Preparation method of antioxidant selenium-rich peptide and ultrasound-assisted enzymatic hydrolysis

Abstract

The present invention specifically relates to a method for preparing an antioxidant selenium-rich peptide and ultrasound-assisted enzymatic hydrolysis. Due to its poor taste, dark tuna meat is currently mainly used as a feed raw material with low market value. The present invention believes that the dark tuna meat has a high protein content and abundant selenium, and is expected to be used as a biological source of selenium-rich peptides. In order to achieve the above-mentioned technical effects, the present invention provides a method for preparing antioxidant selenium-rich peptides from dark tuna meat by energy-concentrating ultrasound-assisted enzymatic hydrolysis. The water-soluble crude protein in the dark tuna meat is enzymatically hydrolyzed and separated, and finally a polypeptide product with good antioxidant activity and rich selenium content is obtained, which has broad application prospects in the preparation of foods and functional products with antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the technical field of selenium-rich polypeptides, and in particular to an antioxidant selenium-rich peptide, a method for preparing the antioxidant selenium-rich peptide by ultrasound-assisted enzymatic hydrolysis, and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Selenium (Se) is an essential trace element for the human body, boosting immunity and preventing a variety of diseases. Selenium deficiency can induce conditions such as Kashin-Beck disease and Keshan disease. Selenoproteins are the primary carriers of selenium's biological effects, primarily in the form of selenium bound to cysteine ​​and methionine. Selenium and sulfur are congener elements and share certain commonalities. Since methionine and cysteine ​​contain sulfur, the sulfur is likely replaced by selenium, forming selenomethionine and selenocysteine, respectively, which catalyze oxidation-reduction reactions. Compared to selenoproteins, selenopeptides are more easily digested and absorbed by the human body and possess multiple biological functions, including antioxidant and immunomodulatory properties. Selenopeptides possess the dual activities of selenium and bioactive peptides, are non-toxic, have a simple structure, and are relatively stable, making them promising selenium nutritional supplements.

[0004] Ultrasonic-assisted enzymatic hydrolysis is a new, green and environmentally friendly processing technology that increases the contact between substrate and enzyme through mechanical, thermal, and cavitation effects, thereby improving the rate of enzymatic hydrolysis. Furthermore, ultrasonic pretreatment can affect the flavor of protein hydrolysates by increasing the types of volatile compounds by disrupting the substrate. Ultrasonic transducers are categorized into diverging ultrasound and focused ultrasound, depending on their structure and power. Diverging ultrasound has a lower intensity and is primarily used for cleaning and enhanced mass transfer, while focused ultrasound provides greater intensity and is widely used in cell disruption, changing material properties, and enhancing mass transfer.

[0005] Tuna, a representative of deep-sea fish, is known as the "gold of the ocean" and is an ideal food that meets human health standards. However, because dark tuna meat is tougher and less flavorful than regular meat, it is mostly used in industrial feed processing, resulting in low utilization and added value of dark tuna resources. Dark tuna meat is high in protein, low in fat, and contains a complete amino acid profile, including polyunsaturated fatty acids such as DHA and EPA, and is rich in vitamins and minerals. Notably, the selenium content in dark tuna meat ranges from 2.0 to 4.7 mg / kg, far exceeding the selenium content standard for selenium-rich aquatic products (0.10-0.50 mg / kg) in the "Classification Requirements for Selenium Content of Selenium-Rich Agricultural Products" (DB45 / T 1061-2014), making it a good selenium-rich raw material. Summary of the Invention

[0006] Dark meat of tuna is currently mainly used as a feed raw material and has a low market value. In response to the shortcomings of the above-mentioned existing technologies, the inventors believe that dark meat of tuna is rich in protein and selenium, and it is expected to extract highly active selenium-rich peptides from it. Screening for peptide products with good activity is of great significance to improving the market value of dark meat of tuna and developing functional selenium peptide products.

[0007] In order to achieve the above objectives, the present invention screened and separated the active protein in the dark meat of tuna, and finally obtained a selenium-rich peptide product with good antioxidant activity and a corresponding preparation process. The relevant technical solutions are as follows:

[0008] In a first aspect, the present invention provides an antioxidant selenium-rich peptide, wherein the polypeptide is a polypeptide composition having an amino acid sequence as described in SEQ ID NO: 1-9;

[0009] Table 1

[0010]

[0011] The polypeptides shown in SEQ ID NO: 1-9 have the same amino acid sequence as Table 1. According to the identification of the present invention, the combination of the above polypeptides has good antioxidant activity and is rich in selenium. It is a highly active selenium-rich peptide product with high market value.

[0012] In a preferred embodiment, the antioxidant selenium-rich peptide is a combination of several of the above 9 polypeptides, such as 2 to 9 types, preferably 3 to 8 types, and more preferably 3 to 6 types.

