Preparation method and application of antioxidant salmon protein oligopeptide

Abstract

The invention discloses a preparation method and application of antioxidant salmon protein oligopeptide, and belongs to the technical field of aquatic product processing. The method comprises the following steps: respectively extracting salmon skin crude protein fluid, salmon swimming bladder crude protein fluid and salmon meat crude protein fluid; the three components are mixed according to the mass ratio of (75-115): (35-55): (65-85), and ascorbyl palmitate, flavourzyme and chymotrypsin are added for synergistic enzymolysis; and separating the enzymatic hydrolysate by using a 1800 Da ultrafiltration membrane, and drying to obtain a target product. Through synergistic utilization of three salmon by-products and integrated innovation of a quaternary enzymolysis system and a three-section type activity protection network, the prepared salmon protein oligopeptide has the molecular weight of less than 1800 Da, the polypeptide purity of more than or equal to 92% and the proportion of 500-1000 Da small molecule peptide of more than or equal to 64%, and shows excellent free radical scavenging capacity (the DPPH scavenging rate is more than or equal to 88%, and the ABTS scavenging rate is more than or equal to 92%). The product can be widely applied to the fields of functional foods, health foods and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of aquatic product processing technology, specifically relating to a method for preparing and applying antioxidant salmon protein oligopeptides. Background Technology

[0002] Salmon is an important economic fish species globally, with a large-scale processing industry that generates substantial amounts of byproducts annually, including fish skin, swim bladders, bones, and meat scraps. These byproducts are rich in protein, and failure to utilize them effectively not only wastes resources but also burdens the environment. Currently, the utilization of salmon byproducts is largely limited to fish oil extraction or processing into animal feed, with insufficient exploration of the bioactive protein peptides they contain. Therefore, achieving high-value utilization of salmon byproducts has become a crucial research direction in the aquatic product processing field.

[0003] Existing technologies for extracting protein peptides from fish by-products mainly include acid extraction, alkaline extraction, salt extraction, and enzymatic hydrolysis. Among these, enzymatic hydrolysis has become the mainstream method due to its mild conditions, high specificity, and low likelihood of generating harmful substances. However, existing enzymatic hydrolysis technologies still have several limitations. First, in terms of raw material utilization, most studies focus on single fish skin or flesh, failing to achieve systematic and synergistic utilization of multiple salmon by-products (such as collagen-rich fish skin, elastin-rich swim bladder, and nutrient-rich fish flesh), thus failing to maximize the value of the raw materials. Second, in terms of enzymatic hydrolysis processes, single proteases or conventional complex proteases are often used, which have limited enzymatic sites, potentially leading to incomplete hydrolysis and an excessively broad molecular weight distribution in the product. The proportion of small-molecule oligopeptides with excellent absorption and activity (especially peptides with a molecular weight below 2000 Da) is low, affecting the bioavailability of the final product. Furthermore, during the preparation process, proteins and enzymes are susceptible to oxidative inactivation, and existing technologies lack a systematic activity protection strategy throughout the entire extraction and enzymatic hydrolysis process. For example, Chinese patent CN119220623B discloses a method for preparing sturgeon protein peptides. Proteins extracted from different parts of sturgeon are mixed in a specific ratio and then enzymatically hydrolyzed. The added neutral protease and papain can work synergistically to more thoroughly hydrolyze the protein, resulting in sturgeon protein peptides with good antioxidant properties. The molecular weight range of the final product is relatively wide (below 3000 Da), but its ability to enrich higher-activity small molecule peptides is unknown, and the stability of its antioxidant activity needs further improvement. Summary of the Invention

[0004] Therefore, this invention urgently needs to develop a method for preparing salmon protein oligopeptides with smaller molecular weight, higher purity, stronger antioxidant activity, and greater stability by comprehensively utilizing various salmon processing byproducts and employing an innovative enzymatic hydrolysis system and full-process activity protection technology. This not only meets the industrial needs of green and sustainable development but also provides a new raw material solution for developing high-value-added marine-derived functional foods and cosmetics.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention discloses a method for preparing antioxidant salmon protein oligopeptides, comprising the following steps:

[0007] S1. Extraction of crude protein solution from salmon skin: Defatted salmon skin is added to a 2%-5% malic acid solution to swell, then tea polyphenols and chymotrypsin are added for enzymatic hydrolysis, and after enzyme inactivation, the mixture is filtered to obtain crude protein solution from salmon skin.

[0008] S2. Extraction of crude protein from salmon swim bladder: After soaking salmon swim bladder in sodium hydroxide solution, it is added to a 0.6-0.9 mol / L propionic acid solution and stirred for extraction. The extract is then salted out and dried to obtain crude protein from salmon swim bladder.

