Antioxidant selenium-rich peptide and ultrasonic-assisted enzymolysis preparation method

Abstract

The invention particularly relates to an antioxidant selenium-rich peptide and an ultrasonic-assisted enzymolysis preparation method. The tuna dark meat is mainly used as a feed raw material with low market value at present due to poor taste. According to the invention, the tuna dark meat has high protein content and abundant selenium element, and is expected to be used as a biological source of selenium-rich peptide. In order to realize the technical effect, the invention provides the method for preparing the antioxidant selenium-enriched peptide by energy-gathered ultrasonic-assisted enzymolysis of the tuna dark meat, and the method is used for carrying out enzymolysis and separation on water-soluble crude protein in the tuna dark meat to finally obtain a polypeptide product with good antioxidant activity and rich selenium element content. The method has a wide application prospect in the aspect of preparing food and functional products with antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the technical field of selenium-rich peptides, 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 the 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 a person skilled in the art.

[0003] Selenium (Se) is an essential trace element for the human body. It can improve the body's immunity and prevent the occurrence of many diseases. Selenium deficiency can induce osteomalacia, Keshan disease, etc. Selenoprotein is the main carrier of selenium to exert its biological effects. The main form of selenium is through the combination with cysteine ​​and methionine. Selenium and sulfur are elements of the same main group and have commonalities to a certain extent. Since methionine and cysteine ​​happen to contain sulfur, the sulfur in them is likely to be replaced by selenium to become selenomethionine and selenocysteine, thereby catalyzing oxidation-reduction reactions. Compared with selenoproteins, selenopeptides are easier to be digested and absorbed by the human body, and also have multiple biological functions such as anti-oxidation and immunomodulation. Selenopeptides have the dual activities of selenium and bioactive peptides, and also have no toxic side effects, simple structure and relatively high stability. They are highly potential selenium nutritional supplements.

[0004] Ultrasonic assisted enzymatic hydrolysis technology is a new type of green and environmentally friendly processing technology, which can increase the chance of substrate and enzyme contact through mechanical, thermal and cavitation effects, thereby increasing the rate of enzymatic hydrolysis. In addition, ultrasonic pretreatment can affect the flavor of protein hydrolysates by increasing the type of volatile compounds by destroying the substrate. According to the structure and power of the ultrasonic transducer, it is divided into divergent ultrasonic and focused ultrasonic. Divergent ultrasonic has a lower intensity and is mainly used for cleaning and enhanced mass transfer, while focused ultrasonic can provide greater intensity and has a wide range of uses in cell disruption, changing material properties and enhancing mass transfer.

[0005] Tuna, as a representative of deep-sea fish, is known as the "gold of the sea" and is an ideal food that meets human health standards. Since dark meat is tougher and has a poor taste than ordinary meat, it is mostly used for feed processing in industry, so the utilization rate and added value of dark meat resources of tuna are not high. Dark meat of tuna is high in protein, low in fat, and has a complete amino acid content. It contains polyunsaturated fatty acids such as DHA and EPA, and is rich in vitamins and minerals. It is worth noting that the selenium content in dark meat of tuna is 2.0-4.7 mg / kg, which is much higher than the selenium content standard (0.10-0.50 mg / kg) of selenium-rich aquatic products in the "Classification Requirements for Selenium Content of Selenium-rich Agricultural Products" (DB45 / T 1061-2014), and is 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 relatively low market value. In view of the deficiencies in the above-mentioned prior art, 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 of peptide products with good activity is of great significance to improving the market value of dark meat of tuna and developing selenium peptide drugs.

[0007] In order to achieve the above-mentioned purpose, the present invention screens and separates the active protein in the dark meat of tuna, and finally obtains a selenium-rich peptide product with good antioxidant activity and a corresponding preparation process. The relevant technical scheme is as follows:

[0008] In a first aspect, the present invention provides an antioxidant selenium-rich peptide, wherein the polypeptide is a polypeptide composition of the amino acid sequence 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 sequences as those in 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, and 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 kinds, preferably 3 to 8 kinds, and more preferably 3 to 6 kinds.

