CN1269877C - Preparation method of undenatured natural fish collagen - Google Patents
Preparation method of undenatured natural fish collagen Download PDFInfo
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- CN1269877C CN1269877C CN 200410040486 CN200410040486A CN1269877C CN 1269877 C CN1269877 C CN 1269877C CN 200410040486 CN200410040486 CN 200410040486 CN 200410040486 A CN200410040486 A CN 200410040486A CN 1269877 C CN1269877 C CN 1269877C
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Abstract
The present invention discloses a method for preparing undenatured natural fish collagen, which is characterized in that 100 portions by weight of refrigerated sea fish skin or fresh stream fish skin rich in collagen protein are taken and then are soaked in 2 to 5 portions by weight of ether or 5 to 10 portions by weight of solution of nonionic surface active agents mixed with water according to an indefinite proportion for 5 to 24 hours, the solution is filtered, and filtered substances are stirred for 24 to 48 hours in 500 to 700 shares by weight of 0.1 to 4 M of EDTA solution; 10 to 20 portions by weight of protease are added into 100 portions by weight of filtered deposits, acid is used for regulating the pH to 2 to 4, and the mixture is stirred at 4 to 6 DEG C for 1 to 3 days; 0.5 to 5 M of NaCl is added for salting-out into collagen solution obtained after centrifugal filtration, the salting-out treatment is repeated for 3 to 5 times, and membrane dialyzing treatment is repeated for 3 times to obtain undenatured natural fish collagen protein solution with the high purity of 0.5 to 2%. A differential thermal meter is utilized to measure and obtain the results that the denaturation temperature of stream fish natural collagen is from 20 to 28 DEG C, and the denaturation temperature of sea fish natural collagen is from 12 to 25 DEG C. An electrophoretic method is adopted to obtain the results that the molecular weights of contained alpha chains, beta chains and gamma chains are respectively 100,000, 200,000 and 300,000.
Description
One, technical field
The present invention relates to the former preparation method of the natural fish glue of a kind of not sex change, belong to the preparation field of bio-medical material.
Two, background technology
Collagen protein or title collagen are a kind of fibrous protein, are one of important animal proteinums.Collagen is present in all kinds of animal bodies widely, from vertebrates to fish, skin, tendon, cartilage, various internal organs, vessel wall and the esophagus etc. of animal such as batrachians, birds and mammals all have collagen.Collagen has occupied 30% of total amount in the interior all proteins of vertebrates body, is main structural protein, and skin contains collagen more than 50%.
Sex change natural collagen (being biological active collagen) is not a kind of biomacromolecule, and molecular weight is about 300,000.Natural structure when its structure still keeps in animal body, i.e. the triple helix structure of collagen, this natural collagen protein is counted as the integral part of organism and is not considered to foreign matter.Natural collagen help cell survival and growth, can promote that thrombocyte condenses, function such as low antigenicity and excellent biological compatibility, be used widely aspect burn, wound, cornea disease, beauty treatment, orthopedic, the hard tissue repair.Chi H.Lee, Anuj Singla andYugyung Lee, Biochemical applications of collagen, International Journal of Pharmaceutics, 2001,221:1-22. because the sex change natural collagen does not also have good performance of keeping humidity and promotes the epidermal growth breeding, thus skin elasticity, minimizing wrinkle can be strengthened effectively, so also be used for the cosmetics of super quality.Chvapil，M.andEckmayer，Z.，Role?of?proteins?in?cosmetics，International?Journal?of?Cosmetic?Science，1985，7：41-49.
The widespread use of sex change natural collagen must be more and more not higher to the requirement of the demand of collagen and quality.Domestic employed not sex change natural collagen mainly comes from the skin or heel string of slaughtered animals such as pig and ox at present, but because factors such as global integration trend, make animal infectious diseases such as mad cow disease and foot and mouth disease generally popular, and still do not have effective methods of treatment at present in the whole world.So seeking more, the source of the natural collagen of high quality and greater security just seems extremely important.Though and the collagen safety of from human body such as placenta, extracting, limited amount.With respect to terrestrial animal collagen proteins such as pig, oxen, the aquatic animal collagen protein shows its advantage aspect safety in utilization.Yuko?Ushiki，ShunjiHattori?and?Shinkichi?Irie，Safety?aspect?of?collagen?as?a?raw?material?for?biomedicalapplication，Fragrance?Journal，1997，7：42-47.