[0009] S3. Extraction of crude salmon protein solution: Mix salmon meat paste with distilled water, add sodium thiosulfate and bromelain, stir and hydrolyze, inactivate the enzyme, centrifuge and collect the supernatant to obtain salmon meat crude protein solution.

[0010] S4. Mixed enzymatic hydrolysis: The salmon skin crude protein solution obtained in step S1, the salmon swim bladder crude protein obtained in step S2, and the salmon meat crude protein solution obtained in step S3 are mixed in a mass ratio of (75-115):(35-55):(65-85). Then, ascorbate palmitate, flavor protease, and chymotrypsin are added for mixed enzymatic hydrolysis. The supernatant is collected by centrifugation to obtain the enzymatic hydrolysis product.

[0011] S5. Post-processing: After inactivating the enzyme in the enzymatic hydrolysis product, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da. The permeate is collected and dried to obtain the antioxidant salmon protein oligopeptide.

[0012] Specifically, in step S1, the amount of tea polyphenols added is 1%-2.2% of the weight of defatted salmon skin, and the amount of chymotrypsin added is 0.6%-2.2% of the weight of defatted salmon skin; and / or,

[0013] In step S3, the amount of sodium thiosulfate added is 1.5%-2.5% of the meat paste weight, and the amount of bromelain added is 1.2%-2.2% of the meat paste weight; and / or,

[0014] In step S4, the amount of ascorbate palmitate added is 0.15%-0.35% of the mass of the mixture, the amount of flavor protease added is 0.12%-0.32% of the mass of the mixture, and the amount of chymotrypsin added is 2.2%-4.2% of the mass of the mixture.

[0015] Adding tea polyphenols in step S1 serves two purposes: firstly, it acts as an antioxidant, protecting the protein from oxidative damage during enzymatic hydrolysis; secondly, it stabilizes the chymotrypsin structure and maintains enzyme activity. The hydrolysis temperature of 38-52 °C matches the optimal activity temperature of chymotrypsin, ensuring maximum hydrolysis efficiency. In step S3, sodium thiosulfate acts as a reducing agent, breaking the disulfide bonds between protein molecules, causing the protein structure to unfold, while simultaneously protecting the sulfhydryl groups of bromelain from oxidation, thus maintaining enzyme activity.

[0016] Specifically, in step S4, the temperature for the mixed enzymatic hydrolysis is 38-48 ℃, and the time is 2.5-4.5 h.

[0017] In step S4, the mixing of three crude protein raw materials in a specific ratio can achieve complementary amino acid compositions, laying the foundation for the generation of highly active oligopeptides; ascorbate palmitate can protect the sulfhydryl groups of flavor protease and chymotrypsin, preventing enzyme molecules from being oxidized and inactivated; flavor protease (which prefers to hydrolyze the carboxyl-terminal peptide bonds of hydrophobic amino acids) and chymotrypsin (which prefers to hydrolyze the carboxyl-terminal peptide bonds of aromatic amino acids) work synergistically to completely break protein peptide bonds and improve the yield of small molecule oligopeptides; the enzymatic hydrolysis temperature of 38-48℃ is the common optimal temperature range for both enzymes, which can ensure synergistic enzymatic hydrolysis efficiency.

[0018] Specifically, in step S1, the mass concentration of the malic acid solution is 3.5%, and the swelling conditions are swelling at 5-11°C for 18-24 hours; and / or,

[0019] In step S2, the concentration of the propionic acid solution is 0.7 mol / L, and the stirring extraction conditions are extraction at 5-9 ℃ for 38-44 h.

[0020] Specifically, in step S1, the enzymatic hydrolysis conditions are enzymatic hydrolysis at 38-52 °C for 2-4 h; and / or,

[0021] In step S3, the enzymatic hydrolysis conditions are: stirring at 38-52 °C and a rate of 90-130 r / min for 4-6 h.

[0022] Specifically, in step S5, the ultrafiltration membrane pressure is 0.15 MPa and the feed temperature is 35 ℃.

[0023] Secondly, the present invention discloses an antioxidant salmon protein oligopeptide prepared by the aforementioned preparation method, which has a molecular weight of less than 1800 Da, a polypeptide purity of not less than 92%, and a scavenging rate of not less than 88% for DPPH free radicals and a scavenging rate of not less than 92% for ABTS free radicals.

[0024] Specifically, peptides with a molecular weight in the range of 500 Da to 1000 Da account for no less than 64% of the total peptide content.

[0025] Thirdly, this invention discloses the application of the aforementioned antioxidant salmon protein oligopeptide in the preparation of antioxidant functional foods, health foods, or cosmetics.

