Fish-derived salty peptide with antioxidant activity as well as screening method and application of fish-derived salty peptide

Abstract

The invention relates to a fish-derived salty peptide with antioxidant activity and a screening method and application thereof. The amino acid sequence of the fish-derived salty peptide is PVGR. The fish-sourced salty peptide has excellent salty taste enhancing capacity, experimental verification shows that the salty taste of 0.1% (w / v) PVGR is equivalent to the flavor generated by 4-5 mmol / L NaCl, and the fish-sourced salty peptide can be used in the fields of seasonings, low-salt foods and the like to achieve the effect of reducing salt but not reducing salty taste, and is healthier and safer. In addition, the fish-derived salty peptide obtained by the invention also has certain DPPH free radical scavenging ability, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biology and food technology, and in particular to a fish-derived salty peptide with antioxidant activity and a screening method and application thereof. Background Art

[0002] As people's awareness of healthy eating continues to increase, finding food ingredients that can both satisfy taste buds and promote health has become an important topic in the field of food science and technology. Traditional condiments, especially salt, have gradually been restricted due to their association with health problems such as hypertension and cardiovascular disease. Therefore, the development of new low-sodium or sodium-free condiments has become the key to solving this public health challenge. At the same time, peptides with both antioxidant activity and salty taste have attracted widespread attention from researchers and the food industry. Antioxidant peptides can scavenge free radicals in the body, reduce oxidative stress, and have a positive effect on anti-aging and the prevention of chronic diseases. They are ideal candidates for food additives.

[0003] Traditionally, the saltiness of food mainly comes from sodium chloride (salt), while natural antioxidants such as vitamin C and E are good for health but cannot provide a salty experience. Therefore, discovering and utilizing polypeptide substances that have both salty and antioxidant properties can not only reduce the use of salt and reduce the burden on the cardiovascular system, but also enhance the nutritional value and health attributes of food, satisfying consumers' dual pursuit of deliciousness and health.

[0004] In recent years, with the progress of biotechnology, especially the development of enzyme engineering technology and proteomics, it has become possible to efficiently prepare these functional peptides from animal and plant protein sources through enzymatic hydrolysis. Combining modern separation and purification technology with the study of structure-function relationship, the continuous exploration of how to efficiently separate salty peptides with specific functions from complex enzymatic hydrolysis products is of great significance to promoting the innovative development of the food industry. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a fish-derived salty peptide with antioxidant activity and a screening method and application thereof.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a fish-derived salty peptide having antioxidant activity, wherein the amino acid sequence of the fish-derived salty peptide is PVGR.

[0008] The present invention proposes a fish-derived salty peptide with antioxidant activity, which has excellent salty taste enhancement ability. Experimental verification shows that the salty taste of 0.1% (w / v) PVGR is equivalent to the flavor produced by 4-5mmol / L NaCl. It can be used in condiments, low-salt foods and other fields to achieve the effect of reducing salt without reducing saltiness, and is healthier and safer. In addition, the fish-derived salty peptide obtained by the present invention also has a certain DPPH free radical scavenging ability, and has broad application prospects.

[0009] Preferably, the molecular weight of the fish-derived salty peptide is 427.55 Da.

[0010] In a second aspect, the present invention provides a method for screening fish-derived salty peptides with antioxidant activity as described in any of the above technical solutions, comprising the following steps: using bonito as raw material, performing proteolysis and separation and purification on it, collecting graded components, using nano-liquid chromatography-tandem mass spectrometry to sequence identify different components, and then using computer-assisted screening to select peptide sequences that are non-toxic, have hydrophilicity > 0, and can bind to the salty receptor ENaC, namely the fish-derived salty peptides.

[0011] The present invention takes bonito as the research object, focusing on exploring the regulation of the protease hydrolysis process to prepare active polypeptides with specific amino acid sequences, thereby achieving efficient stimulation of flavor. The present invention further proposes the use of computer-aided technology to screen polypeptides with salty characteristics, which has the advantages of high efficiency, economy and accuracy, and can improve the flavor and quality of food while meeting consumers' demand for healthy and low-sodium food.

[0012] Preferably, the enzymatic hydrolysis method is: enzymatically hydrolyzing the bonito surimi with alkaline protease, inactivating the enzyme after the enzymatic hydrolysis, and collecting the supernatant by centrifugation to obtain the bonito enzymatic hydrolyzate.

