CN103571901A - Preparation method of macromolecular active collagen - Google Patents
Preparation method of macromolecular active collagen Download PDFInfo
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- CN103571901A CN103571901A CN201210276450.4A CN201210276450A CN103571901A CN 103571901 A CN103571901 A CN 103571901A CN 201210276450 A CN201210276450 A CN 201210276450A CN 103571901 A CN103571901 A CN 103571901A
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Abstract
The invention discloses a preparation method of macromolecular active collagen. The preparation method comprises the following steps: pretreatment of fish skin; extraction of the active collagen; purification of an active collagen solution. According to the preparation method disclosed by the invention, the collagen is extracted through the synergistic effect of ultrasonic waves, acetic acid and a composite enzyme, the macromolecular active collagen solution is prepared by comprehensively applying salting-out, dialysis and ultrafiltration technologies, and the macromolecular active collagen solution can also exist stably.
Description
Technical field
The present invention relates to the preparation field of bio-medical material, be specifically related to a kind of preparation method of macromole active collagen.
Background technology
Collagen protein is gang's protein with unique texture, is extensively present in mammalian tissues, accounts for the more than 30% of gross protein.Collagen protein provides biomechanical property, controls developmental cytogene phenotype for bone, skin, tendon and ligament.The structure of collagen protein is triple helix structure, three polypeptide chains, consists of, and each collagen chain is all left hand helix configuration, and three left hand helix α-chains twine into mutually again right-handed helix structure---and be superhelix.Single α-chain contains at least one Gly-X-Y sequence.Every three amino acid just have a glycine residue.Peculiar structure and the chemical constitution of collagen protein make it have many peculiar properties and function.These character and function make it in every field, be widely applied, as fields such as biomedical material, drug conveying carrier, organizational project, makeup and food.
At present, domestic active collagen Sui You R&D institution is studied and develops, and have patent and the report of some active collagen extraction process aspects, but from industrialization level, still have certain gap at present, be only limited to several companies and can carry out pilot scale or trial production on a small scale.Because active collagen industrialization level is lower, its Downstream Products and application have been limited.At present, the application of active collagen in protective foods, cosmetic field has a large amount of reports and sale, but for the extractive technique of medical grade macromolecule active collagen only in small-scale level.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of preparation method of macromole active collagen is provided.
The technical solution used in the present invention is: a kind of preparation method of macromole active collagen, comprises the following steps:
One, the pre-treatment of fish-skin
Choose fresh fish-skin, 100 weight parts, after washing fleshing, add (1:20-40 in 20-30% NaCI (0.05M Tris-HCI, pH7.5) solution, w/v) homogeneous 5-10min, with rotating speed, be the centrifugal 10-25min of 10000-15000rpm high speed freezing centrifuge, get precipitation, repeat aforesaid operations, fat and Bubble formation until do not swim, then use washed with de-ionized water;
Two, the extraction of active collagen
Pretreated fish-skin is added to (1:50-100 in the deionized water of precooling, w/v), ice bath, add acetic acid to the ultrasonic 10-25min of 0.5-1.0M, ultrasonic power 300-600W, add 0.1-0.5 weight part stomach en-, one or several in acidicenzym 3350 and papoid, enzymolysis 5-8h, centrifugal, get supernatant, gained precipitation adds deionized water (1:50-100), adjust pH is to neutral, add 0.1-0.5 weight part neutral protease, one or both in bromeline, enzymolysis 3-6h, centrifugal, get supernatant, merge two parts of supernatants, obtain collagen solution,
Three, the purifying of active collagen solution
Selecting aperture is the concentrated above-mentioned collagen solution of ultra-filtration membrane of 10-50nm, add NaCl to final concentration be 0.5-1.3M, saltout and spend the night, then centrifugal 10-30min, gets precipitation, after precipitation is dissolved with 0.5-1M acetic acid again, centrifugal 1-2h, gets supernatant, to 0.1-0.2M acetic acid dialysis desalting, with the ultra-filtration membrane in 100-200nm aperture, remove small-molecule substance and polypeptide, obtaining concentration is the macromole active collagen solution of 20-50%.
Beneficial effect: the synergy of the present invention by ultrasonic wave, acetic acid and prozyme extracted collagen protein, and integrated use is saltoutd, dialysis and ultra-filtration technique make macromole active collagen solution, and can stable existence.
Embodiment
Below the specific embodiment of the present invention is described in detail.