[0026] Specifically, the antioxidant salmon protein oligopeptide is used as an active ingredient in the preparation of oral liquids, compressed candies, capsules, lyophilized powders, serums, or facial masks.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention is the first to synergistically utilize salmon skin, salmon swim bladder, and salmon meat, combining a malic acid / propionic acid extraction system, a quaternary enzymatic hydrolysis system of chymotrypsin + bromelain + flavor protease + chymotrypsin, and a three-stage activity protection network of tea polyphenols + sodium thiosulfate + ascorbate palmitate. This achieves high-value and full utilization of salmon by-products and successfully prepares salmon protein oligopeptides with a molecular weight <1800 Da, a small peptide content >93%, and antioxidant activity significantly superior to existing technologies. Detailed Implementation

[0029] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0030] In this invention, the tea polyphenols were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and are green tea extracts with an extraction ratio of 10:1; the chymotrypsin was purchased from Beijing Solarbio Biotechnology Co., Ltd., with an enzyme activity of 50,000 U / g; the flavor protease was purchased from Beijing Solarbio Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; and the chymotrypsin was purchased from Beijing Solarbio Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0031] Example 1: A method for preparing antioxidant salmon protein oligopeptides

[0032] S1. Extraction of crude protein from salmon skin: Salmon skin was cut into pieces (5 mm × 5 mm), soaked in 3.5% NaCl solution at 13℃ for 16 h, washed with distilled water, and defatted with petroleum ether at 7℃ for 26 h to obtain defatted salmon skin. The defatted salmon skin was then added to 3.5% malic acid solution and swelled at 7℃ for 20 h. Then, 1.6% (by weight of defatted salmon skin) of tea polyphenols and 1.3% (by weight of defatted salmon skin) of chymotrypsin were added, and the mixture was enzymatically hydrolyzed at 45℃ for 3.5 h. The enzyme was inactivated by boiling in a water bath for 12 min, cooled, filtered, and the filtrate was collected.

[0033] S2. Extraction of crude protein from salmon swim bladder: Salmon swim bladder was cut into pieces (5 mm × 5 mm), soaked in NaOH, washed with water until neutral, and then extracted with 0.7 mol / L propionic acid solution by stirring. The extract was then salted out and dried to obtain crude protein from salmon swim bladder.

[0034] S3. Extraction of crude salmon protein solution: Salmon meat was cut into pieces (1 cm × 1 cm) and minced. The salmon mince was mixed with distilled water, and 2.2% sodium thiosulfate and 1.6% bromelain (by weight of the mince) were added. The mixture was hydrolyzed at a stirring rate of 110 r / min and a temperature of 45 ℃ for 5.5 h. After enzyme inactivation, the supernatant was collected by centrifugation to obtain the crude salmon protein solution.

[0035] S4. Mixed enzymatic hydrolysis: Mix 95 g of crude fish skin protein solution, 45 g of crude fish swim bladder protein solution, and 75 g of crude fish meat protein solution; add 0.2% ascorbate palmitate, 0.2% flavor protease, and 2.2% chymotrypsin by weight of the mixture, and hydrolyze at 43 ℃ for 3.5 h; centrifuge and collect the supernatant to obtain the enzymatic hydrolysis product;

[0036] S5. Post-processing: The enzyme in the enzymatic hydrolysate was inactivated by boiling in a water bath for 12 min to obtain the enzymatic hydrolysate. The hydrolysate was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da at 0.15 MPa and 35 ℃, and the permeate was collected. Finally, the hydrolysate was freeze-dried at -46 ℃ and 5 Pa until the moisture content was <5%, yielding the antioxidant salmon protein oligopeptides.

[0037] Example 2: A method for preparing antioxidant salmon protein oligopeptides

[0038] S1. Extraction of crude protein from salmon skin: Salmon skin was cut into pieces (5 mm × 5 mm), soaked in 3.5% NaCl solution at 13℃ for 16 h, washed with distilled water, and defatted with petroleum ether at 7℃ for 26 h to obtain defatted salmon skin. The defatted salmon skin was then added to 5% malic acid solution and swelled at 5℃ for 24 h. Then, 1.0% (by weight of defatted salmon skin) of tea polyphenols and 0.6% (by weight of defatted salmon skin) of chymotrypsin were added, and enzymatic hydrolysis was performed at 38℃ for 4 h. The enzyme was inactivated by boiling in a water bath for 12 min, cooled, filtered, and the filtrate was collected.

[0039] S2. Extraction of crude protein from salmon swim bladder: Salmon swim bladder was cut into pieces (5 mm × 5 mm), soaked in NaOH, washed with water until neutral, and then extracted with 0.6 mol / L propionic acid solution by stirring. The extract was then salted out and dried to obtain crude protein from salmon swim bladder.

[0040] S3. Extraction of crude salmon protein solution: Salmon meat was cut into pieces (1 cm × 1 cm) and minced. The salmon mince was mixed with distilled water, and 1.5% sodium thiosulfate and 1.2% bromelain (by weight of the mince) were added. The mixture was enzymatically hydrolyzed at a stirring rate of 90 r / min and a temperature of 38 ℃ for 6 h. After enzyme inactivation, the supernatant was collected by centrifugation to obtain the crude salmon protein solution.

