CN103103242A - Antioxidant active peptide and preparation method thereof - Google Patents

Abstract

The invention discloses an antioxidant active peptide and a preparation method thereof. The antioxidant active peptide is characterized by taking decapterus fish protein as the raw material, alkaline protease generated by bacillus subtilis 2709 is adopted for hydrolysis, after enzyme deactivation of the hydrolysate, the hydrolysate is separated through centrifugation, ultra-filtration, gel chromatography and reverse phase high performance liquid chromatography so as to determine the structures of two natural antioxidant peptide, the amino acid sequences are His-Asp-His-Pro-Val-Cys (histidine-aspartic acid-histidine-proline-valine-cysteine) and His-Glu-Lys-Val-Cys(histidine-glutamic acid-lysine-valine-cysteine), the concentration (EC50) for eliminating DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical of 50% are 0.0310+/-0.0011mu M and 0.0677+/-0.0012mu M, the concentration for eliminating superoxide anion (O2-) radical of 50% are 0.3814+/-0.0017mu M and 0.3744+/-0.0021mu M, and the antioxidant active peptide has good antioxidant activity.

Description

A kind of antioxidant active peptide and preparation method thereof

technical field

The invention belongs to the field of functional foods, and in particular relates to an antioxidative activity peptide of marine low-value fish, blue round trevally, meat protein and a preparation method thereof. the

Background technique

Chemosynthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which are often used as food additives, are restricted in use due to their toxicity and side effects. Natural antioxidants can not only prevent the oxidation of the oil system in food, but also prevent the damage of free radicals in the human body, delay aging and various chronic degenerative diseases such as cancer, coronary heart disease, diabetes, etc. Peptides, which are between proteins and amino acids, have been widely studied due to their special structural characteristics, high safety and significant biological activity. In recent years, people have isolated a large number of natural antioxidant peptides from a variety of animal and plant proteins, among which antioxidant peptides derived from animal proteins are mostly aquatic antioxidant peptides. However, there are many kinds of marine organisms and their quantity is huge, and their proteins are very different from those of terrestrial organisms in terms of amino acid composition and sequence, and their development and utilization are also becoming increasingly active. "Aquatic Science" 2008, No. 7, pages 370-373, reported that hydrolyzed marine proteins such as Alaskan pollock, mackerel, and tuna prepared highly active antioxidant peptides. Chinese patent (ZL201010141974.3) discloses antioxidant peptides derived from collagen and their uses. Four antioxidant peptides with different amino acid sequences are isolated from collagen, and can be well used in food, cosmetics, and health care goods etc. the

Blue round trevally (Decapterus maruadsi), a species of round trevally in the family Trevalidae of Perciformes. Also known as pond fish, balang fish, etc. Distributed in the Indian Ocean and the Pacific Ocean, it is mainly produced in the South China Sea and the East China Sea in my country. It is one of the important economic fish. Due to the low price and backward processing and recycling methods, it is mostly made into salty dry products or feed fish meal, and the economic benefits are low. In recent years, many studies have discussed its comprehensive utilization, such as "Food Research and Development" No. 12, 2007, "Food Industry Science and Technology" No. 1, 2008, and "Journal of Guangdong Ocean University" No. 4, 2009. Different proteases were used to enzymatically hydrolyze the blue trevally protein, and the optimal protease was selected as the experimental enzyme according to the nitrogen recovery rate, amino nitrogen content or polypeptide extraction rate in the enzymatic hydrolysis products, and the enzymatic hydrolysis process conditions were investigated. The first issue of "Food Science" in 2009 studied the effects of temperature, pH value, food accessories and various metal ions on the stability of the antioxidant peptides of blue scads hydrolyzed by Alcalase 2.4. "China Brewing" 2009 No. 9 and "Food Research and Development" 2010 No. 2 selected neutral protease and alkaline protease to enzymatically hydrolyze the blue squid protein, and used the central combination test method to optimize the enzymatic hydrolysis experimental conditions. "Journal of Chinese Food Science" No. 8 in 2012 described the optimization of papain and flavor protease hydrolysis process conditions for the hydrolysis of blue trevally protein by response surface analysis method, and the antioxidant activity of hydrolyzate was studied by free radical scavenging rate. These are only related to enzymatic hydrolysis processing technology or product antioxidant activity and stability determination, etc., and the preparation method of separating and purifying a single antioxidant active peptide from the blue round trevally protein has not been reported yet. the

Contents of the invention

The present invention provides an antioxidant active peptide derived from marine fish blue trevally protein and a preparation method thereof. , gel chromatography, and reversed-phase high-performance liquid chromatography were used to separate and determine the structures of two natural antioxidant peptides. the

The concrete realization steps of the present invention and condition are as follows:

