CN103204906A

CN103204906A - Mussel meat protein antioxidative peptide and preparation method thereof

Info

Publication number: CN103204906A
Application number: CN2013100354694A
Authority: CN
Inventors: 王斌; 马佳卉; 栗丽; 罗红宇
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2013-01-29
Filing date: 2013-01-29
Publication date: 2013-07-17

Abstract

The invention relates to a mussel meat protein antioxidant peptide and a preparation method thereof, characterized in that: the antioxidant peptide is a pentapeptide compound, the amino acid sequence is YPPAK (Tyr-Pro-Pro-Ala-Lys), and ESI-MS detection Gives molecular ion peak m/z [M+H]+575.26. The preparation method comprises the steps of homogenization, degreasing, mixing, enzymolysis, desalting, ultrafiltration, chromatography and the like. The prepared highly active antioxidant peptide YPPAK (Tyr-Pro-Pro-Ala-Lys) has a good scavenging effect on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals; at the same time, YPPAK (Tyr-Pro-Pro-Ala-Lys) Ala-Lys) also showed good lipid peroxidation inhibitory effect. YPPAK (Tyr-Pro-Pro-Ala-Lys) has the advantages of safety, no side effects, strong antioxidant activity and easy digestion and absorption. It can be used as medicine, health food or food additive.

Description

A kind of mussel meat protein antioxidant peptide and preparation method thereof

Technical field

The present invention relates to protein antioxidant peptide, specifically refer to a kind of mussel meat protein antioxidant peptide and preparation method thereof.

Background technology

Antioxidant is that a class can weaken or dispel radical pair human body infringement and protection food is avoided the rotten class material of oxidative damage, mainly is divided into natural and two types of chemosynthesis.At present, with butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT) and tertiarybutylhydroquinone (TBHQ) be representative the chemosynthesis antioxidant since price relatively cheap, anti-oxidant activity good, thereby be widely applied in the foodstuffs industry.But the existing chemosynthesis antioxidant that studies show that all has disadvantageous effect in liver, the kidney and other organs to human body in varying degrees.Therefore, the application of chemosynthesis antioxidant has been subjected to considerable influence, and part developed country has limited the quantity of or banned use of chemosynthesis antioxidant to the human body toxic side effect.Therefore, seek efficient, safe natural antioxidants instead of chemical synthetized oxidation preventive agent and become the research focus.

Mussel belongs to Mollusca, lamellibranchiata, mussel order, Mytilidae, mussel belongs to, be the marine products bivalve shellfishs, be commonly called as blue or green mouthful, Hai Hong, have the title of " marine egg ", its protein contains 8 kinds of indispensable amino acids such as the Xie Ansuan, leucine of human body needs, and content is far above egg, chicken, duck, fish, shrimp and meat.There are some researches show, that mussel extract or mussel meat zymolyte have is antitumor, reducing blood-fat, anticoagulation, hypotensive, anti-oxidant, improve immunizing power isoreactivity function, the patent No. is that the Chinese invention patent of ZL200510110096.8 discloses a kind of " extract of Mytilus crassitesta Lischke, manufacture method and uses thereof ", and it is the application of extract aspect anti influenza and anti-SARS virus that utilizes Mytilus crassitesta Lischke; The patent No. is that the Chinese invention patent of ZL200510024391.1 discloses " a kind of Trachyostracous mussel extract that improves immunity function ", and it is that extract about Mytilus crassitesta Lischke is in the application aspect the immunomodulatory; Application number is that 201010296130.6 Chinese invention patent application discloses a kind of " preparation method and the application of anti-prostate cancer mussel enzymatic hydrolyzed extract ", and it is to utilize mussel meat Sumizyme MP (Alcalase) enzymatic hydrolyzed extract that prostate cancer cell is had good inhibited proliferation.Above patent is significant to intensive processing and the active exploitation of mussel.But the active substance that above patent is mentioned mostly is crude extract or total zymolyte, complicated component, and the quality control difficulty is bigger, thereby is subjected to the restriction of many aspects in application

Summary of the invention

Technical problem to be solved by this invention is that the present situation at prior art provides a kind of mussel meat protein antioxidant peptide that can remove free radical and suppress lipid peroxidation, and it can be used as the safe additive of medicine, protective foods and food.

Another technical problem to be solved by this invention provides a kind of preparation method of mussel meat protein antioxidant peptide.