[0041] S4. Mixed enzymatic hydrolysis: Mix 75 g of crude fish skin protein solution, 35 g of crude fish swim bladder protein solution, and 65 g of crude fish meat protein solution; add 0.15% ascorbate palmitate, 0.12% flavor protease, and 2.2% chymotrypsin by weight of the mixture, and hydrolyze at 38 ℃ for 4.5 h; centrifuge and collect the supernatant to obtain the enzymatic hydrolysis product;

[0042] S5. Post-processing: The enzyme in the enzymatic hydrolysate was inactivated by boiling in a water bath for 12 min to obtain the enzymatic hydrolysate. The hydrolysate was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da at 0.15 MPa and 35 ℃, and the permeate was collected. Finally, the hydrolysate was freeze-dried at -46 ℃ and 5 Pa until the moisture content was <5%, yielding the antioxidant salmon protein oligopeptides.

[0043] Example 3: A method for preparing antioxidant salmon protein oligopeptides

[0044] S1. Extraction of crude protein from salmon skin: Salmon skin was cut into pieces (5 mm × 5 mm), soaked in 3.5% NaCl solution at 13℃ for 16 h, washed with distilled water, and defatted with petroleum ether at 7℃ for 26 h to obtain defatted salmon skin. The defatted salmon skin was then added to 2% malic acid solution and swelled at 11℃ for 18 h. Then, 2.2% (by weight of defatted salmon skin) of tea polyphenols and 2.2% (by weight of defatted salmon skin) of chymotrypsin were added, and enzymatic hydrolysis was performed at 52℃ for 2 h. The enzyme was inactivated by boiling in a water bath for 12 min, cooled, filtered, and the filtrate was collected.

[0045] S2. Extraction of crude protein from salmon swim bladder: Salmon swim bladder was cut into pieces (5 mm × 5 mm), soaked in NaOH, washed with water until neutral, and then extracted with 0.9 mol / L propionic acid solution by stirring. The extract was then salted out and dried to obtain crude protein from salmon swim bladder.

[0046] S3. Extraction of crude salmon protein solution: Salmon meat was cut into pieces (1 cm × 1 cm) and minced. The salmon mince was mixed with distilled water, and 2.5% sodium thiosulfate and 2.2% bromelain (by weight of the mince) were added. The mixture was hydrolyzed at a stirring rate of 130 r / min and a temperature of 52 ℃ for 4 h. After enzyme inactivation, the supernatant was collected by centrifugation to obtain the crude salmon protein solution.

[0047] S4. Mixed enzymatic hydrolysis: Mix 115 g of crude fish skin protein solution, 55 g of crude fish swim bladder protein solution, and 85 g of crude fish meat protein solution; add 0.35% ascorbyl palmitate, 0.32% flavor protease, and 4.2% chymotrypsin by weight of the mixture, and hydrolyze at 48 ℃ for 2.5 h; centrifuge and collect the supernatant to obtain the enzymatic hydrolysis product;

[0048] S5. Post-processing: The enzyme in the enzymatic hydrolysate was inactivated by boiling in a water bath for 12 min to obtain the enzymatic hydrolysate. The hydrolysate was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da at 0.15 MPa and 35 ℃, and the permeate was collected. Finally, the hydrolysate was freeze-dried at -46 ℃ and 5 Pa until the moisture content was <5%, yielding the antioxidant salmon protein oligopeptides.

[0049] Comparative Example 1

[0050] Compared with Example 1, the difference is that salmon skin crude protein solution was not added in step S3 of this comparative example. Instead, 45 g of salmon swim bladder crude protein and 75 g of salmon meat crude protein solution were mixed and enzymatically hydrolyzed. The types and amounts of enzymes were adjusted proportionally. All other experimental steps were the same as in Example 1.

[0051] Comparative Example 2

[0052] Compared with Example 1, the difference is that: in this comparative example, no tea polyphenols were added when preparing the crude salmon skin protein solution in step S1, no sodium thiosulfate was added when preparing the crude salmon meat protein solution in step S3, and no ascorbate palmitate was added when mixing and enzymatically hydrolyzing in step S4. All other experimental steps were the same as in Example 1.

[0053] Comparative Example 3

[0054] Compared with Example 1, the difference is that in this comparative example, only flavor protease (the amount was adjusted to 2.4%) was used during the mixed enzymatic hydrolysis in step S4, and chymotrypsin was not used. All other experimental steps were the same as in Example 1.

[0055] Example 1: Determination of peptide purity and small molecule peptide ratio of antioxidant salmon protein oligopeptides.