[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) Preparing water-soluble crude protein: Dissolve the freeze-dried powder of dark tuna meat in water, extract 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 cutoff of 3000 to 4000 Da;

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

[0016] (3) The protein after enzymatic hydrolysis was screened for a portion with a molecular weight of <3 kDa, and was further separated into seven purified portions F1, F2, F3, F4, F5, F6 and F7 by reverse phase 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 tuna, existing on the surface of the lateral line of the body and between the dorsal and ventral parts, extending from the outside of both sides of the horizontal diaphragm under 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 and 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 heavier fishy and sour taste and a poor taste, so it is usually used as a feed raw material. In this field, freeze-dried powder can be obtained conventionally by tissue crushing and freeze-drying.

[0022] In the above step (1):

[0023] The dissolution ratio of the lyophilized powder to water is 0.8-1.2 g: 8-12 mL, preferably distilled water is used, and the filtrate retained after sufficient stirring is the above-mentioned solution part, and the water-soluble protein in the lyophilized powder is obtained by dissolving. Furthermore, in order to make the dissolution more complete, the dissolution process of the protein in the lyophilized powder can be accelerated by stirring, and a specific example is to add magnetic stirring for 3-5 hours.

[0024] The ammonium sulfate salting-out process is as follows: add acid to adjust the pH of the solution to 4-5, then add ammonium sulfate to saturation, let stand at 3-5°C for 10-14h, and retain 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 tuna dark meat.

[0030] In the above step (2):

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

[0032] Furthermore, the ultrasound can be in the form of focused ultrasound or divergent ultrasound, and the focused ultrasound is more effective, 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 20-25KHz, the power is 25-35W / 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, the trypsin needs to be inactivated, and a high-temperature inactivation method can be used, such as placing the enzymatic hydrolysis solution in boiling water for 13 to 17 minutes and transferring it to ice water for cooling.

[0035] In the above step (3), the enzymatic hydrolysate is filtered by ultrafiltration 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 chromatographic separation outflow time, and the retention time of F7 is 25min to 27min.

[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 mentioned above include medicines, functional foods, health products, skin care products or agricultural products.

[0038] Among them, the above-mentioned drugs, functional foods or 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 lotion product, which 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, which is a combination product of selenium-rich peptides, has significant antioxidant effects, and is rich in selenium: the IC value of DPPH free radical scavenging rate is 50 The value is 4.073mg / 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-enriched peptide product has higher activity and market value as a raw material for medicine, functional food 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 in the specification, 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 diagram and DPPH free radical scavenging rate measurement results of different enzymatic hydrolysates in Example 1 are shown;

[0046] in, Figure 1 A is the hydrolysis degree test result diagram, Figure 1 B is the result of SDS-PAGE gel electrophoresis. Figure 1 C 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 the material-liquid ratio on the hydrolysis degree and DPPH free radical scavenging rate of the enzymatic hydrolyzate;

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

[0050] Figure 2 D is the effect of enzymatic hydrolysis time on the hydrolysis degree and DPPH free 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 4 C is the response surface diagram of the interaction between the solid-liquid ratio and the enzymatic hydrolysis time on the DPPH free radical scavenging rate;

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

[0063] Figure 4 E is the response surface diagram of the interaction between ultrasound 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 free radical scavenging rate.

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

[0066] Figure 6 This is a statistical diagram of the antioxidant activity of different ultrafiltration components in Example 2.

[0067] Figure 7 The figure is a diagram of the reverse 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 result 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 all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those 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 exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates 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 in combination with specific embodiments and comparative examples.

