CN117551186A - Method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis - Google Patents
Method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis Download PDFInfo
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 78
- 102000008186 Collagen Human genes 0.000 title claims abstract description 65
- 108010035532 Collagen Proteins 0.000 title claims abstract description 65
- 229920001436 collagen Polymers 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 53
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 49
- 238000001914 filtration Methods 0.000 claims abstract description 42
- 238000009835 boiling Methods 0.000 claims abstract description 38
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 150000008442 polyphenolic compounds Chemical class 0.000 claims abstract description 17
- 235000013824 polyphenols Nutrition 0.000 claims abstract description 17
- 238000004108 freeze drying Methods 0.000 claims abstract description 14
- 235000018553 tannin Nutrition 0.000 claims abstract description 13
- 229920001864 tannin Polymers 0.000 claims abstract description 13
- 239000001648 tannin Substances 0.000 claims abstract description 13
- 239000012530 fluid Substances 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 4
- 241000251468 Actinopterygii Species 0.000 claims description 59
- 229940088598 enzyme Drugs 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 31
- 239000012466 permeate Substances 0.000 claims description 31
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 30
- 238000004140 cleaning Methods 0.000 claims description 14
- 239000011229 interlayer Substances 0.000 claims description 14
- 239000002994 raw material Substances 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 14
- 239000000706 filtrate Substances 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 10
- 235000013372 meat Nutrition 0.000 claims description 10
- 239000012465 retentate Substances 0.000 claims description 10
- 238000010257 thawing Methods 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 8
- 238000007599 discharging Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 238000001728 nano-filtration Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 108090000145 Bacillolysin Proteins 0.000 claims description 2
- 108091005658 Basic proteases Proteins 0.000 claims description 2
- 102000029816 Collagenase Human genes 0.000 claims description 2
- 108060005980 Collagenase Proteins 0.000 claims description 2
- 108091005507 Neutral proteases Proteins 0.000 claims description 2
- 102000035092 Neutral proteases Human genes 0.000 claims description 2
- 108090000526 Papain Proteins 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 108090000631 Trypsin Proteins 0.000 claims description 2
- 229960002424 collagenase Drugs 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 229940055729 papain Drugs 0.000 claims description 2
- 235000019834 papain Nutrition 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 102000004400 Aminopeptidases Human genes 0.000 claims 1
- 108090000915 Aminopeptidases Proteins 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 17
- 230000003013 cytotoxicity Effects 0.000 abstract description 10
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 10
- 239000000047 product Substances 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 6
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- 239000012716 precipitator Substances 0.000 abstract 1
- 238000006460 hydrolysis reaction Methods 0.000 description 13
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- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
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- 230000001376 precipitating effect Effects 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 235000008694 Humulus lupulus Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 230000006578 abscission Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
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- 239000003513 alkali Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- -1 hydrogen ions Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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Classifications
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- 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]
-
- 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
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for preparing collagen peptide by utilizing countercurrent immobilized enzyme column enzymolysis, which comprises the following steps: s1, obtaining an extracting solution; s2, boiling, centrifuging and filtering; s3, immobilizing an enzyme column for the first time; s4, immobilizing the enzyme column for the second time; s5, adding a precipitator for boiling; s6, membrane treatment; s7, freeze drying: freeze-drying the trapped fluid C in the step S6 to obtain cod collagen peptide; the collagen peptide is obtained by adding a tannin polyphenol precipitant after enzymolysis through a twice boiling collagen extraction process and a countercurrent immobilized enzyme column enzymolysis process. The cytotoxicity of the product is reduced by the twice boiling process, the enzymolysis process of the immobilized enzyme column can greatly shorten the enzymolysis time, improve the enzymolysis efficiency, and the immobilized enzyme columns can be flexibly connected in series and in parallel through valves, so that the method is suitable for the production of collagen peptides with different molecular weight requirements and different functional requirements; for macromolecular proteins which possibly remain after enzymolysis, the immunogenicity and cytotoxicity of the final product are greatly reduced by adding a polyphenol precipitant, boiling and filtering or centrifuging.
Description
Technical Field
The invention belongs to the technical field of collagen peptide preparation, and particularly relates to a method for preparing collagen peptide by utilizing countercurrent immobilized enzyme column enzymolysis.