[0056] Peptide purity determination: Reversed-phase high-performance liquid chromatography (RP-HPLC) was used. The specific steps are as follows:

[0057] First, the solutions were prepared. An ultrapure aqueous solution containing 0.1% trifluoroacetic acid was used as mobile phase A, and an acetonitrile solution containing 0.1% trifluoroacetic acid was used as mobile phase B. Both mobile phases were filtered through a 0.22 μm filter and degassed by sonication. Bovine serum albumin standard was accurately weighed, dissolved and diluted with mobile phase A to prepare a solution containing approximately 0.1 mg per mL, which served as the standard solution. Simultaneously, each analyte sample was accurately weighed, dissolved and diluted to volume with mobile phase A to prepare a sample solution containing approximately 1.0 mg per mL. This sample solution was filtered through a 0.22 μm filter, and the filtrate was used as the test solution.

[0058] Chromatographic analysis was performed under the following conditions: column: octadecylsilane-bonded silica gel (4.6 mm × 250 mm, 5 μm); column temperature: 30℃; detection wavelength: 220 nm; flow rate: 1.0 mL per minute; injection volume: 10 μL.

[0059] Elution procedure: Gradient elution was used. The initial mobile phase B concentration was 5% and maintained for 5 minutes; then, over 30 minutes, the mobile phase B concentration was linearly increased to 40%; subsequently, it was rapidly increased to 95% and maintained over 5 minutes to wash away strongly retained impurities; finally, the concentration was restored to the initial 5% concentration over 2 minutes, and the column was equilibrated for the next injection. The total run time was approximately 40 minutes.

[0060] After the instrument stabilizes, precisely pipette 10 μL each of the standard solution and the test solution and inject them into the liquid chromatograph, recording the chromatograms. Qualitative identification of the peptide components in the sample is achieved by comparing the retention times of the peaks with those of the standard solution. The purity of the peptide is obtained by integrating the results using the chromatography workstation and calculating the percentage of the peak area of ​​the main peak of the test sample relative to the total peak area (excluding solvent peaks).

[0061] Determination of small molecule peptide proportion: High-performance gel filtration chromatography was used. The specific steps are as follows:

[0062] First, a phosphate buffer solution at pH 6.8 containing 10 mmol / L KH₂PO₄ and 100 mmol / L NaCl was prepared as the mobile phase. This solution was then filtered through a 0.22 μm aqueous membrane and degassed. A series of molecular weight standard stock solutions at 1.0 mg / mL were prepared and further diluted and mixed to form a 0.1 mg / mL mixed standard working solution for establishing a molecular weight-retention time standard curve. Simultaneously, each test sample was accurately weighed, dissolved in the above mobile phase, and brought to a final volume to prepare a 1.0 mg / mL sample solution. This sample solution was then filtered through a 0.22 μm aqueous membrane, and the filtrate was used as the test solution.

[0063] Chromatographic analysis was performed under the following conditions: Chromatographic system: high-performance gel filtration chromatography system; Mobile phase: the above-mentioned phosphate buffer (isocratic elution); Flow rate: 0.5 mL / min; Column temperature: 30 ℃; Detection wavelength: 220 nm; Injection volume: 20 μL;

[0064] Accurately pipette 20 μL each of the mixed standard solution and the test solution sequentially into the chromatograph and record the chromatograms within 30 minutes. Establish a standard curve of molecular weight logarithm versus retention time using the mixed standard chromatogram. In the test sample chromatogram, determine the range of chromatographic peaks with a molecular weight less than 1800 Da based on the standard curve, and calculate the percentage of the total peak area of ​​all chromatographic peaks within this range relative to the total peak area of ​​the total peptides (excluding solvent peaks), thus obtaining the proportion of small molecule peptides.

[0065] Table 1. Results of purity and small molecule peptide ratio determination of salmon protein oligopeptides prepared in the examples and comparative examples.

[0066]

[0067] Table 1 shows the results of the purity and small molecule peptide ratio determination of the salmon protein oligopeptides prepared in the above examples and comparative examples. It can be seen that the proportion of oligopeptides with a molecular weight of less than 1800 Da in the antioxidant salmon protein oligopeptides prepared in Examples 1-3 all exceeded 93%, which meets the industry standard for oligopeptides.

[0068] Example 2: Determination of the antioxidant properties of salmon protein oligopeptides

[0069] To verify the performance of the antioxidant salmon protein oligopeptides prepared in this invention, the antioxidant properties of the salmon protein oligopeptides prepared in the examples and comparative examples were tested. The specific test methods are as follows:

[0070] DPPH free radical scavenging capacity determination: Accurately weigh an appropriate amount of salmon protein oligopeptide sample, dissolve it in distilled water to prepare a 4 mg / mL sample solution. Accurately measure 4 mL of this sample solution, add 1 mL of 40 μmol / L DPPH-ethanol solution, vortex to mix, and react at 25℃ in the dark for 30 minutes. Use 4 mL of distilled water instead of the sample solution, and perform the same procedure as a blank control. After the reaction, measure the absorbance values ​​of the sample group and the blank group at a wavelength of 517 nm, and record them as A. 样品 With A 空白 The formula for calculating the DPPH free radical scavenging capacity is: DPPH free radical scavenging rate (%) = (1-A) / (1-A) 样品 / A 空白 )×100.

