Antioxidant peptide from red meat of sturgeon and preparation method and application thereof

Abstract

The application provides a sturgeon red meat antioxidant peptide and a preparation method and application thereof, and relates to the technical field of bioengineering. The preparation method uses sturgeon red meat as raw material, uniformly grinds and vacuum freeze-dries to form loose porous powder, and then carries out the first step of hydrolysis under neutral pH conditions by using a compound enzyme preparation of neutral protease and bromelain. After the reaction is completed, the enzyme is inactivated by heating, the system is adjusted to an acidic environment, and the second step of hydrolysis is carried out by adding pepsin. The supernatant is obtained by centrifugation of the second step of hydrolysis reaction liquid, deodorization is carried out by adding beta-cyclodextrin, and the crude product is obtained by freeze-drying after filtration. The crude product is dissolved and purified by Sephadex G-25 gel chromatography, the elution peak with antioxidant activity is collected and freeze-dried, and the target product is obtained. The above method of the application overcomes the problem of synergistic failure of multiple proteases due to pH incompatibility through step-by-step enzymolysis, significantly improves the antioxidant activity of the sturgeon red meat antioxidant peptide, and effectively reduces the components of fishy substances.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an antioxidant peptide from sturgeon red meat, its preparation method, and its application. Background Technology

[0002] With the development of aquaculture, sturgeon, as an important economic fish, generates a large number of byproducts during processing, such as fish bones, skin, viscera, and meat residue. Among these, sturgeon red meat, due to its high content of myoglobin and heme-like substances, has a strong fishy smell and poor taste, making it unsuitable for direct use in human food processing. Currently, it is mostly used as low-value feed or disposed of as waste. Industry statistics show that sturgeon red meat accounts for 8% to 14% of the whole fish's weight, resulting in serious resource waste and hindering the high-value development of the entire sturgeon industry chain.

[0003] To enhance the comprehensive utilization value of aquatic product by-products, researchers have recently explored extracting functional components from fish tissues. A technique has been disclosed that yields "antioxidant peptides from sturgeon bone hydrolysate and its preparation method." This method includes the following steps: pretreating sturgeon bones to obtain fish bone powder; mixing the fish bone powder with water and adding protease under suitable pH conditions for enzymatic hydrolysis; after the reaction is complete, heating to inactivate the enzyme, filtering and collecting the filtrate, and freeze-drying to obtain the antioxidant peptide product. This technique prepares hydrolysates with certain antioxidant properties from sturgeon bones through enzymatic hydrolysis, providing a research foundation for the development of functional products in the food and pharmaceutical industries.

[0004] However, the aforementioned existing technologies have several limitations: First, the raw material used is sturgeon bone, and it does not involve the effective development of sturgeon red meat, a component with a strong fishy smell and difficult to utilize; second, hydrolysis is usually carried out using a single protease, resulting in insufficient protein hydrolysis and low release efficiency of antioxidant peptides; third, the optimal reaction conditions (such as pH and temperature) of different proteases vary greatly. If multiple proteases are added at the same time, the enzyme activity may decrease or even become inactive due to environmental incompatibility, affecting the antioxidant performance of the final product; fourth, the existing technical solutions do not include an effective deodorization step, and the resulting hydrolysate still has a noticeable fishy smell, which limits its application prospects in the food, health product and other fields.

[0005] Furthermore, experiments showed that when bromelain, papain, neutral protease, alkaline protease, pepsin, trypsin, and flavor protease were used to hydrolyze sturgeon red meat, the DPPH free radical scavenging rates of the resulting hydrolysates varied significantly, as did the clarity and fishy odor of the solutions. For example, while pepsin yielded a relatively clear hydrolysate with a milder fishy odor, its antioxidant capacity was low when used alone. Neutral protease and alkaline protease, although possessing high antioxidant activity, exhibited a decrease in antioxidant capacity when simply combined with other proteases, indicating that synergistic effects between enzymes are difficult to achieve.

[0006] Therefore, selecting sturgeon red meat as the raw material, designing a reasonable multi-enzyme synergistic hydrolysis strategy, and effectively removing the fishy smell while maintaining antioxidant activity have become the technical challenges that urgently need to be solved for the high-value utilization of sturgeon by-products.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide a method for preparing antioxidant peptides from sturgeon red meat. By employing a stepwise hydrolysis process involving initial hydrolysis with a combination of neutral protease and bromelain, followed by acidic hydrolysis with pepsin after enzyme inactivation, the technical obstacle of multiple proteases being unable to work synergistically due to pH conflicts is overcome. This significantly improves the yield and activity of small molecule antioxidant peptides while significantly reducing the fishy odor components of sturgeon red meat antioxidant peptides.

[0009] The second objective of this invention is to provide an antioxidant peptide from sturgeon red meat.

[0010] A third objective of this invention is to provide an application of an antioxidant peptide from sturgeon red meat.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The present invention provides a method for preparing antioxidant peptides from sturgeon red meat, comprising the following steps: (A) Provide pretreated sturgeon red meat, and use a compound enzyme preparation of neutral protease and bromelain to perform the first step of hydrolysis on the protein in the sturgeon red meat under neutral pH conditions; after the first step of hydrolysis is completed, heat to inactivate the compound enzyme preparation. (B) Adjust the system to an acidic pH environment, add pepsin to carry out the second step of hydrolysis, centrifuge after the reaction to obtain the supernatant to obtain a peptide solution, add β-cyclodextrin to the peptide solution for deodorization, filter and freeze dry to obtain crude product; (C) The crude product is dissolved and purified by gel chromatography. The elution peak with antioxidant activity is collected and freeze-dried to obtain sturgeon red meat antioxidant peptides.