The present invention solves the problems of the technologies described above the technical scheme that adopts: this mussel meat protein antioxidant peptide, it is characterized in that this anti-oxidation peptide is pentapeptide compound, aminoacid sequence is YPPAK (Tyr-Pro-Pro-Ala-Lys), and ESI-MS detects and provides molecular ion peak m/z[M+H]+575.26.

The preparation method of above-mentioned mussel meat protein antioxidant peptide is characterized in that comprising the steps:

1) mussel meat that will clean, shells is processed into homogenate with high-speed tissue mashing machine, add Virahol according to solid-liquid ratio 1g:2 ~ 4mL then, degreasing 20 ~ 24h in 30 ~ 35 ℃, centrifugal 10 ~ 15min removes Virahol in 2～6 ℃, 4000 ~ 5000rpm then, collects degreasing mussel meat solid substance;

2) described degreasing mussel meat solid substance is the phosphate buffered saline buffer of 0.05mol/L by solid-to-liquid ratio 1g:20 ~ 25mL adding concentration, and regulating pH is 6.5 ~ 7.5, obtains mixed solution;

3) described mixeding liquid temperature being risen to 55 ~ 65 ℃ and stir preheating 10 ~ 15min, is that benchmark adds 2.0 ~ 4.0% proteolytic enzyme with the quality of described degreasing mussel meat solid substance, at 55 ~ 65 ℃ of enzymolysis 2 ~ 4h, obtains enzymolysis product;

4) after constant temperature keeps 8 ~ 10min after the enzymolysis product of step 3) gained is warming up to 95 ~ 100 ℃, be cooled to 20 ~ 25 ℃, centrifugal then, obtain enzymolysis solution;

5) above-mentioned enzymolysis solution is added the phosphate buffered saline buffer of 0.05mol/L, making its concentration is 20 ~ 25mg/mL, ratio according to macroporous resin weight and enzymolysis solution volume 1g:40 ~ 50mL joins enzymolysis solution in the macroporous resin chromatography column, be that 45～55% ethanol carries out wash-out with purity then, at last in temperature below 40 ℃, vacuum tightness-0.1MPa steams with backspin and removes ethanol, and concentrated solution carries out lyophilize, gets desalination zymolyte dry powder;

6) above-mentioned desalination zymolyte dry powder is dissolved in the phosphate buffered saline buffer that pH is 0.04～0.06mol/L of 6.8～7.2 and is made into the solution that concentration is 8～12mg/mL, under the working temperature of the operating pressure of 0.1 ~ 0.15MPa and 20 ~ 25 ℃, adopt ultra-filtration membrane to carry out uf processing, collect molecular weight less than the 1kDa part, obtain the ultrafiltration enzymolysis solution, after the freeze-drying of ultrafiltration enzymolysis solution, get ultrafiltration enzymolysis solution lyophilized powder;

7) be that the phosphate buffered saline buffer of 0.04～0.06mol/L of 6.8～7.2 is made into the solution that concentration is 8～12mg/mL with above-mentioned ultrafiltration enzymolysis solution lyophilized powder pH, join anion-exchange resin column, water, 0.1mol/L, 0.5mol/L and 1.0mol/L NaCl solution carry out wash-out respectively then, every 5mL elutriant is collected 1 test tube, and according to the absorbancy drafting elution fraction graphic representation of every test tube elutriant under 280nm, wherein, the highest component of DPPH free radical scavenging activity is the ion-exchange enzymolysis solution, freeze-drying gets dry powder; Described ion-exchange enzymolysis solution lyophilized powder is made into the solution of 10mg/mL with 0.05mol/L phosphate buffered saline buffer (pH7.0), join in the gel chromatography column, with phosphate buffered saline buffer (0.05mol/L, pH7.0) carry out wash-out, every 5mL elutriant is collected 1 test tube, and draws the elution fraction graphic representation according to the absorbancy of every test tube elutriant under 280nm, wherein, the highest component of DPPH free radical scavenging activity is the preparing gel zymolyte, and freeze-drying gets dry powder;

Above-mentioned preparing gel zymolyte lyophilized powder is made into the solution of 10mg/mL with 0.05mol/L phosphate buffered saline buffer (pH7.0), carry out purifying with RPLC, collect the highest active polypeptide according to the absorbancy curve under the 280nm and anti-oxidant activity, namely get high reactivity anti-oxidation peptide YPPAK (Tyr-Pro-Pro-Ala-Lys).

Preferably, described mussel meat is Mytilus edulis meat.

Preferably, the proteolytic enzyme described in the step 3) is neutral protease, enzyme activity 〉=2.0 * 105U/g.