Background
With the development of fishery, the processing of aquatic products is attracting more and more attention. The fish skin contains abundant proteins, and can account for more than 70% of the dry weight of the fish skin, wherein the maximum content is collagen.
Collagen is an important functional protein, is closely related to cell proliferation, differentiation, exercise, immunity, joint lubrication, wound healing and the like, and directly affects the growth, aging, health and the like of a human body. In addition, collagen has weak antigenicity and good biocompatibility, and is widely applied to various fields such as medicines, foods, daily chemical industry, biosynthesis and the like.
The collagen extracted from animal tissue has unique triple supercoiled structure, so that it is difficult to digest and utilize, and the collagen is further hydrolyzed into collagen polypeptide to obviously improve absorption and functional characteristics.
The existing method mainly adopts hot water, acid method, alkali method and other methods to extract collagen from raw materials, then further hydrolyzes to obtain collagen peptide, and the collagen hydrolysis is free enzymatic hydrolysis at present, namely the protease is directly added into an enzymolysis tank, reacts for a period of time at a certain temperature, and then is subjected to enzyme deactivation treatment. The method has longer enzymolysis reaction period, is disposable for enzyme use, increases the use cost of the enzyme, and can remain in the product as foreign matters, thereby leading to higher immunogenicity and cytotoxicity of the final product.
Disclosure of Invention
The invention aims to design a method for preparing collagen peptide by utilizing countercurrent immobilized enzyme column enzymolysis, the enzymolysis process of the immobilized enzyme column can greatly shorten the enzymolysis time, and the immobilized enzyme columns can be flexibly connected in series and in parallel through valves, so that the method is suitable for the production of collagen peptide with different molecular weight requirements and different functional requirements.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for preparing collagen peptide by utilizing countercurrent immobilized enzyme column enzymolysis is characterized in that: the method comprises the following steps:
s1, obtaining an extracting solution: extracting the fish skin raw material to obtain collagen extracting solution;
s2, first boiling centrifugal filtration: boiling at 100deg.C for 2-100min, centrifuging or filtering to obtain filtrate or supernatant, and removing grease or other floating substances and impurity protein precipitate; collagen is a class of proteins that are soluble in hot water, while most of the proteins precipitate after boiling. Centrifuging or filtering to remove impurity protein or impurity insoluble in hot water;
s3, immobilizing the enzyme column for the first time: loading the filtrate or supernatant into a column in a countercurrent mode, wherein the flow rate is 8.5-25000 ml/min, the time required for 50kg of raw materials to flow through the enzyme column is 0.5-2h, the temperature of the interlayer insulation of the immobilized column is 10-80 ℃, and the enzymolysis liquid is discharged from the top of the first immobilized enzyme column;
s4, a second immobilized enzyme column: s3, allowing the enzymolysis liquid in the S3 to enter a second immobilized enzyme column in a countercurrent mode, keeping the temperature of the interlayer of the immobilized enzyme column at 10-80 ℃, and discharging the enzymolysis liquid from the top;
the immobilized enzyme is adopted in the step of the invention, which has good process stability, and can greatly reduce the use cost of the enzyme for some enzymes with higher prices; the protease can be reused in the repeated use process after being immobilized by simple flushing.
The enzyme column is adopted to have the synergistic effect of various proteases, so that the substrate can be subjected to catalytic reaction at different enzyme active centers, the catalytic efficiency is improved, and the hydrolysis effect which is difficult to achieve by single enzyme catalysis can be achieved; the continuous catalytic reaction of the multi-enzyme column reduces the loss of the substrate in the reaction process, catalyzes the substrate into smaller molecular weight and improves the substrate utilization rate. The collagen peptide obtained by the hydrolysis reaction of the enzyme columns is different in products, the amino acid sequences of the products are different, the corresponding antioxidant activity, the functions of enhancing bone density and calcium supplement, reducing photooxidation and the like are different, and the multi-enzyme column is used in series-parallel recombination, so that convenient selection is provided for the production of various functional collagen proteins.
