CN113201567A - Method for extracting antioxidant polypeptide from fish scales by using compound protease - Google Patents
Method for extracting antioxidant polypeptide from fish scales by using compound protease Download PDFInfo
- Publication number
- CN113201567A CN113201567A CN202011072932.9A CN202011072932A CN113201567A CN 113201567 A CN113201567 A CN 113201567A CN 202011072932 A CN202011072932 A CN 202011072932A CN 113201567 A CN113201567 A CN 113201567A
- Authority
- CN
- China
- Prior art keywords
- fish scales
- fish
- compound protease
- scales
- centrifuging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 129
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 26
- 239000004365 Protease Substances 0.000 title claims abstract description 26
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 26
- 150000001875 compounds Chemical class 0.000 title claims abstract description 26
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 25
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 25
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 25
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 25
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 21
- 238000003756 stirring Methods 0.000 claims abstract description 16
- 230000009849 deactivation Effects 0.000 claims abstract description 13
- 238000012545 processing Methods 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 6
- 238000005185 salting out Methods 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 239000007853 buffer solution Substances 0.000 claims description 25
- 239000011780 sodium chloride Substances 0.000 claims description 25
- 239000002244 precipitate Substances 0.000 claims description 23
- 239000006228 supernatant Substances 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 20
- 238000001914 filtration Methods 0.000 claims description 15
- 238000002791 soaking Methods 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 239000012266 salt solution Substances 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 238000001291 vacuum drying Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000005119 centrifugation Methods 0.000 abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 4
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 108091005899 fibrous proteins Proteins 0.000 description 1
- 102000034240 fibrous proteins Human genes 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021190 leftovers Nutrition 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108010048734 sclerotin Proteins 0.000 description 1
Classifications
-
- 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
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Water Supply & Treatment (AREA)
- Meat, Egg Or Seafood Products (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of antioxidant polypeptide extraction, in particular to a method for extracting antioxidant polypeptide from fish scales by using compound protease, which comprises the following steps: s1: fish scale selection and processing, S2: crushing, S3: stirring, S4: first centrifugation, S5: salting out, S6: drying, S7: pretreatment, S8: enzymolysis, S9: enzyme deactivation treatment, S10: second centrifugation, S11: elution, S12: the method for extracting the antioxidant polypeptide from the fish scales by using the compound protease can effectively extract the antioxidant polypeptide from the fish scales, not only improves the economic value of fish scale processing, but also effectively utilizes the fish scales, and reduces the requirement of extracting the antioxidant polypeptide by using other plants.
Description
Technical Field
The invention relates to the technical field of antioxidant polypeptide extraction, in particular to a method for extracting antioxidant polypeptide from fish scales by using compound protease.
Background
China is a big aquatic product country, and aquatic products have wide international markets and are listed as key industrial development projects by the ministry of agriculture. The total yield of aquatic products in China can reach ten thousand tons every year, so that the rapid development of the deep processing technology of the aquatic products is promoted. Especially for processing fish, a large amount of leftovers such as fish scales are produced in the processing process, and the fish scales produced in the processing process are generally discarded, however, the fish scales have excellent efficacy, firstly, the fish scales account for 2-3% of the weight of the fish, and the main characteristics of the scales are as follows: it is transparent in appearance, like petals, with slightly curled edges, white luster, slightly fishy, firm and soft texture, and has a water content ranging from 16.4% to 17.8%, with an average of 17.5%. Moreover, the research of nutriologists finds that the fish scales are special health-care food. It contains more egg scale fat, and can decompose fat globule into emulsion and dissolve with water, and has effects of improving human brain memory and delaying cell aging. Meanwhile, fish scales are sclerotin generated by collagen in the dermis of fish and are known as fish scale scleroprotein. In summary, fish scales have high nutritive value and can not be effectively utilized because the fish scales are directly discarded, and therefore, a method for extracting antioxidant polypeptide from the fish scales by using compound protease is provided.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the method for extracting the antioxidant polypeptide from the fish scales by using the compound protease, and the method can effectively extract the antioxidant polypeptide from the fish scales, thereby not only improving the economic value of fish scale processing, but also effectively utilizing the fish scales, and on the other hand, reducing the demand of extracting the antioxidant polypeptide by using other plants.