[0012] Furthermore, the preprocessing method in step (A) includes: Sturgeon red meat is homogenized and then freeze-dried under vacuum to produce a loose, porous powder.

[0013] Furthermore, step (A) also includes: before the first hydrolysis step, mixing the pretreated powdered material with water and then subjecting it to a water bath treatment; Preferably, the ratio of the powdered material to water in the mixture is 1:9~10; Preferably, the water bath treatment is performed at a temperature of 90-100°C for 25-40 minutes.

[0014] Furthermore, in step (A), the first hydrolysis step has a pH of 6-7, a reaction temperature of 50-60°C, and a reaction time of 6-9 hours. Preferably, in the compound enzyme preparation, the mass ratio of neutral protease to bromelain is 5-8:1; Preferably, the total amount of the compound enzyme preparation added is 5000~6000 U / g dry fish weight.

[0015] Furthermore, in step (B), the second step of hydrolysis has a pH of 2 to 2.5, a reaction temperature of 50 to 55°C, and a reaction time of 6 to 9 hours. Preferably, in the second step of hydrolysis in step (B), the amount of pepsin added is 2500~3000 U / g of dry fish meat.

[0016] Furthermore, in step (B), the amount of β-cyclodextrin added is 0.5~10 mg / 20 mL, relative to the volume of the peptide solution.

[0017] Furthermore, in step (B), after adding β-cyclodextrin to the peptide solution, the mixture is continuously shaken or stirred at room temperature for 40-50 minutes to achieve full inclusion and deodorization.

[0018] Furthermore, the method for gel chromatography purification in step (C) includes: The lyophilized crude product was dissolved in buffer or water to obtain a solution with a concentration of 75 mg / mL. After pre-filtration through a 0.45 μm filter membrane, the solution was used as the loading solution. The loading solution was separated using a Sephadex G25 gel column with an injection volume of 100–200 mg and an elution mobile phase of water or buffer solution at a flow rate of 0.6–2 mL / min. The absorbance of the eluent was monitored online at a wavelength of 280 nm. The separation peaks were determined based on the elution curves, and the fractions corresponding to the first and / or second main elution peaks were collected. At least one of the components in the first and / or second main elution peaks had antioxidant activity.

[0019] This invention provides a method for preparing antioxidant peptides from sturgeon red meat using the above-mentioned method; The antioxidant peptide from sturgeon red meat exhibits a DPPH free radical scavenging rate of ≥80% at a concentration of 1 mg / mL.

[0020] The present invention provides the application of the above-mentioned sturgeon red meat antioxidant peptide in the preparation of functional products for scavenging free radicals.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing antioxidant peptides from sturgeon red meat. This method uses sturgeon red meat as raw material to prepare antioxidant peptides, achieving high-value utilization of aquatic processing byproducts. This application overcomes the technical obstacle of multiple proteases failing to work synergistically due to pH conflicts by employing a stepwise hydrolysis process involving initial hydrolysis with a neutral protease and bromelain, followed by acidic hydrolysis with pepsin after enzyme inactivation. This significantly improves the yield and activity of small-molecule antioxidant peptides. The final peptide product exhibits a DPPH free radical scavenging rate of 80.8% at a concentration of 1 mg / mL, demonstrating excellent antioxidant performance. Simultaneously, this application introduces β-cyclodextrin for deodorization during the peptide solution stage, effectively removing fishy odor components without affecting antioxidant activity. Further purification by Sephadex G-25 gel chromatography separates and collects highly active components, resulting in a white to pale yellow powder with no obvious fishy odor. This method not only improves the functional quality of the product but also enhances its sensory characteristics.

[0022] The present invention provides a sturgeon red meat antioxidant peptide with excellent in vitro antioxidant activity. The DPPH free radical scavenging rate at a concentration of 1 mg / mL is not less than 80%.

[0023] The above-mentioned sturgeon red meat antioxidant peptides provided by this invention can be widely used in the preparation of functional products that scavenge free radicals. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 The free radical scavenging rate of the antioxidant peptide solution after hydrolysis of the complex enzyme preparation of pepsin and neutral protease, trypsin or bromelain provided by the present invention; Figure 2 The free radical scavenging rate of antioxidant peptide solutions after hydrolysis by different monomeric proteases provided in Example 1 of this invention; Figure 3 The free radical scavenging rate of the antioxidant peptide solutions obtained with different feed-to-liquid ratios provided in Example 2 of this invention; Figure 4 The free radical scavenging rate of the antioxidant peptide solutions obtained at different hydrolysis times provided in Example 2 of this invention; Figure 5 The free radical scavenging rate of the antioxidant peptide solutions obtained with different enzyme addition amounts provided in Example 2 of this invention; Figure 6This is an example of the absorbance curves of the effluent under different injection volumes provided in Example 3 of the present invention. Figure 7 This is an example of absorbance curves of the effluent at different flow rates provided in Embodiment 3 of the present invention. Figure 8 Sensory profiles of β-cyclodextrin deodorization at different addition ratios provided in Example 4 of the present invention; Figure 9 This is a comparison chart of the free radical scavenging rates of the products from Examples 4-6 provided in Example 5 of the present invention. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] According to one aspect of the present invention, a method for preparing antioxidant peptides from sturgeon red meat includes the following steps: (A) Provide pretreated sturgeon red meat, and use a compound enzyme preparation of neutral protease and bromelain to perform the first step of hydrolysis on the protein in the sturgeon red meat under neutral pH conditions; after the first step of hydrolysis is completed, heat to inactivate the compound enzyme preparation. (B) Adjust the system to an acidic pH environment, add pepsin to carry out the second step of hydrolysis, centrifuge after the reaction to obtain the supernatant to obtain a peptide solution, add β-cyclodextrin to the peptide solution for deodorization, filter and freeze dry to obtain crude product; (C) The crude product is dissolved and purified by gel chromatography. The elution peak with antioxidant activity is collected and freeze-dried to obtain sturgeon red meat antioxidant peptides.