[0013] In a second aspect, the present invention provides a method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis, the method comprising the following steps:

[0014] (1) Preparation of water-soluble crude protein: Dissolve the freeze-dried powder of dark tuna meat in water, extract it under a magnetic stirrer, filter the retained solution, obtain protein precipitate by ammonium sulfate salting out, dialyze the precipitate, and freeze-dry to obtain water-soluble crude protein with a molecular weight cut-off of 3000~4000 Da;

[0015] (2) ultrasonically treating the aqueous solution of the water-soluble crude protein, and then adding trypsin for enzymatic hydrolysis;

[0016] (3) The protein after enzymatic hydrolysis was screened for the fraction with a molecular weight <3 kDa and further separated into seven purified fractions F1, F2, F3, F4, F5, F6 and F7 by reverse chromatography. The chromatographic conditions were as follows:

[0017] Chromatographic column: C18 column

[0018] Mobile phase: Mobile phase A is water, mobile phase B is acetonitrile;

[0019] Elution program: 0-5 min, 92% mobile phase A; 5-16 min, 92-65% mobile phase A; 16-30 min, 65-55% mobile phase A; 30-32 min, 55% mobile phase A;

[0020] Among them, F7 is the above-mentioned antioxidant selenium-rich peptide.

[0021] The above process uses dark meat of tuna as the raw material for extraction. The dark meat refers to the dark meat distributed on the back of the tuna, existing on the surface of the lateral line and between the dorsal and ventral parts of the body. It extends from the outside of both sides of the horizontal diaphragm below the lateral line of the fish to the periphery of the spine. Its muscle fibers are slightly thinner and rich in pigment proteins such as hemoglobin and myoglobin, as well as various enzyme proteins. In addition to containing more pigment proteins than ordinary meat, dark meat also contains more lipids, glycogen, vitamins and enzymes. This part has a higher protein content and less fat, but as a food it has a stronger fishy and sour smell and a poor taste, so it is usually used as a feed raw material. In this field, its freeze-dried powder can be obtained by conventional tissue crushing and freeze-drying.

[0022] In the above step (1):

[0023] The lyophilized powder is dissolved in water at a ratio of 0.8-1.2 g: 8-12 mL, preferably using distilled water. After thorough stirring, the filtrate is filtered and retained as the solution, thereby obtaining the water-soluble protein in the lyophilized powder through dissolution. Furthermore, to achieve more complete dissolution, stirring can be used to accelerate the dissolution of the protein in the lyophilized powder. For example, magnetic stirring can be applied for 3-5 hours.

[0024] The ammonium sulfate salting-out process is as follows: adding acid to adjust the pH of the solution to 4-5, then adding ammonium sulfate to saturation, standing at 3-5° C. for 10-14 hours, and retaining the precipitate after high-speed centrifugation, which is the protein precipitate.

[0025] Furthermore, the acid added is preferably a simple carboxylic acid, such as formic acid or acetic acid, more preferably acetic acid.

[0026] Furthermore, the rotation speed of the high-speed centrifugation is 8000-12000 r / min, and the centrifugation time is 8-12 min.

[0027] The specific operation of the dialysis is as follows: the precipitate is dissolved in Tris-HCl buffer and dialyzed against water for 40 to 50 hours.

[0028] Furthermore, the concentration of the Tris-HCl buffer is 45-55 mmol / L, and the pH is 8-9.

[0029] Furthermore, the dialysis interception portion is freeze-dried to obtain the water-soluble crude protein of the tuna dark meat.

[0030] In step (2) above:

[0031] The above-mentioned water-soluble crude protein was added to water and dissolved at a material-liquid ratio of 0.8-1.2 g: 40-60 mL. The aqueous solution was ultrasonically treated for 25-35 min, and then 3-5% trypsin was added. The pH of the solution was adjusted to 7-8, and enzymatic hydrolysis was carried out at 50-60°C for 7.3 h.

[0032] Furthermore, the ultrasound can be used in the form of focused ultrasound or divergent ultrasound, and the more effective one is focused ultrasound, which can effectively improve the efficiency of protein enzymatic hydrolysis and make the enzymatic hydrolysis more thorough; in one embodiment verified by the present invention, the frequency of the above-mentioned focused ultrasound treatment is 20-25 KHz, the power is 25~35 W / L, and it is achieved by an ultrasonic cell disruptor.

[0033] Furthermore, the trypsin activity is 250,000 U / g.

[0034] Furthermore, after the above enzymatic hydrolysis is completed, trypsin needs to be inactivated. High-temperature inactivation can be used, such as placing the enzymatic hydrolysis solution in boiling water for 13 to 17 minutes and then transferring it to ice water for cooling.

[0035] In the above step (3), the enzymatic hydrolysate is filtered by ultrafiltration to filter the portion with a molecular weight of <3 kDa, and the molecular weight of the ultrafiltration membrane is 3 kDa; F1 to F7 are divided according to the time of chromatographic separation outflow, and the retention time of F7 is 25 min~27 min.

[0036] The third aspect of the present invention provides the use of the antioxidant selenium-rich peptide described in the first aspect or the antioxidant selenium-rich peptide prepared by the method described in the second aspect in the preparation of functional products.

[0037] Possible examples of the functional products include health care products, skin care products or agricultural products.

[0038] Among them, the above-mentioned health products have the effects and functions of preventing, improving or enhancing immunity, anti-oxidation, anti-aging, improving cardiovascular and cerebrovascular diseases, regulating blood sugar, etc., and the feasible dosage forms include tablets, capsules or oral liquid preparations.

