CN105200104B - Preparation method of crab shell leftover antioxidant peptide chain - Google Patents
Preparation method of crab shell leftover antioxidant peptide chain Download PDFInfo
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- CN105200104B CN105200104B CN201510399380.5A CN201510399380A CN105200104B CN 105200104 B CN105200104 B CN 105200104B CN 201510399380 A CN201510399380 A CN 201510399380A CN 105200104 B CN105200104 B CN 105200104B
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Abstract
The invention discloses a preparation method of an antioxidant peptide chain of crab shell leftovers, which is characterized in that enzymolysis liquid is prepared by degreasing and enzymolysis of neutral protease and papain, the enzymolysis liquid is separated and purified by adopting ultrafiltration, macroporous resin purification, anion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain the antioxidant peptide chain Ile-Trp-Met-Glu-Cys-Asn-Trp, and the molecular weight of ESI-MS (electronic signature verification system) is 979.15 Da. The antioxidant peptide chain prepared by the invention has good scavenging effect on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals; meanwhile, Ile-Trp-Met-Glu-Cys-Asn-Trp also shows good lipid peroxidation inhibition effect.
Description
Technical Field
The invention relates to a preparation method of protein antioxidant peptide, in particular to a preparation method of crab shell leftover antioxidant peptide chain.
Background
The yield of Chinese aquatic products accounts for about one third of the total yield in the world, but the processing rate is about 30 percent, the difference with 80 percent of countries such as the United states is large, and the aquatic product processing and comprehensive utilization technology needs to be further improved. At present, the utilization rate of the processing by-products of aquatic products in China is low, the processing by-products are effectively classified and utilized according to the chemical compositions and biochemical characteristics of the by-products, and the processing value even exceeds that of fish by several times to dozens of times. A large amount of leftovers such as shrimp heads, shrimp shells, crab legs and the like are generated in the processing process of shrimp and crab aquatic products, still contain a large amount of protein, unsaturated fatty acid, organic calcium, chitin and other various nutritional ingredients and active substances, and have a good utilization prospect.
The antioxidant can reduce or eliminate free radical damage to human body, or protect food from oxidative damage and deterioration. Chemically synthesized antioxidants, such as t-butylhydroquinone, butylhydroxyanisole, propyl gallate, dibutylhydroxytoluene, and the like, are inexpensive and widely used in the food industry. However, chemically synthesized antioxidants damage organs such as liver and kidney of human body to different degrees, and some countries and regions have been limited or prohibited to use them. Therefore, the development of efficient and safe natural antioxidants is a hot spot. The antioxidant peptide is widely regarded for its advantages of edible safety, high utilization rate, no toxic and side effects, etc.
The applicant finds that the process research for preparing the crab shell leftover antioxidant peptide by using the crab shell leftovers as the raw materials and utilizing the enzymolysis technology is in a blank stage, and the preparation of the high-activity antioxidant peptide by using the crab shell leftover enzymolysis products as the materials and the application thereof are not reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of crab shell leftover antioxidant peptide chain which has antioxidant activity and is safe to human body aiming at the current situation of the prior art.
The preparation method of the crab shell leftover antioxidant peptide chain is characterized by comprising the following steps:
1) crab shell leftovers pretreatment: taking crab shell leftovers which are sieved by a 100-mesh sieve, adding isopropanol according to the material-liquid ratio of 1g: 5-10 mL, degreasing for 8-10 h at 40-50 ℃, then centrifuging for 10-15 min at 8000-10000 rpm to remove the isopropanol, and collecting degreased crab shell leftovers solid matter.
2) Enzymolysis of crab shell leftovers: adding phosphate buffer (0.05 mol/L, pH 6.5-7.5) into the degreased crab shell leftover solid according to the solid-to-liquid ratio of 1g: 10-15 mL, raising the temperature of the mixed solution to 45-50 ℃, and carrying out ultrasonic treatment for 10-15 min; then adding neutral protease according to 1.0-1.5% of the solid matter weight of the degreased crab shell leftovers, and carrying out enzymolysis for 4-6 h at 45-50 ℃; heating the enzymolysis liquid to 90-95 ℃, keeping the temperature at the constant temperature for 10-15 min, cooling the temperature of the enzymolysis liquid to 50-60 ℃, adding papain according to 2.0-2.5% of the solid matter amount of the degreased crab shell leftovers, carrying out enzymolysis for 4-6 h at 50-60 ℃, cooling to room temperature, centrifuging at 9000-10000 rpm for 10-15 min, and removing precipitates to obtain the enzymolysis liquid.