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

[0074] Tuna dark meat: The species is yellowfin tuna, caught in the South China Sea. Fresh yellowfin tuna is dissected and the dark meat near the lateral line is taken and stored in a -80℃ refrigerator for later 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:

[0077] Table 1 Instrument and equipment statistics

[0078]

[0079]

[0080] The research methods involved in the following examples are as follows:

[0081] 1. Determination of hydrolysis degree

[0082] The degree of hydrolysis of the enzymatic hydrolyzate was determined by the o-phthalaldehyde (OPA) method.

[0083] Preparation of OPA reagent: weigh 7.620g sodium tetraborate and 200mg sodium dodecyl sulfate and dissolve them in 150mL distilled water. After fully dissolved, add 4mL 0.04% (w / v) OPA ethanol solution. After mixing the two, add 0.4mL β-mercaptoethanol and dilute the solution to 200mL. Prepare and use it now. Preparation of serine standard solution: dissolve 50mg serine in 500mL distilled water. The Serine-NH2 in this solution is about 0.9516meqv / L.

[0084] Drawing of standard curve: Pipette 0, 50, 100, 200, 400 μL L-serine standard solution into test tubes respectively, make up with water if less than 400 μL, add 3 mL of LOP A reagent respectively, mix well, react for 2 minutes, measure the absorbance at 340 nm, and draw the standard curve with serine concentration as the horizontal axis and absorbance as the vertical axis.

[0085] Determination of the degree of hydrolysis of the enzymatic hydrolyzate: first add 3 mL of OPA reagent to each test tube; 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.

[0086] 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):

[0087]

[0088]

[0089]

[0090] 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 of fish protein are 1 and 0.4 respectively.

[0091] 2. Determination of DPPH free radical scavenging rate

[0092] Prepare 0.25, 0.5, 1, 2.5, 5, and 10 mg / mL peptide solutions. Sample group (A x ): 100μL 0.10mmol / LDPPH-anhydrous ethanol solution and 100μL sample solutions of different mass concentrations were added in sequence; anhydrous ethanol group (A0): 100μL anhydrous ethanol and 100μL sample solutions of different concentrations were added in sequence; control group (A1): 100μL 0.10mmol / LDPPH-anhydrous ethanol solution and 100μL distilled water were added in sequence. Mix well, react in the dark for 0.5h, and measure the absorbance at 517nm. Calculate the clearance rate according to formula (4):

[0093]

[0094] 3. ABTS + Determination of free radical scavenging rate

[0095] Prepare 0.25, 0.5, 1, 2.5, 5, 10 mg / mL peptide solutions according to the total antioxidant capacity detection kit (ABTS method) of Nanjing Jiancheng Biotechnology Institute. For specific steps, refer to the instructions of the ABTS assay kit. + The free radical scavenging rate was calculated according to formula (5):

[0096]

[0097] 4. FRAP free radical scavenging rate determination method

[0098] A total antioxidant capacity detection kit (FRAP method) was used, and the specific steps were referred to the instructions of the FRAP assay kit.

[0099] 5. Determination method of selenium content

[0100] The selenium content in the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to GB65009.268.

[0101] Example 1

[0102] In this embodiment, an antioxidant selenium-rich peptide is provided, and the preparation process of the antioxidant selenium-rich peptide is optimized:

[0103] 1. Optimization of enzymatic hydrolysis process

[0104] (1) Raw material pretreatment: thawing the dark meat of tuna at 4° C., grinding it into minced meat in a tissue disruptor, freeze-drying it, grinding it in a grinder to make dry powder, packaging it in sealed containers, and storing it in a desiccator to obtain freeze-dried tuna dark meat powder;

[0105] (2) Extraction of dark meat water-soluble protein of tuna: the freeze-dried dark meat powder of tuna obtained in step (1) was dissolved in distilled water at a solid-liquid ratio of 1 g:10 mL, and the mixture was extracted by stirring with a magnetic stirrer for 4 h, and filtered. The residue was extracted once and washed with a small amount of deionized water for 3 times, and filtered to dryness. The filtrate was combined, acidified to pH = 4.5 with acetic acid, and ammonium sulfate was added to adjust to saturation. The mixture was allowed to stand at 4°C for 12 h, and centrifuged at 10000 r / min for 10 min. The precipitate was dissolved in 10 mL of 50 mmol / L Tris-HCl buffer (pH = 8.5), and dialyzed with deionized water for 48 h. The dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 Da. The obtained protein solution was freeze-dried under vacuum to obtain the dark meat water-soluble crude protein of tuna.