[0039] The skin care product is an external-use lotion product that has the effect of delaying skin aging.

[0040] Possible examples of the agricultural products include fertilizers, feeds and the like.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The present invention is designed to prepare a selenium-rich peptide product using dark tuna meat as raw material, and provides an antioxidant selenium-rich peptide. The selenium-rich peptide is a combination product of selenium-rich polypeptides, has significant antioxidant effect, and is rich in selenium: IC of DPPH free radical scavenging rate 50 The value is 4.073 mg / mL, ABTS + IC of free radical scavenging rate 50 The value is 1.110 mg / mL, and the selenium content can reach 4.89±0.16 mg / kg. This selenium-rich peptide product has higher activity and market value as a raw material for health care products or skin care products.

[0043] 2. The present invention also provides a preparation process for the above-mentioned selenium-rich peptide product. Through focused ultrasound-assisted enzymatic hydrolysis, the preparation process is simple, easy to implement and cost-effective for corporate production, and can convert raw materials with originally low market value into high-net-value products, with significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0045] Figure 1 The hydrolysis degree, SDS-PAGE gel electrophoresis and DPPH free radical scavenging rate of different enzymatic hydrolysates in Example 1 are shown in FIG.

[0046] in, Figure 1 A is the hydrolysis degree test result diagram, Figure 1 B is the result of SDS-PAGE gel electrophoresis. Figure 1C is the result of DPPH free radical scavenging rate determination.

[0047] Figure 2 This is a graph showing the results of a single-factor experiment in Example 1;

[0048] in, Figure 2 A is the effect of ultrasonic power density on the hydrolysis degree and DPPH free radical scavenging rate of the enzymatic solution, Figure 2 B is the effect of material-liquid ratio on the hydrolysis degree and DPPH radical scavenging rate of the enzymatic hydrolyzate;

[0049] Figure 2 C is the effect of ultrasonic time on the hydrolysis degree and DPPH radical scavenging rate of the enzymatic solution;

[0050] Figure 2 D is the effect of enzymatic hydrolysis time on the hydrolysis degree and DPPH radical scavenging rate of the enzymatic hydrolyzate.

[0051] Figure 3 is the response surface diagram of the degree of hydrolysis in Example 1;

[0052] in, Figure 3 A is the response surface diagram of the interaction between solid-liquid ratio and ultrasonic power density on hydrolysis degree;

[0053] Figure 3 B is the response surface diagram of the interaction between solid-liquid ratio and ultrasonic time on hydrolysis degree;

[0054] Figure 3 C is the response surface diagram of the interaction between the solid-liquid ratio and the enzymatic hydrolysis time on the hydrolysis degree;

[0055] Figure 3 D is the response surface diagram of the interaction between ultrasonic power density and ultrasonic time on hydrolysis degree;

[0056] Figure 3 E is the response surface diagram of the interaction between ultrasonic power density and enzymatic hydrolysis time on hydrolysis degree;

[0057] Figure 3 F is the response surface diagram of the interaction between ultrasound time and enzymatic hydrolysis time on hydrolysis degree.

[0058] Figure 4 Response surface diagram of DPPH free radical scavenging rate in Example 1;

[0059] in, Figure 4 A is the response surface diagram of the interaction between solid-liquid ratio and ultrasonic power density on DPPH free radical scavenging rate;

[0060] Figure 4 B is the response surface diagram of the interaction between solid-liquid ratio and ultrasonic time on DPPH free radical scavenging rate;

[0061] Figure 4C is the response surface diagram of the interaction between the solid-liquid ratio and the enzymatic hydrolysis time on the DPPH radical scavenging rate;

[0062] Figure 4 D is the response surface diagram of the interaction between ultrasonic power density and ultrasonic time on DPPH free radical scavenging rate;

[0063] Figure 4 E is the response surface diagram of the interaction between ultrasonic power density and enzymatic hydrolysis time on DPPH free radical scavenging rate;

[0064] Figure 4 F is the response surface diagram of the interaction between ultrasound time and enzymatic hydrolysis time on DPPH radical scavenging rate.

[0065] Figure 5 This is a graph showing the antioxidant activity test results of the tuna dark meat protein hydrolysate in Example 1.

[0066] Figure 6 Statistical diagram of the antioxidant activity of different ultrafiltration fractions in Example 2.

[0067] Figure 7 1 is a diagram showing the reversed-phase high performance liquid chromatography purification results of different purified components in Example 2;

[0068] in, Figure 7 A is the elution curve. Figure 7 B is the statistical results of antioxidant activity.

[0069] Figure 8 This is the total ion current chromatogram (TIC) of component F7 in Example 2. DETAILED DESCRIPTION

[0070] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0071] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0072] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0073] The sources of materials in the following examples are as follows:

[0074] Dark Tuna Meat: This species is yellowfin tuna, caught in the South China Sea. Dissect fresh yellowfin tuna and remove the dark meat near the lateral line. Store in a -80°C freezer until ready to use.

[0075] Trypsin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., ABTS + , FRAP detection kit was purchased from Nanjing Jiancheng Biotechnology Institute.