[0071] ABTS free radical scavenging capacity determination: Accurately weigh an appropriate amount of salmon protein oligopeptide sample, dissolve it in distilled water to prepare a 4 mg / mL sample solution. Dilute the ABTS free radical stock solution with phosphate buffer at pH 7.4, adjusting its absorbance at 734 nm to 0.700 ± 0.020, to prepare the ABTS assay working solution. Accurately measure 4 mL of the ABTS assay working solution, add 40 μL of the above sample solution, mix rapidly, and react at room temperature in the dark for a certain time (usually 6 minutes). Measure the absorbance at 734 nm and take the average of multiple readings, recorded as A. The formula for calculating the ABTS scavenging rate is: ABTS free radical scavenging rate (%) = (1 - A / 0.700) × 100.

[0072] Hydroxyl radical scavenging capacity determination: Weigh the sample, dissolve it in distilled water to prepare a 4 mg / mL sample solution. The Fenton reaction system and the o-phenanthroline colorimetric method were used for determination. The specific steps are as follows:

[0073] Damage tube: Add 1 mL of o-phenanthroline ethanol solution, 2 mL of phosphate buffer (PBS), 1 mL of sample solution, 1 mL of ferrous sulfate solution and 1 mL of hydrogen peroxide in sequence.

[0074] Undamaged tube: Replace the hydrogen peroxide in the damaged tube with 1 mL of distilled water.

[0075] Sample tube: Replace the distilled water in the damaged tube with 1 mL of sample solution.

[0076] Mix all tubes thoroughly and react in a water bath at 37 ℃ for 60 minutes. Measure the absorbance of each tube at a wavelength of 536 nm and record it as A. 损 A 未 and A 样 .

[0077] The hydroxyl radical scavenging rate is calculated using the following formula:

[0078] • OH removal rate (%) = [(A 样 -A 损 ) / (A 未 -A 损 )]×100.

[0079] Table 2 shows the antioxidant properties of the salmon protein oligopeptides prepared in the examples and comparative examples.

[0080]

[0081] The test results are shown in Table 2. The salmon protein oligopeptides prepared in Examples 1-3 of this invention showed significantly higher scavenging rates of DPPH free radicals, ABTS free radicals, and hydroxyl free radicals than those in Comparative Examples 1-3. This indicates that the crude protein mixture of salmon skin, salmon swim bladder, and salmon meat in this invention has a synergistic effect. Furthermore, the addition of tea polyphenols, sodium thiosulfate, ascorbate palmitate, and the synergistic enzymatic hydrolysis by flavor protease and chymotrypsin can effectively improve the overall antioxidant properties of salmon protein oligopeptides. Further analysis revealed that Comparative Example 1, lacking the addition of crude salmon skin protein solution, lacked the collagen and tea polyphenols-protected active peptides abundant in salmon skin, resulting in a 13%-16% decrease in its antioxidant performance compared to Example 1. Comparative Example 2, without the addition of tea polyphenols, sodium thiosulfate, and ascorbate palmitate, lacked enzyme activity protection and its protein peptides were susceptible to oxidative damage, with the scavenging rates of the three free radicals all dropping below 83%. Comparative Example 3, using only a single flavor protease, could not fully hydrolyze the protein to generate highly active oligopeptides, and its hydroxyl free radical scavenging rate decreased by more than 12% compared to Example 1. This fully demonstrates the rationality and necessity of the technical solution of the present invention in terms of raw material combination, addition of active protective agents, and synergistic selection of enzymes.

[0082] Example 3: In vivo antioxidant and anti-aging experiments

[0083] Experimental animals: SPF-grade male ICR mice, 6-8 weeks old, weighing 20±2 g, were housed in an SPF environment with a temperature of 22±2 ℃, humidity of 50±5%, and a 12-hour light-dark cycle, with free access to food and water.

[0084] Test samples: salmon protein oligopeptides prepared in Examples 1–3 were prepared into three dosage groups of 200, 400, and 800 mg / kg·d with physiological saline.

[0085] Reagents and instruments: D-galactose, SOD, MDA, and GSH-Px detection kits; Morris water maze system.