[0028] This invention provides a method for preparing antioxidant peptides from sturgeon red meat. This method uses sturgeon red meat as raw material to prepare antioxidant peptides, achieving high-value utilization of aquatic processing byproducts. This application overcomes the technical obstacle of multiple proteases being unable to work synergistically due to pH conflicts by employing a stepwise hydrolysis process involving initial hydrolysis with a neutral protease and bromelain, followed by acidic hydrolysis with pepsin after enzyme inactivation. This significantly improves the yield and activity of small-molecule antioxidant peptides. The final peptide product exhibits a DPPH free radical scavenging rate of up to 80.8% at a concentration of 1 mg / mL, demonstrating excellent antioxidant performance.

[0029] Simultaneously, this application introduces β-cyclodextrin in the peptide solution stage for deodorization, effectively removing fishy odor components without affecting antioxidant activity; followed by Sephadex G-25 gel chromatography purification to separate and collect highly active components, resulting in a final product that is a white to pale yellow powder with no obvious fishy odor. This method not only improves the functional quality of the product but also enhances its sensory characteristics.

[0030] In a preferred embodiment of the present invention, the pretreatment method in step (A) includes: homogenizing sturgeon red meat and then freeze-drying it under vacuum to produce a loose and porous powder.

[0031] In a preferred embodiment, the red meat of sturgeon is first homogenized and then freeze-dried under vacuum to produce a loose, porous powder, which is beneficial for the full progress of subsequent enzymatic hydrolysis. Homogenization allows for more thorough disruption of tissue cells and more complete exposure of proteins; vacuum freeze-drying removes moisture at low temperatures, avoiding excessive denaturation of the protein structure at high temperatures, while simultaneously forming a porous and loose structure, increasing the specific surface area and water swelling performance of the material.

[0032] In a preferred embodiment of the present invention, step (A) further includes: before the first step of hydrolysis, mixing the pretreated powdered material with water and then subjecting it to a water bath treatment; As an optional implementation, the ratio of the powdered material to water is 1:9 to 10, for example, it can be 1:9, 1:9.2, 1:9.5, 1:9.8 or 1:10, or any ratio between 1:9 and 1:10.

[0033] As an optional implementation, the temperature of the water bath treatment is 90-100°C, for example, it can be 90°C, 92°C, 95°C, 98°C, 100°C, or any value between 90-100°C; the treatment time is 25-40 min, for example, it can be 25 min, 30 min, 35 min, 40 min, or any value between 25-40 min.

[0034] In a preferred embodiment of the present invention, the pH of the first step of hydrolysis in step (A) is 6-7, the reaction temperature is 50-60°C, and the time is 6-9 hours. As an optional implementation, the pH of the first hydrolysis step in step (A) is 6 to 7, for example, it can be 6.0, 6.2, 6.5, 6.8, 7.0, or any value between 6 and 7; the reaction temperature is 50 to 60°C, for example, it can be 50°C, 52°C, 55°C, 58°C, 60°C, or any value between 50 and 60°C; the hydrolysis time is 6 to 9 h, for example, it can be 6 h, 7 h, 8 h, 9 h, or any value between 6 and 9 h.

[0035] As an optional implementation, the mass ratio of neutral protease to bromelain in the compound enzyme preparation is 5 to 8:1, for example, it can be 5:1, 6:1, 7:1, 8:1, or any ratio between 5 and 8:1; wherein, a mass ratio of 5:1 is a preferred embodiment within this range.

[0036] As an optional implementation, the total amount of the compound enzyme preparation added is 5000-6000 U / g (based on the dry weight of fish meat), for example, it can be 5000 U / g, 5200 U / g, 5500 U / g, 5800 U / g, 6000 U / g, or any value between 5000 and 6000 U / g.

[0037] In a preferred embodiment of the present invention, the pH of the second hydrolysis step (B) is 2-2.5, the reaction temperature is 50-55°C, and the reaction time is 6-9 hours. As an optional implementation, the pH of the second hydrolysis step (B) is 2 to 2.5, for example, it can be 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any value between 2 and 2.5; the reaction temperature is 50 to 55°C, for example, it can be 50°C, 52°C, 53°C, 55°C, or any value between 50 and 55°C; the reaction time is 6 to 9 h, for example, it can be 6 h, 7 h, 8 h, 9 h, or any value between 6 and 9 h.

