CN107176973B - Antioxidative enzymolysis oligopeptide from peri-pacific squid oothecal gland - Google Patents
Antioxidative enzymolysis oligopeptide from peri-pacific squid oothecal gland Download PDFInfo
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Abstract
The invention discloses an antioxidant enzymolysis oligopeptide from the periwinkle glands of northern pacific squid. The invention takes the peripherical glands of the northern Pacific squid as raw materials, and prepares the antioxidant enzymolysis oligopeptide of the peripherical glands of the northern Pacific squid through ultrafiltration and HPLC purification, the amino acid sequence of the oligopeptide is Ala-Tyr-Ala-Ser-Ser, the molecular weight is 497.50Da, and the molecular structure is
Description
Technical Field
The invention relates to a marine bioactive substance, and in particular relates to an antioxidative enzymolysis oligopeptide for peri-pacific squid periwinkle glands.
Background
Intermediates such as superoxide anion (O) produced by the body during metabolism2-）、H2O2Peroxygen Radical (ROO) and hydroxyl radical (-OH) have strong oxidizing action. Under normal physiological conditions, the free radicals can be effectively eliminated by antioxidant enzymes and non-enzyme factors in the body, so that the balance state of the free radicals is maintained. In pathological conditions, however, lipid peroxidation can rapidly cause damage to cell membranes, DNA and protein activities, resulting in a variety of human disease-related events. Thus, the elimination of free radicals in the body is an important way to defend the body against the development of diseases. Because most of the synthetic antioxidants have potential safety hazards, screening high-efficiency and low-toxicity antioxidants becomes a new trend for biological, medical and food science research. Research has shown that active peptides derived from various proteins all have antioxidant activity, and the enzymatic hydrolysis method is one of the effective methods for obtaining bioactive peptides from proteins, and has been widely used as a method for improving the active function and nutritive value of proteins. Egg in dietThe white matter is absorbed in the intestinal tract mainly in the form of polypeptide and amino acid, and the polypeptide plays an important role in the antioxidant process of the body. In recent years, research on marine bioactive peptides, particularly antioxidant bioactive peptides, has been greatly advanced. However, the marine active peptide has not formed a large-scale industry, so that by taking advantage of the enzyme engineering technology developed by the land active peptide, the marine biological protein resource is taken as a raw material, and the comprehensive research on the processes such as enzymolysis, preparation and the like is carried out, so that the series of natural, efficient and novel biological active peptides which can not be produced by the land protein source and chemical synthesis can be developed.
The squid is an important marine cephalopod animal in the world, has short growth cycle, strong reproductive capacity and quick resource recovery, and is a sustainable marine fishery resource. In recent years, with the development of deep sea fishing technology at home and abroad, squids have become the main marine fishing and aquatic product processing varieties in China. At present, the processing amount of squid in China is up to 40-50 ten thousand tons every year, and the squid is the first in the world, and the processed variety is mainly North Pacific ocean squid, Argentina squid and Peru squid. The squid is favored by consumers because of the fine and nutritional meat quality, delicious flavor, high protein and low fat, and rich in various essential amino acids needed by human body. In the processing process of the squid, the body of the squid is generally processed to generate about 50 percent of viscera and other byproducts, wherein gonads (oviparotid, spermary, ovary and the like) account for 20 percent of the viscera, and the viscera and other byproducts are generally processed into feed and used as baits of the fish; burying the squid liver after extracting squid oil; the obtained squid ootheca usually only needs to be eaten by cooking, the biological added value is low, and certain pollution is caused to the environment if the squid ootheca is not treated in time. The extraction of squid periwinal gland enzymolysis polypeptide is not reported at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing an antioxidant enzymolysis oligopeptide from the periwinkle glands of northern Pacific ocean squids with antioxidant effect.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an antioxidative enzymolysis oligopeptide for peri-pacific squid ootheca, which has a molecular structure as follows:
namely, the amino acid sequence of the enzymolysis oligopeptide is Ala-Tyr-Ala-Ser-Ser, and the molecular mass is 497.50 Da.