Preferred used macroporous resin is D101, and anionite-exchange resin is the DEAE-52 Mierocrystalline cellulose, and used gel is sephadex G-25; Described RPLC condition is: sample size 20 μ L; Chromatographic column is Zorbax C18(250mm * 4.6mm, 5 μ m); Column temperature is 30 ℃; Moving phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile (containing 0.1% trifluoroacetic acid); Gradient elution: 0 ~ 55min acetonitrile concentration from 0 to 50%, every 5min increases by 5%; Elution speed 1.0mL/min; The ultraviolet detection wavelength is 280nm.

Compared with prior art, mussel meat protein antioxidant peptide provided by the present invention has good scavenging(action) to DPPH free radical (EC502.62mg/mL), hydroxyl radical free radical (EC500.228mg/mL) and ultra-oxygen anion free radical (EC500.072mg/mL); Simultaneously, YPPAK also demonstrates good lipid peroxidation restraining effect; YPPAK (Tyr-Pro-Pro-Ala-Lys) has good scavenging(action) to DPPH free radical, hydroxyl radical free radical and ultra-oxygen anion free radical; Simultaneously, YPPAK (Tyr-Pro-Pro-Ala-Lys) also demonstrates good lipid peroxidation restraining effect; YPPAK (Tyr-Pro-Pro-Ala-Lys) can be used as the additive of medicine, protective foods and food, and has advantages such as safe without toxic side effect, anti-oxidant activity is strong and be easy to digest and assimilate.Preparation technology is scientific and reasonable for this mussel meat protein antioxidant peptide, select for use neutral protease (neutrase) economical and practical with enzyme as enzymolysis, merge macroporous resin desalination, ultrafiltration classification and chromatographic refining simultaneously by biologic enzymolysis method, enzymolysis process is easily monitored, and the anti-oxidation peptide that makes simultaneously has higher activity.

Description of drawings

The RP-HPLC of Fig. 1 sephadex G-25 preparation zymolyte analyzes.

The mass spectrum of Fig. 2 YPPAK (Tyr-Pro-Pro-Ala-Lys).

The DPPH free radical scavenging activity of Fig. 3 YPPAK (Tyr-Pro-Pro-Ala-Lys).

The hydroxyl radical free radical of Fig. 4 YPPAK (Tyr-Pro-Pro-Ala-Lys) is removed active.

The ultra-oxygen anion free radical of Fig. 5 YPPAK (Tyr-Pro-Pro-Ala-Lys) is removed active.

It is active that the lipid peroxidation of Fig. 6 YPPAK (Tyr-Pro-Pro-Ala-Lys) suppresses.

Embodiment

Describe in further detail below in conjunction with the present invention of accompanying drawing embodiment.

The preparation method of mussel meat protein antioxidant peptide, preparation technology's flow process is as follows: mussel meat → degreasing → enzymolysis → zymolyte → macroporous resin desalination → ultrafiltration → ion exchange chromatography → gel permeation chromatography → high performance liquid chromatography preparation → anti-oxidation peptide.

Concrete steps are:

1) the meat high-speed tissue mashing machine of the Mytilus edulis that will clean, shells is processed into homogenate with it, adds Virahol in 30 ~ 35 ℃ of degreasing 24h according to solid-liquid ratio 1g:3mL then, and centrifugal 15min removes Virahol in 4 ℃, 5000rpm, collects solid substance.

2) degreasing mussel meat solid substance adds 0.05mol/L phosphate buffered saline buffer (pH7.0) by solid-to-liquid ratio 1g:25mL, gets mixed solution;

3) mixeding liquid temperature is risen to 60 ℃ and stir preheating 10min, add neutral protease (neutrase) according to 3% of degreasing mussel meat solid quality, hydrolysis temperature is 60 ℃, and enzymolysis time 3h gets enzymolysis product;

4) enzymolysis product of step 3) gained is gone out enzyme is handled:

1. enzyme goes out: enzymolysis product is warming up to 90 ~ 95 ℃, and after this temperature keeps 10min, is cooled to 20 ~ 25 ℃, with the centrifugal 15min of 5000rpm, gets enzymolysis solution then;

2. desalination: will add the 0.05mol/L phosphate buffered saline buffer in the enzymolysis solution, making its concentration is 20mg/mL, ratio according to macroporous resin weight and enzymolysis solution volume 1g:50mL joins enzymolysis solution in the macroporous resin chromatography column, be that 50% ethanol carries out wash-out with purity then, absorbancy is less than 0.05 under 280nm to elutriant, and at last in temperature below 40 ℃, vacuum tightness-0.1MPa steams the ethanol of removing in the elutriant with backspin, the surplus solution lyophilize gets desalination zymolyte dry powder;