[0076] The instruments and equipment involved in the following examples are shown in Table 1 below:

[0077] Table 1 Instrument and equipment statistics

[0078]

[0079] The research methods involved in the following examples are as follows:

[0080] 1. Determination of hydrolysis degree

[0081] The degree of hydrolysis of the enzymatic hydrolyzate was determined by the o-phthalaldehyde (OPA) method.

[0082] To prepare the OPA reagent: Weigh 7.620 g of sodium tetraborate and 200 mg of sodium dodecyl sulfate and dissolve them in 150 mL of distilled water. Once fully dissolved, add 4 mL of 0.04% (w / v) OPA ethanol solution. Mix thoroughly, then add 0.4 mL of β-mercaptoethanol and bring the solution to 200 mL. Prepare the serine standard solution: Dissolve 50 mg of serine in 500 mL of distilled water. The Serine-NH2 content in this solution is approximately 0.9516 meqv / L.

[0083] Preparation of the standard curve: Pipette 0, 50, 100, 200, and 400 µL of L-serine standard solution into a test tube, respectively. Add water to make up for the amount less than 400 µL. Add 3 mL of OPA reagent to each tube, mix well, and react for 2 minutes. Measure the absorbance at 340 nm and draw a standard curve with serine concentration as the horizontal axis and absorbance as the vertical axis.

[0084] Determination of the degree of hydrolysis of the enzymatic hydrolyzate: First, add 3 mL of OPA reagent to each test tube; then take 400 μL of the enzymatic hydrolyzate and add it to the test tube, mix evenly, accurately time the reaction for 2 minutes, and then measure the absorbance at 340 nm.

[0085] Find C on the standard curve serine-NH2 (mmol / g), and the degree of hydrolysis of the sample was calculated using formulas (1) to (3):

[0086] (1);

[0087] (2);

[0088] (3);

[0089] Where, X: weight of sample; h: number of peptide bonds broken during hydrolysis, mmol / g; W serine-NH2 That is H hot , is the total number of peptides in the sample, mmol / g. The correction factors a and β for fish protein are 1 and 0.4, respectively.

[0090] 2. Determination of DPPH free radical scavenging rate

[0091] Prepare 0.25, 0.5, 1, 2.5, 5, and 10 mg / mL peptide solutions. Sample group (A x ): 100 μL of 0.10 mmol / L DPPH-anhydrous ethanol solution and 100 μL of sample solutions of different mass concentrations were added sequentially; the anhydrous ethanol group (A0): 100 μL of anhydrous ethanol and 100 μL of sample solutions of different concentrations were added sequentially; the control group (A1): 100 μL of 0.10 mmol / L DPPH-anhydrous ethanol solution and 100 μL of distilled water were added sequentially. Mix well, react in the dark for 0.5 h, and measure the absorbance at 517 nm. The clearance rate was calculated according to formula (4):

[0092] (4).

[0093] 3. ABTS + Determination of free radical scavenging rate

[0094] Prepare 0.25, 0.5, 1, 2.5, 5, and 10 mg / mL peptide solutions according to the total antioxidant capacity test kit (ABTS method) of Nanjing Jiancheng Biotechnology Institute. For specific steps, refer to the ABTS assay kit instructions. + The free radical scavenging rate was calculated according to formula (5):

[0095] (5).

[0096] 4. FRAP free radical scavenging rate determination method

[0097] A total antioxidant capacity detection kit (FRAP method) was used. For specific steps, please refer to the FRAP assay kit instructions.

[0098] 5. Determination of selenium content

[0099] The selenium content in the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to GB65009.268.

[0100] Example 1

[0101] In this embodiment, an antioxidant selenium-rich peptide is provided, and the preparation process of the antioxidant selenium-rich peptide is optimized:

[0102] 1. Enzymatic hydrolysis process optimization

[0103] (1) Raw material pretreatment: thaw the dark meat of tuna at 4°C, grind it into minced meat in a tissue crusher, freeze-dry it, grind it into dry powder in a grinder, package it in sealed containers, and store it in a desiccator to obtain freeze-dried tuna dark meat powder;

[0104] (2) Extraction of tuna dark meat water-soluble protein: The tuna dark meat freeze-dried powder obtained in step (1) was dissolved in distilled water at a material-liquid ratio of 1 g:10 mL, stirred and extracted with a magnetic stirrer for 4 h, filtered, and the residue was repeatedly extracted once and washed with a small amount of deionized water 3 times, filtered to dryness, and the filtrate was combined. The filtrate was acidified to pH = 4.5 with acetic acid, and ammonium sulfate was added to adjust to saturation. The filtrate was allowed to stand at 4°C for 12 h, centrifuged at 10000 r / min for 10 min, and the precipitate was dissolved in 10 mL of 50 mmol / L Tris-HCl buffer (pH = 8.5), and dialyzed against deionized water for 48 h. The dialysis was carried out through a dialysis bag with a molecular weight cutoff of 3500 Da. The obtained protein solution was freeze-dried under vacuum to obtain the tuna dark meat water-soluble crude protein.

