CN107629122A - The collagen of catfish fish guts protease extraction - Google Patents
The collagen of catfish fish guts protease extraction Download PDFInfo
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- CN107629122A CN107629122A CN201710950845.0A CN201710950845A CN107629122A CN 107629122 A CN107629122 A CN 107629122A CN 201710950845 A CN201710950845 A CN 201710950845A CN 107629122 A CN107629122 A CN 107629122A
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- fish
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Abstract
The collagen extracted with catfish fish guts protease, using fish-skin as raw material, non-collagen soluble in peeling fish skin is removed by adding inorganic agent pretreatment, then add mixed liquor and carry out degreasing, add catfish visceral protein enzyme to be digested, the filtrate after enzymolysis is added into sodium chloride saltouts, and obtains sediment, after acetate dissolution being added in sediment, bag filter dialysis is transferred to, pours out the collagen solution in bag filter, is freeze-dried, off-white color collagen is obtained, is had the beneficial effect that：Purity is high, impurity is few, colorless and odorless, it is short with extraction time, the features such as yield is high, and environmental pollution is smaller, collagen is damaged small, the collagen with good biological activity can be obtained, food and cosmetics manufacture field are applicable to, the increment to catfish fish-skin is realized and utilizes, improve the economic value added of catfish.
Description
Technical field
The present invention relates to field of processing of aquatic products, more particularly, to the refinement technique of collagen.
Background technology
With biochemical development, people are gradually stepped up to the understanding of collagen, and different tissues are extracted from animal
In different types of collagen, its function and effect are also different, collagen as immunoglobulin, rich in diversity and
The specificity of Tissue distribution, be with each tissue, the relevant functional protein of organ function, with culture fishery and processing industry
Develop rapidly, it is big to produce substantial amounts of leftover bits and pieces fish head, fish-skin, fin, fish tail, fish-bone and its residual flesh of fish, wherein fish-skin, fish scale
7.5%~10% is accounted for, collagen accounts for fish-skin, 1/3rd or so of squama raw material, if making full use of, can not only improve fish
The added value of processing, while can reduce environmental pollution, good economic benefit and social benefit are obtained, contains what is enriched in fish-skin
Collagen, therefore fish-skin can be processed into collagen and gelatin and its addition product, and collagen and its modified product category boiomacromolecule
Material, nutrient and healthcare products, superior cosmetics, food, biological pesticide, bio-feritlizer etc. can be further processed into, had higher
Economic value.
Prior art such as Authorization Notice No. is CN103131746B Chinese invention patent, is disclosed in a kind of band squama fish skin
The extracting method of collagen peptide, the step of cleaning can be simplified and use hydrochloric acid, reduce water consumption, reduce cost.This method
By weight, including step：A：0.003-0.01 parts sodium carbonate and 2-3 part water, stirring, and draining are added in 1 part of band squama fish skin,
Afterwards plus 1-3 parts water cleans, and obtains the band squama fish skin of degreasing；B：2-5 part water is added into the band squama fish skin of degreasing, mixes simultaneously powder
It is broken, obtain slurries；C：The temperature of the slurries is adjusted to 50-65 DEG C, and the pH value of the slurries is adjusted to 8-9, it is rear to add
0.001-0.004 part protease, is digested, the slurries digested；D：The slurries of the enzymolysis are filtrated to get clear liquid, and
0.02-0.05 part activated carbons are added to clear liquid, is filtered after stirring, obtains filtrate and filtering medium；E：The filtrate is concentrated, spraying is dry
It is dry, obtain collagen peptide；But the collagen purity of this method extraction is not high, efficiency is low, inconvenient for operation.The content of the invention
It is an object of the invention to provide a kind of DNA purity is high, extraction is convenient, activity stabilized catfish fish guts protease carries
The collagen taken.