The marine site is vast in the world, the seashore line length, and marine fishery resources is abundant, and is of a great variety.The quantity of the catch of squid only, annual world total amount just reaches more than 2,500,000 tons, and the quantity of the catch of Chinese annual squid also has nearly 250,000 tons.Halobiontic diversity, volume have all determined the utilization of marine organisms aspect collagen, and fish, particularly marine fishes are subjected to environmental pollution little, can obtain the most natural safe natural collagen.China is an aquatic products big country, and albacore still is that freshwater fish is all abundant relatively.Several whole China has been run through in the great river, and freshwater fish is one of main food of hinterland, although also be subjected to some influences of environment aspect, along with the development that science is breeded fish, various fish quantity are increasing.Therefore, the former extraction starting material of fish glue are appreciable.
The Isin glue collagen that extracts from fish is mainly used in the finings the production processes such as (1) beer, grape wine at present; Roger B.Boulton, Vernon L.Singleton, Linda F.Bisson, Ralph E.Kunkee work, Zhao Guangao, Yin Zhuorong, Zhang Jimin, Yang Ming, Li Jiming translates, " enology---principle and application ", China Light Industry Press, 2001, P276-277.(2) beauty-care beverage.Qin Yuqing, Liu Chengchu, the mensuration of collagen from squid skin and recovery, " Shanghai Aquatic Products Univ. 9CN)'s journal ", 2002,11 (2): 138-144.These Isin glue collagen main components are Collagen Hydrolysates, and Collagen Hydrolysate be sex change has taken place collagen protein, it is biologically active not, does not belong to the not category of sex change natural collagen.
Because fish is grown in the water, the environment that sashimi (raw fish) is lived has determined the former denaturation temperature of fish glue to be lower than the denaturation temperature of collagens such as terrestrial animal pig, ox.Generally the not sex change natural collagen denaturation temperature from the tissue extraction of pig, ox is 38-44 ℃, and the denaturation temperature of freshwater fish natural collagen is 20-28 ℃, the denaturation temperature of seafood fish natural collagen is lower, generally has only 12-25 ℃ (because of the kind difference difference of fish).Because fish collagenous degeneration temperature is low, existing from terrestrial animal tissue extraction such as pig, ox not the sex change natural collagen technology of preparing and to be not suitable for preparing the natural fish glue of not sex change former.
Three, summary of the invention
The objective of the invention is provides the natural fish glue of a kind of not sex change former preparation method at the deficiencies in the prior art, be characterized in organic solvent and nonionogenic tenside degreasing, the deliming of EDTA solution, it is former to obtain the natural fish glue of not sex change with the proteolytic enzyme extraction under low temperature and acidic conditions.
Purpose of the present invention is realized that by following technical measures wherein said raw material umber is parts by weight except that specified otherwise.
The former preparation method of the natural fish glue of sex change not:
1, the pre-treatment of raw material
Select the freezing marine products fish-skin or the fresh freshwater fish skin that are rich in collagen for use, scale, fleshing, be cut into fritter 100 weight parts of 0.2-2cm, with ether 2-5 weight part, soaked 5-24 hour or soaked 5-24 hour with arbitrary proportion blended nonionogenic tenside 5-10 weight part, filter, add 0.1-4M EDTA solution 500-700 weight part and soaked 24-4g hour with water, stir, filter.The temperature of all operations is 4-6 ℃.The gained sediment is as following extraction raw material for standby.
2, the not former extraction of the natural fish glue of sex change
Pretreated fish-skin fragment 100 weight parts, proteolytic enzyme 10-20 weight part with acid for adjusting pH=2-4, was handled 1-3 days under 4-6 ℃ of low temperature, obtained the natural Isin glue collagen liquid of not sex change.