[0105] (3) Preparation of tuna dark meat water-soluble protein hydrolysate: Weigh a certain amount of tuna dark meat water-soluble protein obtained in step (2), add distilled water at a material-liquid ratio of 1 g:40 mL, and stir evenly; after treating with different methods ① to ④ as described below, place each hydrolysate in boiling water for 15 min, immediately transfer to ice water for cooling, centrifuge at 8000 g at 4°C for 20 min, collect the supernatant, and store at -20°C; the trypsin activity is 250,000 U / g;

[0106] ① Untreated: Stir the tuna dark meat water-soluble protein solution evenly, add 4.0% trypsin by weight, adjust to the optimal pH 7.5, and perform enzymatic hydrolysis at 55°C for 3 h;

[0107] ② Stirring pretreatment: Stirring treatment was performed for 30 min. After the treatment, 4.0% trypsin was added, adjusted to the optimal pH of 7.5, and enzymatic hydrolysis was performed at 55°C for 3 h.

[0108] ③ Divergent ultrasound: Place the tuna dark meat water-soluble protein solution in an ultrasonic cleaning machine for 30 minutes, set the ultrasonic power density to 30 W / L, and after the treatment, add 4.0% by weight of trypsin, adjust to the optimal pH of 7.5, and perform enzymatic hydrolysis at 55°C for 3 hours;

[0109] ④ Focused ultrasound: Place the tuna dark meat water-soluble protein solution in an ultrasonic cell disruptor and ultrasonicate for 30 minutes. Set the ultrasonic power density to 30 W / L. After the treatment, add 4.0% trypsin by mass, adjust to the optimal pH 7.5, and perform enzymatic hydrolysis at 55°C for 3 hours.

[0110] The results of hydrolysis degree, SDS-PAGE gel electrophoresis and antioxidant activity were as follows: Figure 1 As shown, Figure 1 A is the hydrolysis degree test result, which shows that the hydrolysis degree of tuna dark meat water-soluble protein hydrolysate prepared by focused ultrasound-assisted enzymatic hydrolysis is the highest, followed by divergent ultrasound-assisted enzymatic hydrolysis, stirring pretreatment, and untreated groups; Figure 1 B is the SDS-PAGE gel electrophoresis diagram. As shown in the figure, the bands of the four treatment methods are basically the same, but at 63 kDa, the protein assisted by focused ultrasound is more thoroughly digested; Figure 1 C shows the antioxidant activity test results, which show that the enzymatic hydrolyzate obtained by focused ultrasound also has better antioxidant activity than the other three hydrolyzates. Therefore, focused ultrasound-assisted enzymatic hydrolysis was selected as the preparation method, and the next step was to optimize the extraction process.

[0111] 2. Extraction process optimization

[0112] A certain amount of the tuna dark meat water-soluble protein obtained in step (2) was weighed, and distilled water was added according to different material-liquid ratios, and the mixture was stirred evenly; the tuna dark meat water-soluble protein solution was treated at a certain ultrasonic power density and ultrasonic time, and after the treatment, 4.0% by weight of trypsin was added, and the solution was adjusted to an optimum pH of 7.5 and an optimum temperature of 55°C, and enzymatic hydrolysis was carried out for a certain enzymatic hydrolysis time; after the treatment, the enzymatic hydrolyzate was placed in boiling water for 15 minutes, immediately transferred to ice water for cooling, centrifuged at 4°C and 8000 g for 20 minutes, and the supernatant was collected and stored at -20°C.

[0113] ①Determination of optimal ultrasonic power density

[0114] The solid-liquid ratio was 1:40 (g / mL), the ultrasonic time was 30 min, the enzymatic hydrolysis time was 3 h, and the ultrasonic power density was set to 20, 25, 30, 35, and 40 W / L.

[0115] ②Determination of the optimal material-liquid ratio

[0116] The ultrasonic time was 30 min, the ultrasonic power density was 30 W / L, the enzymatic hydrolysis time was 3 h, and the solid-liquid ratio was set to 1:20, 1:30, 1:40, 1:50, and 1:60 (g / mL).

[0117] ③Determination of the optimal ultrasound time

[0118] The solid-liquid ratio was 1:40 (g / mL), the ultrasonic power density was 30 W / L, the enzymatic hydrolysis time was 3 h, and the ultrasonic time was set to 10, 20, 30, 40, and 50 min.

[0119] ④Determination of the optimal enzymatic hydrolysis time

[0120] The solid-liquid ratio was 1:40 (g / mL), the ultrasonic time was 30 min, the ultrasonic power density was 30 W / L, and the enzymatic hydrolysis time was set to 1, 3, 5, 7, and 9 h.

[0121] Figure 2 This is a statistical chart of the single-factor experimental results. Figure 2 A represents the effect of ultrasonic power density on the degree of hydrolysis and DPPH radical scavenging rate of the enzymatic hydrolyzate. With increasing ultrasonic power density, the degree of hydrolysis and DPPH radical scavenging rate of dark tuna meat water-soluble protein both initially increased and then decreased. Appropriate ultrasonic power can increase the contact rate between substrate and enzyme, thereby improving the efficiency of the enzymatic reaction. When the ultrasonic power reaches a certain value, saturation occurs, weakening the ultrasonic effect. Furthermore, the active sites of the protein molecules are damaged, leading to a decrease in the degree of hydrolysis and DPPH radical scavenging rate. Therefore, the optimal ultrasonic power density is set at 30 W / L.