[0106] (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 solid-liquid ratio of 1 g:40 mL, and stir evenly; after treating in different ways ① to ④ as described below, place each hydrolysate in boiling water for 15 min, immediately transfer to ice water for cooling, centrifuge at 4°C and 8000 g for 20 min, collect the supernatant, and store at -20°C; the enzyme activity of trypsin is 250,000 U / g;

[0107] ① Untreated: Stir the dark meat tuna water-soluble protein solution evenly, add 4.0% trypsin by mass, adjust to the optimum pH 7.5, and perform enzymolysis at 55°C for 3 hours;

[0108] ② Stirring pretreatment: Stirring treatment for 30 minutes. After the treatment, 4.0% trypsin was added, adjusted to the optimum pH of 7.5, and enzymolysis was performed at 55°C for 3 hours.

[0109] ③ Divergent ultrasound: Place the dark tuna meat water-soluble protein solution in an ultrasonic cleaning machine for 30 minutes, set the ultrasonic power density to 30W / L, add 4.0% trypsin by mass, adjust to the optimal pH 7.5, and perform enzymatic hydrolysis at 55°C for 3 hours;

[0110] ④ Focused ultrasound: Place the dark meat water-soluble protein solution of tuna in an ultrasonic cell disruptor for 30 minutes, set the ultrasonic power density to 30W / 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.

[0111] The results of hydrolysis degree, SDS-PAGE gel electrophoresis and antioxidant activity are shown in Figure 1 As shown, Figure 1A is the hydrolysis degree test result, and the results show that the hydrolysis degree of tuna dark meat water-soluble protein hydrolysate prepared by concentrated 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 hydrolyzed more thoroughly; Figure 1 C is the result of the antioxidant activity test, and the results show that the enzymatic hydrolysate obtained by focused ultrasound also has better antioxidant activity than the other three hydrolysates. Therefore, focused ultrasound-assisted enzymatic hydrolysis was selected as the preparation method, and the next step of process extraction optimization was carried out.

[0112] 2. Extraction process optimization

[0113] A certain amount of the dark meat water-soluble protein of tuna obtained in step (2) is weighed, and distilled water is added according to different material-liquid ratios, and the mixture is stirred evenly; the dark meat water-soluble protein solution of tuna is treated at a certain ultrasonic power density and ultrasonic time, and after the treatment, 4.0% of trypsin is added, and the solution is adjusted to an optimum pH of 7.5 and an optimum temperature of 55° C., and enzymolysis is performed at a certain enzymolysis time; after the treatment, the enzymolysis solution is placed in boiling water for 15 minutes, and immediately transferred to ice water for cooling, centrifuged at 4° C. and 8000 g for 20 minutes, and the supernatant is collected and stored at -20° C.

[0114] ① Determination of optimal ultrasonic power density

[0115] 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.

[0116] ②Determination of the optimal material-liquid ratio

[0117] 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).

[0118] ③Determination of the optimal ultrasound time

[0119] 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.

[0120] ④Determination of the optimal enzymatic hydrolysis time

[0121] 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.

[0122] Figure 2 This is a statistical chart of the single-factor experimental results. Figure 2 A is the effect of ultrasonic power density on the degree of hydrolysis and DPPH radical scavenging rate of enzymatic hydrolysate. With the increase of ultrasonic power density, the degree of hydrolysis and DPPH radical scavenging rate of dark tuna meat water-soluble protein both showed a trend of first increasing and then decreasing. When appropriate ultrasound can increase the contact rate between substrate and enzyme, the efficiency of enzymatic reaction can be improved. When the power of ultrasound increases to a certain value, saturation will occur and the ultrasonic effect will be weakened. In addition, the active sites of protein molecules will also be damaged, resulting in a decrease in the degree of hydrolysis and DPPH radical scavenging rate. Therefore, the optimal ultrasonic power density is set to 30W / L.

