CN108178794B - Superoxide radical-resistant bone collagen polypeptide and preparation method thereof - Google Patents
Superoxide radical-resistant bone collagen polypeptide and preparation method thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 46
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention relates to a superoxide radical resistant bone collagen polypeptide and a preparation method thereof, which solves the technical problems of single source and immunogenicity of the existing products, and the amino acid sequence of the collagen polypeptide is shown in a sequence table 1. The invention also provides a preparation method of the peroxyl radical resistant bone collagen polypeptide, which takes tuna bones as raw materials to extract collagen, then adopts pancreas-minced fillet complex enzyme to carry out enzymolysis, separation and purification, and freeze drying to obtain the peroxyl radical resistant bone collagen polypeptide. The invention can be used in the field of preparation of the collagen polypeptide resisting peroxy radicals.
Description
Technical Field
The invention relates to a polypeptide and a preparation method thereof, in particular to a peroxy radical resistant bone collagen polypeptide and a preparation method thereof.
Background
The oxidation of biomolecules is a free radical mediated process that can have many adverse effects on food and biological systems. Powerful free radicals associated with various diseases such as arteriosclerosis and cancer are inevitably generated along with the process of metabolism. In food, oxidation of food nutrients produces peroxides which not only affect the edible value, cause a reduction in the quality of the food, but can even cause physical illness in the ingesters. Therefore, research on biochemical nutrition is focused on finding safe antioxidants consistent. Although synthetic antioxidants are much cheaper than natural antioxidants, they are harmful to the human body, and therefore, it is necessary to find a safe natural antioxidant.
Collagen is a macromolecular protein ubiquitous in the animal body, and is found primarily in the connective tissue of animals. Collagen is widely used in the fields of food, medicine, tissue engineering, cosmetics, etc. because of its excellent biocompatibility, bioactivity and biodegradability. The collagen hydrolysate is a hydrolysate obtained by moderately hydrolyzing collagen under certain conditions. Compared with collagen, the collagen hydrolysate has low molecular weight and good water solubility, and is easy to be absorbed by human body. Meanwhile, the hydrolysate of collagen also has more biological activity, such as: resisting oxidation, delaying aging, supplementing calcium, promoting skeleton development, improving immunity, promoting skin cell regeneration, etc.
At present, collagen hydrolysate is mainly derived from animals such as pigs, cows and the like. However, these collagen hydrolysates may suffer from immunogenicity, religious beliefs, and the like. With the development of the domestic aquatic product processing industry, leftovers such as fish heads, fish skins, fish scales, fish bones, fish fins and the like generated by aquatic product processing enterprises are increased year by year. However, the aquatic product processing leftovers are not fully utilized, which causes resource waste.
Disclosure of Invention
The invention aims to solve the technical problems of single source and immunogenicity of the existing products, and provides the peroxygen radical-resistant bone collagen polypeptide with good oxidation resistance and no immunogenicity and the preparation method thereof.
Therefore, the invention provides a superoxide radical resistant bone collagen polypeptide, the amino acid sequence of which is shown in a sequence table 1, and the C end of the polypeptide chain of the superoxide radical resistant bone collagen polypeptide is a phenylalanine residue.
The invention also provides a preparation method of the peroxyl radical resistant bone collagen polypeptide, which takes tuna bones as raw materials to extract collagen, then adopts pancreas-minced fillet complex enzyme to carry out enzymolysis, separation and purification, and freeze drying to obtain the peroxyl radical resistant bone collagen polypeptide.
Preferably, the enzymolysis conditions are as follows: the pH value is 8, the temperature is 37 ℃, the enzymolysis time is 2h, the mass concentration of trypsin is 0.5 percent, and the mass concentration of chymotrypsin is 0.1 percent.
Preferably, the separation and purification means comprises DEAE-52 cellulose ion exchange chromatography and RP-HPLC reversed phase high performance liquid chromatography.