[0086] Experimental methods:

[0087] (1) Establishment of aging model and grouping of drugs

[0088] Sixty mice were randomly divided into six groups (n=10): a blank control group (intraperitoneal injection + gavage with saline); a model control group (intraperitoneal injection of D-galactose 120 mg / kg + gavage with saline); low, medium, and high dose groups of Example 1 (intraperitoneal injection of D-galactose + gavage with different doses of the Example 1 sample); and a positive control group (intraperitoneal injection of D-galactose + gavage with vitamin E 100 mg / kg·d). The intervention lasted for 60 days, and subsequent tests were conducted after the last administration.

[0089] (2) Serum antioxidant index detection

[0090] After fasting for 12 hours following the last administration, blood was collected from the orbital cavity, and serum was separated by centrifugation. Serum SOD activity, GSH-Px activity, and MDA content were measured according to the kit instructions.

[0091] (3) Morris water maze experiment

[0092] Orientation and navigation experiment: The mice were trained for 5 consecutive days, and the escape latency was recorded.

[0093] Space exploration experiment: On day 6, the platform was removed, and the time the mice spent in the original platform quadrant and the number of times they crossed the original platform were recorded within 5 minutes.

[0094] Table 3 shows that the serum SOD and GSH-Px activities of mice in the model control group were significantly reduced, while the MDA content was significantly increased (P < 0.01), indicating that the aging model was successfully established. In Example 1, each dose group dose-dependently increased SOD and GSH-Px activities and decreased MDA content. The high-dose group showed no significant difference in any indicators compared to the blank control group (P > 0.05), and its effect was superior to the vitamin E positive control group, demonstrating that the product of this invention can effectively enhance antioxidant capacity and alleviate oxidative stress damage in vivo.

[0095] Meanwhile, the results in Table 4 show that the model control group mice exhibited a significantly prolonged escape latency, a significantly reduced time spent in the original platform quadrant, and a significantly reduced number of crossings in the Morris water maze test (P < 0.01). After intervention in Example 1, all dosage groups improved the above behavioral indicators, and there was no significant difference between the high-dose group and the blank control group (P > 0.05), indicating that the product of this invention can effectively improve the learning and memory abilities of aging model mice and has a clear in vivo anti-aging effect.

[0096] Table 3 Results of serum antioxidant index determination in mice of each group

[0097]

[0098] Table 4 Results of the Morris water maze test in each group of mice

[0099]

[0100] Example 4: Safety Experiment

[0101] 1. Acute toxicity test (maximum tolerated dose (MTD) determination)

[0102] Experimental animals: SPF-grade ICR mice, half male and half female, 6-8 weeks old, weighing 20±2 g.

[0103] Test sample: salmon protein oligopeptides prepared in Example 1, prepared with physiological saline to a maximum feasible concentration of 200 mg / mL.

[0104] Experimental Methods: Acute toxicity was evaluated using the maximum dose method. Mice were administered Example 1 (dose of 2000 mg / kg) via gavage at a volume of 10 mL / kg, and were subsequently observed for 14 days. Their general condition (mental state, appetite, defecation, etc.), weight changes, and mortality were recorded. After the observation period, the mice were sacrificed, and the morphology of the major organs (heart, liver, spleen, lungs, and kidneys) was dissected and observed.

[0105] The results showed that none of the mice died, and their mental state, diet, water intake, defecation, and weight gain were normal, with no significant difference compared to the blank control group (P > 0.05). Upon dissection, no swelling, atrophy, congestion, or abnormal color changes were observed in the major organs. Therefore, the maximum tolerated dose (MTD) of salmon protein oligopeptides in mice is not less than 2000 mg / kg. Based on body surface area, this dose is equivalent to more than 60 times the recommended daily intake (2 g / person) for an adult (60 kg), indicating that the sample has high acute safety.

[0106] 2. Long-term toxicity study (3-month repeated-dose toxicity assay)

[0107] Experimental animals: SPF-grade SD rats, 30 males and 30 females, 6-8 weeks old, weighing 180±20 g;

[0108] Test sample: salmon protein oligopeptides prepared in Example 1, with three dosage groups: low (200 mg / kg·d), medium (400 mg / kg·d), and high (800 mg / kg·d), and a blank control group (administered by gavage with physiological saline).

[0109] Experimental methods: Ten male and ten female rats were used in each group. The drug was administered by gavage once a day for 90 consecutive days. During the administration period, the rats' body weight and food intake were recorded weekly. After the administration, blood was collected from the orbital sinus to measure hematological parameters (red blood cell count, white blood cell count, hemoglobin content, platelet count) and blood biochemical parameters (ALT, AST, BUN, Cr, TC, TG). The rats were sacrificed, and the weights of the heart, liver, spleen, lungs, and kidneys were measured. The organ coefficient (organ weight / body weight × 100%) was calculated.

[0110] Table 5. Results of hematological parameters in rats of each group.