[0122] Figure 2 B represents the effect of the solid-liquid ratio on the degree of hydrolysis and DPPH radical scavenging rate of the enzymatic hydrolyzate. As the solid-liquid ratio increases, the degree of hydrolysis of the enzymatic hydrolyzate first increases and then decreases, while the DPPH radical scavenging rate first increases, then decreases, then increases again, and finally decreases. Between 1:20 and 1:50, the gradual increase in the solid-liquid ratio allows for full contact between the enzyme and substrate, promoting the hydrolysis reaction. As the solid-liquid ratio continues to increase to 1:60, the increased amount of pure water in the reaction system leads to a decrease in enzyme concentration, preventing full reaction between the enzyme and substrate, resulting in a lower degree of hydrolysis. Taking all factors into consideration, the optimal degree of hydrolysis was selected as 1:50.

[0123] Figure 2C represents the effect of ultrasound duration on the degree of hydrolysis and DPPH radical scavenging rate of the enzymatic hydrolyzate. The degree of hydrolysis and DPPH radical scavenging rate of dark tuna meat water-soluble protein increased with increasing ultrasound duration. They peaked at 30 minutes and then gradually declined. This may be due to the mechanical and cavitation effects of ultrasound, which exposed more enzyme binding sites in dark tuna meat water-soluble protein. However, prolonged ultrasound duration gradually disrupted the binding sites and enzyme structure, impairing protein hydrolysis. Therefore, 30 minutes was the optimal ultrasound duration.

[0124] Figure 2 D represents the effect of enzymatic hydrolysis time on the degree of hydrolysis and DPPH radical scavenging rate of the hydrolyzate. Both the degree of hydrolysis and the DPPH radical scavenging rate increased significantly during the initial 1-3 h of enzymatic hydrolysis (P < 0.05). At 7 h, the degree of hydrolysis and the DPPH radical scavenging rate reached their maximum values ​​of 35.03% and 23.52%, respectively. This may be due to the fact that, in the early stages of hydrolysis, the reaction between the substrate and the enzyme became more thorough with increasing enzymatic hydrolysis time, leading to an increase in the degree of hydrolysis. At 9 h, the degree of hydrolysis and the DPPH radical scavenging rate showed a decreasing trend. Excessive enzymatic hydrolysis time may lead to a decrease in substrate concentration or other negative effects, thereby reducing the degree of hydrolysis. Therefore, 7 h was selected as the optimal enzymatic hydrolysis time.

[0125] 3. Response surface methodology optimization experiment

[0126] Combined with the results of the above single factor investigation, this part takes the hydrolysis degree and DPPH free radical scavenging rate as the response value, analyzes the effects of material-liquid ratio, ultrasonic power density, ultrasonic time and enzymatic hydrolysis time on the response value, designs a four-factor three-level response surface experiment, and explores the optimal preparation process for the preparation of antioxidant selenium-rich peptides. The experimental data and results are shown in Table 2:

[0127] Table 2 Response surface design scheme and experimental results

[0128]

[0129] Using Design-Expert software, we performed quadratic regression analysis by fitting and obtained the regression equations of hydrolysis degree and DPPH free radical scavenging rate, as shown in formulas (6) to (7):

[0130] Y1=44.91+0.4383A-0.0767B-0.3142C+0.8108D-0.175AB+1.3AC+1.64AD-0.76BC+1.49BD+1.6CD-3.54A 2 -3.86B 2 -1.89C 2 -4.23D2 (6)

[0131] Y2=33.04-0.74A-0.1333B+0.245C+0.985D+0.6875AB+1.27AC-1.33AD+0.6875BC-0.98BD+1.31CD-2.98A 2 -3.93B 2 -3.41C 2 -2.88D 2 (7)

[0132] In formula (6), Y1 is the response value of the degree of hydrolysis;

[0133] In formula (7), Y2 is the response value of DPPH radical scavenging rate;

[0134] A is the coding value of the material-liquid ratio; B is the coding value of the ultrasonic power density; C is the coding value of the ultrasonic time; and D is the coding value of the enzymatic hydrolysis time.

[0135] The response surface regression model and variance analysis table are shown in Table 3:

[0136] Table 3 Response surface regression model and variance analysis

[0137]

[0138] The results of response surface variance analysis are shown in Table 3. The P test value of the quadratic regression model of Y1 is <0.0001, and the lack of fit term (0.3296) is not significant. The P test value of the quadratic regression model of Y2 is <0.0001, and the lack of fit term (0.1286) is not significant, indicating that the model has significant statistical significance, and the R of the fitting equation is 2 They are 0.9664 and 0.9436 respectively, indicating that the quadratic regression model has a good fit.