3, the purifying of extract
Above-mentioned collagen liquid is separated with the 16000-20000rpm supercentrifuge, remove filter residue, adding 0.5-5M NaCl in the supernatant liquor saltouts, the gained precipitation is dissolved with 0.1-2M acetate after centrifugation, saltout so repeatedly and operate 3-5 time, through film dialysis treatment 3 times, obtaining concentration is the former pure solution of the natural fish glue of not sex change of 0.5-2% again.
Grease-removing agent is at least a in ether, sherwood oil, ethylene glycol ethyl ether or aliphatic acid polyethenoxy ether, the degreasing of alkylphenol polyoxyethylene nonionogenic tenside.
Proteolytic enzyme is any in stomach en-, 537 proteolytic enzyme or the fungi prozyme.
The natural fish glue that makes is former to be tested with difference formula scanning calorimeter, and the former test result of the natural fish glue of not sex change that obtains sees for details shown in Figure 1.The result shows: freshwater fish collagenous degeneration temperature is 20-28 ℃, and albacore collagenous degeneration temperature is 12-25 ℃.Molecule measuring pilot production SDS-PAGE gel electrophoresis sees for details shown in Figure 2.The result shows: its α chain, β chain, γ chain molecular weight are respectively 100,000,200,000 and 300,000.Former pig, the ox natural collagen of replacing of the natural fish glue of not sex change of this biologically active is used for organizational project, surgical operation with biomedical sectors such as stanch fibres, also can be used for senior cosmetics, healthcare products.
Four, description of drawings
Fig. 1 is the former DSC thermal denaturation temperature test pattern of fish glue.
Fig. 2 is the former SDS-PAGE electrophoretic separation figure of fish glue.
Five, embodiment
Below by embodiment the present invention is specifically described; be necessary to be pointed out that at this present embodiment is used for the present invention is further specified; can not be interpreted as restriction to the present invention's protection; the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment 1: the fresh grass carp after will slaughtering, get skin, remove scale, scrape off unnecessary meat tissue, after cleaning with high purity water, be cut into the fine grained chippings of 0.2-2cm, get fine grained chippings 100g, add in ether 2g, the sherwood oil 3g mixed solution and soaked 24 hours, in 0.5M EDTA solution 700g, stirred 24 hours.To 2-4, adding stomach en-12g with acid for adjusting pH, is 4-6 ℃ in temperature, stirs 48 hours.Centrifugal removing do not dissolved part, adds 1MNaCl in the supernatant liquor and saltouts, with 2M acetate dissolution precipitation, the repeated treatments of saltouing 3 times.Obtaining clarifying concentration is the natural fish glue original solution of the thick not sex change 500g of 1-2%.
Embodiment 2: get and wash clean frozen squid skin, after cleaning with high purity water, be cut into the fine grained chippings of 0.2-2cm, get 100g, put into ethylene glycol ethyl ether 3g, aliphatic acid polyethenoxy ether 2g, soaked 24 hours among the alkylphenol polyoxyethylene 3g, in 1M EDTA solution 600g, stirred 24 hours, with acid for adjusting pH to 2.5-3.5, add 537 proteolytic enzyme 10g, stirred 48 hours down in temperature 4-6 ℃, remove through centrifugation and do not dissolve part, add 3M NaCl in the supernatant liquid and saltout, dissolve with 0.5M acetate, the repeated treatments of saltouing 4 times, obtaining concentration is the not former about 400g of the natural fish glue of sex change of 0.5-1.0%.
