CN107815480A - With the method for catfish fish guts protease extraction collagen - Google Patents
With the method for catfish fish guts protease extraction collagen Download PDFInfo
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- CN107815480A CN107815480A CN201710950833.8A CN201710950833A CN107815480A CN 107815480 A CN107815480 A CN 107815480A CN 201710950833 A CN201710950833 A CN 201710950833A CN 107815480 A CN107815480 A CN 107815480A
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 69
- 102000008186 Collagen Human genes 0.000 title claims abstract description 69
- 229920001436 collagen Polymers 0.000 title claims abstract description 69
- 241001233037 catfish Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000004365 Protease Substances 0.000 title claims abstract description 25
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 24
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 23
- 238000000605 extraction Methods 0.000 title claims abstract description 14
- 238000005238 degreasing Methods 0.000 claims abstract description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 42
- 102000004190 Enzymes Human genes 0.000 claims description 30
- 108090000790 Enzymes Proteins 0.000 claims description 30
- 239000012153 distilled water Substances 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 239000011780 sodium chloride Substances 0.000 claims description 21
- 230000009278 visceral effect Effects 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000000706 filtrate Substances 0.000 claims description 15
- 239000003795 chemical substances by application Substances 0.000 claims description 14
- 238000000502 dialysis Methods 0.000 claims description 14
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
- 230000009849 deactivation Effects 0.000 claims description 8
- 238000002203 pretreatment Methods 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000013049 sediment Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 241000370738 Chlorion Species 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000005185 salting out Methods 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 5
- 239000002537 cosmetic Substances 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 2
- 235000013305 food Nutrition 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 230000009965 odorless effect Effects 0.000 abstract 1
- 235000019419 proteases Nutrition 0.000 description 13
- 241000251468 Actinopterygii Species 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 238000005265 energy consumption Methods 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000007654 immersion Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 108010077465 Tropocollagen Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000011001 backwashing Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- KWKQGHSSNHPGOW-BQBZGAKWSA-N Arg-Ala-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)NCC(O)=O KWKQGHSSNHPGOW-BQBZGAKWSA-N 0.000 description 1
- SVHRPCMZTWZROG-DCAQKATOSA-N Arg-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCN=C(N)N)N SVHRPCMZTWZROG-DCAQKATOSA-N 0.000 description 1
- JPAWCMXVNZPJLO-IHRRRGAJSA-N Arg-Ser-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JPAWCMXVNZPJLO-IHRRRGAJSA-N 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- SZQCDCKIGWQAQN-FXQIFTODSA-N Cys-Arg-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O SZQCDCKIGWQAQN-FXQIFTODSA-N 0.000 description 1
- OCEHKDFAWQIBHH-FXQIFTODSA-N Cys-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CS)N)CN=C(N)N OCEHKDFAWQIBHH-FXQIFTODSA-N 0.000 description 1
- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 1
- PYUCNHJQQVSPGN-BQBZGAKWSA-N Gly-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)CN=C(N)N PYUCNHJQQVSPGN-BQBZGAKWSA-N 0.000 description 1
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 1
- UIQGJYUEQDOODF-KWQFWETISA-N Gly-Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 UIQGJYUEQDOODF-KWQFWETISA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000143233 Mytilus coruscus Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 235000018992 Prunus glandulosa Nutrition 0.000 description 1
- 240000001619 Prunus glandulosa Species 0.000 description 1
- 235000013999 Prunus japonica Nutrition 0.000 description 1
- WXUBSIDKNMFAGS-IHRRRGAJSA-N Ser-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WXUBSIDKNMFAGS-IHRRRGAJSA-N 0.000 description 1