Embodiment 1: choose fresh fish-skin, 100 weight parts, after washing fleshing, add (1:20 in 20% NaCI (0.05M Tris-HCI, pH7.5) solution, w/v) homogeneous 5min, with rotating speed, be the centrifugal 10min of 10000rpm high speed freezing centrifuge, get precipitation, repeat aforesaid operations, fat and Bubble formation until do not swim, then use washed with de-ionized water.Pretreated fish-skin is added in the deionized water of precooling (1:50, w/v), ice bath, adds acetic acid to the ultrasonic 10min of 0.5M, ultrasonic power 300W.Add 0.1 weight part stomach en-, enzymolysis 5h, centrifugal, get supernatant.Gained precipitation adds deionized water (1:50), and adjust pH, to neutral, adds 0.1 weight part neutral protease, and enzymolysis 3h is centrifugal, gets supernatant.Merge two parts of supernatants, obtain collagen solution.Selecting aperture is the concentrated above-mentioned collagen solution of ultra-filtration membrane of 10nm, add NaCl to final concentration be 0.5M, saltout and spend the night, then centrifugal 10min, gets precipitation.After precipitation is dissolved with 0.5M acetic acid again, centrifugal 1h, gets supernatant, to 0.1M acetic acid dialysis desalting, with the ultra-filtration membrane in 100nm aperture, removes small-molecule substance and polypeptide, obtains concentration and be 20% macromole active collagen solution.
Embodiment 2: choose fresh fish-skin, 100 weight parts, after washing fleshing, add (1:40 in 30% NaCI (0.05M Tris-HCI, pH7.5) solution, w/v) homogeneous 10min, with rotating speed, be the centrifugal 25min of 15000rpm high speed freezing centrifuge, get precipitation, repeat aforesaid operations, fat and Bubble formation until do not swim, then use washed with de-ionized water.Pretreated fish-skin is added in the deionized water of precooling (1:100, w/v), ice bath, adds acetic acid to the ultrasonic 25min of 1.0M, ultrasonic power 600W.Add 0.5 weight part stomach papoid, enzymolysis 8h, centrifugal, get supernatant.Gained precipitation adds deionized water (1:100), and adjust pH, to neutral, adds 0.5 weight part bromeline, and enzymolysis 6h is centrifugal, gets supernatant.Merge two parts of supernatants, obtain collagen solution.Selecting aperture is the concentrated above-mentioned collagen solution of ultra-filtration membrane of 50nm, add NaCl to final concentration be 1.3M, saltout and spend the night, then centrifugal 30min, gets precipitation.After precipitation is dissolved with 1M acetic acid again, centrifugal 2h, gets supernatant, to 0.2M acetic acid dialysis desalting, with the ultra-filtration membrane in 200nm aperture, removes small-molecule substance and polypeptide, obtains concentration and be 50% macromole active collagen solution.
Embodiment 3: choose fresh fish-skin, 100 weight parts, after washing fleshing, add (1:30 in 25% NaCI (0.05M Tris-HCI, pH7.5) solution, w/v) homogeneous 8min, with rotating speed, be the centrifugal 20min of 12000rpm high speed freezing centrifuge, get precipitation, repeat aforesaid operations, fat and Bubble formation until do not swim, then use washed with de-ionized water; Pretreated fish-skin is added to (1:70 in the deionized water of precooling, w/v), ice bath, add acetic acid to the ultrasonic 20min of 0.8M, ultrasonic power 400W, adds one or several in 0.3 weight part stomach en-, acidicenzym 3350 and papoid, enzymolysis 6h, centrifugal, get supernatant, gained precipitation adds deionized water (1:80), and adjust pH is to neutral, add one or both in 0.3 weight part neutral protease, bromeline, enzymolysis 5h, centrifugal, get supernatant, merge two parts of supernatants, obtain collagen solution; Selecting aperture is the concentrated above-mentioned collagen solution of ultra-filtration membrane of 30nm, add NaCl to final concentration be 0.9M, saltout and spend the night, then centrifugal 20min, gets precipitation, after precipitation is dissolved with 0.8M acetic acid again, centrifugal 1.5h, gets supernatant, to 0.15M acetic acid dialysis desalting, with the ultra-filtration membrane in 150nm aperture, remove small-molecule substance and polypeptide, obtain concentration and be 30% macromole active collagen solution.
It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.In the present embodiment not clear and definite each integral part all available prior art realized.