[0038] As an optional implementation, the amount of pepsin added during the second hydrolysis step (B) is 2500-3000 U / g (based on the dry weight of the fish), for example, it can be 2500 U / g, 2600 U / g, 2700 U / g, 2800 U / g, 2900 U / g, 3000 U / g, or any value between 2500-3000 U / g.

[0039] In a preferred embodiment of the present invention, the amount of β-cyclodextrin added in step (B) is 0.5~10 mg / 20 mL, relative to the volume of the peptide solution.

[0040] As an optional implementation, the amount of β-cyclodextrin added in step (B) is 0.5 to 10 mg / 20 mL (based on the volume of the peptide solution), for example, it can be 0.5 mg / 20 mL, 2 mg / 20 mL, 5 mg / 20 mL, 8 mg / 20 mL, 10 mg / 20 mL, or any value between 0.5 and 10 mg / 20 mL.

[0041] In a preferred embodiment of the present invention, after adding β-cyclodextrin to the peptide solution in step (B), the mixture is continuously shaken or stirred at room temperature for 40-50 minutes to achieve full encapsulation and deodorization.

[0042] In a preferred embodiment of the present invention, the method for step (C) gel chromatography purification includes: The lyophilized crude product was dissolved in buffer or water to obtain a solution with a concentration of 75 mg / mL. After pre-filtration through a 0.45 μm filter membrane, the solution was used as the loading solution. The loading solution was separated using a Sephadex G25 gel column with an injection volume of 100–200 mg and an elution mobile phase of water or buffer solution at a flow rate of 0.6–2 mL / min. The absorbance of the eluent was monitored online at a wavelength of 280 nm. The separation peaks were determined based on the elution curves, and the fractions corresponding to the first and / or second main elution peaks were collected. At least one of the components in the first and / or second main elution peaks had antioxidant activity.

[0043] According to one aspect of the present invention, a sturgeon red meat antioxidant peptide prepared by the above-described method is provided. The antioxidant peptide from sturgeon red meat exhibits a DPPH free radical scavenging rate of ≥80% at a concentration of 1 mg / mL.

[0044] The sturgeon red meat antioxidant peptide provided by this invention has excellent in vitro antioxidant activity. The DPPH free radical scavenging rate at a concentration of 1 mg / mL is not less than (≥) 80%.

[0045] According to one aspect of the present invention, the application of the above-mentioned sturgeon red meat antioxidant peptide in the preparation of free radical scavenging functional products.

[0046] The above-mentioned sturgeon red meat antioxidant peptides provided by this invention can be widely used in the preparation of functional products that scavenge free radicals.

[0047] The technical solution of the present invention will be further described below with reference to the embodiments.

[0048] It should be noted that in the research on the preparation of antioxidant peptides from sturgeon red meat in this application, the applicant initially attempted to enhance the product activity by using a combination of multiple proteases for hydrolysis. However, experiments showed that although pepsin has a strong ability to degrade proteins and the resulting hydrolysate has high clarity and a mild fishy smell, its antioxidant activity is low when used alone. Furthermore, when pepsin was combined with common proteases such as neutral proteases, trypsin, or bromelain in the same reaction system for co-hydrolysis, the DPPH free radical scavenging rate of the resulting product not only did not show a synergistic enhancement effect, but was actually lower than that of some single-enzyme treatment groups, and even lower than the theoretical average. See [link to relevant documentation] for details. Figure 1 .

[0049] Figure 1 The DPPH free radical scavenging rate of the complex enzyme preparation of pepsin with neutral protease, trypsin or bromelain provided by the present invention.

[0050] The above phenomenon may stem from the significant differences in the optimal reaction conditions among different proteases, especially their incompatible pH environments (e.g., the optimal pH for neutral proteases is 6-7, while for pepsin it is 2-2.5), making it impossible for them to coexist and function effectively in the same reaction system. Forcibly mixing them can lead to enzyme activity inhibition or inactivation, thereby reducing the overall hydrolysis efficiency.

[0051] Therefore, the applicant proposes a stepwise enzymatic hydrolysis strategy: first, under neutral conditions, neutral protease and bromelain are used to perform preliminary enzymatic hydrolysis of sturgeon red meat protein; then, the first-stage reaction is terminated by heating to inactivate the enzymes; finally, the system is adjusted to an acidic environment, and pepsin is introduced for deep hydrolysis. This avoids the problem of pH conflict among multiple enzymes and achieves the efficient action of each enzyme under its optimal conditions. Specific corresponding embodiments are as follows: Example 1 Enzyme Screening Experiment To screen for proteases suitable for efficient hydrolysis of sturgeon red meat protein, this embodiment compares the hydrolysis effects of various single proteases under their respective optimal conditions, and uses DPPH free radical scavenging rate as an evaluation index to determine the preliminary dominant enzymes.

[0052] The experimental methods include: 1. Raw material pretreatment: Fresh sturgeon red meat is taken, rinsed with clean water to remove blood and impurities, drained, and then subjected to high-speed homogenization to obtain a uniform fish meat paste. The paste is then portioned into trays and... Pre-freeze at 80℃ for 4 hours, then place in a vacuum freeze dryer. After continuous drying at 80℃ and 10Pa for 30 hours, a sponge-like, brittle, loose, porous powder was obtained, namely freeze-dried sturgeon red meat powder.