3) Preparing the crab shell leftover antioxidant peptide chain: performing ultrafiltration treatment on the enzymolysis liquid by using a 1 kDa ultrafiltration membrane, and collecting the part with the molecular weight less than 1 kDa to obtain ultrafiltration enzymolysis liquid; adding the ultrafiltration enzymolysis liquid into a chromatographic column filled with 6-8 times of DA201-C macroporous resin according to the volume ratio, eluting with double-distilled water with 4-5 times of column volume to remove impurities, eluting with 70% ethanol with 5-8 times of column volume, removing the ethanol by low-pressure rotary evaporation of ethanol eluent at the temperature of below 50 ℃, freezing and drying to obtain a polypeptide mixture, and purifying the polypeptide mixture by anion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the crab shell leftover antioxidant peptide chain.
Preferably, the crab shells in the step 1) are from Portunus trituberculatus (blue crab)Portunus trituberculatus）。
Preferably, the specific processes of anion exchange resin chromatography, gel column chromatography and RP-HPLC purification in the step 3) are as follows:
ion exchange resin chromatography: dissolving the polypeptide mixture in double distilled water to prepare a solution with the concentration of 45-50 mg/mL, separating by an anion exchange resin (DEAE Sepharose FF) chromatographic column, eluting by water, 0.25-0.35 mol/L and 0.45-0.55 mol/L NaCl solution, and collecting an elution component according to an absorbance curve under 215 nm, wherein the component with the highest hydroxyl radical scavenging activity is an ion exchange chromatography zymolyte;
gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 15-20 mg/mL, separating by Sephadex LH-20 column chromatography, eluting by the double distilled water, and collecting an elution component according to an absorbance curve under 215 nm, wherein the peak with the highest hydroxyl radical scavenging activity is the gel chromatography zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution of 80-100 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 polypeptide with high antioxidant activity, namely Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW), according to the scavenging activity of hydroxyl free radicals.
More preferably, the RP-HPLC conditions are: the sample injection amount is 8-10 mu L; chromatographic column Hypersil BDS C18(250 mm. times.4.6 mm, 5 μm); mobile phase: 25% acetonitrile (containing 0.1% trifluoroacetic acid); the elution speed is 0.5-0.8 mL/min; the ultraviolet detection wavelength is 215 nm.
Compared with the prior art, the crab shell leftover antioxidant peptide chain has good scavenging effect on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals; meanwhile, Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW) also shows good lipid peroxidation inhibition effect; Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW) has the advantages of safety, no toxic or side effect, strong antioxidant activity, easy digestion and absorption and the like.
Drawings
FIG. 1 is a chromatogram of an anion exchange resin of the present invention;
FIG. 2 is a sephadex chromatogram of the present invention;
FIG. 3 is an RP-HPLC analysis of the Sephadex preparation of the invention for the zymolyte Fr.5-III;
FIG. 4 is a mass spectrum of Ile-Trp-Met-Glu-Cys-Asn-Trp of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
A preparation method of an antioxidant peptide chain of crab shell leftovers comprises the following preparation process flows: crab shell leftovers, degreasing, enzymolysis, zymolyte, ultrafiltration, macroporous resin purification, anion exchange chromatography, gel filtration chromatography, reversed phase high performance liquid chromatography and antioxidant peptide preparation.
The method comprises the following specific steps:
1) crab shell leftovers pretreatment: taking portunus trituberculatus which is sieved by a 100-mesh sieve (Portunus trituberculatus) Adding isopropanol into crab shell leftovers at a feed-liquid ratio of 1g: 8mL, degreasing at 45 ℃ for 8 h, then centrifuging at 9000 rpm for 10min to remove the isopropanol, collecting degreased materialsCrab shell leftover solid.
2) Enzymolysis of crab shell leftovers: adding phosphate buffer solution (0.05 mol/L, pH 7.0) into the defatted crab shell leftover solid according to the solid-to-liquid ratio of 1g:10mL, heating the mixed solution to 45 ℃, and carrying out ultrasonic treatment for 15 min; then adding neutral protease according to 1.2 percent of the mass of the solid matters of the defatted crab shell leftovers, and carrying out enzymolysis for 5 h at 45 ℃; heating the enzymolysis solution to 90 deg.C, maintaining at constant temperature for 15min, cooling to 55 deg.C, adding papain in an amount of 2.2% of solid matter of the defatted crab shell leftovers, performing enzymolysis at 55 deg.C for 4 h, cooling to room temperature, centrifuging at 10000rpm for 10min, and removing precipitate to obtain enzymolysis solution.