Claims (1)
1. a preparation method for macromole active collagen, is characterized in that, comprises the following steps:
One, the pre-treatment of fish-skin
Choose fresh fish-skin, 100 weight parts, after washing fleshing, add (1:20-40 in 20-30% NaCI (0.05M Tris-HCI, pH7.5) solution, w/v) homogeneous 5-10min, with rotating speed, be the centrifugal 10-25min of 10000-15000rpm high speed freezing centrifuge, get precipitation, repeat aforesaid operations, fat and Bubble formation until do not swim, then use washed with de-ionized water;
Two, the extraction of active collagen
Pretreated fish-skin is added to (1:50-100 in the deionized water of precooling, w/v), ice bath, add acetic acid to the ultrasonic 10-25min of 0.5-1.0M, ultrasonic power 300-600W, add 0.1-0.5 weight part stomach en-, one or several in acidicenzym 3350 and papoid, enzymolysis 5-8h, centrifugal, get supernatant, gained precipitation adds deionized water (1:50-100), adjust pH is to neutral, add 0.1-0.5 weight part neutral protease, one or both in bromeline, enzymolysis 3-6h, centrifugal, get supernatant, merge two parts of supernatants, obtain collagen solution,
Three, the purifying of active collagen solution
Selecting aperture is the concentrated above-mentioned collagen solution of ultra-filtration membrane of 10-50nm, add NaCl to final concentration be 0.5-1.3M, saltout and spend the night, then centrifugal 10-30min, gets precipitation, after precipitation is dissolved with 0.5-1M acetic acid again, centrifugal 1-2h, gets supernatant, to 0.1-0.2M acetic acid dialysis desalting, with the ultra-filtration membrane in 100-200nm aperture, remove small-molecule substance and polypeptide, obtaining concentration is the macromole active collagen solution of 20-50%.
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Cited By (8)
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| CN105524965A (en) * | 2016-02-26 | 2016-04-27 | 福建华灿制药有限公司 | Extraction method of bovine tendon collagen |
| CN105541964A (en) * | 2016-03-04 | 2016-05-04 | 河南省农业科学院 | Method for preparing sesame 11S protein from cold-pressed sesame cake meal |
| CN105801666A (en) * | 2014-12-30 | 2016-07-27 | 浙江海洋学院 | Preparation method of miichthys miiuy maw protein peptide and application thereof |
| CN107586818A (en) * | 2017-09-11 | 2018-01-16 | 浙江海洋大学 | A kind of extracting method of Japanese croaker collagen and application |
| CN108324676A (en) * | 2018-04-28 | 2018-07-27 | 广东巴松那生物科技有限公司 | A kind of promotion of compacting, anti-aging face cream and preparation method thereof |
| WO2019169646A1 (en) * | 2018-03-07 | 2019-09-12 | 广州创尔生物技术股份有限公司 | Quality control method for biologically active collagen |
| CN114766537A (en) * | 2022-05-24 | 2022-07-22 | 河南工业大学 | High-temperature-resistant chicken skin collagen edible casing film and preparation method thereof |
| CN116284341A (en) * | 2023-02-20 | 2023-06-23 | 中国石油大学(华东) | Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance |
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2012
- 2012-08-06 CN CN201210276450.4A patent/CN103571901A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105801666A (en) * | 2014-12-30 | 2016-07-27 | 浙江海洋学院 | Preparation method of miichthys miiuy maw protein peptide and application thereof |
| CN105801666B (en) * | 2014-12-30 | 2021-06-04 | 浙江海洋学院 | Preparation method and application of miichthys miiuy swim bladder protein peptide |
| CN105524965A (en) * | 2016-02-26 | 2016-04-27 | 福建华灿制药有限公司 | Extraction method of bovine tendon collagen |
| CN105541964A (en) * | 2016-03-04 | 2016-05-04 | 河南省农业科学院 | Method for preparing sesame 11S protein from cold-pressed sesame cake meal |
| CN105541964B (en) * | 2016-03-04 | 2019-05-17 | 河南省农业科学院 | A method of preparing sesame 11S albumen from cold press sesame cake meal |
| CN107586818A (en) * | 2017-09-11 | 2018-01-16 | 浙江海洋大学 | A kind of extracting method of Japanese croaker collagen and application |
| WO2019169646A1 (en) * | 2018-03-07 | 2019-09-12 | 广州创尔生物技术股份有限公司 | Quality control method for biologically active collagen |
| CN108324676A (en) * | 2018-04-28 | 2018-07-27 | 广东巴松那生物科技有限公司 | A kind of promotion of compacting, anti-aging face cream and preparation method thereof |
| CN114766537A (en) * | 2022-05-24 | 2022-07-22 | 河南工业大学 | High-temperature-resistant chicken skin collagen edible casing film and preparation method thereof |
| CN116284341A (en) * | 2023-02-20 | 2023-06-23 | 中国石油大学(华东) | Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance |
| CN116284341B (en) * | 2023-02-20 | 2024-05-03 | 中国石油大学(华东) | Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance |
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Application publication date: 20140212 |