[0053] 2. Preparation of the reaction system: Weigh out 7 equal portions of the above freeze-dried sturgeon red meat powder, add distilled water at a ratio of 1:10 (g / mL) and mix thoroughly to form a suspension; place each mixture in a boiling water bath (95~100℃) and heat for 30 minutes, gently shaking during the process to promote wetting and swelling, and then let it cool naturally to room temperature.

[0054] 3. Enzymatic hydrolysis reaction: The cooled suspension was divided into 7 groups. Each group was adjusted to the optimal pH value for the corresponding protease, and one of the following proteases was added to each group, with a final concentration of 6000 U / g of dried fish meat: Table 1. Optimal pH and reaction temperature for different proteases:

[0055] Each reaction system was reacted in a constant-temperature water bath for 6 hours at the corresponding temperature and pH.

[0056] 4. Enzyme inactivation treatment: After enzymatic hydrolysis, each reaction solution was transferred to a 95°C water bath and heated for 15 minutes to completely inactivate the protease. After cooling to room temperature, the solution was centrifuged (4°C, 10,000 rpm, 20 min) and the supernatant was collected to obtain the "crude antioxidant peptide solution".

[0057] 5. Antioxidant activity assay (DPPH method): After diluting each crude solution obtained from enzymatic hydrolysis by 10 times, 2 mL of the solution was mixed with 2 mL of 0.1 mM DPPH ethanol solution and reacted in the dark for 30 minutes. The absorbance was measured at a wavelength of 517 nm using a UV-Vis spectrophotometer. The DPPH solution without the sample was used as a blank control (A0), and the absorbance after adding the sample was measured as A1.

[0058] The formula for calculating DPPH free radical scavenging rate is: DPPH free radical scavenging rate (%) = (A0-A1) / A0×100%.

[0059] III. Results (See attached) Figure 2 And Table 2.

[0060] Figure 2 The free radical scavenging rate of red meat solutions hydrolyzed by different proteases is shown in this embodiment.

[0061] Table 2:

[0062] As shown in Table 2, among the seven single proteases tested, the neutral protease hydrolysate exhibited the highest antioxidant activity (45%), followed by trypsin and alkaline protease. Therefore, subsequent process optimization should prioritize using neutral protease as a base.

[0063] Example 2: Investigation of process parameters for neutral protease hydrolysis.

[0064] Based on the determination that neutral protease has high hydrolytic activity on sturgeon red meat, this embodiment systematically investigated the effects of three key process parameters—material-liquid ratio, hydrolysis time, and enzyme dosage—on the antioxidant activity of the hydrolysate through a single-factor experimental system. The optimal reaction conditions were screened to provide support for subsequent synergistic and stepwise hydrolysis using compound enzymes.

[0065] I. Materials and Methods: 1. Raw material pretreatment: Same as in Example 1; 2. Single-factor experimental design: (1) Experiment on optimization of material-liquid ratio: Fixed conditions: enzyme addition of 6000 U / g dry fish weight, pH 6.5, temperature 55℃, hydrolysis time 6 h; Variation factors: The material-to-liquid ratios were set to 1:3, 1:5, 1:8, 1:10, and 1:15 (g / mL); Add distilled water according to the above ratio, mix thoroughly, and then perform water bath swelling treatment (95~100℃, 30 min). After cooling to room temperature, adjust the pH to 6.5 and add neutral protease to start the reaction.

[0066] (2) Hydrolysis time optimization experiment: Fixed conditions: material-to-liquid ratio 1:10 (g / mL), enzyme addition 6000 U / g, pH 6.5, temperature 55℃; Factors affecting the process: hydrolysis time was set to 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and 9 h respectively; other operations were the same as in step (1).

[0067] (3) Optimization experiment of enzyme addition amount: Fixed conditions: material-to-liquid ratio 1:10 (g / mL), pH 6.5, temperature 55℃, hydrolysis time 6 h; Factors affecting the variation: the amount of neutral protease added was 1000, 3000, 6000, 8000, and 12000 U / g of dry fish meat; other operations were the same as in step (1).

[0068] 3. Reaction and Post-processing: Each experiment was conducted in a constant temperature shaking water bath. After the reaction was completed, the mixture was transferred to a 95°C water bath and heated for 15 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant was then used as the crude antioxidant peptide solution.

[0069] 4. Antioxidant activity assay (DPPH method) Each group of crude product solutions was diluted 10 times, and 2 mL of each solution was mixed with 2 mL of 0.1 mM DPPH ethanol solution. After reacting in the dark for 30 minutes, the absorbance was measured at a wavelength of 517 nm, and the DPPH free radical scavenging rate was calculated. The formula for calculating the DPPH free radical scavenging rate is the same as in Example 1.

[0070] II. Results Analysis: Figure 3 The free radical scavenging rate of the antioxidant peptide solutions obtained with different feed-to-liquid ratios provided in this embodiment.

[0071] Depend on Figure 3 It can be seen that as the material-to-liquid ratio increases (i.e., the amount of water added increases), the fluidity of the system improves, but too low a substrate concentration may affect the enzyme's efficiency. When the material-to-liquid ratio is 1:10, the free radical scavenging rate reaches 44%, while the hydrolysate concentration is economical.

[0072] Figure 4 The free radical scavenging rate of the antioxidant peptide solutions obtained at different hydrolysis times provided in this embodiment.