The present invention is directed to the problem of being mentioned in above-mentioned technology, and the technical scheme taken is：Carried with catfish fish guts protease
The collagen taken, using fish-skin as raw material, obtained by following steps:
Bulk processing：Fish-skin is subjected to stripping and slicing pretreatment, is soaked, is filtered with degreasing cotton yarn, fish with 25-35 times of inorganic agent of its quality
Skin is washed repeatedly with distilled water, is fully drained, then adds 30-45 times times of mixed liquor of its quality in 2-8 DEG C of leaching in fish-skin
12-18h is steeped, is washed with distilled water, fully drained repeatedly, mixed liquor composition is ether, isopropanol, distilled water, the content of three
Than for 2:2:8-10, inorganic agent are concentration 1-2.5%NaCl and 0.1-0.4mol/L NaOH mixed liquors, soak duration 6-12h,
Inorganic agent is added in fish-skin after stripping and slicing, increases the contact area of inorganic agent and fish-skin, accelerates reaction rate, is removed in fish-skin
Muscle fibril, destroy fish-skin in non-collagen composition so that fish-skin soften, foreign protein separate out.The addition of neutral salt is also
Collagen hydro can be reduced, collagen purification, preferably can be separated the collagen in fish-skin and foreign protein, so as to fully remove
Soluble non-collagen, obtain higher recovery rate；Mixed liquor is added in fish-skin, is mixing the lipolyse in fish-skin
In liquid, so as to remove fish-skin superabundant fats in immersion process, until fat eliminates, then cleaned and drained with distilled water, made
Obtained fish-skin degreasing, decontamination, facilitate the extraction of collagen；
Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, digests 6-7h at 50-65 DEG C, fish-skin,
W/v between catfish visceral protein enzyme, distilled water three is 1g:0.01-0.02g：5-10ml, after enzymolysis terminates,
100-120 DEG C of enzyme deactivation 8-12min, filtering, collect filtrate；Catfish visceral protein enzyme makes the particular peptide of the intercellular protein of fish-skin
Key is hydrolyzed so that cell dissociation, the specific peptide bond of protein is because of flexural deformation and is activated hydrolyzing, and forms amino and carboxyl respectively,
Micromolecule polypeptide or amino acid are obtained, discharges from fish-skin cell, is operated by enzyme deactivation, makes unnecessary catfish visceral protein
Enzyme terminating reaction, prevent that enzymolysis is excessive, the filtrate containing collagen is obtained after filtering；
Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.2-2.7mol/L, at 4-9 DEG C,
12-17min is centrifuged under 11000-13000r/min, abandons supernatant, obtains sediment；By the way that collagen filtrate is mixed with sodium chloride
Closing, centrifugation, obtain salt precipitation thing, the sodium chloride added in the step makes the solubility of collagen reduce and Precipitation,
Above method advantage is that sodium chloride sedimentation effect of saltouing is good, saves energy consumption；
Dialysis：After precipitating with 0.2-0.8mol/L acetate dissolutions, bag filter dialysis is transferred to, detects, pours out to without chlorion
The collagen solution in bag is analysed, freeze-drying, obtains off-white color collagen；Above-mentioned dialysis procedure will dissolve with sediment
Acetate solution is fitted into the bag filter containing distilled water, and sealing is dialysed, by the collagen solution cooling treatment in bag filter
Afterwards, off-white color collagen is produced, the above method in the solution can be by bag filter using small-molecule substance, and macromolecular substances
The purpose of separation small molecular weight impurity by the property of bag filter, can not be reached, simple to operate, effect is good, saves energy consumption.
Preferably, catfish visceral protein enzyme contains 0.1-0.5% active peptides, the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKRAGGYACRCRSFAGRGRCKCFRC；The active peptides added in catfish visceral protein enzyme, Ke Yiti
The solubility of high protein, exposure restriction enzyme site, be advantageous to the close of protease, alternative cuts off non-helical end peptide and dissolved
Go out tropocollagen molecule, and the triple helix structure of collagen in enzymolysis with higher stability, be not easy to be removed, so as to aobvious
Write the yield for improving collagen.