The technical scheme of the invention is as follows:
a method for extracting antioxidant polypeptide from fish scales by using compound protease is characterized by comprising the following steps: the method comprises the following steps:
s1: selecting and processing fish scales, selecting fresh fish scales, repeatedly washing the fish scales in water to remove impurities attached to the fish scales, soaking the fish scales in warm water at the temperature of 60-80 ℃ for 60-100min after washing is finished, removing part of fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales, soaking the fish scales in a salt solution with the concentration of 60-90%, soaking the fish scales for 3-6h, removing the rest fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales for later use;
s2: crushing, namely putting the fish scales for later use in S1 into a crusher for crushing, filtering clear water after crushing, and draining for later use after filtering;
s3: stirring, namely putting the crushed fish scales in the S2 into a stirrer, adding water while stirring, controlling the temperature at 30-40 ℃ for 1-2 days;
s4: centrifuging for the first time, namely placing the stirred fish scales in S3 in a high-speed centrifuge, wherein the revolution of the centrifuge is 5000-12000 r/min, centrifuging for 30-60min, and collecting supernatant;
s5: salting out, adding NaCl into the supernatant in S4 to enable the concentration of NaCl to be 4-5 mol/L, stirring for 12-24 h, standing for 12-24 h, centrifuging for 20-40 min at the rotation number of 5000-10000 r/min, removing the supernatant, dissolving the precipitate in a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, wherein each 1L of the buffer solution contains 1mol of NaCl and 0.1mol of tris (hydroxymethyl) aminomethane, adding HCl to adjust the pH value to 7.2-7.6, enabling the mass volume ratio of the fish scales to the buffer solution to be 1: 20-50 g/mL, adding NaCl into the buffer solution to enable the concentration of NaCl to be 1.7-2.4 mol/L, standing for 12h, and centrifuging for 30-60min at the rotation number of 5000-10000 r/min to obtain the precipitate;
s6: drying, namely pre-freezing the precipitate, and putting the precipitate into a freeze dryer for vacuum freeze drying to obtain fish scale protein;
s7: pretreating, namely preparing the fish scale protein prepared in the S6 into an aqueous solution with the substrate concentration of 3-6%, quickly heating the solution to 80-85 ℃ after dissolving, and keeping the temperature for 10-15 min;
s8: performing enzymolysis, adding phosphoric acid buffer solution to adjust pH to 7.0-8.5 after pretreatment, adding compound protease, performing enzymolysis at 45-60 deg.C with enzyme dosage of 6-9% of substrate concentration for 60-120 min;
s9: enzyme deactivation, namely rapidly heating the enzymolysis liquid to 90 ℃, and keeping for 20min for enzyme deactivation;
s10: centrifuging for the second time, rapidly cooling to room temperature after enzyme deactivation, centrifuging for 15-20min at 3000-4000r/min, and collecting supernatant;
s11: elution, as 100 g: adding 120ml of trypsin enzymolysis liquid into wet macroporous adsorption resin DA201-C in the proportion of 100-;
s12: vacuum drying and concentrating, collecting the eluent prepared in S11, and vacuum drying and concentrating.
Further, in the step S1, the ratio between the fish scales and the salt solution is 1: 40-60.
Furthermore, the power of the crusher in S2 is 2000-3000W, the rotating speed is 100-300 r/min, and the filtering is carried out by adopting a multi-layer ultrafiltration membrane.
Further, the rotating speed of the stirrer in the S3 is 30-60 r/min.
Further, the temperature of the freeze dryer in the S6 is-10-0 ℃, and the time is 100-180 h.
The invention has the beneficial effects that: the method for extracting the antioxidant polypeptide from the fish scales by using the compound protease can effectively extract the antioxidant polypeptide from the fish scales, not only improves the economic value of fish scale processing, but also effectively utilizes the fish scales, and reduces the demand of extracting the antioxidant polypeptide by using other plants.