(1) Mix the protein powder of blue round trevally with water to prepare a reaction raw material solution with a protein content of 1-5wt%; Hydrolyze for 3 to 6 hours, and finally heat to 100°C to terminate the enzymolysis reaction;

(2) Centrifuge the enzymolysis solution obtained in step 1 at 4°C and 8000r/min for 10-15min to obtain the supernatant and collect the permeate by ultrafiltration with an ultrafiltration membrane with a molecular weight of 5000Da;

(3) Use deionized water as the mobile phase to adopt Sephedex G-15 gel chromatography column to separate the ultrafiltration permeate obtained in step 2), and detect and measure the eluate corresponding to each peak at 280nm with a protein nucleic acid detector Antioxidant activity, collect highly active No. 2 absorption peak (see Figure 1) component and lyophilize;

(4) The freeze-dried components obtained in step 3) are further separated by reversed-phase HPLC, using an ODS column (Hedera ODS-C2, 10 × 250mm), using water and acetonitrile (0-25-25 mm) containing 0.1% trifluoroacetic acid 0～21～50% acetonitrile) within 60min for linear elution of the mobile phase, detect the elution peak at a wavelength of 220nm and collect and measure the antioxidant activity of each peak, the flow rate is 1.5mL·min ^-1 , according to the detection spectrum (see Figure 2) Collect the eluate corresponding to the No. 1 absorption peak with the highest activity; then use a C18 column (HypersilODS-C18, 4.6×250mm) to purify the No. 1 absorption peak eluate again, with an elution gradient of 0-20% within 40 min Acetonitrile, flow rate 0.5mL·min ^-1 , according to the detection spectrum (see Figure 3), collect the eluent corresponding to the absorption peak No. 1 and No. 2 with higher activity respectively and freeze-dry to obtain the antioxidant peptide; use tandem flight The amino acid sequences obtained by time mass spectrometry are His-Asp-His-Pro-Val-Cys (histidine-aspartic acid-histidine-proline-valine-cysteine) respectively (see figure 4) and His-Glu-Lys-Val-Cys (histidine-glutamic acid-lysine-valine-cysteine) (see Figure 5) two antioxidant peptides.

The antioxidant capacity of the two antioxidant peptides was determined by superoxide anion free radical and DPPH·free radical scavenging methods, respectively. The required concentration (EC ₅₀ ) of the antioxidant peptide His-Asp-His-Pro-Val-Cys to scavenge 50% DPPH free radicals is 0.0310±0.0011μM, which is equivalent to the scavenging ability of the natural antioxidant peptide glutathione; scavenging 50 The required concentration (EC ₅₀ ) for % superoxide anion radicals was 0.3814±0.0017 μM. Antioxidant peptide His-Glu-Lys-Val-Cys required concentration (EC ₅₀ ) for scavenging 50% DPPH·free radicals was 0.0677±0.0012 μM, and required concentration (EC ₅₀ ) for scavenging 50% superoxide anion free radicals was 0.3744± 0.0021 μM. It can be seen from the results that the two antioxidant peptides derived from the meat of the blue trevally of the present invention have good antioxidant activities in the two detection systems.

The source of raw materials for the preparation of antioxidant peptides in the present invention is cheap, rich in resources, the preparation method is simple, low in cost and easy to operate, and has strong applicability; the prepared antioxidant peptides have strong antioxidant activity, good water solubility, and are safe and non-toxic. Toxicity, no side effects, etc., suitable for use in food, cosmetics, medicine and other fields. the

Adopt respectively DPPH method and superoxide anion free radical method to measure the free radical scavenging ability of blue round trevally protein antioxidant peptide in the present invention, these methods are all conventional detection methods, concrete steps are as follows:

1. Determination of the ability of antioxidant peptides to scavenge 1,1-diphenyltrinitrophenylhydrazine (DPPH·) free radicals

Take 500 μL of the sample to be tested and add 500 μL of 0.1mM DPPH·methanol solution. After mixing, let stand in the dark at 30°C for 30 min, and measure the absorbance at 515 nm. The DPPH radical scavenging ability was calculated according to the following formula, wherein, the sample was replaced by deionized water, and the measured absorbance value was used as a control, and the DPPH solution was replaced by methanol, and the measured absorbance value was used as a blank. the

DPPH free radical scavenging ability (%) = (control sample + blank sample - test sample) × 100% / control sample

2. Determination of the ability of antioxidant peptides to scavenge superoxide anion (O ₂ ^- ·) free radicals

Take 200 μL of the sample to be tested, add 5.7 mL of tris-HCl buffer solution (50 mM, pH 8.2), and 100 μL of 5 mM pyrogallol solution, mix well, measure A 320nm at 320 _nm every 30 s, and finish the reaction for 4 minutes to obtain _Sample V (that is, the average rate of change of light absorption per minute). In the blank tube, 10mmol/L HCl was used to replace the pyrogallol solution, and the rate of pyrogallol autoxidation within 4min was V _white .