Preferably, the purification steps are: (1) separating the enzymolysis product by DEAE-52 cellulose anion exchange chromatography, washing off unadsorbed components after sample loading, eluting by 0.1, 0.5 and 1.0mol/L NaCl solution with the flow rate of 1.0mL/min by pH 7.2 phosphate buffer solution, measuring at 280nm, and determining the antioxidant activity of the eluted components corresponding to each absorption peak; (2) collecting peak with optimal antioxidant activity, separating by RP-HPLC reversed-phase high performance liquid chromatography with Zorbax SB-C18 as chromatographic column, loading amount of 20 μ L, flow rate of 1.0mL/min, detection wavelength of 280nm, gradient eluting from mixed solution containing 2% acetonitrile and 98% water by volume to 98% acetonitrile and 2% water by volume; collecting the elution peak at 27.6min to obtain the collagen polypeptide resisting peroxy radicals. The amino acid sequence of the polypeptide is then identified using MS/MS.
The invention also provides application of the superoxide radical resistant bone collagen polypeptide in scavenging superoxide radicals.
Preferably, the application of the superoxide radical-resistant bone collagen polypeptide in scavenging superoxide radicals, provided by the invention, is used for resisting the semi-inhibitory concentration IC of the superoxide radical-scavenging superoxide radical of the bone collagen polypeptide50The value was 4.025 mM.
The collagen of tuna contains a large amount of amino acids with antioxidant activity, such as aromatic amino acids, lysine, arginine, etc. The amino acids exposed at one end of the polypeptide can enhance the antioxidant property of the polypeptide. Trypsin cleaves lysine, arginine sites of the protease. Chymotrypsin cleaves aromatic amino acid sites. Therefore, the pancreatic-chymotrypsin complex protease is selected to hydrolyze the bone collagen of the tuna to obtain a hydrolysate with high oxidation resistance, and the polypeptide with high oxidation resistance is further separated and purified.
Superoxide radicals generated in the human body can induce lipid peroxidation in the human body, accelerate the aging process of the whole body from the skin to internal organs, induce skin diseases, cardiovascular diseases, cancers and the like, and seriously damage the health of the human body. The invention can effectively eliminate superoxide radical and is a potential antioxidant.
The invention aims to find an antioxidant of natural colleges and universities, which takes tuna ossein as a starting point and cuts the collagen into active polypeptide with a specific structural composition by controlling the cutting condition of trypsin-chymotrypsin, so that the antioxidant performance is efficiently realized; the invention also improves the condition that the utilization rate of tuna bones is low in China, can solve the problem of high-efficiency utilization of a large amount of tuna bone resources, can also contact the worry of consumers on the aspect of food safety of antioxidants, and has profound significance on the development of science and technology, economy and food industry.
Description of the drawings:
FIG. 1 is an RP-HPLC profile of a collagen polypeptide resistant to peroxy radicals;
FIG. 2 is a graph showing the relationship between "dose-effect" of superoxide radical scavenging by purified superoxide radical-resistant collagen polypeptides.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
The preparation method comprises the following steps:
(1) extraction of tuna bone collagen hydrolysate
The extraction process conditions are as follows:
the tuna bones are cleaned, soaked in 1.25mol/L hydrochloric acid for 12 hours, and the acid is changed every 1.5 hours to remove inorganic salts. And then, cleaning the tuna bones until the pH value is 7.0, carrying out enzymolysis for 2 hours under the enzymolysis conditions of pH value of 8, temperature of 37 ℃, trypsin accounting for 0.5% by volume and chymotrypsin accounting for 0.1% by volume, collecting supernatant, and filtering, concentrating and freeze-drying the supernatant to obtain the tuna bone collagen hydrolysate.