Detailed Description
The following further illustrates embodiments of the invention:
example 1
A method for extracting antioxidant polypeptide from fish scales by using compound protease comprises the following steps:
s1: selecting and processing fish scales, namely selecting fresh fish scales, repeatedly washing the fresh fish scales in water to remove impurities attached to the fish scales, soaking the fish scales in warm water at 60 ℃ for 60min after washing is finished, removing part of fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales, soaking the fish scales in a salt solution with the concentration of 60%, soaking the fish scales for 3h, removing the residual fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales for later use;
s2: crushing, namely putting the fish scales for later use in S1 into a crusher for crushing, filtering clear water after crushing, and draining for later use after filtering;
s3: stirring, namely putting the crushed fish scales in the S2 into a stirrer, adding water while stirring, and controlling the temperature at 30 ℃ for 1 day;
s4: centrifuging for the first time, namely placing the stirred fish scales in the S3 into a high-speed centrifuge, wherein the revolution number of the centrifuge is 5000r/min, centrifuging for 30min, and collecting supernatant;
s5: salting out, adding NaCl into the supernatant in S4 to make the concentration of NaCl 4mol/L, stirring for 12h, standing for 12h, centrifuging for 20min at the revolution of 5000r/min, removing the supernatant, taking the precipitate, dissolving the precipitate in a tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution, wherein each 1L of the buffer solution contains 1mol of NaCl and 0.1mol of tris-hydroxymethyl aminomethane, adding HCl to adjust the pH value to 7.2, and the mass-to-volume ratio of the fish scales to the buffer solution is 1:20g/mL, adding NaCl into the buffer solution to make the concentration of NaCl 1.7mol/L, standing for 12h, centrifuging for 30min at the revolution of 5000r/min to obtain the precipitate;
s6: drying, namely pre-freezing the precipitate, and putting the precipitate into a freeze dryer for vacuum freeze drying to obtain fish scale protein;
s7: pretreating, namely preparing the fish scale protein prepared in the S6 into an aqueous solution with the substrate concentration of 3%, quickly heating the solution to 80 ℃ after dissolving, and keeping the temperature for 10 min;
s8: performing enzymolysis, adding a phosphoric acid buffer solution to adjust the pH value to 7.0 after pretreatment, adding compound protease, performing enzymolysis at 45 ℃ for 60min, wherein the enzyme dosage is 6% of the substrate concentration;
s9: enzyme deactivation, namely rapidly heating the enzymolysis liquid to 90 ℃, and keeping for 20min for enzyme deactivation;
s10: centrifuging for the second time, inactivating enzyme, rapidly cooling to room temperature, centrifuging at 3000r/min for 15min, and collecting supernatant;
s11: elution, as 100 g: adding 100ml of the trypsin enzymolysis solution into wet macroporous adsorption resin DA201-C, keeping in a constant temperature shaking table at 25 ℃ for 180min at the rotating speed of 150r/min, and sequentially eluting with 25%, 50%, 75% and 100% ethanol solution for 120 min;
s12: vacuum drying and concentrating, collecting the eluent prepared in S11, and vacuum drying and concentrating.
The ratio of fish scales to the salt solution in the S1 is 1: 40.
and in the S2, the power of the crusher is 2000W, the rotating speed is 100r/min, and the filtering is carried out by adopting a multi-layer ultrafiltration membrane.
And the rotating speed of the stirrer in the S3 is 30 r/min.
The temperature of the freeze dryer in the S6 is-10 ℃, and the time is 100 h.