[0123] Figure 2 B is the effect of the solid-liquid ratio on the hydrolysis degree of the enzymatic hydrolyzate and the scavenging rate of DPPH free radicals. With the increase of the solid-liquid ratio, the hydrolysis degree of the enzymatic hydrolyzate first increased and then decreased, while the scavenging rate of DPPH free radicals first increased and then decreased, then increased, and finally decreased. At 1:20-1:50, as the solid-liquid ratio gradually increased, the enzyme and the substrate were fully in contact, thereby promoting the hydrolysis reaction; the solid-liquid ratio continued to increase to 1:60, and the amount of pure water in the reaction system increased, resulting in a decrease in the concentration of the enzyme, and the enzyme and the substrate could not fully react, resulting in a decrease in the hydrolysis degree. After comprehensive consideration, the optimal hydrolysis degree was selected as 1:50.

[0124] Figure 2 C is the effect of ultrasound time on the hydrolysis degree and DPPH free radical scavenging rate of the enzymatic hydrolysate. The hydrolysis degree and DPPH free radical scavenging rate of the dark meat water-soluble protein of tuna increase with the increase of ultrasound time. When the ultrasound time reaches 30 minutes, the hydrolysis degree and DPPH free radical scavenging rate reach a peak value and then gradually decrease. This may be due to the mechanical and cavitation effects of ultrasound, which expose more enzyme binding sites of the dark meat water-soluble protein of tuna. However, when the ultrasound time is too long, the binding sites and enzyme structure are gradually destroyed, thus affecting protein hydrolysis. Therefore, the optimal ultrasound time is selected as 30 minutes.

[0125] Figure 2 D is the effect of enzymatic hydrolysis time on the hydrolysis degree and DPPH free radical scavenging rate of the enzymatic hydrolyzate. The hydrolysis degree and DPPH free radical scavenging rate increased significantly in the initial 1h to 3h of enzymatic hydrolysis (P<0.05). When the enzymatic hydrolysis time was 7h, the hydrolysis degree and DPPH free radical scavenging rate reached the maximum values ​​of 35.03% and 23.52%, respectively. This may be because in the early stage of hydrolysis, as the enzymatic hydrolysis time increased, the substrate and enzyme reacted more thoroughly, thereby increasing the hydrolysis degree. When the enzymatic hydrolysis time was 9h, the hydrolysis degree and DPPH free radical scavenging rate showed a downward trend. Too long enzymatic hydrolysis time may lead to a decrease in substrate concentration or other negative effects, thereby reducing the hydrolysis degree. Therefore, the optimal enzymatic hydrolysis time was selected as 7h.

[0126] 3. Response surface methodology optimization experiment

[0127] 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 solid-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:

[0128] Table 2 Response surface design scheme and experimental results

[0129]

[0130]

[0131] Using Design-Expert software, a quadratic regression analysis was performed by fitting, and the regression equations of hydrolysis degree and DPPH free radical scavenging rate were obtained, as shown in formulas (6) to (7):

[0132] 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 -4.23D 2 (6)

[0133] 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)

[0134] In formula (6), Y1 is the response value of the degree of hydrolysis;

[0135] In formula (7), Y2 is the response value of DPPH free radical scavenging rate;

[0136] A is the coding value of the solid-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.

[0137] The response surface regression model and variance analysis table are shown in Table 3:

[0138] Table 3 Response surface regression model and variance analysis

[0139]

[0140] Note: *P<0.05 indicates significant; **P<0.01 indicates extremely significant

[0141] 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.