- KCFKKAQKRZBWJB-ZLUOBGJFSA-N Ser-Cys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O KCFKKAQKRZBWJB-ZLUOBGJFSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 235000019835 bromelain Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 108010079202 tyrosyl-alanyl-cysteine Proteins 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- -1 viscosity is high Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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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- Life Sciences & Earth Sciences (AREA)
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- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
With the method for catfish fish guts protease extraction collagen, including pretreatment, degreasing, digest, saltout, dialysing, having 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, discloses one kind and utilizes compound egg
The method that white enzyme prepares active collagen, comprises the following steps：1）Fish-skin mixed liquor by pretreatment adds compound protein
Enzyme is digested；Wherein described compound protease comprises at least papain, bromelain, trypsase, neutral protein
Two kinds in enzyme, animal proteolytic enzyme, acid protease and pepsin；2）Obtained enzymolysis liquid is subjected to centrifugal treating, taken
Supernatant；3）Saltoutd after supernatant is concentrated by ultrafiltration；4）Ultrafiltration desalination is carried out again and is concentrated；Wherein, fish-skin mixed liquor
Concentration be 2.0-8.0wt%, pH 2-6, the addition of the compound protease is the 2.0- of fish-skin mixed liquor weight
8.0wt%.The inventive method is safe, has no side effect, preparation-obtained active collagen have recovery rate, purity and
The advantages that thermal denaturation temperature, but it is inconvenient for operation in practical application, and the collagen activity of extraction is not high.The content of the invention
It is an object of the invention to provide a kind of DNA purity height, extraction convenience, activity stabilized use catfish fish guts protease
The method for extracting collagen.
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 method for taking collagen, including pretreatment, degreasing, digest, saltout, dialysing, concretely comprise the following steps：
Pretreatment：Fish-skin is subjected to stripping and slicing pretreatment, is soaked, is filtered with degreasing cotton yarn, fish with 25-30 times of inorganic agent of its quality
Skin is washed repeatedly with distilled water, is fully drained, and inorganic agent is concentration 2-2.5%NaCl and 0.1-0.3mol/L NaOH mixed liquors,
Duration 6-10h is soaked, inorganic agent is added in the fish-skin after stripping and slicing, increases the contact area of inorganic agent and fish-skin, accelerates reaction
Speed, the muscle fibril in fish-skin is removed, the non-collagen composition destroyed in fish-skin so that fish-skin softens, and foreign protein separates out.
The addition of neutral salt can also reduce collagen hydro, and collagen purification can be preferably by the collagen in fish-skin and foreign protein point
Open, so as to fully remove soluble non-collagen, obtain higher recovery rate；
Degreasing：30-40 times of the mixed liquor that its quality is added in fish-skin after the pre-treatment soaks 12-15h in 4-8 DEG C, with steaming
Distilled water is washed repeatedly, is fully drained, and mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:8-10,
By adding mixed liquor in fish-skin after the pre-treatment, make the lipolyse in fish-skin in mixed liquor, so as in immersion process
Middle removal fish-skin superabundant fats, until fat eliminates, then are cleaned with distilled water and are drained, so as to get fish-skin degreasing, impurity elimination
Matter, 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 50-60 DEG C, fish-skin,
W/v between catfish visceral protein enzyme, distilled water three is 1g:0.01-0.02g：5-9ml, after enzymolysis terminates,
100-110 DEG C of enzyme deactivation 10-12min, filtering, collect filtrate；Catfish visceral protein enzyme makes the specific of the intercellular protein of fish-skin
For peptide bond hydrolysis so that cell dissociation, the specific peptide bond of protein is because of flexural deformation and is activated hydrolyzing, and forms amino and carboxylic respectively