Example 2
A method for extracting antioxidant polypeptide from fish scales by using compound protease comprises the following steps:
s1: selecting and processing fish scales, namely selecting fresh fish scales, repeatedly washing the fresh fish scales in water to remove impurities attached to the fish scales, soaking the fish scales in warm water at 80 ℃ for 100min after washing is finished, removing part of fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales, soaking the fish scales in a salt solution with the concentration of 90%, soaking the fish scales for 6h, removing the residual fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales for later use;
s2: crushing, namely putting the fish scales for later use in S1 into a crusher for crushing, filtering clear water after crushing, and draining for later use after filtering;
s3: stirring, namely putting the crushed fish scales in the S2 into a stirrer, adding water while stirring, and controlling the temperature at 40 ℃ for 2 days;
s4: centrifuging for the first time, namely placing the stirred fish scales in S3 in a high-speed centrifuge, wherein the revolution number of the centrifuge is 12000r/min, centrifuging for 60min, and collecting supernatant;
s5: salting out, adding NaCl into the supernatant in S4 to make the concentration of NaCl 5mol/L, stirring for 24h, standing for 24h, centrifuging for 40min at the rotation number of 10000r/min, removing the supernatant, taking the precipitate, dissolving the precipitate in a tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution, wherein each 1L of the buffer solution contains 1mol of NaCl and 0.1mol of tris-hydroxymethyl aminomethane, adding HCl to adjust the pH value to 7.6, and the mass-volume ratio of the fish scales to the buffer solution is 1:50g/mL, adding NaCl into the buffer solution to make the concentration of NaCl 2.4mol/L, standing for 12h, centrifuging for 60min at the rotation number of 10000r/min to obtain the precipitate;
s6: drying, namely pre-freezing the precipitate, and putting the precipitate into a freeze dryer for vacuum freeze drying to obtain fish scale protein;
s7: pretreating, namely preparing the fish scale protein prepared in the S6 into an aqueous solution with the substrate concentration of 6%, and quickly heating the solution to 85 ℃ after dissolving, and keeping the temperature for 15 min;
s8: performing enzymolysis, adding a phosphoric acid buffer solution to adjust the pH value to 8.5 after pretreatment, adding compound protease, performing enzymolysis at 60 ℃ for 120min, wherein the enzyme dosage is 9% of the substrate concentration;
s9: enzyme deactivation, namely rapidly heating the enzymolysis liquid to 90 ℃, and keeping for 20min for enzyme deactivation;
s10: centrifuging for the second time, inactivating enzyme, rapidly cooling to room temperature, centrifuging at 4000r/min for 20min, and collecting supernatant;
s11: elution, as 100 g: adding 120ml of the trypsin enzymolysis solution into wet macroporous adsorption resin DA201-C, maintaining in a constant temperature shaking table at 25 ℃ for 240min at the rotation speed of 200r/min, and sequentially eluting with 25%, 50%, 75% and 100% ethanol solution for 150 min;
s12: vacuum drying and concentrating, collecting the eluent prepared in S11, and vacuum drying and concentrating.
The ratio of fish scales to the salt solution in the S1 is 1: 60.
and in the S2, the power of the crusher is 3000W, the rotating speed is 300r/min, and the filtering is carried out by adopting a multi-layer ultrafiltration membrane.
And the rotating speed of the stirrer in the S3 is 60 r/min.
The temperature of the freeze dryer in the S6 is 0 ℃, and the time is 180 h.