(2) Separation and purification of enzymolysis product
Separating the enzymolysis product by DEAE-52 cellulose anion collagen chromatography, washing off unadsorbed components after sample loading, eluting by 0.1, 0.5 and 1.0mol/L NaCl solution with the flow rate of 1.0mL/min by using a pH 7.2 phosphate buffer solution, measuring at 280nm, and determining the antioxidant activity of the eluted components corresponding to each absorption peak; collecting peak with optimal antioxidant activity, separating by RP-HPLC reversed phase high performance liquid chromatography with Zorbax SB-C18 as chromatographic column, loading amount of 20 μ L, flow rate of 1.0mL/min, detection wavelength of 280nm, and gradient eluting from mixed solution containing 2% acetonitrile and 98% water by volume to 98% acetonitrile and 2% water by volume. And collecting the elution peak at 27.6min to obtain the polypeptide of the invention.
(3) Determination of amino acid sequence of superoxide radical-resistant collagen polypeptide
The amino acid sequence of the collagen polypeptide resisting peroxy radicals is identified to be GEQGSTGPAGF by utilizing LC-MS/MS and searching MASCOT spectral library
(4) Determination of superoxide radical scavenging Capacity
Superoxide radical clearance assays were used to study the superoxide radical resistant collagen polypeptides. A50 mM Tris-HCl buffer (pH 8.5) containing 1mM EDTA was prepared. A1.5 mM pyrogallol solution was prepared using 10mM HCl solution. mu.L of the sample, 80. mu.L of Tris-HCl, and 40. mu.L of pyrogallol solution were mixed in a 96-well plate, left at room temperature for 10min, and the absorbance thereof was measured at 405 nm.
Example 1
10g of tuna bones are taken and cleaned, soaked in 1.25mol/L hydrochloric acid for 12 hours, and the acid is changed every 1.5 hours to remove inorganic salts. Then, the tuna bone is cleaned until the pH value is 7.0, then the enzymolysis is carried out for 2 hours under the conditions that the enzymolysis condition is that the pH value is 8, the temperature is 37 ℃, trypsin and chymotrypsin are added according to the condition that the volume concentration of the trypsin is 0.5 percent and the volume concentration of the chymotrypsin is 0.1 percent, the supernatant is collected, and the tuna bone collagen hydrolysate is obtained after filtration, concentration and freeze drying.
(2) Separation and purification of enzymolysis product
Separating the enzymolysis product by DEAE-52 cellulose anion collagen chromatography, washing off unadsorbed components after sample loading, eluting by 0.1, 0.5 and 1.0mol/L NaCl solution with the flow rate of 1.0mL/min by using a pH 7.2 phosphate buffer solution, measuring at 280nm, and determining the antioxidant activity of the eluted components corresponding to each absorption peak; collecting peak with optimal antioxidant activity, separating by RP-HPLC reversed phase high performance liquid chromatography with Zorbax SB-C18 as chromatographic column, loading amount of 20 μ L, flow rate of 1.0mL/min, detection wavelength of 280nm, and gradient eluting from mixed solution containing 2% acetonitrile and 98% water by volume to 98% acetonitrile and 2% water by volume. And collecting the elution peak at 27.6min to obtain the polypeptide of the invention.
The purified polypeptide has strong antioxidant capacity, as shown in FIG. 2.
As can be seen from FIG. 2, the clearance rate of peroxy radicals was 38.1% at the concentration of antioxidant collagen polypeptide of 3mM and 69.9% at the concentration of antioxidant collagen polypeptide of 5 mM. Clearance of peroxy radicals is greater than 50% at concentrations greater than 4 mM. The calculated half inhibitory concentration IC of the compound on the peroxy radical50The value was 4.025 mM. Has a lower semi-inhibitory concentration relative to other polypeptides, such as GVPLT (IC)50 5.93mM)[1]. The antioxidant collagen polypeptide has stronger antioxidant capacity.
[1]Wang B,Li L,Chi C F,et al.Purification and characterisation of a novel antioxidant peptide derived from blue mussel(Mytilus edulis)protein hydrolysate[J].Food Chemistry,2013,138(2-3):1713.