Compared with prior art, the advantage of the invention is that：The collagen that the present invention extracts, viscosity is high, impurity is few, nothing
Color is tasteless, has the features such as extraction time is short, and yield is high, and environmental pollution is smaller, collagen is damaged it is small, remain compared with
Complete fibrillar meshwork structure, triple-helix structure is complete, can obtain the collagen with good biological activity, be applicable to eat
Product and cosmetics manufacture field, realize the increment to catfish fish-skin and utilize, improve the economic value added of catfish.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The collagen extracted with catfish fish guts protease, using fish-skin as raw material, the preparation method of collagen
Including bulk processing, digest, saltout, dialyse, concretely comprise the following steps：
1）Bulk processing：Fish-skin is subjected to stripping and slicing pretreatment, is soaked with 25-35 times of inorganic agent of its quality, is filtered with degreasing cotton yarn,
Fish-skin is washed repeatedly with distilled water, is fully drained, then adds 30-45 times times of mixed liquor of its quality in 2-8 DEG C in fish-skin
12-18h is soaked, is washed with distilled water, fully drained repeatedly, mixed liquor composition is ether, isopropanol, distilled water, and three's contains
Amount is than being 2:2:8-10, inorganic agent are concentration 1-2.5%NaCl and 0.1-0.4mol/L NaOH mixed liquors, soak duration 6-
12h, inorganic agent is added in the fish-skin after stripping and slicing, increase the contact area of inorganic agent and fish-skin, accelerate reaction rate, remove fish
Muscle fibril in skin, the non-collagen composition destroyed in fish-skin so that fish-skin softens, and foreign protein separates out.Neutral salt adds
Enter to reduce collagen hydro, collagen purification, preferably can separate the collagen in fish-skin and foreign protein, so as to fully
Soluble non-collagen is removed, obtains higher recovery rate；Mixed liquor is added in fish-skin, the lipolyse in fish-skin is existed
In mixed liquor, so as to remove fish-skin superabundant fats in immersion process, until fat eliminates, then drip is cleaned with distilled water
It is dry, so as to get fish-skin degreasing, decontamination, facilitate the extraction of collagen；
2）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 6-7h, fish are digested at 50-65 DEG C
W/v between skin, catfish visceral protein enzyme, distilled water three is 1g:0.01-0.02g：5-10ml, enzymolysis terminate
Afterwards, 100-120 DEG C of enzyme deactivation 8-12min, filtering, filtrate is collected；Catfish visceral protein enzyme makes the spy of the intercellular protein of fish-skin
Determine peptide bond hydrolysis so that cell dissociation, the specific peptide bond of protein is because of flexural deformation and is activated hydrolyzing, formed respectively amino and
Carboxyl, micromolecule polypeptide or amino acid are obtained, discharge from fish-skin cell, operated by enzyme deactivation, made in unnecessary catfish
Dirty protease terminating reaction, prevent that enzymolysis is excessive, the filtrate containing collagen is obtained after filtering；
3）Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.2-2.7mol/L, at 4-9 DEG C,
12-17min is centrifuged under 11000-13000r/min, abandons supernatant, obtains sediment；By the way that collagen filtrate is mixed with sodium chloride
Closing, centrifugation, obtain salt precipitation thing, the sodium chloride added in the step makes the solubility of collagen reduce and Precipitation,
Above method advantage is that sodium chloride sedimentation effect of saltouing is good, saves energy consumption；
4）Dialysis：After precipitating with 0.2-0.8mol/L acetate dissolutions, bag filter dialysis is transferred to, detects, pours out to without chlorion
Collagen solution in bag filter, freeze-drying, obtains off-white color collagen；Above-mentioned dialysis procedure is will to be dissolved with sediment
Acetate solution be fitted into the bag filter containing distilled water, sealing dialysed, at the collagen solution cooling in bag filter
After reason, off-white color collagen is produced, the above method in the solution can be by bag filter using small-molecule substance, and macromolecular complex
Matter by the property of bag filter, can not reach the purpose of separation small molecular weight impurity, and simple to operate, effect is good, save energy consumption.
Catfish visceral protein enzyme contains 0.1-0.5% active peptides, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKRAGGYACRCRSFAGRGRCKCFRC；The active peptides added in catfish visceral protein enzyme, Ke Yiti
The solubility of high protein, exposure restriction enzyme site, be advantageous to the close of protease, alternative cuts off non-helical end peptide and dissolved
Go out tropocollagen molecule, and the triple helix structure of collagen in enzymolysis with higher stability, be not easy to be removed, so as to aobvious
Write the yield for improving collagen.
