response to obtain the response surface model. Based on the current raw material information of bullfrog by-products, and combining the linear regression model and the response surface model, the target parameter set corresponding to the current raw material is obtained.

8. The method for preparing bullfrog protein peptides according to claim 7, characterized in that, The step of obtaining the target parameter set corresponding to the current raw materials based on the current bullfrog by-product information, combined with the linear regression model and the response surface model, includes: Obtain the model parameters of the linear regression model and the contour information of the response surface model. The model parameters include the significance test results of the main effect coefficients and interaction term coefficients and the goodness-of-fit index. The contour information includes the boundary of the target high-response region. Based on the significance test results, the main effects of the independent variables are ranked and significant two-factor interaction terms are identified to obtain a list of dominant factors and a list of interaction constraints. Based on the contour information, extract the target high-response region and convert it into the candidate range of each variable; Based on the raw material information of the current bullfrog by-products, feature extraction is performed to obtain raw material feature data, which includes at least tissue composition, liquid composition parameters and initial pH value; Based on the raw material characteristic data, a process label is generated, wherein the process label includes a compound enzyme ratio direction label, a composition level label, and a pH deviation label; Based on the list of dominant factors and the candidate range, combined with the interaction constraints, the value ranges of each independent variable are cross-filtered to obtain the initial range of each independent variable. Determine the initial values ​​of each variable based on the initial range and the pH deviation label; Based on the compound enzyme ratio direction label and the composition level label, the initial ratio of alkaline protease and neutral protease in the compound protease is set to obtain the initial ratio of the compound enzyme. Based on the target high-response region, the initial values ​​of each independent variable and the initial ratio of the compound enzyme are checked for compliance, and the target parameter set corresponding to the current raw materials is obtained.

9. The method for preparing bullfrog protein peptides according to claim 2, characterized in that, The steps of inactivating the protease, separating soluble bullfrog protein peptides from the enzymatic hydrolysate, and freeze-drying to obtain solid bullfrog protein peptides include: The enzymatic hydrolysate was inactivated in a boiling water bath for 5 to 15 minutes and then cooled to room temperature. The cooled enzymatic hydrolysate was centrifuged at 4000-6000 rpm for 5 to 15 minutes to separate the soluble bullfrog protein peptides from the precipitate. The supernatant was then filtered to obtain the soluble bullfrog protein peptides. The soluble bullfrog protein peptides were frozen and dried to obtain solid bullfrog protein peptides.

10. The application of the bullfrog protein peptide as described in claim 1, characterized in that: The bullfrog protein peptides are used to prepare feed for aquaculture species.

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