[0013] Preferably, the enzymatic hydrolysis conditions are: pH 9.0, enzyme substrate ratio 5.25% w / w, enzymatic hydrolysis temperature 50° C., enzymatic hydrolysis time 3.09 h, and substrate concentration 31.6% w / v.

[0014] Preferably, the separation and purification method is: Sephadex G-25 gel column chromatography is used to separate and purify the bonito hydrolysate obtained by the protein hydrolysis, the sample volume is 5 mL, the eluent is deionized water, the flow rate is 0.33 mL / min, the detection wavelength is 214 nm, the graded components are collected, and freeze-dried.

[0015] Preferably, the computer-assisted screening method is: the multiple peptide sequences obtained by the sequence identification are first screened out using the ToxinPred database to obtain non-toxic peptide sequences with hydrophilicity>0, and then the Chem3D Pro software is used to generate a peptide model for the non-toxic peptide sequence with hydrophilicity>0, and then the peptide model is imported into Discovery Studio 2019, and molecular docking is performed with the salty taste receptor ENaC model to further screen out peptide sequences that can bind to the salty taste receptor ENaC.

[0016] In a third aspect, the present invention provides a fish-derived salty peptide with antioxidant activity as described in any of the above technical solutions or a fish-derived salty peptide obtained by the screening method for fish-derived salty peptide with antioxidant activity as described in any of the technical solutions, and its application in the preparation of condiments and foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the secondary mass spectrum of the fish-derived salty peptide of Example 1;

[0018] Figure 2 The structural model diagram of the fish-derived salty peptide of Example 1 and the schematic diagram of its docking structure with the ENaC receptor molecule;

[0019] Figure 3 The antioxidant activity of the bonito hydrolysate in Example 1 is characterized (with the DPPH free radical scavenging ability as an indicator);

[0020] Figure 4 This is the characterization of the salty taste activity of the bonito hydrolysate in Example 1, where 1, 2, 3, 4, and 5 represent sodium chloride solutions with concentrations of 10, 20, 30, 40, and 50 mmol / L, respectively;

[0021] Figure 5 This is a characterization of the salty activity of the fish-derived salty peptide of Example 1, where 1, 2, 3, 4, and 5 represent sodium chloride solutions with concentrations of 1, 2, 3, 4, and 5 mmol / L, respectively. DETAILED DESCRIPTION

[0022] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0023] The embodiment of the present invention provides a fish-derived salty peptide with antioxidant activity, and the amino acid sequence of the fish-derived salty peptide is PVGR.

[0024] In a preferred embodiment of the present invention, the molecular weight of the fish-derived salty peptide is 427.55 Da.

[0025] The screening is carried out by the following method, comprising the following steps: using bonito as raw material, performing proteolysis and separation and purification on it, collecting graded components, using nano-liquid chromatography-tandem mass spectrometry to sequence identify different components, and then using computer-assisted screening to select peptide sequences that are non-toxic, have a hydrophilicity greater than 0, and can bind to the salty taste receptor ENaC, namely, fish-derived salty peptides.

[0026] In a preferred embodiment of the present invention, the method of proteolysis is: enzymolyzing the bonito surimi with alkaline protease, inactivating the enzyme after the enzymolysis, and collecting the supernatant by centrifugation to obtain the bonito enzymolysis solution.

[0027] In a more preferred embodiment of the present invention, the enzymatic hydrolysis conditions are: pH 9.0, enzyme substrate ratio 5.25% w / w, enzymatic hydrolysis temperature 50°C, enzymatic hydrolysis time 3.09h, and substrate concentration 31.6% w / v.

[0028] In a preferred embodiment of the present invention, the separation and purification method is: Sephadex G-25 gel column chromatography is used to separate and purify the bonito hydrolysate obtained by proteolysis, the sample volume is 5 mL, the eluent is deionized water, the flow rate is 0.33 mL / min, the detection wavelength is 214 nm, and the graded components are collected and freeze-dried.

[0029] In a preferred embodiment of the present invention, the computer-assisted screening method is: the multiple peptide sequences obtained by sequence identification are first screened out using the ToxinPred database to obtain non-toxic peptide sequences with hydrophilicity>0, and then the Chem3D Pro software is used to generate peptide models for the non-toxic peptide sequences with hydrophilicity>0, and then the peptide models are imported into Discovery Studio 2019, and molecular docking is performed with the salty taste receptor ENaC model to further screen out peptide sequences that can bind to the salty taste receptor ENaC.