Example 3
A method for extracting antioxidant polypeptide from fish scales by using compound protease comprises the following steps:
s1: selecting and processing fish scales, namely selecting fresh fish scales, repeatedly washing the fresh fish scales in water to remove impurities attached to the fish scales, soaking the fish scales in warm water at 70 ℃ for 80min after washing is finished, removing part of fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales, soaking the fish scales in a salt solution with the concentration of 75%, soaking the fish scales for 4.5h, removing the residual fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales for later use;
s2: crushing, namely putting the fish scales for later use in S1 into a crusher for crushing, filtering clear water after crushing, and draining for later use after filtering;
s3: stirring, namely putting the crushed fish scales in the S2 into a stirrer, adding water while stirring, and controlling the temperature at 35 ℃ for 1.5 days;
s4: centrifuging for the first time, namely placing the stirred fish scales in the S3 into a high-speed centrifuge, wherein the revolution number of the centrifuge is 8500r/min, centrifuging for 45min, and collecting supernatant;
s5: salting out, adding NaCl into the supernatant in S4 to make the concentration of NaCl be 4.5mol/L, stirring for 18h, standing for 18h, centrifuging for 30min at the revolution of 7500r/min, removing the supernatant, taking the precipitate, dissolving the precipitate in a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, wherein each 1L of the buffer solution contains 1mol of NaCl and 0.1mol of tris (hydroxymethyl) aminomethane, adding HCl to adjust the pH value to 7.4, and the mass-volume ratio of the fish scales to the buffer solution is 1:35g/mL, adding NaCl into the buffer solution to make the concentration of NaCl be 2.0mol/L, standing for 12h, centrifuging for 45min at the revolution of 7500r/min to obtain the precipitate;
s6: drying, namely pre-freezing the precipitate, and putting the precipitate into a freeze dryer for vacuum freeze drying to obtain fish scale protein;
s7: pretreating, namely preparing the fish scale protein prepared in the S6 into an aqueous solution with the substrate concentration of 4.5%, quickly heating the solution to 82.5 ℃ after dissolving, and keeping the temperature for 12.5 min;
s8: performing enzymolysis, adding a phosphoric acid buffer solution to adjust the pH value to 7.75 after pretreatment, adding compound protease, performing enzymolysis at the temperature of 52.5 ℃ and the enzyme dosage of 7.5 percent of the substrate concentration for 90 min;
s9: enzyme deactivation, namely rapidly heating the enzymolysis liquid to 90 ℃, and keeping for 20min for enzyme deactivation;
s10: centrifuging for the second time, inactivating enzyme, rapidly cooling to room temperature, centrifuging at 3500r/min for 17.5min, and collecting supernatant;
s11: elution, as 100 g: adding 110ml of the trypsin enzymolysis solution into wet macroporous adsorption resin DA201-C, keeping the temperature in a constant temperature shaking table at 25 ℃ for 210min, keeping the rotation speed at 175r/min, and sequentially eluting with 25%, 50%, 75% and 100% ethanol solution for 135 min;
s12: vacuum drying and concentrating, collecting the eluent prepared in S11, and vacuum drying and concentrating.
The ratio of fish scales to the salt solution in the S1 is 1: 50.
and in the S2, the power of the crusher is 2500W, the rotating speed is 200r/min, and the filtering is carried out by adopting a plurality of layers of ultrafiltration membranes.
And the rotating speed of the stirrer in the S3 is 45 r/min.
The temperature of the freeze dryer in the S6 is-5 ℃, and the time is 140 h.
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
Claims (5)
1. A method for extracting antioxidant polypeptide from fish scales by using compound protease is characterized by comprising the following steps: the method comprises the following steps:
s1: selecting and processing fish scales, selecting fresh fish scales, repeatedly washing the fish scales in water to remove impurities attached to the fish scales, soaking the fish scales in warm water at the temperature of 60-80 ℃ for 60-100min after washing is finished, removing part of fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales, soaking the fish scales in a salt solution with the concentration of 60-90%, soaking the fish scales for 3-6h, removing the rest fishy smell, repeatedly washing the fish scales with clean water and draining the fish scales for later use;
