CN1858229A - Process for preparing oligomeric peptide capable of clearing superoxide anion - Google Patents
Process for preparing oligomeric peptide capable of clearing superoxide anion Download PDFInfo
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- CN1858229A CN1858229A CN 200610044482 CN200610044482A CN1858229A CN 1858229 A CN1858229 A CN 1858229A CN 200610044482 CN200610044482 CN 200610044482 CN 200610044482 A CN200610044482 A CN 200610044482A CN 1858229 A CN1858229 A CN 1858229A
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- constant temperature
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Abstract
The preparation process of oligomeric peptide capable of eliminating superoxide anion includes the following steps: soaking java tilapia skin successively in sodium hydroxide aqua, sulfuric acid aqua and citric acid aqua; water washing to neutrality after each soaking, pulping skin with distilled water, stirring at constant temperature to extract, centrifuging, freeze drying supernatant to obtain java tilapia skin glue, dissolving in buffering citric acid-disodium hydrogen phosphate solution, adding bromelin through stirring at constant temperature to enzymolyze, deactivating enzyme, cooling, regulating pH with alkali solution, adding Alcalase enzyme through stirring at constant temperature to enzymolyze, deactivating enzyme, cooling, centrifuging to reach required superoxide anion eliminating index IC50 value, ultrafiltering the supernatant, chromatographic separating, desalting, chromatographic purification, and freeze drying.
Description
Technical field:
The present invention relates to a kind of producing and have the method for removing the active oligomeric peptide of ultra-oxygen anion free radical with prozyme directional enzymatic Java tilapia skin.
Background technology:
Recent study is found, many disorders such as cancers that are detrimental to health, aging, cardiovascular disorder, Metabolic disorder, light injury, cataract, muscle injury, poisonous substance infringement, even fatigue etc. is all relevant with the intravital free radical of people, how to remove the research focus that the intravital free radical of people becomes the nutritive health-care subject.Therefore antioxidant not only is used for the anti-oxidant of fat-containing food, and what is more important is used for the exploitation of protective foods and makeup etc. as functional factor.At present, the kind of free-radical scavengers is a lot, but because the insecurity of the free-radical scavengers of chemosynthesis and the enhancing of people's health care consciousness, more and more stricter to the requirement that free-radical scavengers is used in food, people progressively turn to the natural free-radical scavengers of extraction from each kind of plant, animal tissues to sight, decompose extract of the soybean peptides that obtains and arrowhead, scallop, LIUYUESHUANG or the like as the tea-polyphenol in the tealeaves, the soybean isoflavones in the soybean, the carnosine in the skeletal muscle, lycium barbarum polysaccharide, soybean protein in the matrimony vine.But the shortcoming of these products is cost height, removes the weak effect of ultra-oxygen anion free radical.The at present domestic report that also from collagen of fish skin, does not extract the oligomeric peptide that to remove superoxide anion.
Summary of the invention:
The purpose of this invention is to provide a kind of method for preparing oligomeric peptide with prozyme directional enzymatic Java tilapia skin with free radical activity of removing superoxide anion, this oligomeric peptide has low toxicity, characteristics of high efficiency, can overcome the above-mentioned defective of existing free-radical scavengers.
A kind of preparation method that can remove the oligomeric peptide of superoxide anion, it is characterized in that Java tilapia skin successively respectively at aqueous sodium hydroxide solution, soak in aqueous sulfuric acid and the aqueous citric acid solution, each equal water in back that soaks cleans to neutral, then fish-skin is pulled an oar with distilled water, and constant temperature stirring down extracts, centrifugal then, the freeze-drying of gained supernatant liquor is obtained the tilapia hide glue, this tilapia hide glue with citric acid-Sodium phosphate dibasic damping fluid dissolving, is added bromeline, constant temperature stirs enzymolysis, the enzyme that goes out then, adjusting PH with base is used in cooling, add the Alcalase enzyme again, constant temperature stirs enzymolysis, the enzyme that goes out, cooling, centrifugal, to remove the IC of superoxide anion
50Value is got supernatant liquor for index, after ultrafiltration, gel filtration chromatography, cation exchange column chromatography, gel column desalination, the separation and purification of anti-phase high phase liquid chromatography, and freeze-drying.
The present invention is a raw material with the Java tilapia skin of low value, utilizes bromeline and Alcalase enzyme complex enzyme hydrolysis to prepare oligomeric peptide, and products therefrom has superoxide anion removes effect preferably.