[0030] The fish-derived salty peptide with antioxidant activity according to any of the above embodiments or the fish-derived salty peptide obtained by the method for screening fish-derived salty peptide with antioxidant activity according to any of the above embodiments can be applied to the preparation of condiments and foods.

[0031] The technical solution of the present invention is further described and illustrated below based on specific embodiments.

[0032] Example 1

[0033] A method for screening fish-derived salty peptides with antioxidant activity comprises the following steps:

[0034] S1. Proteinase hydrolysis: Use a stainless steel meat grinder to grind the bonito powder into minced fish. Weigh 100 g of bonito minced fish, add 0.316 L of deionized water, adjust the pH to 9.00, add 5.25 g of alkaline protease, place in a 50°C shaker for enzymolysis for 3.09 h, boil in a boiling water bath for 10 min to inactivate the enzyme, cool to room temperature, centrifuge at 10,000 r / min for 20 min, collect the supernatant to obtain bonito hydrolysate, and freeze-dry.

[0035] S2. Separation and purification: Weigh 1 g of the lyophilized powder of the above-mentioned bonito hydrolysate, add 5 mL of deionized water, stir until completely dissolved, pass through a 0.22 μm filter membrane, and separate and purify it by Sephadex G-25 gel column chromatography, with a sample volume of 5 mL, deionized water as the eluent, a flow rate of 0.33 mL / min, and a detection wavelength of 214 nm. Collect the fractionated components and freeze-dry them.

[0036] S3. Sequence identification: Nano-liquid chromatography-tandem mass spectrometry was used to perform sequence identification on the different components collected above (100 μg each).

[0037] S4. Computer-assisted screening: The multiple peptide sequences obtained by the above sequence identification were first screened out using the ToxinPred database to select peptide sequences with non-toxicity and hydrophilicity>0, and then the Chem3D Pro software was used to generate peptide models for the screened peptide sequences with non-toxicity and hydrophilicity>0. The peptide models were then imported into Discovery Studio 2019 for molecular docking with the salty taste receptor ENaC model to further screen out peptide sequences that can bind to the salty taste receptor ENaC, thus obtaining fish-derived salty peptides (amino acid sequence PVGR, molecular weight 427.55Da, hydrophilicity 0.38);

[0038] Among them, the salty taste receptor ENaC model was obtained by the following method: the amino acid sequence of the salty taste receptor ENaC was obtained from the NCBI website, and homology modeling was performed on the SWISS-MODEL website based on the sequence, and the receptor model with the highest score was screened as the molecular docking receptor model.

[0039] The secondary mass spectrum of fish-derived salty peptides can be found at Figure 1 , the structural model of fish-derived salty peptides and the schematic diagram of their docking structure with ENaC receptors can be found in Figure 2 .

[0040] The bonito hydrolysate obtained in step S1 (denoted as KPSPs) was subjected to DPPH antioxidant activity and electronic tongue saltiness assay, and the results were recorded in Figure 3 and Figure 4 .

[0041] (1) Determination of antioxidant activity of bonito hydrolysate

[0042] The determination method is as follows:

[0043] Dissolve 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) in anhydrous ethanol to obtain a 0.1mmol / L DPPH solution. The sample group was prepared by mixing equal volumes of bonito hydrolysate of different concentrations with DPPH solution, storing them in the dark at room temperature for 30 minutes, and then measuring their absorbance at 517nm; the blank group was prepared by replacing the DPPH solution with an equal volume of anhydrous ethanol solution; the control group was prepared by mixing equal volumes of anhydrous ethanol solution and DPPH solution. The DPPH free radical scavenging rate of the sample was calculated as follows:

[0044]

[0045] Depend on Figure 3 It can be seen that KPSPs has a certain DPPH free radical scavenging ability, and its IC 50 It is 5.08mg / mL.

[0046] (2) Determination of salty taste activity of bonito hydrolysate

[0047] The determination method is as follows:

[0048] Different concentrations of KPSPs (10, 20, 30, 40, 50 mg / mL) and NaCl (10, 20, 30, 40, 50 mmol / L) were dissolved in ultrapure water and measured by cTongue-8 electronic tongue. The electronic tongue consists of 6 sensors. The sensors are preheated and cleaned before sample detection. The sensor magnification is 30 times, the sample volume is 10-20 mL, and each sample is measured 4-6 times in parallel.