Embodiment 2：
The collagen extracted with catfish fish guts protease, using fish-skin as raw material, its preparation method comprises the following steps：
1）Bulk processing：Fish-skin is subjected to stripping and slicing pretreatment, is soaked, is filtered with degreasing cotton yarn, fish with 25 times of inorganic agent of its quality
Skin is washed repeatedly with distilled water, is fully drained, and inorganic agent is concentration 2.2%NaCl and 0.2mol/L NaOH, immersion duration 8h；
30 times of mixed liquor for adding its quality in fish-skin again soaks 12h in 8 DEG C, is washed with distilled water, fully drained repeatedly, mixes
Liquid composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:8；
2）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in the broken fish-skin after degreasing, digests 6h at 50 DEG C, fish-skin,
W/v between catfish visceral protein enzyme, distilled water three is 1g:0.01g：5ml, after enzymolysis terminates, 100 DEG C of enzyme deactivations
8min, filtering, collect filtrate；
3）Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.3mol/L, at 4 DEG C,
12min is centrifuged under 12000r/min, abandons supernatant, obtains sediment；
4）Dialysis：After precipitating with 0.2mol/L acetate dissolutions, bag filter dialysis is transferred to, is detected to without chlorion, pours out dialysis
Collagen solution in bag, freeze-drying, obtains off-white color collagen.
Catfish visceral protein enzyme contains 0.2% active peptides, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKRAGGYACRCRSFAGRGRCKCFRC。
Embodiment 3：
The collagen extracted with catfish fish guts protease, using fish-skin as raw material, comprises the following steps：
1）Bulk processing：Fish-skin is subjected to stripping and slicing pretreatment, is soaked, is filtered with degreasing cotton yarn, fish with 26 times of inorganic agent of its quality
Skin is washed repeatedly with distilled water, is fully drained, and inorganic agent is concentration 2.3%NaCl and 0.3mol/L NaOH, immersion duration 9h；
36 times of mixed liquor for adding its quality in fish-skin again soaks 12-18h in 7 DEG C, is washed with distilled water, fully drained repeatedly,
Mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:10；
2）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 7h, fish-skin, Nian are digested at 65 DEG C
W/v between fish guts protease, distilled water three is 1g:0.02g:10ml, after enzymolysis terminates, 20 DEG C of enzyme deactivations
12min, filtering, collect filtrate；
3）Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.5mol/L, at 4 DEG C,
14min is centrifuged under 12500r/min, abandons supernatant, obtains sediment；
4）Dialysis：After precipitating with 0.3mol/L acetate dissolutions, bag filter dialysis is transferred to, is detected to without chlorion, pours out dialysis
Collagen solution in bag, freeze-drying, obtains off-white color collagen；
Catfish visceral protein enzyme contains 0.25% active peptides, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKRAGGYACRCRSFAGRGRCKCFRC；
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
Sequence table
<110>Ningbo City Yinzhou District Chao Rui food science and technologies Co., Ltd
<120>The collagen extracted with catfish fish guts protease
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> PRT
<213>Artificial synthesized (Mytilus coruscus)
<400> 1
Ser Cys Ala Ser Arg Tyr Gly Arg Arg Cys Arg Ala Gly Arg Cys Lys
1 5 10 15
Arg Ala Gly Gly Tyr Ala Cys Arg Cys Arg Ser Phe Ala Gly Arg Gly
20 25 30
Arg Cys Lys Cys Phe Arg Cys
35
Claims (5)
1. the collagen extracted with catfish fish guts protease, it is characterised in that：Described uses catfish fish guts albumen
The collagen of enzyme extraction is obtained using fish-skin as raw material by following steps:
1）Bulk processing：Fish-skin is subjected to stripping and slicing pretreatment, is soaked with 25-35 times of inorganic agent of its quality, is filtered with degreasing cotton yarn,
Fish-skin is washed repeatedly with distilled water, is fully drained, then adds 30-45 times times of mixed liquor of its quality in 2-8 DEG C in fish-skin
12-18h is soaked, is washed with distilled water, fully drained repeatedly；
2）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 6-7h, enzyme are digested at 50-65 DEG C
After solution terminates, 100-120 DEG C of enzyme deactivation 8-12min, filtering, filtrate is collected；
3）Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.2-2.7mol/L, at 4-9 DEG C,
12-17min is centrifuged under 11000-13000r/min, abandons supernatant, obtains sediment；
4）Dialyse and be：After precipitating with 0.2-0.8mol/L acetate dissolutions, bag filter dialysis is transferred to, is detected to without chlorion, is fallen
The collagen solution gone out in bag filter, freeze-drying, obtains off-white color collagen.