3. ultrafiltration: above-mentioned desalination zymolyte dry powder is dissolved in 0.05mol/L phosphate buffered saline buffer (pH7.0) is made into the solution that concentration is 10mg/mL, under the working temperature of the operating pressure of 0.1 ~ 0.15MPa and 20 ~ 25 ℃, adopt ultra-filtration membrane to carry out uf processing, collect molecular weight less than the 1kDa part, get the ultrafiltration enzymolysis solution, with its freeze-drying, get ultrafiltration enzymolysis solution lyophilized powder;

4. anion-exchange chromatography: the solution that above-mentioned ultrafiltration enzymolysis solution lyophilized powder is made into 10mg/mL with 0.05mol/L phosphate buffered saline buffer (pH7.0), separate through DEAE-52 Mierocrystalline cellulose anionite-exchange resin, the difference water, 0.1mol/L, 0.5mol/L and 1.0mol/L NaCl solution carries out wash-out, every 5mL elutriant is collected 1 test tube, and according to the absorbancy drafting elution fraction graphic representation of every test tube elutriant under 280nm, each chromatographic peak is a component, wherein, the highest component of DPPH free radical scavenging activity is the ion-exchange enzymolysis solution, with its freeze-drying, get ion-exchange enzymolysis solution lyophilized powder;

5. gel chromatography chromatography: above-mentioned ion-exchange enzymolysis solution lyophilized powder is made into the solution that concentration is 10mg/mL with 0.05mol/L phosphate buffered saline buffer (pH7.0), through sephadex G-25 column chromatography for separation, carry out wash-out with 5 times of column volume phosphate buffered saline buffers, every 5mL elutriant is collected 1 test tube, and according to the absorbancy drafting elution fraction graphic representation of every test tube elutriant under 280nm, each chromatographic peak is a component, wherein, the highest component of DPPH free radical scavenging activity is the preparing gel zymolyte, with its freeze-drying, get preparing gel zymolyte lyophilized powder.

6. high performance liquid chromatography is refining: above-mentioned preparing gel zymolyte lyophilized powder is made into the solution that concentration is 10mg/mL with 0.05mol/L phosphate buffered saline buffer (pH7.0), and (RP-HPLC) carries out purifying with RPLC; Condition: sample size 20 μ L; Chromatographic column is Zorbax C18(250mm * 4.6mm, 5 μ m; Column temperature is 30 ℃; Moving phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile (containing 0.1% trifluoroacetic acid); Gradient elution: 0-55min acetonitrile concentration from 0 to 50%, every 5min increases by 5%; Elution speed 1.0mL/min; UV detection wavelength 280nm gets 1 high reactivity anti-oxidation peptide according to the absorbancy curve under the 280nm and anti-oxidant activity.

7. structure detection: collect active 1 the highest anti-oxidation peptide, detecting through RPLC (RP-HPLC) is simple spike (Fig. 1), utilize albumen/peptide sequence analysis-e/or determining aminoacid sequence to be YPPAK (Tyr-Pro-Pro-Ala-Lys), ESI-MS detects and provides molecular ion peak m/z[M+H]+575.26(Fig. 2).

The mussel meat protein antioxidant peptide YPPAK (Tyr-Pro-Pro-Ala-Lys) that makes is carried out DPPH free radical scavenging experiment, and test-results is seen Fig. 3; Carry out hydroxyl radical free radical and remove experiment, test-results is seen Fig. 4; Carry out ultra-oxygen anion free radical and remove experiment, test-results is seen Fig. 5; Carry out lipid peroxidation and suppress experiment, test-results is seen Fig. 6.

Experimental result by Fig. 3 to Fig. 6 can be learnt: YPPAK (Tyr-Pro-Pro-Ala-Lys) has good scavenging(action) to DPPH free radical (EC502.62mg/mL), hydroxyl radical free radical (EC500.228mg/mL) and ultra-oxygen anion free radical (EC500.072mg/mL); Simultaneously, YPPAK also demonstrates good lipid peroxidation restraining effect.

Claims

1. A mussel meat protein antioxidant peptide, characterized in that the antioxidant peptide is a pentapeptide compound, the amino acid sequence is YPPAK (Tyr-Pro-Pro-Ala-Lys), and the ESI-MS detection gives the molecular ion peak m /z[M+H]+575.26.