[0049] from Figure 4 It can be seen that the cumulative variance contribution rate of PC1 and PC2 is 98.2%, which can be considered to represent the overall taste characteristics of the sample. The saltiness of KPSPs increases with the increase of concentration (the projection of the sample in the PC1 direction represents the intensity of the saltiness). The saltiness of 50 mg / mL KPSPs is equivalent to 50 mmol / L NaCl. This result shows that KPSPs has salty characteristics.

[0050] The fish-derived salty peptide sequence obtained in step S4 is synthesized by a solid phase synthesis method, and the salty activity of the fish-derived salty peptide is measured by an electronic tongue, and the result is as follows: Figure 5 shown.

[0051] The determination method is as follows:

[0052] The bonito hydrolysate obtained in step S1 was used as a control, the synthesized fish-derived salty peptide was prepared into an aqueous solution with the same concentration as the test sample, and different concentrations of sodium chloride solution (1, 2, 3, 4, 5 mmol / L) were used as standard saltiness intensity references, and measured by cTongue-8 electronic tongue. The electronic tongue consists of 6 sensors. The sensors are preheated and cleaned before sample detection. The sensor magnification is 100 times, the sample volume is 10-20 mL, and each sample is measured in parallel 4-6 times. The obtained sample signal data reflects the difference in saltiness between samples through principal component analysis.

[0053] Depend on Figure 5 It can be seen that the figure shows the flavor information of different samples. The reference group is a sodium chloride solution of different concentrations. The saltiness of 0.1% (w / v) KPSPs is equivalent to the saltiness of 1mmol / L NaCl. Compared with the control group, the saltiness intensity of the fish-derived salty peptide PVGR screened is further improved, and the saltiness of 0.1% (w / v) PVGR is equivalent to the flavor produced by 4-5mmol / L NaCl. This result shows that the fish-derived salty peptide obtained in Example 1 has better salty activity and can be used in the field of condiments and food.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fish-derived salty peptide with antioxidant activity, characterized in that: The amino acid sequence of the fish-derived salty peptide is PVGR.

2. The fish-derived salty peptide with antioxidant activity according to claim 1, characterized in that: The molecular weight of the fish-derived salty peptide is 427.55 Da.

3. A method for screening fish-derived salty peptides with antioxidant activity as claimed in claim 1, comprising the following steps: Taking bonito as raw material, it is subjected to proteolysis and separation and purification, and the graded components are collected. Nano-liquid chromatography-tandem mass spectrometry is used to identify the sequences of different components, and then computer-assisted screening is used to select peptide sequences that are non-toxic, have a hydrophilicity greater than 0, and can bind to the salty taste receptor ENaC, namely the fish-derived salty peptide.

4. The method for screening fish-derived salty peptides with antioxidant activity according to claim 3, characterized in that: The enzymatic hydrolysis method comprises the following steps: enzymatically hydrolyzing the bonito surimi with alkaline protease, killing the enzyme after the enzymatic hydrolysis is completed, and collecting the supernatant by centrifugation to obtain the bonito enzymatic hydrolyzate.

5. The method for screening fish-derived salty peptides with antioxidant activity according to claim 4, characterized in that: The conditions for the enzymatic hydrolysis are: pH 9.0, enzyme-substrate ratio 5.25% w / w, enzymatic hydrolysis temperature 50° C., enzymatic hydrolysis time 3.09 h, and substrate concentration 31.6% w / v.

6. The method for screening fish-derived salty peptides with antioxidant activity according to claim 3, characterized in that: The separation and purification method comprises: using Sephadex G-25 gel column chromatography to separate and purify the bonito hydrolysate obtained by protein hydrolysis, the sample volume is 5 mL, the eluent is deionized water, the flow rate is 0.33 mL / min, the detection wavelength is 214 nm, and the graded components are collected and freeze-dried.

7. The method for screening fish-derived salty peptides with antioxidant activity according to claim 3, characterized in that: The computer-assisted screening method is: the multiple peptide sequences obtained by the sequence identification are first screened out using the ToxinPred database to obtain non-toxic peptide sequences with hydrophilicity>0, and then the Chem3D Pro software is used to generate peptide models for the non-toxic peptide sequences with hydrophilicity>0, and then the peptide models are imported into Discovery Studio 2019, and molecular docking is performed with the salty taste receptor ENaC model to further screen out peptide sequences that can bind to the salty taste receptor ENaC.

8. Use of the fish-derived salty peptide with antioxidant activity as described in any one of claims 1 to 2 or the fish-derived salty peptide with antioxidant activity obtained by the screening method as described in any one of claims 3 to 7 in the preparation of condiments and foods.

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