An application preparation of the antioxidant enzymolysis oligopeptide from the periploca gigas periploca comprises, by weight, 0.01-0.5 part of the antioxidant enzymolysis oligopeptide from the periploca gigas periploca, 1-3 parts of epimedium herb, 1-5 parts of astragalus membranaceus, 1-10 parts of parasitic loranthus, 10-20 parts of salvia miltiorrhiza, 1-10 parts of semen raphani, 2-10 parts of oldenlandia diffusa and 3-20 parts of radix puerariae according to the parts by weight.
Compared with the prior art, the preparation method of the antioxidant enzymolysis oligopeptide from the periwinkle glands of the northern Pacific squid, provided by the invention, has the advantages that: the invention has scientific and reasonable process, simple operation, high product activity and stable product yield. Meanwhile, the antioxidant enzymolysis oligopeptide of the periploca squids prepared by the invention is prepared by enzymolysis of natural raw materials, is safe, has no toxic or side effect, has obvious antioxidant activity, and can be further researched and developed as antioxidant medicines and functional foods.
Drawings
FIG. 1 is a molecular structural formula of an antioxidative enzymolysis oligopeptide amino acid in example 1 of the present invention;
FIG. 2 is the absorbance at 280nm of 3-5 KD molecular segments of the ultrafiltration component of example 1 of the present invention (absorbance-number of tubes);
FIG. 3 is a graph showing DPPH clearance of the different peaks of FIG. 2 according to the present invention;
FIG. 4 shows the superoxide anion removal rates of different molecular fragments of the enzymatic polypeptide of example 1 of the present invention;
FIG. 5 shows the DPPH clearance of different molecular fragments of the enzymatic polypeptide of example 1;
FIG. 6 shows the reducing power of different molecular fragments of the enzymatic polypeptide of example 1 of the present invention;
FIG. 7 shows hydroxyl radical scavenging rates for different molecular segments of the enzymatic polypeptide of example 1 of the present invention;
FIG. 8 is a high performance liquid chromatogram of peak I at 280nm of example 1 according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1:
an antioxidative enzymolysis oligopeptide for peri-pacific squid ootheca, which has a molecular structure as follows:
namely, the amino acid sequence of the enzymolysis oligopeptide is Ala-Tyr-Ala-Ser-Ser, and the molecular mass is 497.50 Da.
An application preparation of the antioxidant enzymolysis oligopeptide from the periploca gigas periploca comprises, by weight, 0.01-0.5 part of the antioxidant enzymolysis oligopeptide from the periploca gigas periploca, 1-3 parts of epimedium herb, 1-5 parts of astragalus membranaceus, 1-10 parts of parasitic loranthus, 10-20 parts of salvia miltiorrhiza, 1-10 parts of semen raphani, 2-10 parts of oldenlandia diffusa and 3-20 parts of radix puerariae according to the parts by weight.
The preparation method of the antioxidant enzymolysis oligopeptide from the periwiny glands of the northern Pacific squid comprises the following steps:
1) mashing perivitelline gland of northern Pacific ocean squid, homogenizing, adding protease under optimum enzymolysis temperature and pH value with active enzyme, and performing enzymolysis under heat preservation; then inactivating the mixture at 0-4 DEG CoCCentrifuging at 6000-12000 r/min, and taking the supernatant;
2) taking supernatant, and ultrafiltering with molecular weight cutoff of 3-5 KD;
3) separating the ultrafiltration component by agarose gel chromatography, and collecting the component with the first peak of three peaks at 280 nm;
4) and (3) further separating the components of the first peak by HPLC to obtain antioxidant enzymolysis oligopeptide from the periwinkle glands of the pacific squid, wherein the molecular structure of the oligopeptide is as follows:
the active enzyme is alkaline protease, preferably, the enzymolysis temperature is between 40 and 50 ℃, the hydrolysis time is between 5 and 7 hours, the pH is between 8.0 and 10.0, the enzyme adding amount is 2500 to 3500U/g, and the material-liquid ratio is 1: 3-1: 5.