2. the preparation method of mussel meat protein antioxidant peptide as claimed in claim 1, is characterized in that comprising the steps:

1) Use a high-speed tissue masher to process the washed and shelled mussel meat into a homogenate, then add isopropanol according to the ratio of solid to liquid 1g:2~4mL, degrease at 30~35℃ for 20~24h, then Centrifuge at 2-6°C, 4000-5000rpm for 10-15min to remove isopropanol, and collect defatted mussel meat solids;

2) Add 0.05 mol/L phosphate buffer to the solid matter of the defatted mussel meat according to the solid-to-liquid ratio of 1 g: 20-25 mL, and adjust the pH to 6.5-7.5 to obtain a mixed solution;

3) Raise the temperature of the mixture to 55-65°C, stir and preheat for 10-15 minutes, add 2.0-4.0% protease based on the mass of the defatted mussel meat solids, and enzymolyze 2 at 55-65°C ~4h, the enzymatic hydrolysis product was obtained;

4) Heat the enzymolysis product obtained in step 3) to 95-100°C, keep the temperature for 8-10 minutes, cool to 20-25°C, and then centrifuge to obtain the enzymolysis solution;

5) Add the above enzymolysis solution to 0.05mol/L phosphate buffer to make the concentration 20~25mg/mL, and add the enzymolysis solution to the Macroporous resin chromatography column, then elute with ethanol with a purity of 45-55%, and finally remove ethanol by rotary evaporation at a temperature below 40°C and a vacuum degree below -0.1MPa, and freeze-dry the concentrated solution to obtain desalination and enzymatic hydrolysis Dry powder;

6) Dissolve the dry powder of the above-mentioned desalted enzymatic hydrolyzate in 0.04-0.06mol/L phosphate buffer solution with a pH of 6.8-7.2 to prepare a solution with a concentration of 8-12mg/mL, and then use it at a working pressure of 0.1-0.15MPa and Under the working temperature of 20 ~ 25 ℃, use ultrafiltration membrane to carry out ultrafiltration treatment, collect the part with molecular weight less than 1kDa to obtain ultrafiltration enzymatic hydrolysis solution, and freeze-dry the ultrafiltration enzymatic hydrolysis solution to obtain lyophilized powder;

7) Prepare the above freeze-dried powder with 0.04-0.06mol/L phosphate buffer solution with a pH of 6.8-7.2 to make a solution with a concentration of 8-12mg/mL, add it to an anion exchange resin column, and then use water, 0.1mol /L, 0.5mol/L and 1.0mol/L NaCl solution for elution, collect 1 test tube for every 5mL eluent, and draw the elution component curve according to the absorbance of each test tube eluate at 280nm, wherein, DPPH The component with the highest free radical scavenging activity is the ion-exchange enzymatic hydrolyzate, which is freeze-dried to obtain a dry powder; the freeze-dried powder of the ion-exchange enzymatic hydrolyzate is formulated with 0.05mol/L phosphate buffer (pH7.0) to make 10mg/mL solution, added to a gel chromatography column, and eluted with phosphate buffer (0.05mol/L, pH7.0), collecting 1 test tube for every 5mL eluent, and according to the eluent in each test tube at 280nm The absorbance of the elution component curve is drawn, wherein, the component with the highest DPPH free radical scavenging activity is the enzymatic hydrolyzate prepared from the gel, which is freeze-dried to obtain a dry powder;

The lyophilized powder of the enzymatic hydrolyzate prepared from the above gel was prepared into a 10mg/mL solution with 0.05mol/L phosphate buffer (pH7.0), and purified by reverse-phase high-performance liquid chromatography. According to the absorbance curve at 280nm and Antioxidant activity The polypeptide with the highest activity is collected, namely the highly active antioxidative peptide YPPAK (Tyr-Pro-Pro-Ala-Lys).

3. The preparation method according to claim 2, characterized in that the mussel meat described in step 1) is blue mussel meat.

4. The preparation method according to claim 2, characterized in that the protease described in step 3) is a neutral protease, and the enzyme activity is ≥2.0×105U/g.

5. The chromatography method according to claim 2, wherein the macroporous resin used is D101, the anion exchange resin is DEAE-52 cellulose, and the gel used is Sephadex G-25; The reverse-phase high-performance liquid chromatography conditions are: injection volume 20 μL; chromatographic column is Zorbax C18 (250mm×4.6mm, 5μm); column temperature is 30°C; mobile phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile (containing 0.1% trifluoroacetic acid); gradient elution: 0~55min acetonitrile concentration from 0 to 50%, increasing by 5% every 5min; elution rate 1.0mL/min; UV detection wavelength is 280nm.