Protein concentration measurement the amount of protein obtained was 1:1-100 (immobilized enzyme to protein mass ratio) is subjected to enzymolysis at 40-70 ℃ for 0.5-6h,
s5, adding a precipitant for secondary boiling: adding tannin polyphenol precipitant or hops polyphenol into the solution after enzymolysis, boiling at 100 ℃ for 5-100 minutes, and centrifuging or filtering; obtaining filtered liquid or supernatant, and removing impurity proteins and sediment; the precipitant adopted in the step has good reducibility and complexation, can be combined with macromolecules such as protein, loses part of hydrogen ions on hydroxyl groups under acidic conditions, forms ions with negative charges, combines the ions with amino acid residues on macromolecular proteins or a small amount of abscission enzymes, forms insoluble complexes, and is removed by centrifugation or filtration;
s6, film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; continuously performing membrane filtration on the permeate A by using a 1000 Da ultrafiltration membrane to obtain trapped fluid B and permeate B; filtering the permeate B with a 100D nanofiltration membrane to obtain a retentate C and a permeate; the filter Tao Lvmo has a decoloring function, and the filter 5000 Da is used for intercepting a solution with a molecular weight greater than 5000 and transmitting a solution with a molecular weight less than 5000; ultrafiltration through 1000 Da is to entrap more than 1000 molecular weight, permeate less than 1000 molecular weight solution; ultrafiltration through 100D is to intercept more than 100 molecular weight, and to concentrate through less than 100 molecular weight solution (water); the small molecular collagen peptide can obviously improve the absorbability and the functional property;
s7, freeze drying: and (3) freeze-drying the trapped fluid C in the step (S6) to obtain the cod collagen peptide.
Further, the obtained extracting solution is specifically used for thawing the fish skin, preprocessing the fish skin and extracting the fish skin at a controlled temperature.
Further, the specific method for obtaining the extracting solution comprises the following steps:
the first step is thawing of the fish skin: thawing fresh frozen fish skin raw material at 0-15deg.C, and removing fish scales and fish meat;
the second step is pretreatment of the fish skin: firstly, treating the fishskin in NaOH with the temperature of 0-15 ℃ and the temperature of 0.01-1.0M for 1-24 hours, then treating the fishskin with a detergent for 1-24 hours, cleaning the fishskin to be neutral, and crushing the fishskin;
the third step is temperature control extraction: the pretreated fish skin is prepared by the mass and volume ratio of 1: heating and extracting at 50-120deg.C for 0.5-12 hr at 5-100 deg.C.
Further, the fish skin is treated with dilute acid solution including but not limited to acetic acid, citric acid and hydrochloric acid to eliminate acid soluble protein.
Further, the enzymes of the immobilized enzyme column comprise one or more of immobilized papain, immobilized alkaline protease, immobilized collagenase, immobilized trypsin and immobilized neutral protease.
Further, the immobilized enzyme column enzymolysis is performed at least once again after the second immobilized enzyme column enzymolysis.
Furthermore, the immobilized enzyme columns are connected in series or in parallel.
The following beneficial effects can be obtained through the technical scheme:
the collagen peptide is finally obtained through twice boiling collagen extraction process and countercurrent immobilized enzyme column enzymolysis process, adding tannin polyphenol precipitant after enzymolysis, and membrane treatment. The obtained collagen peptide has small molecular weight and obvious absorption and functional characteristics improvement; the cytotoxicity of the product is reduced by the twice boiling process, the enzymolysis time of the immobilized enzyme column is greatly shortened, the enzymolysis efficiency is improved, the immobilized enzyme columns can be flexibly connected in series and in parallel through valves, and the method is suitable for the production of collagen peptides with different molecular weight requirements and different functional requirements; for macromolecular proteins which possibly remain after enzymolysis, the immunogenicity and cytotoxicity of the final product are greatly reduced by adding a polyphenol precipitant, boiling and filtering or centrifuging.
The invention adopts the countercurrent immobilized enzyme column enzymolysis technology, well makes up the defect of free enzyme, shortens the reaction period, can be repeatedly used (more than 10 times), greatly improves the use efficiency of enzyme, reduces the use cost, has high product yield and good quality, and has low immunogenicity and cytotoxicity of the final product.
Drawings
FIG. 1 is a graph showing the effect of different types of enzyme column flow rates on the degree of hydrolysis.