O ₂ ⁻ Free radical scavenging ability (%)=[( _Vbai -V _sample )/ _Vbai ]×100%

Description of drawings

Figure 1 is the protein detection spectrum of the eluate when the ultrafiltration permeate is separated by gel chromatography

Figure 2 is the HPLC detection spectrum of the No. 2 absorption peak in the eluent of Figure 1

Figure 3 is the HPLC detection spectrum of the No. 1 absorption peak in the eluent of Figure 2

Figure 4 is the amino acid sequence mass spectrum of the No. 1 absorption peak in the eluate of Figure 3

Figure 5 is the amino acid sequence mass spectrum of the No. 2 absorption peak in the eluate of Figure 3

Detailed ways:

Example 1

Mix the protein powder of blue trevally with water to make a reaction raw material solution with a protein content of 1wt%, then add 0.03wt% alkaline protease, hydrolyze at 50°C and pH 8.0 for 3 hours, and finally heat to 100°C to stop enzymatic reaction. Centrifuge the obtained mixture at 4°C and 8000r/min for 10-15min, take the supernatant of enzymatic hydrolysis and use an ultrafiltration membrane with a molecular weight of 5000Da to separate and collect the permeate, and use deionized water as the mobile phase to coagulate with Sephedex G-15. Gel chromatography separates the ultrafiltration permeate, and detects and measures the antioxidant activity of the eluent corresponding to each peak with a protein and nucleic acid detector at 280nm, and collects highly active No. 2 absorption peaks (see Figure 1) for further analysis by reversed-phase HPLC. Separation, using ODS column (Hedera ODS-C2, 10×250mm), using water containing 0.1% trifluoroacetic acid and acetonitrile (5-60% acetonitrile within 55min) as the mobile phase for linear elution, and detecting the elution peak at a wavelength of 220nm And collect and measure the antioxidant activity of each peak, the flow rate is 1.5mL·min ^-1 , collect the eluent corresponding to No. 1 absorption peak with the highest activity according to the detection spectrum (see Figure 2); then use C18 column (Hypersil ODS-C18 , 4.6×250mm) to purify the No. 1 absorption peak eluent again, the elution gradient is 0-20% acetonitrile within 30min, the flow rate is 0.5mL·min ^-1 , and the higher ones were collected according to the detection spectrum (see Figure 3). The eluate corresponding to the active No. 1 and No. 2 absorption peaks was freeze-dried to obtain the antioxidant peptide; the amino acid sequences obtained by tandem time-of-flight mass spectrometry were His-Asp-His-Pro-Val-Cys (histidine -Aspartate-Histidine-Proline-Valine-Cysteine) and His-Glu-Lys-Val-Cys (Histidine-Glu-Lysine-Valine- Cysteine) two antioxidant peptides.

Example 2

Mix the protein powder of blue scorpion fish meat with water to make a reaction raw material solution with a protein content of 3wt%; then add 0.005wt% alkaline protease, hydrolyze at 55°C and pH 10 for 6 hours, and finally heat to 100°C to stop enzymatic reaction. Centrifuge the obtained mixture at 4°C and 8000r/min for 10-15min, take the supernatant of enzymatic hydrolysis and use an ultrafiltration membrane with a molecular weight of 5000Da to separate and collect the permeate, and use deionized water as the mobile phase to coagulate with Sephedex G-15. Gel chromatography separates the ultrafiltration permeate, and detects and measures the antioxidant activity of the eluent corresponding to each peak with a protein and nucleic acid detector at 280nm, and collects highly active No. 2 absorption peaks (see Figure 1) for further analysis by reversed-phase HPLC. Separation, using ODS column (Hedera ODS-C2, 10×250mm), using water containing 0.1% trifluoroacetic acid and acetonitrile (0-60% acetonitrile within 60min) as the mobile phase for linear elution, and detecting the elution peak at a wavelength of 220nm And collect and measure the antioxidant activity of each peak, the flow rate is 1.5mL·min ^-1 , collect the eluent corresponding to No. 1 absorption peak with the highest activity according to the detection spectrum (see Figure 2); then use C18 column (Hypersil ODS-C18 , 4.6×250mm) to repurify the eluent from the No. 1 absorption peak, the elution gradient is 5-20% acetonitrile within 40min, the flow rate is 0.5mL·min ^-1 , and the higher The eluate corresponding to the active No. 1 and No. 2 absorption peaks was freeze-dried to obtain the antioxidant peptide; the amino acid sequences obtained by tandem time-of-flight mass spectrometry were His-Asp-His-Pro-Val-Cys (histidine -Aspartate-Histidine-Proline-Valine-Cysteine) and His-Glu-Lys-Val-Cys (Histidine-Glu-Lysine-Valine- Cysteine) two antioxidant peptides.