Embodiment
With Java tilapia skin 40～60 grams (preferably 50 grams), 0.2% (the concentration expressed in percentage by weight that adds 6～8 times of (preferably 7 times) tare weights successively respectively, down together) aqueous sodium hydroxide solution, 0.2% aqueous sulfuric acid and 1% aqueous citric acid solution respectively soak 30～50 minutes (preferably 40 minutes), each equal water in back that soaks cleans to neutral, then fish-skin is added 8～12 times of (preferably 10 times) distilled water making beating, under 40-50 ℃, preferably 45 ℃ of following constant temperature stir and extract 10～14 hours (preferably 12 hours), centrifugal then, the supernatant liquor freeze-drying is obtained the tilapia hide glue.Citric acid-Sodium phosphate dibasic damping fluid the dissolving that this tilapia hide glue is added its 18～22 times (preferably 20 times) weight, ratio according to 3800～4200u/g (preferably 4000u/g) tilapia hide glue adds bromeline, under 40-50 ℃, preferably 45 ℃ of following constant temperature stir enzymolysis 3～5 hours (preferably 4 hours), the enzyme that goes out then, cooling.With lye pH adjustment to 7.5, according to the ratio adding Alcalase enzyme of 5800～6200u/g (preferably 6000u/g) with the tilapia hide glue, under 50-60 ℃, preferably 55 ℃ of following constant temperature stir enzymolysis 1～3 hour (preferably 2 hours), the enzyme that goes out, cooling, centrifugal, supernatant liquor is removed the IC of superoxide anion through ultrafiltration with each component
50Value is index, collect the part of molecular weight less than 1000Da, after passing through Sephadex G-25 gel filtration chromatography, SP Sephadex C-25 cation exchange column chromatography, the desalination of Sephadex G-15 gel column, the separation and purification of anti-phase high phase liquid chromatography again, promptly obtain the oligomeric peptide that to remove superoxide anion of the present invention.
Adopting pyrogallol autoxidation method to measure the removing effect of each component to superoxide anion, is IC so that the superoxide anion inhibiting rate is reached 50% o'clock required concentration
50Value is index, and experimental result is as follows: the gained oligomeric peptide is removed the IC of superoxide anion
50Value is 4.95 μ g/mL, less than the IC of free-radical scavengers Vc commonly used at present
50Be worth 6.80 μ g/mL, this free radical scavenging effect that oligomeric peptide of the present invention is described is better than Vc.Through mass spectroscopy, the molecular weight of this oligomeric peptide mainly concentrates on 311.3～932.8Da.
Claims (1)
1, a kind of preparation method that can remove the oligomeric peptide of superoxide anion, it is characterized in that Java tilapia skin successively respectively at aqueous sodium hydroxide solution, soak in aqueous sulfuric acid and the aqueous citric acid solution, each equal water in back that soaks cleans to neutral, then fish-skin is pulled an oar with distilled water, and constant temperature stirring down extracts, centrifugal then, the freeze-drying of gained supernatant liquor is obtained the tilapia hide glue, this tilapia hide glue with citric acid-Sodium phosphate dibasic damping fluid dissolving, is added bromeline, constant temperature stirs enzymolysis, the enzyme that goes out then, adjusting PH with base is used in cooling, add the Alcalase enzyme again, constant temperature stirs enzymolysis, the enzyme that goes out, cooling, centrifugal, to remove the IC of superoxide anion
50Value is got supernatant liquor for index, after ultrafiltration, gel filtration chromatography, cation exchange column chromatography, gel column desalination, the separation and purification of anti-phase high phase liquid chromatography, and freeze-drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610044482 CN1858229A (en) | 2006-03-03 | 2006-03-03 | Process for preparing oligomeric peptide capable of clearing superoxide anion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610044482 CN1858229A (en) | 2006-03-03 | 2006-03-03 | Process for preparing oligomeric peptide capable of clearing superoxide anion |
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| CN1858229A true CN1858229A (en) | 2006-11-08 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN 200610044482 Pending CN1858229A (en) | 2006-03-03 | 2006-03-03 | Process for preparing oligomeric peptide capable of clearing superoxide anion |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607982B (en) * | 2008-06-19 | 2012-01-04 | 陈栋梁 | Fish skin collagen peptide with whitening effect and preparation process and application thereof |
| CN105255972A (en) * | 2015-11-13 | 2016-01-20 | 汪恒森 | Method for preparing antioxidant active peptide by means of fermenting tilapia skin through aspergillus oryzae |
| CN105274171A (en) * | 2015-11-13 | 2016-01-27 | 潘发全 | Method of preparing antioxidant peptides by hydrolyzing skin of tilapias using compound protease |
| CN105907823A (en) * | 2016-05-05 | 2016-08-31 | 广东海洋大学 | Tilapia skin protein peptide and preparation method and application thereof |
-
2006
- 2006-03-03 CN CN 200610044482 patent/CN1858229A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607982B (en) * | 2008-06-19 | 2012-01-04 | 陈栋梁 | Fish skin collagen peptide with whitening effect and preparation process and application thereof |
| CN105255972A (en) * | 2015-11-13 | 2016-01-20 | 汪恒森 | Method for preparing antioxidant active peptide by means of fermenting tilapia skin through aspergillus oryzae |
| CN105274171A (en) * | 2015-11-13 | 2016-01-27 | 潘发全 | Method of preparing antioxidant peptides by hydrolyzing skin of tilapias using compound protease |
| CN105907823A (en) * | 2016-05-05 | 2016-08-31 | 广东海洋大学 | Tilapia skin protein peptide and preparation method and application thereof |
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