FIG. 2 shows the effect of the flow rate of the immobilized enzyme column on the degree of hydrolysis.
FIG. 3 shows the effect of the temperature of the immobilized column interlayer on the degree of hydrolysis.
FIG. 4 shows the molecular weight distribution of each of the enzyme columns at 12500 and 25000 ml/min.
FIG. 5 shows that the molecular weight of each enzyme column is less than 1000 at 12500 and 25000 ml/min.
Fig. 6 is a process flow diagram.
FIG. 7 is a graph showing cytotoxicity analysis of collagen peptide obtained by two boiling and polyphenol precipitant processes.
FIG. 8 is a graph of cytotoxicity analysis of collagen peptide in a single boiling process.
Detailed Description
The invention is further described below with reference to fig. 1-8:
in the invention, as shown in figure 6, the collagen extracting solution is obtained by the steps of fish skin raw material-low-temperature running water thawing-fish skin pretreatment, crushing and temperature control extraction, namely, the collagen extracting solution is boiled at 100 ℃ for 2-100min, centrifugated or filtered, immobilized enzyme column 1, immobilized enzyme column 2, immobilized enzyme column 3, enzymatic hydrolysate is added with polyphenol precipitant, and boiled for 5-100min, centrifugated or filtered, subjected to double-membrane treatment and freeze-dried, and the fish skin collagen peptide is obtained.
Examples
A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis comprises the following specific steps:
1) Fish skin raw material: selecting fresh fish skin raw materials, thawing and cleaning at 0 ℃, and removing fish scales and fish meat for later use;
2) Pre-treating fish skin, namely soaking the fish skin in 0.1M NaOH solution at the temperature of 0 ℃ for 1h, cleaning to be neutral, treating the fish skin once by 1h in 0.1M NaOH solution at the temperature of 0 ℃, and then treating the fish skin by a detergent for 1h, and cleaning to be neutral for later use;
3) Crushing: crushing the fish skin by a meat grinder with a pore diameter of 2 mm;
4) And (3) temperature control extraction: accurately weighing 50kg of pretreated fishskin, and mixing with 1:10 proportion of feed liquid, heating for 12h at 50 ℃;
5) First boiling and filtering: boiling the extractive solution at 100deg.C for 2 min, and filtering to remove oil or other floating substances and precipitate;
6) Immobilized enzyme column: loading the filtrate on a column in a countercurrent mode, wherein the flow rate is 85 ml/min, the temperature of the interlayer insulation of the immobilized column is 10 ℃, and discharging the filtrate from the top of the immobilized enzyme column to obtain enzymolysis liquid;
7) Adding tannin polyphenol precipitant for secondary boiling: adding tannin polyphenol precipitant into the enzymatic hydrolysate after enzymatic hydrolysis, heating and boiling for 30 minutes at 100 ℃, and filtering;
8) Filtering with a filter flask and a filter paper plate to remove foreign proteins and sediments to obtain filtrate or supernatant;
9) Film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; filtering the permeate A with 1000 Da ultrafiltration membrane to obtain retentate B and permeate B; filtering the permeate B with 100D nanofiltration membrane to obtain retentate C and permeate;