2. the collagen according to claim 1 extracted with catfish fish guts protease, it is characterised in that：It is described
Catfish visceral protein enzyme contains 0.1-0.5% active peptides in enzymolysis step, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKRAGGYACRCRSFAGRGRCKCFRC。
3. the collagen according to claim 1 extracted with catfish fish guts protease, it is characterised in that：It is described
Mixed liquor composition is ether, isopropanol, distilled water in defatting step, and the content ratio of three is 2:2:8-10.
4. the collagen according to claim 1 extracted with catfish fish guts protease, it is characterised in that：It is described
Inorganic agent is concentration 1-2.5%NaCl and 0.1-0.4mol/L NaOH mixed liquors in pre-treatment step, soaks duration 6-12h.
5. the collagen according to claim 1 extracted with catfish fish guts protease, it is characterised in that：It is described
W/v in enzymolysis step between fish-skin, catfish visceral protein enzyme, distilled water three is 1g:0.01-0.02g：5-
10ml。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710950845.0A CN107629122A (en) | 2017-10-13 | 2017-10-13 | The collagen of catfish fish guts protease extraction |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710950845.0A CN107629122A (en) | 2017-10-13 | 2017-10-13 | The collagen of catfish fish guts protease extraction |
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| CN201710950845.0A Withdrawn CN107629122A (en) | 2017-10-13 | 2017-10-13 | The collagen of catfish fish guts protease extraction |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653288A (en) * | 2017-10-13 | 2018-02-02 | 金华市川璞农业科技有限公司 | With the purposes of catfish fish guts protease extraction collagen |
| CN114214384A (en) * | 2021-12-22 | 2022-03-22 | 海南三元星生物科技股份有限公司 | Marine organism-derived collagen peptide, and extraction method and application thereof |
| CN117209338A (en) * | 2023-11-07 | 2023-12-12 | 四川大学 | Ammonium polyphosphate type organic fertilizer containing amino acid and preparation method thereof |
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| CN100345867C (en) * | 2005-10-11 | 2007-10-31 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
| CN102558345A (en) * | 2012-02-13 | 2012-07-11 | 江苏大学 | Method for extracting collagen from sturgeon skin |
| CN103131746A (en) * | 2013-03-12 | 2013-06-05 | 程云开 | Extraction method of collagen peptide contained in scaly fish skin |
| CN103789381A (en) * | 2014-02-14 | 2014-05-14 | 钟春燕 | Method for rapidly extracting fish collagen |
| CN105969832A (en) * | 2016-07-26 | 2016-09-28 | 渤海大学 | Preparation method of plaice skin collagen powder |
| CN107236777A (en) * | 2017-06-21 | 2017-10-10 | 兰溪市沉默生物科技有限公司 | The method that collagen is extracted from crucian fish-skin |
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2017
- 2017-10-13 CN CN201710950845.0A patent/CN107629122A/en not_active Withdrawn
Patent Citations (6)
| 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 |
| CN102558345A (en) * | 2012-02-13 | 2012-07-11 | 江苏大学 | Method for extracting collagen from sturgeon skin |
| CN103131746A (en) * | 2013-03-12 | 2013-06-05 | 程云开 | Extraction method of collagen peptide contained in scaly fish skin |
| CN103789381A (en) * | 2014-02-14 | 2014-05-14 | 钟春燕 | Method for rapidly extracting fish collagen |
| CN105969832A (en) * | 2016-07-26 | 2016-09-28 | 渤海大学 | Preparation method of plaice skin collagen powder |
| CN107236777A (en) * | 2017-06-21 | 2017-10-10 | 兰溪市沉默生物科技有限公司 | The method that collagen is extracted from crucian fish-skin |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653288A (en) * | 2017-10-13 | 2018-02-02 | 金华市川璞农业科技有限公司 | With the purposes of catfish fish guts protease extraction collagen |
| CN114214384A (en) * | 2021-12-22 | 2022-03-22 | 海南三元星生物科技股份有限公司 | Marine organism-derived collagen peptide, and extraction method and application thereof |
| CN117209338A (en) * | 2023-11-07 | 2023-12-12 | 四川大学 | Ammonium polyphosphate type organic fertilizer containing amino acid and preparation method thereof |
| CN117209338B (en) * | 2023-11-07 | 2024-01-02 | 四川大学 | Ammonium polyphosphate type organic fertilizer containing amino acid and preparation method thereof |
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Application publication date: 20180126 |