[0139] According to the response surface optimization model, the optimal extraction conditions of the sample were as follows: solid-liquid ratio 1:49.783 g / mL, ultrasonic power density 29.952 W / L, ultrasonic time 30.28 min, and enzymatic hydrolysis time 7.268 h. At this time, the predicted value of hydrolysis degree was 44.918%, and the predicted value of DPPH free radical scavenging rate was 33.145%. To facilitate the subsequent experiments, the optimized parameters were rounded off; that is, at a solid-liquid ratio of 1:50 g / mL, ultrasonic power density 30 W / L, ultrasonic time 30 min, and enzymatic hydrolysis time 7.3 h. The experimental verification showed that the hydrolysis degree of the sample was 44.56±0.20%, the DPPH free radical scavenging rate was 32.52±0.44%, and the IC value of DPPH free radical scavenging rate was 0.04. 50The value was 4.073 mg / mL, and the relative standard deviation from the predicted value was less than 1%, proving that the model can achieve the purpose of optimizing extraction. + IC of free radical scavenging rate 50 The value is 1.110 mg / mL, and the FRAP value is shown in Figure 5 , indicating that the water-soluble protein hydrolysate of tuna dark meat has good antioxidant activity. At the same time, the selenium content of the water-soluble protein hydrolysate of tuna dark meat was measured to be 4.89±0.16 mg / kg.

[0140] Therefore, in this embodiment, a preparation process of an antioxidant selenium-rich peptide is provided, comprising the following steps:

[0141] (1) Raw material pretreatment: thaw the dark meat of tuna at 4°C, grind it into minced meat in a tissue crusher, freeze-dry it, grind it into dry powder in a grinder, package it in sealed containers, and store it in a desiccator to obtain freeze-dried tuna dark meat powder;

[0142] (2) Extraction of tuna dark meat water-soluble protein: The tuna dark meat freeze-dried powder obtained in step (1) was dissolved in distilled water at a material-liquid ratio of 1 g:10 mL, stirred and extracted with a magnetic stirrer for 4 h, filtered, and the residue was repeatedly extracted once and washed with a small amount of deionized water 3 times, filtered to dryness, and the filtrate was combined. The filtrate was acidified to pH = 4.5 with acetic acid, and ammonium sulfate was added to adjust to saturation. The filtrate was allowed to stand at 4°C for 12 h, centrifuged at 10000 r / min for 10 min, and the precipitate was dissolved in 10 mL of 50 mmol / L Tris-HCl buffer (pH = 8.5), and dialyzed against deionized water for 48 h. The dialysis was carried out through a dialysis bag with a molecular weight cutoff of 3500 Da. The obtained protein solution was freeze-dried under vacuum to obtain the tuna dark meat water-soluble crude protein.

[0143] (3) Preparation of tuna dark meat water-soluble protein hydrolysate: Weigh a certain amount of tuna dark meat water-soluble protein obtained in step (2), add distilled water at a material-liquid ratio of 1 g:50 mL, and stir evenly to obtain a tuna dark meat water-soluble protein solution; then place the solution in an ultrasonic cell disruptor and sonicate for 30 min at an ultrasonic power density of 30 W / L for 30 min. After the treatment, add 4.0% trypsin by weight, adjust the pH to 7.5, and perform enzymatic hydrolysis at 55°C for 7.3 h.

[0144] Example 2

[0145] In order to obtain a polypeptide component with better antioxidant effect, this example further separated and purified the antioxidant selenium-rich peptide prepared in Example 1 above, and the steps are as follows:

[0146] (1) Ultrafiltration separation: The tuna dark meat water-soluble protein hydrolysate obtained in Example 1 was separated into three components with different molecular weights using 3 kDa and 10 kDa molecular weight ultrafiltration membranes, namely component 1 (>10 kDa), component 2 (3-10 kDa) and component 3 (<3 kDa). The components were freeze-dried and their activities were measured respectively to screen the ultrafiltration component with the highest antioxidant activity.

[0147] Figure 6 The figure is a statistical chart of the antioxidant activity of fractions with different molecular weights. The results show that compared with fractions 1 and 2, the ultrafiltration fraction with a molecular weight <3 kDa has the highest antioxidant activity, and fraction 3 was selected for the next step of purification.

[0148] (2) Reverse-phase HPLC purification: The ultrafiltration group component 3 was divided into seven purified fractions F1, F2, F3, F4, F5, F6 and F7 by preparative reverse-phase HPLC. The chromatographic column was a C18 column (20×250 mm, 10 μm, Daisogel). The mobile phases were solution A (deionized water) and solution B (acetonitrile). The elution program was: 0-5 min, 92% mobile phase A; 5-16 min, 92-65% mobile phase A; 16-30 min, 65-55% mobile phase A; 30-32 min, 55% mobile phase A. The preparative reverse-phase HPLC fractions were collected, freeze-dried, and the antioxidant activity was determined at the same concentration (2.5 mg / mL). The fraction with the strongest antioxidant activity was collected and freeze-dried to obtain the fraction containing the target active selenopeptide. Figure 7 The figure shows the results of reversed-phase high performance liquid chromatography purification of different purified components. Figure 7 A is the elution curve. Figure 7 B is the statistical results of antioxidant activity, which shows that purified fraction 7 (F7) has the strongest antioxidant activity.