10 Freeze drying: and freeze-drying the trapped liquid C to obtain the cod collagen peptide.
Examples
A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis comprises the following specific steps:
1) Fish skin raw material: selecting fresh fish skin raw materials, thawing and cleaning at 15 ℃, and removing fish scales and fish meat for later use;
2) Pre-treating fish skin, namely soaking the fish skin in a 15 ℃ 0.1M NaOH solution for 12h, cleaning to neutrality, treating the fish skin with the 15 ℃ 0.1M NaOH solution for 12h, and then treating the fish skin with a detergent for 12 hours, and cleaning to neutrality for standby;
3) Crushing: crushing the fish skin by a meat grinder with the aperture of 10 mm;
4) And (3) temperature control extraction: accurately weighing 50kg of pretreated fishskin, and mixing with 1:90, heating for 1h at 100 ℃;
5) Boiling and filtering: filtering with filter paper to remove grease or other floating matters and sediment;
6) Immobilized enzyme column 1 (first immobilized enzyme column): loading the filtrate on a column in a countercurrent mode, wherein the flow rate is 25000ml/min, the heat preservation temperature of an immobilized column interlayer is 55 ℃, and discharging the filtrate from the top of an immobilized enzyme column 1;
7) Immobilized enzyme column 2 (second immobilized enzyme column): the enzymolysis liquid flowing out of the immobilized enzyme column 1 enters the immobilized enzyme column 2 in a countercurrent mode, the heat preservation temperature of the interlayer of the immobilized enzyme column is 75 ℃, and the enzymolysis liquid is discharged from the top;
8) Boiling to inactivate enzyme (adding tannin polyphenol precipitant): adding tannin precipitant into the solution after enzymolysis, heating and boiling for 30 minutes, and filtering;
9) Filtering or centrifuging with a plate filter or a tubular centrifuge to remove foreign proteins and precipitate to obtain filtrate or clear liquid;
10 Film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; filtering the permeate A with 1000 Da ultrafiltration membrane to obtain retentate B and permeate B; filtering the permeate B with 100D nanofiltration membrane to obtain retentate C and permeate;
(11) And (3) freeze drying: and freeze-drying the trapped liquid C to obtain the fish skin collagen peptide.
Examples
A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis comprises the following specific steps:
1) Fish skin raw material: selecting fresh fish skin raw materials, thawing and cleaning at 8 ℃ to remove fish scales and fish meat for later use;
2) Pre-treating fish skin, namely soaking the fish skin in a 0.2M NaOH solution at 8 ℃ for 3h, cleaning to be neutral, treating the fish skin once in the 0.2M NaOH solution at 8 ℃ and then treating the fish skin for 3 hours by using a detergent, and cleaning to be neutral for standby;
3) Crushing: crushing the fish skin by a meat grinder with a pore diameter of 5 mm;
4) And (3) temperature control extraction: accurately weighing 50kg of pretreated fishskin, and mixing with 1:50, heating at 80 ℃ for 1.5h;
5) Boiling and filtering: boiling at 100deg.C for 1 hr, filtering with plate filter or tube centrifuge or centrifuging, removing oil or other floating substances, and precipitating to obtain clear liquid;
6) Immobilized enzyme column 1 (first immobilized enzyme column): loading the filtered liquid or the centrifugated clear liquid on a column in a countercurrent mode, wherein the flow rate is 12500ml/min, the heat preservation temperature of an immobilization column interlayer is 40 ℃, and discharging from the top of an immobilization enzyme column 1;
7) Immobilized enzyme column 2 (second immobilized enzyme column): the enzymolysis liquid flowing out of the immobilized enzyme column enters the immobilized enzyme column 2 in a countercurrent mode, the heat preservation temperature of the interlayer of the immobilized column is 40 ℃, and the enzymolysis liquid is discharged from the top;
8) Boiling to inactivate enzyme (adding tannin polyphenol precipitant): adding tannin precipitant into the solution after enzymolysis, heating and boiling for 20 minutes, and filtering or centrifuging to obtain clear liquid;
9) Film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; filtering the permeate A with 1000 Da ultrafiltration membrane to obtain retentate B and permeate B; filtering the permeate B with 100D nanofiltration membrane to obtain retentate C and permeate;