[0142] According to the response surface optimization model, the optimal extraction conditions of the sample are: solid-liquid ratio 1:49.783g / mL, ultrasonic power density 29.952W / L, ultrasonic time 30.28min, enzymatic hydrolysis time 7.268h, at which the predicted value of hydrolysis degree is 44.918%, and the predicted value of DPPH free radical scavenging rate is 33.145%; to facilitate the subsequent experiments, the optimized parameters are rounded; that is, when the solid-liquid ratio is 1:50g / mL, the ultrasonic power density is 30W / L, the ultrasonic time is 30min, and the enzymatic hydrolysis time is 7.3h. The experiment verified that the hydrolysis degree of the sample is 44.56±0.20%, the DPPH free radical scavenging rate is 32.52±0.44%, and the IC value of DPPH free radical scavenging rate is 33.145%. 50 The 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. In addition, ABTS + 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±0.16mg / kg.

[0143] Therefore, in this embodiment, a preparation process of an antioxidant selenium-rich peptide is provided, comprising the following steps:

[0144] (1) Raw material pretreatment: thawing the dark meat of tuna at 4° C., grinding it into minced meat in a tissue disruptor, freeze-drying it, grinding it in a grinder to make dry powder, packaging it in sealed containers, and storing it in a desiccator to obtain freeze-dried tuna dark meat powder;

[0145] (2) Extraction of dark meat water-soluble protein of tuna: the freeze-dried dark meat powder of tuna obtained in step (1) was dissolved in distilled water at a solid-liquid ratio of 1 g:10 mL, and the mixture was extracted by stirring with a magnetic stirrer for 4 h, and filtered. The residue was extracted once and washed with a small amount of deionized water for 3 times, and filtered to dryness. The filtrate was combined, acidified to pH = 4.5 with acetic acid, and ammonium sulfate was added to adjust to saturation. The mixture was allowed to stand at 4°C for 12 h, and centrifuged at 10000 r / min for 10 min. The precipitate was dissolved in 10 mL of 50 mmol / L Tris-HCl buffer (pH = 8.5), and dialyzed with deionized water for 48 h. The dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 Da. The obtained protein solution was freeze-dried under vacuum to obtain the dark meat water-soluble crude protein of tuna.

[0146] (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 according to the material-liquid ratio of 1g:50mL, stir evenly to obtain tuna dark meat water-soluble protein solution; place it in an ultrasonic cell disruptor for 30 minutes, set the ultrasonic power density to 30W / L, and the ultrasonic time to 30 minutes. After the treatment, add 4.0% trypsin by mass, adjust to the optimal pH of 7.5, and perform enzymolysis at 55°C for 7.3 hours.

[0147] Example 2

[0148] In order to obtain a polypeptide component with better antioxidant effect, the antioxidant selenium-rich peptide prepared in the above Example 1 was further separated and purified in this example, and the steps are as follows:

[0149] (1) Ultrafiltration separation: The tuna dark meat water-soluble protein hydrolysate obtained in Example 1 was separated into three components of 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.

[0150] Figure 6 The figure is a statistical chart of the antioxidant activity of components with different molecular weights. The results show that compared with components 1 and 2, the ultrafiltration fraction with a molecular weight <3 kDa has the highest antioxidant activity, and component 3 is selected for the next step of purification.

[0151] (2) Reverse-phase HPLC purification: The ultrafiltration group component 3 was divided into seven purification parts 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 procedure 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 component with the strongest antioxidant activity was collected and freeze-dried to obtain the component containing the target active selenopeptide. Figure 7 The figure shows the results of reverse phase high performance liquid chromatography purification of different purified components. Figure 7 A is the elution curve. Figure 7 B is a statistical result diagram of antioxidant activity, and the results show that purified fraction 7 (F7) has the strongest antioxidant activity.

[0152] (3) Amino acid sequence analysis of F7 component: UPLC-MS / MS technology was used to search the amino acid sequence library and obtain 9 seleno-peptides with a credibility of ≥70%, as shown in Table 4.