[0073] Depend on Figure 4 It can be seen that the antioxidant activity first increases and then tends to stabilize with the extension of hydrolysis time; it reaches its highest value within 7 hours; and there is no significant increase after 7 hours. Therefore, the optimal hydrolysis time was selected as 7 hours.

[0074] Figure 5 The free radical scavenging rate of the antioxidant peptide solutions obtained with different enzyme addition amounts provided in this embodiment.

[0075] Depend on Figure 5 It was found that the clearance rate continued to increase when the enzyme dosage increased from 1000 U / g to 6000 U / g; however, the increase slowed down when the dosage was increased to above 8000 U / g, and the cost increased significantly. Considering both activity performance and economic efficiency, the optimal enzyme dosage was determined to be 6000 U / g of dry fish meat.

[0076] Example 3 Investigation of Gel Chromatography Process Parameters To determine the optimal operating conditions for purifying crude antioxidant peptides from sturgeon red meat using Sephadex G-25 gel columns, the effects of different injection volumes and elution flow rates on the separation effect were systematically investigated to obtain elution peaks with high resolution and concentrated activity, thereby improving the purification efficiency and yield of the target peptides.

[0077] I. Materials and Methods: 1. Preparation of crude product: The crude antioxidant peptides from sturgeon red meat obtained in Example 1 were freeze-dried under vacuum for later use.

[0078] 2. Preparation of sample loading solution: The lyophilized crude product was dissolved in ultrapure water to prepare a solution with a concentration of 75 mg / mL, and then pre-filtered through a 0.45 μm aqueous filter membrane to obtain a clear loading solution.

[0079] 3. Gel column separation conditions: Chromatography column: Sephadex G-25 gel column (2.0 cm × 30 cm); Detection wavelength: 280 nm (online monitoring of effluent absorbance using a UV spectrophotometer); Elution phase: ultrapure water; Temperature: Room temperature (25℃); (1) Experiment on optimization of injection volume: With a fixed flow rate of 2 mL / min, the injection volumes were set to 100 mg, 150 mg, and 200 mg, respectively, while keeping other conditions consistent. Elution curves were recorded, and the separation degree and tailing of the main peak were compared under each condition.

[0080] (2) Elution flow rate optimization experiment: Based on the UV detection data of the elution effluent, the injection volume was fixed at 150 mg, and the elution flow rate was set to 0.6 mL / min, 1 mL / min, and 2 mL / min, respectively, with other conditions remaining the same. The elution curves were recorded, and the peak symmetry and separation efficiency at different flow rates were evaluated.

[0081] 4. Separation effect evaluation indicators: The fractions corresponding to each major elution peak were collected, freeze-dried, and their DPPH free radical scavenging activity was measured to confirm the location of the highly active components.

[0082] Taking into account separation efficiency, peak shape quality, and process feasibility, the optimal conditions for Sephadex G-25 gel column purification were determined to be: injection volume: 150 mg; elution flow rate: 1 mL / min.

[0083] II. Results: Figure 6 The absorbance curves of the effluent under different injection volumes are provided in this embodiment.

[0084] Depend on Figure 6 It can be seen that when the injection volume is 200 mg, the sample concentration is too high, resulting in severe overlap of the two elution peaks; when the injection volume is 100 mg, the sample concentration is too low, and the response value of the second elution peak is too low. Choosing an injection volume of 150 mg is the optimal value, which not only results in higher response values ​​for both peaks but also better separation.

[0085] Figure 7 The absorbance curves of the effluent at different flow rates are provided in this embodiment.

[0086] Depend on Figure 7 As shown in the figure, at an elution rate of 2 mL / min, the two peaks were not completely separated due to the relatively fast elution speed. Slowing down the elution rate, both 0.6 mL / min and 1 mL / min effectively separated the two peaks; however, considering time and economic factors, an elution rate of 1 mL / min was chosen.

[0087] Example 4 A method for preparing antioxidant peptides from sturgeon red meat includes the following steps: (1) Raw material pretreatment: Fresh sturgeon red meat is taken, rinsed with clean water to remove surface blood stains and impurities, drained, and then subjected to high-speed homogenization to obtain a uniform fish meat slurry. The slurry is divided into trays and pre-frozen at -80℃ for 4 hours, and then placed in a vacuum freeze dryer and continuously dried at -80℃ and 10 Pa pressure for 30 hours to obtain a sponge-like, brittle, loose, porous powder, i.e., loose, porous powder material.

[0088] (2) Construction of the system before the first step of hydrolysis: Weigh the above powdered material and add distilled water at a material-to-liquid ratio of 1:10 (g / mL). Mix thoroughly to form a suspension. Place the mixture in a boiling water bath and heat for 0.5 hours, gently shaking during the process to promote full wetting and swelling of the material. Allow it to cool naturally to room temperature.

[0089] (3) First step of enzymatic hydrolysis: Adjust the pH of the suspension to 6.5, add a compound enzyme preparation of neutral protease and bromelain, with a total enzyme activity of 6000 U / g dry fish meat, wherein the mass ratio of neutral protease to bromelain is 5:1; place the reaction system in a constant temperature shaking water bath and enzymatically hydrolyze at 55℃ for 6 hours.

[0090] (4) Enzyme inactivation treatment: After enzymatic hydrolysis, the reaction solution is transferred to a 95°C water bath and heated for 15 minutes to completely inactivate the protease; then it is removed and cooled to room temperature.