11 Freeze drying: and freeze-drying the trapped liquid C to obtain the cod collagen peptide.
Examples
A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis comprises the following specific steps:
1) Fish skin raw material: selecting fresh fish skin raw materials, thawing and cleaning at 4 ℃ to remove fish scales and fish meat for later use;
2) Pre-treating fish skin, namely soaking the fish skin in a NaOH solution of 0.1M at 4 ℃ for 6h, cleaning to be neutral, treating the fish skin once in the NaOH solution of 0.1M at 4 ℃ and then treating the fish skin for 6 hours by using a detergent, and cleaning to be neutral for standby;
3) Crushing: crushing the fish skin by using a meat grinder with the aperture of 1-10 mm;
4) And (3) temperature control extraction: taking pretreated fish skin, and mixing with 1:20, heating at 90 ℃ for 1.5h;
5) Boiling and filtering: boiling at 100deg.C for 1 hr, filtering with plate filter or tube centrifuge or centrifuging, removing oil or other floating substances, and precipitating to obtain clear liquid;
6) Immobilized enzyme column 1 (first immobilized enzyme column): loading the filtrate into a column in a countercurrent mode, wherein the flow rate is 6250ml/min, the temperature of the interlayer insulation of the immobilized column is 45 ℃, and discharging the filtrate from the top of the immobilized enzyme column 1;
7) Immobilized enzyme column 2 (second immobilized enzyme column): the enzymolysis liquid flowing out of the immobilized enzyme column enters the immobilized enzyme column 2 in a countercurrent mode, the heat preservation temperature of the interlayer of the immobilized column is 45 ℃, and the enzymolysis liquid is discharged from the top;
8) Boiling to inactivate enzyme (adding tannin polyphenol precipitant): adding tannin precipitant into the solution after enzymolysis at 100deg.C, heating and boiling for 10 min, and filtering or centrifuging;
9) Film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; filtering the permeate A with 1000 Da ultrafiltration membrane to obtain retentate B and permeate B; filtering the permeate B with 100D nanofiltration membrane to obtain retentate C and permeate;
11 Freeze drying: and freeze-drying the trapped liquid C to obtain the cod collagen peptide.
Compared with other examples, the example 1 shows that the collagen polypeptide content obtained by enzymolysis at the same flow rate is higher (as shown in figure 5), so that the enzymolysis process can be conveniently recombined according to market or target requirements, the aim of really realizing the function requirements according to the molecular weight requirements of the protein is fulfilled, and the absorption and the function characteristics of the micromolecular collagen peptide can be obviously improved by the recombination production process.
The hydrolysis degree values of the single enzyme columns and the enzyme columns passing through the two times are shown in the attached figure 1, which shows that the hydrolysis degree of the enzyme columns passing through the two times and more is higher than that of the single enzyme columns, and the hydrolysis degree is increased by adopting the enzyme columns at least twice.
FIG. 2 shows the degree of hydrolysis of the enzyme column at different flow rates, showing the optimal flow rate values for the immobilized enzyme column, namely: the hydrolysis degree effect is best when 6250 ml/min-25000 ml/min.
FIG. 3 shows that the enzyme column is kept at a proper temperature in the interlayer heat preservation temperature, and the hydrolysis effect of the immobilized column interlayer at 45-50 ℃ is good as shown in the data of FIGS. 1-5.
Fig. 4 and 5 show the distribution of the obtained enzymatic hydrolysate in different molecular weight ranges under different flow rates when the enzymatic hydrolysate flows through the enzyme column at the flow rate of 12500-25000 ml/min.
Finally obtaining optimal parameters: the temperature of the immobilized column interlayer is kept at 45-50 ℃, and the hydrolysis effect is good when the flow rate of the immobilized enzyme column is controlled at 6250-25000 ml/min.
The results shown in fig. 7 and 8 show that the collagen peptide obtained by the combination process of the twice boiling and the polyphenol precipitant in the patent has much lower cytotoxicity than the collagen peptide obtained by the traditional process of the once boiling, and the corresponding cell activity is also higher than that of the collagen peptide obtained by the traditional process of the once boiling, which proves that the technical scheme adopted by the invention has remarkable effect.
According to the invention, collagen peptide is obtained by adding tannin polyphenol precipitant after enzymolysis through a specific combined collagen extraction process and a countercurrent immobilized enzyme column enzymolysis process. The enzymolysis process of the immobilized enzyme column can greatly shorten the enzymolysis time, and the immobilized enzyme columns can be flexibly connected in series and in parallel through valves, so that the method is suitable for the production of collagen peptides with different molecular weight requirements and different functional requirements; for macromolecular proteins which possibly remain after enzymolysis, the immunogenicity and cytotoxicity of the final product are greatly reduced by adding a polyphenol precipitant, boiling and filtering or centrifuging.
The purity of the collagen peptide is greatly improved by using the collagen peptide, the obtained collagen peptide has white color and luster and no peculiar smell and impurity taste, and can be widely applied to a plurality of industrial fields such as medicines, daily chemical industry, biosynthesis and the like.
The foregoing is a preferred embodiment of the present invention, and modifications of the invention in various equivalent forms will occur to those skilled in the art without departing from the principles of the invention, and are intended to be within the scope of the appended claims.