[0153] Table 4 Peptide identification of component F7

[0154]

[0155] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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, characterized in that The antioxidant selenium-rich peptide is a combination of 2 to 9 of the 9 polypeptides shown in SEQ ID NOs: 1-9, preferably 3 to 8, and more preferably 3 to 6.

3. A method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis, characterized in that: The method comprises the following steps: (1) Preparing water-soluble crude protein: Dissolve the freeze-dried powder of dark tuna meat in water, extract 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 cutoff of 3000 to 4000 Da; (2) treating the aqueous solution of the water-soluble crude protein by ultrasonic treatment, and then adding trypsin for enzymatic hydrolysis; (3) The enzymatic hydrolysate was filtered by ultrafiltration for the portion with a molecular weight of less than 3 kDa, and further separated into seven purified portions 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 above-mentioned antioxidant selenium-rich peptide.

4. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis as claimed in claim 3, 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 above-mentioned solution. The water-soluble protein in the lyophilized powder is obtained by dissolution. The stirring is carried out by magnetic stirring for 3-5 hours.

5. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis as claimed in claim 3, characterized in that: In step (1): The ammonium sulfate salting-out operation 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 10-14h, and retaining the precipitate after high-speed centrifugation, which is the protein precipitate; Further, the acid addition is the addition of a simple carboxylic acid selected from formic acid or acetic acid; Furthermore, the speed of the high-speed centrifugation is 8000-12000 r / min, and the centrifugation time is 8-12 min; Alternatively, the specific operation of the dialysis is as follows: the precipitate is dissolved in a Tris-HCl buffer and dialyzed against water for 40 to 50 hours; the concentration of the Tris-HCl buffer is 45 to 55 mmol / L, and the pH is 8 to 9; the dialyzed retentate is freeze-dried to obtain the dark meat water-soluble crude protein of tuna.

6. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis as claimed in claim 3, characterized in that: In step (2): The above water-soluble crude protein is added into water and dissolved, with the solid-liquid ratio being 0.8-1.2 g:40-60 mL. The aqueous solution is ultrasonically treated for 25-35 min, and then 3-5% trypsin is added. The pH value of the solution is adjusted to 7-8, and enzymatic hydrolysis is performed at 50-60° C. for 7.3 h.

7. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis as claimed in claim 6, characterized in that: The ultrasound is focused ultrasound or divergent ultrasound, preferably focused ultrasound, the frequency of the focused ultrasound treatment is 20-25KHz, the power is 25-35W / L, and it is achieved by an ultrasonic cell disruptor; Furthermore, the trypsin activity is 250,000 U / g. Furthermore, after the above enzymatic hydrolysis is completed, the trypsin needs to be inactivated, and a high-temperature inactivation method can be used, such as placing the enzymatic hydrolysis solution in boiling water for 13 to 17 minutes and transferring it to ice water for cooling.

8. The method for preparing antioxidant selenium-rich peptides by ultrasound-assisted enzymatic hydrolysis as claimed in claim 3, characterized in that: In step (3), the enzymatic hydrolysate is filtered by ultrafiltration to obtain a 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 chromatographic separation outflow time, and the retention time of F7 is 25 min to 27 min.

9. Use of the antioxidant selenium-rich peptide according to any one of claims 1 or 2 or the antioxidant selenium-rich peptide prepared by the method according to any one of claims 3 to 8 in the preparation of functional products.

10. The use according to claim 9, characterized in that Functional products include, but are not limited to, medicines, functional foods, health products, skin care products or agricultural products; The medicine, functional food or health product has the functions and effects of preventing, improving or enhancing immunity, anti-oxidation, anti-aging, improving cardiovascular and cerebrovascular diseases, and regulating blood sugar, and the dosage form is selected from tablets, capsules or oral liquid preparations; The skin care product is an external lotion product that has the effect of delaying skin aging; The agricultural products are selected from fertilizers and feeds.

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