Base, micromolecule polypeptide or amino acid are obtained, discharge from fish-skin cell, operated by enzyme deactivation, make unnecessary catfish internal organ
Protease 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 2.2-2.5mol/L, at 4-7 DEG C,
12-15min is centrifuged under 12000-13000r/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 0.2-0.5mol/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 0.2-0.5% active peptides, the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKSFARAGGYACRCRSFAGRGRCKCFRC；The active peptides added in catfish visceral protein enzyme, can
To improve the solubility of albumen, exposure restriction enzyme site is advantageous to the close of protease, alternative cut off non-helical end peptide and
Dissolve tropocollagen molecule, and the triple helix structure of collagen in enzymolysis with higher stability, be not easy to be removed, from
And significantly improve the yield of 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：
Include pretreatment, degreasing with the preparation method of catfish fish guts protease extraction collagen, digest, saltout, thoroughly
Analysis, is concretely comprised the following steps：
1）Pretreatment：Fish-skin is subjected to stripping and slicing pretreatment, is soaked with 25-30 times of inorganic agent of its quality, is filtered with degreasing cotton yarn,
Fish-skin is washed repeatedly with distilled water, is fully drained, and inorganic agent is that concentration 2-2.5%NaCl and 0.1-0.3mol/L NaOH are mixed
Liquid, duration 6-10h is soaked, inorganic agent is added in the fish-skin after stripping and slicing, increases the contact area of inorganic agent and fish-skin, accelerated anti-
Speed is answered, removes the muscle fibril in fish-skin, the non-collagen composition destroyed in fish-skin so that fish-skin softens, foreign protein analysis
Go out.The addition of neutral salt can also reduce collagen hydro, and collagen purification can be preferably by the collagen and foreign protein in fish-skin
Separate, so as to fully remove soluble non-collagen, obtain higher recovery rate；
2）Degreasing：30-40 times of the mixed liquor that its quality is added in fish-skin after the pre-treatment soaks 12-15h in 4-8 DEG C, uses
Distilled water washs repeatedly, fully drains, and mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:8-
10, by adding mixed liquor in fish-skin after the pre-treatment, make the lipolyse in fish-skin in mixed liquor, so as to soak
Superabundant fats are removed in journey, until fat eliminates, then is cleaned and drained with distilled water, so as to get fish-skin degreasing, decontamination,
Facilitate the extraction of collagen；
3）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 6-7h, fish are digested at 50-60 DEG C
W/v between skin, catfish visceral protein enzyme, distilled water three is 1g:0.01-0.02g：5-9ml, after enzymolysis terminates,
100-110 DEG C of enzyme deactivation 10-12min, filtering, collect filtrate；After enzymolysis terminates, 100-110 DEG C of enzyme deactivation 10-12min, filter, receive
Collect filtrate；Catfish visceral protein enzyme makes the specific peptide bond hydrolysis of the intercellular protein of fish-skin so that cell dissociation, protein
Specific peptide bond is because of flexural deformation and is activated hydrolyzing, and forms amino and carboxyl respectively, micromolecule polypeptide or amino acid is obtained, from fish
Discharge in chrotoplast, operated by enzyme deactivation, make unnecessary catfish visceral protein enzyme terminating reaction, prevent that enzymolysis is excessive, mistake
The filtrate containing collagen is obtained after filter；
4）Saltout：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.2-2.5mol/L, at 4-7 DEG C,
12-15min is centrifuged under 12000-13000r/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；
5）Dialysis：After precipitating with 0.2-0.5mol/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 0.2-0.5% active peptides, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKSFARAGGYACRCRSFAGRGRCKCFRC；The active peptides added in catfish visceral protein enzyme, can
To improve the solubility of albumen, exposure restriction enzyme site is advantageous to the close of protease, alternative cut off non-helical end peptide and
Dissolve tropocollagen molecule, and the triple helix structure of collagen in enzymolysis with higher stability, be not easy to be removed, from
And significantly improve the yield of collagen.