[0111]

[0112] Table 6 Results of blood biochemical indicators in rats of each group

[0113]

[0114] Table 7 Results of major organ coefficients in rats of each group

[0115]

[0116] As shown in Tables 5-7, there were no significant differences in hematological indicators, blood biochemical indicators (liver and kidney function, lipid metabolism), and major organ coefficients between the rats in each dosage group and the blank control group (P>0.05). The salmon protein oligopeptide prepared in this invention showed no obvious toxicity to rats at a dose of 800 mg / kg·d (equivalent to 24 times the recommended daily intake for adults) for 90 consecutive days, and its long-term safety was good.

[0117] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made using the present invention specification, or directly / indirectly applied to other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing antioxidant salmon protein oligopeptides, characterized in that, Includes the following steps: S1. Extraction of crude protein solution from salmon skin: Defatted salmon skin is added to a 2%-5% malic acid solution to swell, then tea polyphenols and chymotrypsin are added for enzymatic hydrolysis, and after enzyme inactivation, the mixture is filtered to obtain crude protein solution from salmon skin. S2. Extraction of crude protein from salmon swim bladder: After soaking salmon swim bladder in sodium hydroxide solution, it is added to a 0.6-0.9 mol / L propionic acid solution and stirred for extraction. The extract is then salted out and dried to obtain crude protein from salmon swim bladder. S3. Extraction of crude salmon protein solution: Mix salmon meat paste with distilled water, add sodium thiosulfate and bromelain, stir and hydrolyze, inactivate the enzyme, centrifuge and collect the supernatant to obtain salmon meat crude protein solution. S4. Mixed enzymatic hydrolysis: The salmon skin crude protein solution obtained in step S1, the salmon swim bladder crude protein obtained in step S2, and the salmon meat crude protein solution obtained in step S3 are mixed in a mass ratio of (75-115):(35-55):(65-85). Then, ascorbate palmitate, flavor protease, and chymotrypsin are added for mixed enzymatic hydrolysis. The supernatant is collected by centrifugation to obtain the enzymatic hydrolysis product. S5. Post-processing: After inactivating the enzyme in the enzymatic hydrolysis product, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da. The permeate is collected and dried to obtain the antioxidant salmon protein oligopeptide.

2. The preparation method according to claim 1, characterized in that, In step S1, the amount of tea polyphenols added is 1%-2.2% of the weight of defatted salmon skin, and the amount of chymotrypsin added is 0.6%-2.2% of the weight of defatted salmon skin; and / or, In step S3, the amount of sodium thiosulfate added is 1.5%-2.5% of the meat paste weight, and the amount of bromelain added is 1.2%-2.2% of the meat paste weight; and / or, In step S4, the amount of ascorbate palmitate added is 0.15%-0.35% of the mass of the mixture, the amount of flavor protease added is 0.12%-0.32% of the mass of the mixture, and the amount of chymotrypsin added is 2.2%-4.2% of the mass of the mixture.

3. The preparation method according to claim 1, characterized in that, In step S4, the temperature for the mixed enzymatic hydrolysis is 38-48 °C, and the time is 2.5-4.5 h.

4. The preparation method according to claim 1, characterized in that, In step S1, the malic acid solution has a mass concentration of 3.5%, and the swelling conditions are swelling at 5-11°C for 18-24 hours; and / or, In step S2, the concentration of the propionic acid solution is 0.7 mol / L, and the stirring extraction conditions are extraction at 5-9 ℃ for 38-44 h.

5. The preparation method according to claim 1, characterized in that, In step S1, the enzymatic hydrolysis conditions are: enzymatic hydrolysis at 38-52℃ for 2-4 hours; and / or, In step S3, the enzymatic hydrolysis conditions are: stirring at 38-52 °C and a rate of 90-130 r / min for 4-6 h.

6. The preparation method according to claim 1, characterized in that, In step S5, the ultrafiltration membrane pressure is 0.15 MPa and the feed temperature is 35 ℃.

7. An antioxidant salmon protein oligopeptide prepared by the preparation method according to any one of claims 1-6, characterized in that, Its molecular weight is less than 1800 Da, the peptide purity is not less than 92%, and the scavenging rate of DPPH free radicals is not less than 88%, and the scavenging rate of ABTS free radicals is not less than 92%.

8. The antioxidant salmon protein oligopeptide according to claim 7, characterized in that, Peptides with a molecular weight in the range of 500 Da to 1000 Da account for no less than 64% of the total peptide content.

9. The use of the antioxidant salmon protein oligopeptide according to claim 7 or 8 in the preparation of antioxidant functional foods, health foods or cosmetics.

10. The application according to claim 9, characterized in that, The antioxidant salmon protein oligopeptide is used as an active ingredient in the preparation of oral liquids, compressed candies, capsules, lyophilized powders, serums, or facial masks.

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