Claims (8)
1. A method for preparing collagen peptide by utilizing countercurrent immobilized enzyme column enzymolysis is characterized in that: the method comprises the following steps:
s1, obtaining an extracting solution: extracting the fish skin raw material to obtain collagen extracting solution;
s2, first boiling centrifugal filtration: boiling at 100deg.C for 2-100min, centrifuging or filtering to obtain filtrate or supernatant;
s3, immobilizing the enzyme column for the first time: loading the filtrate or supernatant onto column in countercurrent mode at flow rate of 8.5-25000 ml/min and temperature of 10-80deg.C for heat preservation of the immobilized column, and discharging enzymolysis liquid from the top of the first immobilized enzyme column;
s4, a second immobilized enzyme column: s3, allowing the enzymolysis liquid in the S3 to enter a second immobilized enzyme column in a countercurrent mode, keeping the temperature of the interlayer of the immobilized enzyme column at 10-80 ℃, and discharging the enzymolysis liquid from the top;
s5, adding a precipitant for secondary boiling: adding tannin polyphenol precipitant into the solution after enzymolysis, boiling for 5-100 minutes at 100 ℃, and filtering;
s6, film treatment: filtering the solution at room temperature by using a 5000 Da ultrafiltration membrane under Tao Lvmo and the transmembrane pressure of the effluent liquid of 0.2 Mpa, and separating into a trapped fluid A and a permeate A; continuously performing membrane filtration on the permeate A by using a 1000 Da ultrafiltration membrane to obtain trapped fluid B and permeate B; filtering the permeate B with a 100D nanofiltration membrane to obtain a retentate C and a permeate;
s7, freeze drying: and (3) freeze-drying the trapped fluid C in the step (S6) to obtain the cod collagen peptide.
2. The method for preparing collagen peptide by using countercurrent immobilized enzyme column enzymolysis according to claim 1, wherein the method comprises the following steps: the obtained extract is specifically prepared by thawing fish skin, pretreating fish skin and extracting at controlled temperature.
3. A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis according to claim 1 or 2, wherein: the specific method for obtaining the extracting solution comprises the following steps:
the first step is thawing of the fish skin: thawing fresh frozen fish skin raw material at 0-15deg.C, and removing fish scales and fish meat;
the second step is pretreatment of the fish skin: firstly, treating the fishskin in NaOH with the temperature of 0-15 ℃ and the temperature of 0.01-1.0M for 1-24 hours, then treating the fishskin with a detergent for 1-24 hours, cleaning the fishskin to be neutral, and crushing the fishskin;
the third step is temperature control extraction: the pretreated fish skin is prepared by the mass and volume ratio of 1: heating and extracting at 50-120deg.C for 0.5-12 hr at 5-100 deg.C.
4. A method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis according to claim 3, wherein: the fish skin is pretreated by dilute acid solution including but not limited to acetic acid, citric acid and hydrochloric acid, and acid-soluble impurity proteins are removed.
5. The method for preparing collagen peptide by using countercurrent immobilized enzyme column enzymolysis according to claim 1, wherein the method comprises the following steps: enzymes of the immobilized enzyme column include, but are not limited to, one or more of immobilized papain, immobilized alkaline protease, immobilized collagenase, immobilized trypsin, immobilized neutral protease, immobilized aminopeptidase.
6. The method for preparing collagen peptide by using countercurrent immobilized enzyme column enzymolysis according to claim 1, wherein the method comprises the following steps: and carrying out the enzymolysis of the immobilized enzyme column at least once again after the enzymolysis of the immobilized enzyme column for the first time.
7. The method for preparing collagen peptide by using countercurrent immobilized enzyme column enzymolysis according to claim 1 or 6, wherein the method comprises the following steps: the immobilized enzyme columns are connected in series or in parallel.
8. The method for preparing collagen peptide by using countercurrent immobilized enzyme column enzymolysis according to claim 1, wherein the method comprises the following steps: protein concentration measurement the amount of the obtained protein was measured as an immobilized enzyme to protein mass ratio 1:1-100 enzymolysis at 50deg.C for 0.5-6 hr.
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