3) Preparing the crab shell leftover antioxidant peptide chain: performing ultrafiltration treatment on the enzymolysis liquid by using a 1 kDa ultrafiltration membrane, and collecting the part with the molecular weight less than 1 kDa to obtain ultrafiltration enzymolysis liquid; adding the ultrafiltration enzymolysis liquid into a chromatographic column filled with 8 times of DA201-C macroporous resin according to the volume ratio, eluting with double distilled water with 5 times of column volume to remove impurities, then eluting with 70% ethanol with 8 times of column volume, removing ethanol by low-pressure rotary evaporation of ethanol eluent at the temperature of below 50 ℃, freezing and drying to obtain a polypeptide mixture, and purifying the polypeptide mixture by anion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the crab shell leftover antioxidant peptide chain.
Anion exchange resin chromatography: dissolving the polypeptide mixture in double distilled water to prepare a solution with the concentration of 50 mg/mL, separating by an anion exchange resin (DEAE Sepharose FF) chromatographic column, eluting by water, 0.30 mol/L and 0.50mol/L NaCl solution, and collecting an eluted component according to an absorbance curve at 215 nm, wherein the component with the highest hydroxyl radical scavenging activity is an ion exchange chromatography zymolyte Fr.5 (figure 1).
② gel chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 20 mg/mL, separating by Sephadex LH-20 column chromatography, eluting by double distilled water, and collecting an elution component according to an absorbance curve under 215 nm, wherein a peak with the highest hydroxyl radical scavenging activity is gel chromatography zymolyte Fr.5-III (figure 2).
③ refining by high performance liquid chromatography: preparing the gel chromatography zymolyte into a solution of 80-100 mu g/mL by using double distilled water, and purifying by using RP-HPLC (conditions are that the sample amount is 8-10 mu L, and a chromatographic column Hypersil BDS C18(250 mm. times.4.6 mm, 5 μm); mobile phase: 25% acetonitrile (containing 0.1% trifluoroacetic acid); the elution speed is 0.5-0.8 mL/min; ultraviolet detection wavelength is 215 nm), and 1 polypeptide with high antioxidant activity is obtained according to the scavenging activity to hydroxyl free radicals (figure 3).
Fourthly, structure detection: collecting 1 antioxidant peptide with highest hydroxyl free radical scavenging activity, detecting by RP-HPLC to obtain single peak, determining amino acid sequence by protein/polypeptide sequence analyzer to be Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW), determining molecular weight by ESI-MS to be 979.15 Da ([ M + H)]+980.11 Da) (FIG. 4).
The prepared crab shell leftover antioxidant peptide chain Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW) is subjected to a free radical scavenging experiment and a lipid peroxidation inhibition experiment, and the experiment results show that: Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW) vs DPPH free radical (EC)501.561 mg/mL), hydroxyl radical (EC)500.145 mg/mL) and superoxide anion radical (EC)500.102 mg/mL) has good scavenging effect; meanwhile, Ile-Trp-Met-Glu-Cys-Asn-Trp (IWMECNW) also shows good lipid peroxidation inhibition effect.