the preferred conditions for enzymatic hydrolysis are: the temperature is 50 ℃, the enzymolysis time is 7h, the enzyme adding amount is 3500U/g, the pH value is 9.0, the material-liquid ratio is 1: 6 is the optimum enzymolysis condition
The elution conditions in the step 3) are as follows: column size: 10X 300 and 310 mm; column material: agarose; column particle size: 10 +/-2 mu m; sample loading amount: 500 muL; mobile phase: ultrapure water; eluent flow rate: 0.5 mL/min; detection wavelength: 280 nm; automatic volume collection: 3.2 mL/tube.
The application of the antioxidant enzymolysis oligopeptide for the periwinkle glands of the northern Pacific squid, wherein the amino acid sequence of the enzymolysis oligopeptide is Ala-Tyr-Ala-Ser-Ser, the molecular mass is 497.50Da, and the molecular structural formula of the enzymolysis oligopeptide is as follows:
the use of the enzymolysis oligopeptide in preparing antioxidant health food or food additive. The preparation containing 0.01-0.5 part of antioxidant enzymolysis oligopeptide from ootheca of northern Pacific squid, 1-3 parts of epimedium herb, 1-5 parts of astragalus, 1-10 parts of parasitic loranthus, 10-20 parts of salvia miltiorrhiza, 1-10 parts of radish seed, 1-10 parts of angelica and 3-20 parts of kudzu root is used as a food additive.
Example 2:
materials and methods
Test material
The northern Pacific ocean squid oothecal gland is purchased and placed in local aquatic enterprises; alkaline protease is purchased from Atalangium biotechnology (Beijing) Co., Ltd; DPPH purchased in west asian reagent; felazine, phenanthroline, ferric trichloride, disodium hydrogen phosphate were purchased from alatin reagent; the other reagents are analytically pure and purchased from chemical reagents of national drug group, Inc.
Main instrument
SSW-420-2S constant temperature water bath, Shanghai Min appearance electronics, Inc.; BSA model 124S electronic balance, SartoriusAG, germany; DS-1 organization triturator, Shanghai Biao Ben model factory; PHS-250PH meter, Shanghai Lida Instrument factory; model CF16RX II high speed low temperature centrifuge, Hitachi, Japan; Direct-Q5 UV-R model ultrapure water systems, Millipore, USA; cogentu Scale type ultrafiltration System, Millipore, USA; ALPHA 1-4/LDplus type freeze dryer, Beijing Songyuan Huaxing science and technology development Co., Ltd; ultraviolet spectrophotometer (752 FC), shanghai spectrometer limited; agilent-1260 type HPLC, Agilent, USA.
Method of producing a composite material
Single factor experiment
And (3) selecting alkaline protease to carry out an enzymolysis test, and considering enzymolysis temperature, enzymolysis time, enzyme adding amount, solution pH and material-liquid ratio. The hydrolysis process comprises the following steps:
mashing the periwiny gland of the squid → homogenizing → adjusting the pH value by 1 mol/L sodium hydroxide → preserving heat → hydrolyzing after adding enzyme → inactivating enzyme (heating at 90 ℃ for 15 min) → centrifuging (10000 r/min, 15 min) → fixing the volume of the supernatant → measuring the content of amino nitrogen (AAN).
When the temperature is between 40 and 50 ℃, the hydrolysis time is between 5 and 7 hours, the pH is between 8.0 and 10.0, the enzyme adding amount is 2500 to 3500U/g, and the material-liquid ratio is 1: 3-1: and 5, the hydrolysis effect is better, the obtained free ANN has the largest amount, and the hydrolysis degree is higher. However, the temperature is 50 ℃, the enzymolysis time is 7h, the enzyme adding amount is 3500U/g, the pH value is 9.0, and the feed-liquid ratio is 1: 6 is the optimal enzymolysis condition.