s2: crushing, namely putting the fish scales for later use in S1 into a crusher for crushing, filtering clear water after crushing, and draining for later use after filtering;
s3: stirring, namely putting the crushed fish scales in the S2 into a stirrer, adding water while stirring, controlling the temperature at 30-40 ℃ for 1-2 days;
s4: centrifuging for the first time, namely placing the stirred fish scales in S3 in a high-speed centrifuge, wherein the revolution number of the centrifuge is 5000-12000 r/min, centrifuging for 30-60min, and collecting supernatant;
s5: salting out, adding NaCl into the supernatant in S4 to enable the concentration of NaCl to be 4-5 mol/L, stirring for 12-24 h, standing for 12-24 h, centrifuging for 20-40 min at the rotation number of 5000-10000 r/min, removing the supernatant, dissolving the precipitate in a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, wherein each 1L of the buffer solution contains 1mol of NaCl and 0.1mol of tris (hydroxymethyl) aminomethane, adding HCl to adjust the pH value to 7.2-7.6, enabling the mass volume ratio of the fish scales to the buffer solution to be 1: 20-50 g/mL, adding NaCl into the buffer solution to enable the concentration of NaCl to be 1.7-2.4 mol/L, standing for 12h, and centrifuging for 30-60min at the rotation number of 5000-10000 r/min to obtain the precipitate;
s6: drying, namely pre-freezing the precipitate, and putting the precipitate into a freeze dryer for vacuum freeze drying to obtain fish scale protein;
s7: pretreating, namely preparing the fish scale protein prepared in the S6 into an aqueous solution with the substrate concentration of 3-6%, quickly heating the solution to 80-85 ℃ after dissolving, and keeping the temperature for 10-15 min;
s8: performing enzymolysis, adding phosphoric acid buffer solution to adjust pH to 7.0-8.5 after pretreatment, adding compound protease, performing enzymolysis at 45-60 deg.C with enzyme dosage of 6-9% of substrate concentration for 60-120 min;
s9: enzyme deactivation, namely rapidly heating the enzymolysis liquid to 90 ℃, and keeping for 20min for enzyme deactivation;
s10: centrifuging for the second time, rapidly cooling to room temperature after enzyme deactivation, centrifuging for 15-20min at 3000-4000r/min, and collecting supernatant;
s11: elution, as 100 g: adding 120ml of trypsin enzymolysis liquid into wet macroporous adsorption resin DA201-C in the proportion of 100-;
s12: vacuum drying and concentrating, collecting the eluent prepared in S11, and vacuum drying and concentrating.
2. The method for extracting antioxidant polypeptide from fish scales by using compound protease as claimed in claim 1, wherein the compound protease is selected from the group consisting of: the ratio of fish scales to the salt solution in the S1 is 1: 40-60.
3. The method for extracting antioxidant polypeptide from fish scales by using compound protease as claimed in claim 1, wherein the compound protease is selected from the group consisting of: the power of the crusher in the S2 is 2000-3000W, the rotating speed is 100-300 r/min, and the filtering is carried out by adopting a multi-layer ultrafiltration membrane.
4. The method for extracting antioxidant polypeptide from fish scales by using compound protease as claimed in claim 1, wherein the compound protease is selected from the group consisting of: and the rotating speed of the stirrer in the S3 is 30-60 r/min.
5. The method for extracting antioxidant polypeptide from fish scales by using compound protease as claimed in claim 1, wherein the compound protease is selected from the group consisting of: the temperature of the freeze dryer in the S6 is-10-0 ℃, and the time is 100-180 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011072932.9A CN113201567A (en) | 2020-10-09 | 2020-10-09 | Method for extracting antioxidant polypeptide from fish scales by using compound protease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011072932.9A CN113201567A (en) | 2020-10-09 | 2020-10-09 | Method for extracting antioxidant polypeptide from fish scales by using compound protease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113201567A true CN113201567A (en) | 2021-08-03 |
Family
ID=77025058