Sequence listing
<110> Beijing university of chemical industry
<120> collagen polypeptide resisting peroxy radicals and preparation method thereof
<130> WPFC1180021
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> tuna bone (tuna bone)
<400> 1
Gly Glu Gln Gly Ser Thr Gly Pro Ala Gly Phe
1 5 10
Claims (5)
1. A superoxide radical-resistant collagen polypeptide, characterized by: the amino acid sequence of the superoxide radical resistant bone collagen polypeptide is shown in a sequence table 1, and the C end of the polypeptide chain of the superoxide radical resistant bone collagen polypeptide is a phenylalanine residue.
2. The method for producing a peroxy radical-resistant collagen polypeptide according to claim 1, wherein: extracting collagen from tuna bones as a raw material, performing enzymolysis on the collagen by using pancreas-minced fillet complex enzyme, separating, purifying, and freeze-drying to obtain the peroxygen free radical-resistant collagen polypeptide.
3. The method for producing a peroxy radical-resistant collagen polypeptide according to claim 2, wherein: the enzymolysis conditions are as follows: the pH value is 8, the temperature is 37 ℃, the enzymolysis time is 2h, the mass concentration of trypsin is 0.5 percent, and the mass concentration of chymotrypsin is 0.1 percent.
4. The method for preparing peroxy radical-resistant collagen polypeptide according to claim 2, wherein the separation and purification means comprises DEAE-52 cellulose ion exchange chromatography, RP-HPLC reverse phase high performance liquid chromatography.
5. The method for producing a peroxy radical-resistant collagen polypeptide according to claim 2, wherein: the purification steps are as follows:
(1) separating the enzymolysis product by DEAE-52 cellulose anion exchange chromatography, washing off unadsorbed components after sample loading, eluting by 0.1, 0.5 and 1.0mol/L NaCl solution with the flow rate of 1.0mL/min by pH 7.2 phosphate buffer solution, measuring at 280nm, and determining the antioxidant activity of the eluted components corresponding to each absorption peak;
(2) collecting peak with optimal antioxidant activity, separating by RP-HPLC reversed-phase high performance liquid chromatography with Zorbax SB-C18 as chromatographic column, loading amount of 20 μ L, flow rate of 1.0mL/min, detection wavelength of 280nm, gradient eluting from mixed solution containing 2% acetonitrile and 98% water by volume to 98% acetonitrile and 2% water by volume; collecting the elution peak at 27.6min to obtain the collagen polypeptide resisting peroxy radicals.
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| CN201810064254.8A CN108178794B (en) | 2018-01-23 | 2018-01-23 | Superoxide radical-resistant bone collagen polypeptide and preparation method thereof |
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| CN201810064254.8A CN108178794B (en) | 2018-01-23 | 2018-01-23 | Superoxide radical-resistant bone collagen polypeptide and preparation method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789630A (en) * | 2015-05-11 | 2015-07-22 | 北京化工大学 | Bluefin tuna collagen hydrolyzate and preparation method thereof |
| CN105037494A (en) * | 2015-04-08 | 2015-11-11 | 浙江海洋学院 | Application of tuna dark flesh protein anti-oxidizing iron-chelating peptide |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037494A (en) * | 2015-04-08 | 2015-11-11 | 浙江海洋学院 | Application of tuna dark flesh protein anti-oxidizing iron-chelating peptide |
| CN104789630A (en) * | 2015-05-11 | 2015-07-22 | 北京化工大学 | Bluefin tuna collagen hydrolyzate and preparation method thereof |
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| Title |
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| 《Genbank》;Tanaka,T等;《Genbank》;20130611;第620-630位 * |
| 《Isolation and identification of an antioxidantcollagen peptide from skipjack tuna (Katsuwonuspelamis) bone》;Ding Ding等;《RSC Advances》;20190828;第9卷;第27032-27041页 * |
| 《金枪鱼骨抗氧化酶解物的工艺优化》;胡静等;《食品研究与开发》;20150831;第36卷(第15期);第64-69页 * |
| 《金枪鱼鱼骨胶原肽的制备及抗氧化活性研究》;谭洪亮等;《水产学报》;20140131;第38卷(第1期);第143-148页 * |
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