Sequence listing
SEQUENCE LISTING
<110> Zhejiang ocean academy
<120> preparation method of crab shell leftover antioxidant peptide chain
<130>zjou-wb07-02
<160>1
<170>PatentIn version 3.5
<210>1
<211>7
<212>PRT
<213> Artificial Synthesis
<400>1
Ile Trp Met Glu Cys Asn Trp
1 5
Claims (3)
1. The preparation method of the crab shell leftover antioxidant peptide chain is Ile-Trp-Met-Glu-Cys-Asn-Trp, and comprises the following steps:
1) crab shell leftovers pretreatment: taking crab shell leftovers which are sieved by a 100-mesh sieve, adding isopropanol according to the feed-liquid ratio of 1g: 5-10 mL, degreasing for 8-10 h at 40-50 ℃, then centrifuging for 10-15 min at 8000-10000 rpm to remove the isopropanol, and collecting degreased crab shell leftovers solid matter;
2) enzymolysis of crab shell leftovers: adding phosphate buffer solution into the degreased crab shell leftover solid according to the solid-to-liquid ratio of 1g: 10-15 mL, heating the temperature of the mixed solution to 45-50 ℃, and carrying out ultrasonic treatment for 10-15 min; then adding neutral protease according to 1.0-1.5% of the solid matter weight of the degreased crab shell leftovers, and carrying out enzymolysis for 4-6 h at 45-50 ℃; heating the enzymolysis liquid to 90-95 ℃, keeping the temperature at the constant temperature for 10-15 min, cooling the temperature of the enzymolysis liquid to 50-60 ℃, adding papain according to 2.0-2.5% of the solid matter amount of the degreased crab shell leftovers, carrying out enzymolysis for 4-6 h at 50-60 ℃, cooling to room temperature, centrifuging at 9000-10000 rpm for 10-15 min, and removing precipitates to obtain the enzymolysis liquid; step 2), the phosphate buffer solution is 0.05 mol/L, and the pH value is 6.5-7.5;
3) preparing the crab shell leftover antioxidant peptide chain: carrying out ultrafiltration treatment on the enzymolysis liquid prepared in the step 2) by using a 1 kDa ultrafiltration membrane, and collecting the part with the molecular weight less than 1 kDa to obtain ultrafiltration enzymolysis liquid; adding the ultrafiltration enzymolysis liquid into a chromatographic column filled with 6-8 times of DA201-C macroporous resin according to the volume ratio, eluting with double-distilled water with 4-5 times of column volume to remove impurities, eluting with 70% ethanol with 5-8 times of column volume, removing the ethanol by low-pressure rotary evaporation of ethanol eluent at the temperature of below 50 ℃, freezing and drying to obtain a polypeptide mixture, and purifying the polypeptide mixture by anion exchange resin chromatography, gel column chromatography and reversed-phase high-performance liquid chromatography in sequence to obtain a crab shell leftover antioxidant peptide chain;
the specific processes of anion exchange resin chromatography, gel column chromatography and RP-HPLC purification in the step 3) are as follows:
ion exchange resin chromatography: dissolving the polypeptide mixture in double distilled water to prepare a solution with the concentration of 45-50 mg/mL, separating by using a DEAE Sepharose FF anion exchange resin chromatographic column, eluting by using water, 0.25-0.35 mol/L and 0.45-0.55 mol/L NaCl solution, and collecting an elution component according to an absorbance curve under 215 nm, wherein the component with the highest hydroxyl radical scavenging activity is an ion exchange chromatography zymolyte;
gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 15-20 mg/mL, separating by Sephadex LH-20 column chromatography, eluting by the double distilled water, and collecting an elution component according to an absorbance curve under 215 nm, wherein the peak with the highest hydroxyl radical scavenging activity is the gel chromatography zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution of 80-100 mu g/mL by using double distilled water, purifying by using RP-HPLC, collecting 1 antioxidant peptide with the highest hydroxyl free radical scavenging activity, detecting by using RP-HPLC to obtain a single peak, and determining the amino acid sequence to be Ile-Trp-Met-Glu-Cys-Asn-Trp by using a protein/polypeptide sequence analyzer.
2. The method according to claim 1, wherein the crab shell in step 1) is derived from Portunus trituberculatus.
3. The preparation method according to claim 1, wherein the RP-HPLC condition in the step 3) is a sample size of 8-10 μ L; the chromatographic column is Hypersil BDS C with specification of 250 mm multiplied by 4.6mm and 5 mu m18(ii) a The mobile phase was 25% acetonitrile containing 0.1% trifluoroacetic acid; the elution speed is 0.5-0.8 mL/min; the ultraviolet detection wavelength is 215 nm.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4188264A (en) * | 1977-06-14 | 1980-02-12 | Seikagaku Kogyo Co. Ltd. | Process for determining bacterial endotoxin and reagents used therefor |
| CN103204906A (en) * | 2013-01-29 | 2013-07-17 | 浙江海洋学院 | Mussel meat protein antioxidative peptide and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4188264A (en) * | 1977-06-14 | 1980-02-12 | Seikagaku Kogyo Co. Ltd. | Process for determining bacterial endotoxin and reagents used therefor |
| CN103204906A (en) * | 2013-01-29 | 2013-07-17 | 浙江海洋学院 | Mussel meat protein antioxidative peptide and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 梭子蟹下脚料综合加工技术的研究;陶学明;《中国优秀硕士学位论文全文数据库 工程科技1辑》;20091115(第11期);摘要、正文第12页第1段、第25页第3.3.1节，第26页第3.3.3节、表3-1，第54页第5.3.1节，第57页第5.3.5节，第65页第5.5节、第9页第1.5.1节 * |
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