Embodiment 2：
With the method for catfish fish guts protease extraction collagen, concretely comprise the following steps：
1）Pretreatment：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；
2）Degreasing：30 times of the mixed liquor that its quality is added in fish-skin after the pre-treatment soaks 12h in 8 DEG C, anti-with distilled water
After backwashing is washed, and is fully drained, and mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:8；
3）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 6h, fish-skin, Nian are digested at 50 DEG C
W/v between fish guts protease, distilled water three is 1g:0.01g：5ml, after enzymolysis terminates, 100 DEG C of enzyme deactivations
10min, filtering, collect filtrate；
4）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；
5）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
SCASRYGRRCRAGRCKSFARAGGYACRCRSFAGRGRCKCFRC。
Embodiment 3：
With the method for catfish fish guts protease extraction collagen, concretely comprise the following steps：
1）Pretreatment：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；
2）Degreasing：36 times of the mixed liquor that its quality is added in fish-skin after the pre-treatment soaks 15h in 7 DEG C, anti-with distilled water
After backwashing is washed, and is fully drained, and mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:10；
3）Enzymolysis：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, digests 6-7h at 60 DEG C, fish-skin,
W/v between catfish visceral protein enzyme, distilled water three is 1g:0.02g：9ml, after enzymolysis terminates, 110 DEG C of enzyme deactivations
12min, filtering, collect filtrate；
4）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；
5）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
SCASRYGRRCRAGRCKSFARAGGYACRCRSFAGRGRCKCFRC；
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>Jinhua Nader's Chinese bush cherry bio tech ltd
<120>With the method for catfish fish guts protease extraction collagen
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<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
Ser Phe Ala Arg Ala Gly Gly Tyr Ala Cys Arg Cys Arg Ser Phe Ala
20 25 30
Gly Arg Gly Arg Cys Lys Cys Phe Arg Cys
35 40
Claims (8)
1. with the method for catfish fish guts protease extraction collagen, including pretreatment, degreasing, digest, saltout, thoroughly
Analysis, it is characterised in that：The pre-treatment step is that fish-skin is carried out into stripping and slicing pretreatment, is soaked with 25-30 times of inorganic agent of its quality
Bubble, is filtered with degreasing cotton yarn, and fish-skin is washed, fully drained repeatedly with distilled water.
2. the method according to claim 1 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The inorganic agent is concentration 2-2.5%NaCl and 0.1-0.3mol/L NaOH mixed liquors, soaks duration 6-10h.
3. the method according to claim 1 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The degreasing step is：30-40 times of the mixed liquor that its quality is added in fish-skin after the pre-treatment soaks 12- in 4-8 DEG C
15h, washed with distilled water, fully drained repeatedly.
4. the method according to claim 3 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The mixed liquor composition is ether, isopropanol, distilled water, and the content ratio of three is 2:2:8-10.
5. the method according to claim 1 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The enzymolysis step is：Catfish visceral protein enzyme and distilled water are added in broken fish-skin after degreasing, 6- is digested at 50-60 DEG C
7h, the w/v between fish-skin, catfish visceral protein enzyme, distilled water three are 1g:0.01-0.02g：5-9ml, enzymolysis knot
Shu Hou, 100-110 DEG C of enzyme deactivation 10-12min, filtering, collect filtrate.
6. the method according to claim 5 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The catfish visceral protein enzyme contains 0.2-0.5% active peptides, and the amino acid sequence of active peptides is
SCASRYGRRCRAGRCKSFARAGGYACRCRSFAGRGRCKCFRC。
7. the method according to claim 1 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The salting-out step is：Sodium chloride is added in the filtrate of collection to saltout, and sodium chloride final concentration is reached 2.2-2.5mol/L, in 4-
7 DEG C, 12-15min is centrifuged under 12000-13000r/min, supernatant is abandoned, obtains sediment.
8. the method according to claim 1 that collagen is extracted with catfish fish guts protease, it is characterised in that：
The dialysis is：After precipitating with 0.2-0.5mol/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.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN108396050A (en) * | 2018-03-28 | 2018-08-14 | 通化百泉保健食品有限公司 | A kind of industrialized producing technology of trotter albumen oligopeptide |
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| 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN108396050A (en) * | 2018-03-28 | 2018-08-14 | 通化百泉保健食品有限公司 | A kind of industrialized producing technology of trotter albumen oligopeptide |
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