Sephadex G-25 separation and purification result
Taking a freeze-dried sample of the 3-5 KD molecular segment to carry out Sephadex G-25 chromatography, generating three peaks at 280nm, namely a peak I, a peak II and a peak III, respectively collecting three peak components, carrying out freeze drying, detecting the DPPH clearance rate, obtaining that the peak I has better cleaning capacity, and taking the molecular segment of the peak I to carry out HPLC purification, and referring to figure 3.
HPLC separation, purity detection and target peptide sequence determination results
The preparation result of the peak I by an HPLC chromatographic column is shown in figure 8, when the retention time is about 18min, a single peak appears, the peak height is 161.2, the detection of an N-terminal sequence of an amino acid sequencer shows that the amino acid sequence is Ala-Tyr-Ala-Ser-Ser, the relative molecular mass is 497.50Da, and the structural formula is as follows:
after agarose gel G-25 chromatography, three peaks are obtained, the effect of the peak I is obvious according to the determination of DPPH clearance rate, the DPPH clearance rate reaches 61.8%, and then the amino acid sequence of the target peptide is Ala-Tyr-Ala-Ser-Ser after HPLC separation and purification and detection of the N-end sequence of an amino acid sequencer. Oligopeptide with good antioxidant activity can be obtained from the periwinkle glands of the northern Pacific squid through enzymolysis, the deep processing of squid byproducts can be developed, and the important significance in improving the biological additional value is achieved.
Finally, it should also be noted that the above-mentioned list is only one specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
SEQUENCE LISTING
<110> Zhejiang ocean university
<120> an antioxidative enzymolysis oligopeptide from periwiny glands of northern Pacific ocean squid
<130>zjou-yzs-20161201
<160>1
<170>PatentIn version 3.5
<210>1
<211>5
<212>PRT
<213> Artificial Synthesis
<400>1
Ala Tyr Ala Ser Ser
1 5
Claims (2)
1. An antioxidative enzymolysis oligopeptide for peri-pacific squid oothecal gland is characterized in that: the amino acid sequence of the enzymolysis oligopeptide is Ala-Tyr-Ala-Ser-Ser, and the molecular mass is 497.50 Da.
2. A preparation of antioxidant enzymolysis oligopeptide from squids in North Pacific ocean is characterized in that: the composition comprises, by weight, 0.01-0.5 part of antioxidant enzymolysis oligopeptide from squids of North Pacific ocean according to claim 1, 1-3 parts of epimedium, 1-5 parts of astragalus membranaceus, 1-10 parts of parasitic loranthus, 10-20 parts of salvia miltiorrhiza, 1-10 parts of radish seeds, 2-10 parts of oldenlandia diffusa and 3-20 parts of radix puerariae.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101983968A (en) * | 2010-09-26 | 2011-03-09 | 浙江海洋学院 | Preparation method of sepia oligopeptide by enzymatic hydrolysis and application thereof |
| CN103451257A (en) * | 2013-06-17 | 2013-12-18 | 浙江海洋学院 | Method for preparing polypeptide with high antioxidant activity through enzymolysis of internal organs of squids |
| CN106244657A (en) * | 2016-08-29 | 2016-12-21 | 岭南师范学院 | A kind of squid antioxidation polypeptide and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101983968A (en) * | 2010-09-26 | 2011-03-09 | 浙江海洋学院 | Preparation method of sepia oligopeptide by enzymatic hydrolysis and application thereof |
| CN103451257A (en) * | 2013-06-17 | 2013-12-18 | 浙江海洋学院 | Method for preparing polypeptide with high antioxidant activity through enzymolysis of internal organs of squids |
| CN106244657A (en) * | 2016-08-29 | 2016-12-21 | 岭南师范学院 | A kind of squid antioxidation polypeptide and its preparation method and application |
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| 北太平洋鱿鱼缠卵腺抗氧化酶解寡肽的制备;刘倩茹 等;《湖北农业科学》;20180731;全文 * |
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