[0091] (5) Second step of enzymatic hydrolysis: Adjust the pH of the system to 2.2 with dilute hydrochloric acid, add pepsin at a rate of 3000 U / g dry weight of fish meat, and continue enzymatic hydrolysis at a constant temperature of 52℃ for 7 hours.

[0092] (6) Solid-liquid separation and deodorization treatment: Centrifuge the reaction solution after the second enzymatic hydrolysis at 4℃ and 10000 rpm for 20 minutes, and take the supernatant as the peptide solution; take 20 mL of the peptide solution, add β-cyclodextrin to the final concentration of 1 mg / 20 mL, and continuously shake the reaction at room temperature for 40 minutes to perform deodorization treatment.

[0093] (7) Filtration and preparation of crude product: The deodorized solution was filtered through a 0.22 μm aqueous filter membrane to remove insoluble impurities. The filtrate was collected and freeze-dried under vacuum to obtain crude sturgeon red meat antioxidant peptides.

[0094] (8) Gel chromatography purification: Weigh an appropriate amount of crude product, dissolve it in ultrapure water to prepare a solution with a concentration of 75 mg / mL, and pre-filter it through a 0.45 μm filter membrane; perform chromatography separation using a Sephadex G-25 gel column (2.0 cm × 30 cm), with a sample loading volume of 2 mL (equivalent to an injection volume of approximately 150 mg), ultrapure water as the elution mobile phase, and a flow rate controlled at 1 mL / min; use a UV-Vis spectrophotometer to monitor the absorbance of the eluent in real time at a wavelength of 280 nm, and record the elution curve.

[0095] Two main peaks were observed based on the elution curve. The fractions corresponding to the first and second peaks were collected and freeze-dried separately.

[0096] (9) Product activity detection: The lyophilized products of the two peaks were prepared into 1 mg / mL aqueous solutions, and their antioxidant activity was determined by the DPPH free radical scavenging method: using 0.1 mM DPPH ethanol solution as the reaction system, 2 mL of sample solution was mixed with 2 mL of DPPH solution, and the reaction was carried out in the dark for 30 minutes. The absorbance was measured at a wavelength of 517 nm, and the free radical scavenging rate was calculated after background subtraction.

[0097] The formula for calculating DPPH free radical scavenging rate is: DPPH free radical scavenging rate (%) = (A0-A1) / A0×100%.

[0098] According to the detection data in this embodiment, A0 is 0.387, where the first peak detection value A1 is 0.213 and the second peak detection value A1 is 0.074.

[0099] The calculation results show that: The DPPH radical scavenging rate of the first peak product was 45.3%; The second peak product exhibited a DPPH free radical scavenging rate of 80.8%, significantly higher than that of antioxidant peptides derived from conventional fish.

[0100] (10) Amino acid composition analysis: The amino acid analysis of the second peak product was performed according to the method of GB 5009.124-2016 "Determination of Amino Acids in Food".

[0101] Table 3. Amino acid analysis of the freeze-dried sample corresponding to the second peak in this embodiment:

[0102] The results showed that it was rich in hydrophobic amino acids (such as leucine, valine, and isoleucine) and reducing amino acids (such as tyrosine and phenylalanine), which were closely related to its high antioxidant activity. The finally obtained highly active antioxidant peptide was a white to pale yellow powder with no obvious fishy smell, and could be used in the development of functional foods, health foods, or pharmaceuticals.

[0103] (11) In order to objectively evaluate the fishy smell characteristics of the enzymatic hydrolysate and the deodorizing effect of β-cyclodextrin, this application adopted a standardized sensory evaluation method for systematic determination. The evaluation team consisted of 8 reviewers who were professionally trained, had rich experience in sensory analysis, and were sensitive to fishy smell. There were 4 males and 4 females, aged 25–35 years, to ensure that the evaluation results were representative and gender balanced.

[0104] Test samples included untreated raw enzymatic hydrolysate and deodorized hydrolysate with different concentrations of β-cyclodextrin. 20 mL of each was placed in odorless polypropylene centrifuge tubes, randomly numbered, and presented blinded to avoid subjective bias.

[0105] During the evaluation process, each evaluator smelled one sample in turn, and then cleared their nasal cavity of residual odor by smelling black coffee powder. This served as an effective taste / olfactory cleansing method, ensuring the independence and accuracy of the evaluation of the next sample. Each evaluation round was spaced at least 30 seconds apart to prevent sensory fatigue.

[0106] Before the formal testing, the review team discussed and determined the main fishy odor properties of the enzymatic hydrolysate, and divided them into four sensory description dimensions: 1. Aquatic fishy smell (fishy smell originating from aquatic products); 2. Metallic smell (similar to the smell of blood or rust); 3. Oxidized grease has a rancid smell; 4. Ammonia / sulfur-based putrid smell (fermented or rotten odor).

[0107] A 9-point scale (0-10 points) was used to score each indicator: 0 points indicated no odor, and 10 points indicated an extremely strong odor. The final data was the arithmetic mean of the scores from 8 reviewers, and a sensory profile map was drawn in the form of a radar chart to visually compare the distribution characteristics of fishy odor under different treatment conditions.

[0108] Figure 8 Sensory profiles of β-cyclodextrin for deodorizing with different addition ratios provided in Example 4 of the present invention.