[0149] (3) Amino acid sequence analysis of F7 component: UPLC-MS / MS technology was used to search the amino acid sequence library and analyze 9 selenium-containing peptides with a confidence level of ≥70%, as shown in Table 4.

[0150] Table 4 Peptide identification of component F7

[0151]

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An antioxidant selenium-rich peptide, characterized in that The polypeptide is a polypeptide composition of the amino acid sequence described in SEQ ID NO: 1-9.

2. The antioxidant selenium-rich peptide according to claim 1, wherein The antioxidant selenium-rich peptide is a combination of 2 to 9 of the 9 polypeptides shown in SEQ ID NO: 1-9.

3. The antioxidant selenium-rich peptide according to claim 2, wherein The antioxidant selenium-rich peptide is a combination of 3 to 8 of the 9 polypeptides shown in SEQ ID NO: 1-9.

4. The antioxidant selenium-rich peptide according to claim 2, wherein The antioxidant selenium-rich peptide is a combination of 3 to 6 of the 9 polypeptides shown in SEQ ID NO: 1-9.

5. A method for preparing the antioxidant selenium-rich peptide according to any one of claims 1 to 4 by ultrasound-assisted enzymatic hydrolysis, characterized in that: The method comprises the following steps: (1) Preparation of water-soluble crude protein: Dissolve the freeze-dried powder of dark tuna meat in water, extract it under a magnetic stirrer, filter the retained solution, obtain protein precipitate by ammonium sulfate salting out, dialyze the precipitate, and freeze-dry to obtain water-soluble crude protein with a molecular weight cut-off of 3000~4000 Da; (2) ultrasonically treating the aqueous solution of the water-soluble crude protein, and then adding trypsin for enzymatic hydrolysis; (3) The enzymatic hydrolysate was filtered by ultrafiltration to screen the fraction with a molecular weight of <3 kDa, and further separated into seven purified fractions F1, F2, F3, F4, F5, F6 and F7 by reverse chromatography. The chromatographic conditions were as follows: Chromatographic column: C18 column Mobile phase: Mobile phase A is water, mobile phase B is acetonitrile; Elution program: 0-5 min, 92% mobile phase A; 5-16 min, 92-65% mobile phase A; 16-30 min, 65-55% mobile phase A; 30-32 min, 55% mobile phase A; Among them, F7 is the antioxidant selenium-rich peptide described in any one of claims 1-4.

6. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: In step (1): the dissolution ratio of the lyophilized powder to water is 0.8~1.2 g: 8~12 mL. After sufficient stirring, the filtrate is filtered and retained as the solution portion. The water-soluble protein in the lyophilized powder is obtained by dissolution. The stirring is performed by magnetic stirring for 3~5 h.

7. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: In step (1): The ammonium sulfate salting-out process is as follows: adding acid to adjust the pH of the solution to 4-5, then adding ammonium sulfate to saturation, standing at 3-5° C. for 10-14 hours, and centrifuging at high speed to retain the precipitate, which is the protein precipitate.

8. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 7, characterized in that: The acid addition is the addition of a simple carboxylic acid selected from formic acid or acetic acid.

9. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 7, wherein: The high-speed centrifugation speed is 8000-12000 r / min, and the centrifugation time is 8-12 min; The specific operation of the dialysis is as follows: dissolving the precipitate in a Tris-HCl buffer solution and dialyzing it against water for 40 to 50 hours; the concentration of the Tris-HCl buffer solution is 45 to 55 mmol / L, and the pH is 8 to 9; and freeze-drying the dialyzed fraction to obtain water-soluble crude protein from tuna dark meat.

10. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: In step (2): Dissolve the water-soluble crude protein in water at a material-liquid ratio of 0.8-1.2 g: 40-60 mL. Ultrasonicate the aqueous solution for 25-35 min, then add 3-5% trypsin, adjust the pH of the solution to 7-8, and perform enzymatic hydrolysis at 50-60°C for 7.3 h.

11. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: The ultrasound is focused ultrasound or divergent ultrasound.

12. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 11, characterized in that: The focused ultrasound treatment has a frequency of 20-25 KHz and a power of 25-35 W / L, and is achieved by an ultrasonic cell disruptor.

13. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: The trypsin activity is 250,000 U / g. After the above enzymatic hydrolysis is completed, the trypsin needs to be inactivated at high temperature. The enzymatic hydrolysis solution is placed in boiling water for 13 to 17 minutes and then transferred to ice water for cooling.

14. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis according to claim 5, characterized in that: In step (3), the enzymatic hydrolysate is filtered by ultrafiltration to remove the portion with a molecular weight of less than 3 kDa, and the molecular weight of the ultrafiltration membrane is 3 kDa; F1 to F7 are divided according to the time of chromatographic separation outflow, and the retention time of F7 is 25 min~27 min.

15. Use of the antioxidant selenium-rich peptide according to any one of claims 1 to 4 or the antioxidant selenium-rich peptide prepared by the method according to any one of claims 5 to 14 in the preparation of functional products; the functional products are health products, skin care products or agricultural products; The skin care product is an external-use lotion product; The agricultural products are selected from fertilizers and feeds.

Images