Embodiment 3: get freezing salmon skin, scrape sub-cloud meat tissue with cutter as far as possible, and remove most scale, washing is clean, is cut into broken shape fritter.Get 100g, soaked 48 hours in the 500ml aqueous solution that the compound nonionogenic tenside of adding ethylene glycol ethyl ether 4g, aliphatic acid polyethenoxy ether 3g, alkylphenol polyoxyethylene 3g is made into.Filtering the back stirred 40 hours in containing the solution 500g of 2M EDTA, filter, use acid for adjusting pH value 2-3.5, add Mycophyta compound protease 15g, stirred 72 hours down, remove through centrifugation and do not dissolve part in temperature 4-6 ℃, supernatant liquid adds 5M NaCl and saltouts, the dissolving of 1M acetate, the repeated treatments of saltouing 5 times, obtaining concentration is the natural fish glue stoste of the not sex change 450g of 0.5-1%.
Claims (3)
1. the former preparation method of the natural fish glue of sex change not is characterized in that:
(1) pre-treatment of raw material
Select the freezing marine products fish-skin or the fresh freshwater fish skin that are rich in collagen for use, scale, fleshing, be cut into fritter 100 weight parts of 0.2-2cm,, soaked 5-24 hour or soaked 5-24 hour with arbitrary proportion blended nonionogenic tenside 5-10 weight part with water with ether, ethylene glycol ethyl ether or sherwood oil 2-5 weight part, filter, add 0.1-4M EDTA solution 500-700 weight part and soaked 24-48 hour, stir, filter, the temperature of all operations is 4-6 ℃, and the gained sediment is as following extraction raw material for standby;
(2) the not former extraction of the natural fish glue of sex change
With above-mentioned pretreated raw material 100 weight parts, proteolytic enzyme 10-20 weight part with acid for adjusting pH=2-4, slowly stirred 1-3 days down in temperature 4-6 ℃, obtained the natural Isin glue collagen liquid of not sex change;
(3) purifying of extract
With above-mentioned Isin glue collagen liquid, separate with the 16000-20000rpm supercentrifuge, remove filter residue, supernatant liquid adds 0.5-5M NaCl and saltouts, the gained precipitation is dissolved with 0.1-2M acetate after centrifugation, saltout so repeatedly and handle 3-5 time, again through film dialysis treatment 3 times, acquisition concentration is the high purity natural fish glue original solution of sex change not of 0.5-2%.
2. the former preparation method of the natural fish glue of sex change not according to claim 1 is characterized in that nonionogenic tenside is aliphatic acid polyethenoxy ether or alkylphenol polyoxyethylene.
3. the former preparation method of the natural fish glue of sex change not according to claim 1 is characterized in that proteolytic enzyme is any in stomach en-, 537 aspartic proteases or the fungi prozyme.
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| CN 200410040486 CN1269877C (en) | 2004-08-19 | 2004-08-19 | Preparation method of undenatured natural fish collagen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154425A (en) * | 2011-03-18 | 2011-08-17 | 北京华达杰瑞生物技术有限公司 | Production method of non-denatured II-type collagen |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345867C (en) * | 2005-10-11 | 2007-10-31 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
| CN101186786B (en) * | 2006-11-06 | 2010-06-09 | 四川大学 | Method for extracting unmodified natural collagen from tanning waste leather scrap |
| CN101642263B (en) * | 2008-08-07 | 2012-01-18 | 关德智 | Process for producing fish glue |
| CN106032546A (en) * | 2015-03-18 | 2016-10-19 | 上海市水产研究所 | Method for extracting medical material-based collagen from fish scales |
| CN106893329B (en) * | 2017-03-22 | 2019-02-19 | 四川大学 | A method of using fish processing fent leather for natural collagen film |
| CN109232908A (en) * | 2018-09-21 | 2019-01-18 | 陕西科技大学 | A method of extracting undenatured natural collagen from pickled skin |
| CN114106148B (en) * | 2020-08-31 | 2023-12-01 | 中国海洋大学 | Medical-grade non-denatured collagen with fish skin source and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154425A (en) * | 2011-03-18 | 2011-08-17 | 北京华达杰瑞生物技术有限公司 | Production method of non-denatured II-type collagen |
| CN102154425B (en) * | 2011-03-18 | 2013-05-01 | 北京华达杰瑞生物技术有限公司 | Production method of non-denatured II-type collagen |
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