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011072932.9A Pending CN113201567A (en) | 2020-10-09 | 2020-10-09 | Method for extracting antioxidant polypeptide from fish scales by using compound protease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113201567A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014106A (en) * | 2012-12-03 | 2013-04-03 | 南昌大学 | Extraction method for antioxidant polypeptide in Guangchang nymphaea alba |
| CN106892965A (en) * | 2017-02-08 | 2017-06-27 | 福州大学 | Antioxidation polypeptide prepared by a kind of utilization compound protease |
| CN107312084A (en) * | 2017-07-20 | 2017-11-03 | 温州科技职业学院 | A kind of collagen extraction process based on little yellow croaker fish scale |
| CN108017448A (en) * | 2017-12-11 | 2018-05-11 | 常州市协旺纺织品有限公司 | A kind of preparation method of type soybean nucleotide fertilizer steady in a long-term |
| CN109438571A (en) * | 2018-12-29 | 2019-03-08 | 中港（福建）水产食品有限公司 | A method of collagen fabric body is extracted from marine product leftover bits and pieces |
-
2020
- 2020-10-09 CN CN202011072932.9A patent/CN113201567A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014106A (en) * | 2012-12-03 | 2013-04-03 | 南昌大学 | Extraction method for antioxidant polypeptide in Guangchang nymphaea alba |
| CN106892965A (en) * | 2017-02-08 | 2017-06-27 | 福州大学 | Antioxidation polypeptide prepared by a kind of utilization compound protease |
| CN107312084A (en) * | 2017-07-20 | 2017-11-03 | 温州科技职业学院 | A kind of collagen extraction process based on little yellow croaker fish scale |
| CN108017448A (en) * | 2017-12-11 | 2018-05-11 | 常州市协旺纺织品有限公司 | A kind of preparation method of type soybean nucleotide fertilizer steady in a long-term |
| CN109438571A (en) * | 2018-12-29 | 2019-03-08 | 中港（福建）水产食品有限公司 | A method of collagen fabric body is extracted from marine product leftover bits and pieces |
Non-Patent Citations (2)
| Title |
|---|
| 张丰香等: "鱼鳞明胶生产的浸酸脱钙工艺研究", 食品工业科技, vol. 29, no. 3, pages 199 - 201 * |
| 张俊杰等: "鱼鳞胶的制备及成分测定", 河北理工大学学报, vol. 30, no. 4, pages 127 - 129 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101643767B (en) | Method for preparing almond peptide from almond dregs | |
| CN112062834B (en) | Deep sea fish skin collagen peptide and extraction and preparation method thereof | |
| CN101979532A (en) | Method for comprehensively using pig blood | |
| CN104313096A (en) | Method for preparing beluga swimming bladder protein peptide | |
| CN109400699A (en) | The preparation method of turtle peptide | |
| CN109337949A (en) | A kind of deep-sea fish skin collagen peptide | |
| CN104381990B (en) | The preparation method of rich peptide calcium Acetes beans | |
| CN113201567A (en) | Method for extracting antioxidant polypeptide from fish scales by using compound protease | |
| KR100502653B1 (en) | Method for producing of Ginseng-Toenjang | |
| CN107232613B (en) | Method for extracting soybean fiber and bean dreg protein by combining ultrasonic-assisted hot alkaline process | |
| CN106337074A (en) | Cirrhinus molitorella bone collagen extracting method | |
| CN105614761A (en) | Kelp deep-processing method | |
| CN1164192C (en) | Production process of dewatered mulberry leaf | |
| CN113826886A (en) | Radix puerariae enzyme and preparation method thereof | |
| CN110016077A (en) | A kind of abalone hemocyanin extracting method | |
| CN111675769A (en) | Sea cucumber extract and preparation process thereof | |
| CN106036847B (en) | The method for preparing the low bitter functional food of low raw meat using Tilapia acid-soluble protein | |
| CN110684814A (en) | Preparation method of tuna skin collagen peptide iron chelate | |
| CN110283106A (en) | A method of it is synchronous from Pinctada martensii to extract biological zinc and taurine | |
| AU2021104991A4 (en) | Preparation Method for Tilapia (Oreochromis niloticus) Calcium ion-binding peptides and Application thereof | |
| CN111264676B (en) | Fish gelatin and preparation method thereof, and fish jelly and preparation method thereof | |
| CN113201064A (en) | Method for preparing fish skin collagen powder from fresh fish skin | |
| CN112210579B (en) | Tilapia calcium ion binding peptide and preparation method and application thereof | |
| CN114262728A (en) | Preparation method of hairtail protein peptide chelated ferrous iron | |
| CN106191185A (en) | A kind of method utilizing Rhopilema esculenta processing fent to prepare anti-oxidation peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chen Yibin Inventor after: Yang Wei Inventor before: Chen Yibin |
|
| CB03 | Change of inventor or designer information | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210803 |
|
| WD01 | Invention patent application deemed withdrawn after publication |