Example 3

Mix the protein powder of blue scorpion fish meat with water to make a reaction material solution with a protein content of 5wt%; then add 0.05wt% alkaline protease, hydrolyze at 45°C and pH 8.0 for 4.5 hours, and finally heat to 100°C to stop enzymatic reaction. Centrifuge the obtained mixture at 4°C and 8000r/min for 10-15min, take the supernatant of enzymatic hydrolysis and use an ultrafiltration membrane with a molecular weight of 5000Da to separate and collect the permeate, and use deionized water as the mobile phase to coagulate with Sephedex G-15. Gel chromatography separates the ultrafiltration permeate, and detects and measures the antioxidant activity of the eluent corresponding to each peak with a protein and nucleic acid detector at 280nm, and collects highly active No. 2 absorption peaks (see Figure 1) for further analysis by reversed-phase HPLC. Separation, using ODS column (Hedera ODS-C2, 10 × 250mm), using water containing 0.1% trifluoroacetic acid and acetonitrile (5-55% acetonitrile within 60min) as the mobile phase for linear elution, and detecting the elution peak at a wavelength of 220nm And collect and measure the antioxidant activity of each peak, the flow rate is 1.5mL·min ^-1 , collect the eluent corresponding to No. 1 absorption peak with the highest activity according to the detection spectrum (see Figure 2); then use C18 column (Hypersil ODS-C18 , 4.6×250mm) to purify the No. 1 absorption peak eluent again, the elution gradient is 5-20% acetonitrile within 50min, the flow rate is 0.5mL·min ^-1 , and the higher ones were collected according to the detection spectrum (see Figure 3). The eluate corresponding to the active No. 1 and No. 2 absorption peaks was freeze-dried to obtain the antioxidant peptide; the amino acid sequences obtained by tandem time-of-flight mass spectrometry were His-Asp-His-Pro-Val-Cys (histidine -Aspartate-Histidine-Proline-Valine-Cysteine) and His-Glu-Lys-Val-Cys (Histidine-Glu-Lysine-Valine- Cysteine) two antioxidant peptides.

Claims (2)

1. A method for preparing the anti-oxidative active peptide of the blue-eyed trevally protein is characterized in that the blue-round trevally protein is used as raw material, and the alkaline protease hydrolysis produced by Bacillus subtilis 2709 is adopted, and the specific process is: (1) Mix the protein powder of blue round trevally with water to make a reaction raw material solution with a protein content of 1-5wt%, then add 0.002-0.05wt% alkaline protease, and make it at 45-60°C and pH 7.5-10 Hydrolyze for 3 to 6 hours, and finally heat to 100°C to inactivate the enzyme to obtain the enzymatic hydrolysis solution; (2) Centrifuge the enzymolysis solution at 4°C and 8000r/min for 10-15 minutes to obtain the supernatant and use ultrafiltration membrane with a molecular weight of 5000Da to separate and collect the permeate; (3) Use deionized water as the mobile phase to separate the permeate obtained in step 2 with Sephedex G-15 gel chromatography column, and use a protein and nucleic acid detector to detect and measure the antioxidant activity of the eluate corresponding to each peak at 280nm , collect highly active No. 2 absorption peak (see Figure 1) components and lyophilize to obtain a lyophilized body; (4) The freeze-dried body obtained in step 3 is further separated by reverse-phase HPLC, using an ODS column (Hedera ODS-C2, 10 × 250mm), using water and acetonitrile containing 0.1% trifluoroacetic acid (within 0-25-60min) 0～21～50% acetonitrile) as the mobile phase for linear elution, detect the elution peak at 220nm wavelength and collect and measure the antioxidant activity of each peak, the flow rate is 1.5mL·min ^-1 , collect the activity according to the detection spectrum (see Figure 2) The eluent corresponding to the highest absorption peak No. 1; then use a C18 column (Hypersil ODS-C18, 4.6×250mm) to purify the eluent of No. 1 absorption peak again, and the elution gradient is 0-20% acetonitrile within 40min , with a flow rate of 0.5mL·min ^-1 , according to the detection spectrum (see Figure 3), the eluents corresponding to the absorption peaks No. 1 and No. 2 with higher activity were collected and freeze-dried to obtain two antioxidant peptides. 2. the blue round trevally protein antioxidant activity peptide described in claim 1, its amino acid sequence is respectively His-Asp-His-Pro-Val-Cys (histidine-aspartic acid-histidine-proline acid-valine-cysteine) (see Figure 4 for its mass spectrum) and His-Glu-Lys-Val-Cys (histidine-glutamic acid-lysine-valine-cysteine) (See Figure 5 for its mass spectrum).

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