[0109] See Figure 8Sensory evaluation and solution clarity analysis showed that the intensity of various fishy odors decreased with increasing β-cyclodextrin dosage, especially for ammonia / sulfur-based putrid odors. When the dosage reached 1 mg / 20 mL, the overall improvement in fishy odor stabilized, and further increases in dosage yielded no significant benefit. Therefore, considering both the deodorizing effect and economic cost, the optimal dosage of β-cyclodextrin was determined to be 1 mg / 20 mL.

[0110] Example 5 To demonstrate the technical advantages of the preparation method of the antioxidant peptides from sturgeon red meat in Example 4 of this application, this application replaces "bromelain" in the first enzymatic hydrolysis step (3) with "alkaline protease", and the rest is the same as in Example 4.

[0111] This embodiment describes a technical solution that uses a composite enzyme preparation of neutral protease and alkaline protease for the first step of enzymatic hydrolysis, and pepsin for the second step of enzymatic hydrolysis.

[0112] Example 6 To demonstrate the technical advantages of the preparation method of the sturgeon red meat antioxidant peptide in Example 4 of this application, this application replaces "bromelain" in the first enzymatic hydrolysis step (3) with "trypsin", and the rest is the same as in Example 4.

[0113] This embodiment describes a technical solution that uses a composite enzyme preparation of neutral protease and trypsin for the first step of enzymatic hydrolysis, and pepsin for the second step of enzymatic hydrolysis.

[0114] Figure 9 The graph shows a comparison of the free radical scavenging rates of the products from Examples 4-6 provided in this embodiment.

[0115] Depend on Figure 9 It can be seen that the DPPH clearance rate of the second peak product obtained in Example 4 (neutral protease + bromelain) was 80.8%, which was significantly higher than that of Example 5 (neutral + alkaline protease) (68%) and Example 6 (neutral + trypsin) (50.2%), indicating that the complex enzyme combination selected in Example 1 of the present invention has a special synergistic effect.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing antioxidant peptides from sturgeon red meat, characterized in that, Includes the following steps: (A) Provide pretreated sturgeon red meat, and use a compound enzyme preparation of neutral protease and bromelain to perform the first step of hydrolysis on the protein in the sturgeon red meat under neutral pH conditions. After the first hydrolysis reaction is completed, the complex enzyme preparation is inactivated by heating. (B) Adjust the system to an acidic pH environment, add pepsin to carry out the second step of hydrolysis, centrifuge after the reaction to obtain the supernatant to obtain a peptide solution, add β-cyclodextrin to the peptide solution for deodorization, filter and freeze dry to obtain crude product; (C) The crude product was dissolved and purified by gel chromatography. The elution peak with antioxidant activity was collected and freeze-dried to obtain sturgeon red meat antioxidant peptides.

2. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, The preprocessing method in step (A) includes: Sturgeon red meat is homogenized and then freeze-dried under vacuum to produce a loose, porous powder.

3. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, The step (A) further includes: before the first hydrolysis step, mixing the pretreated powdered material with water and then subjecting it to a water bath treatment; Preferably, the ratio of the powdered material to water in the mixture is 1:9~10; Preferably, the water bath treatment is performed at a temperature of 90-100°C for 25-40 minutes.

4. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, In step (A), the first hydrolysis step has a pH of 6-7, a reaction temperature of 50-60℃, and a time of 6-9 hours. Preferably, in the compound enzyme preparation, the mass ratio of neutral protease to bromelain is 5-8:1; Preferably, the total amount of the compound enzyme preparation added is 5000~6000 U / g of dry fish meat.

5. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, The second step of hydrolysis in step (B) has a pH of 2 to 2.5, a reaction temperature of 50 to 55°C, and a reaction time of 6 to 9 hours. Preferably, in the second step of hydrolysis in step (B), the amount of pepsin added is 2500~3000 U / g of dry fish meat.

6. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, The amount of β-cyclodextrin added in step (B) is 0.5~10 mg / 20 mL, preferably 1 mg / 20 mL.

7. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, In step (B), after adding β-cyclodextrin to the peptide solution, the mixture is continuously shaken or stirred at room temperature for 40-50 minutes to achieve full inclusion and deodorization.

8. The method for preparing sturgeon red meat antioxidant peptides according to claim 1, characterized in that, The method for purification by gel chromatography in step (C) includes: The lyophilized crude product was dissolved in buffer or water to obtain a solution with a concentration of 75 mg / mL. After pre-filtration through a 0.45 μm filter membrane, the solution was used as the loading solution. The loading solution was separated using a Sephadex G25 gel column with an injection volume of 100–200 mg and an elution mobile phase of water or buffer solution at a flow rate of 0.6–2 mL / min. The absorbance of the eluent was monitored online at a wavelength of 280 nm. The separation peaks were determined based on the elution curves, and the fractions corresponding to the first and / or second main elution peaks were collected. At least one of the components in the first and / or second main elution peaks had antioxidant activity.

9. A sturgeon red meat antioxidant peptide prepared by the method according to any one of claims 1 to 8, characterized in that, The antioxidant peptide from sturgeon red meat exhibits a DPPH free radical scavenging rate of ≥80% at a concentration of 1 mg / mL.

10. The application of the sturgeon red meat antioxidant peptide according to claim 9 in the preparation of free radical scavenging functional products.

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