CN112280816A - Large-scale preparation method of marine source type I collagen subunit - Google Patents

Large-scale preparation method of marine source type I collagen subunit Download PDF

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CN112280816A
CN112280816A CN202010991917.8A CN202010991917A CN112280816A CN 112280816 A CN112280816 A CN 112280816A CN 202010991917 A CN202010991917 A CN 202010991917A CN 112280816 A CN112280816 A CN 112280816A
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collagen
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CN112280816B (en
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陈俊德
高然
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Third Institute of Oceanography MNR
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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
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    • 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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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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    • Y02A20/124—Water desalination

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Abstract

The invention discloses a large-scale preparation method of marine source type I collagen subunit, which takes marine organism processing by-products as raw materials, and utilizes acetic acid extraction and salting-out electrodialysis purification integrated technology to prepare type I collagen; taking I-type collagen as a raw material, and preparing a mixed solution of subunit alpha 1 and alpha 2 of the I-type collagen by utilizing a collagenase enzymolysis synergistic multi-stage membrane separation linkage technology; on the basis, the type I collagen subunits alpha 1 and alpha 2 are prepared by using salting out in combination with an electrodialysis technology. Aiming at the technical bottleneck that the existing preparation process of the collagen subunits can only prepare milligram-level and microgram-level samples and cannot realize large-scale production, the invention constructs a technical system integrating a collagenase enzymolysis synergistic multi-level membrane separation linkage technology and a salting-out synergistic electrodialysis technology, and prepares the type I collagen subunits alpha 1 and alpha 2 in a large scale.

Description

Large-scale preparation method of marine source type I collagen subunit
Technical Field
The invention belongs to the technical field of biological extraction, and particularly relates to a large-scale preparation method of marine source type I collagen subunits.
Background
Collagen is the most abundant protein in vertebrates, the most major structural protein in animal skin and bone connective tissue, and accounts for about 30% of total protein. There are 29 types of collagen currently identified, including collagen types I-XXIX, each of which has a unique amino acid sequence, structure and function. Among all collagen types, type I collagen has good biocompatibility, biodegradability and weak antigenicity, and can provide a good source of raw materials for industries such as food, medicine, biofunctional materials and tissue engineering.
In recent years, the global demand of terrestrial biogenic type I collagen has been reduced year by year due to the occurrence of infectious diseases such as avian influenza, mad cow disease, foot and mouth disease and the like. At the same time, the demand of the marine organism-derived type I collagen, especially the fish by-product (mainly fish scales, fish skin, and swim bladder) type I collagen, is rapidly increasing, and has become one of the most important raw material sources of the collagen products in the world.
The I type collagen molecule has a stable triple-helix quaternary structure formed by three collagen alpha subunit peptide chains through a right-handed helix, and the structure causes that collagen cannot be absorbed by human bodies and cannot be developed into medicines, health care products and foods. Food scientists hydrolyze type I collagen by an enzymatic method to obtain type I collagen peptides which can be applied to food, but the type I collagen peptides contain a large amount of polypeptides, the prior art cannot determine the amino acid sequences of all peptides, the product quality is unstable, and the type I collagen peptides cannot become collagen medicines and health care products with high added values. Therefore, the development of marine type I collagen subunits with definite amino acid sequences has become a hot point of research.
The current methods for preparing type I collagen subunits include: (1) chromatography: such as: weng et al uses golden pomfret fish skin as raw material to prepare fish skin collagen, the technical proposal is that the prepared fish skin collagen is dissolved in borate buffer solution, heated for 30min at 45 ℃, separated and purified by CM-52 carboxymethyl cellulose chromatographic column to obtain collagen subunits alpha 1 and alpha 2; (2) electrophoresis method: such as: deyl et al proposed that collagen was dissolved in a boric acid buffer solution at pH 9.2, degraded for 15min, and then separated and purified by capillary electrophoresis to obtain collagen subunits. However, these preparation methods can only obtain milligram-level and microgram-level collagen subunit samples, and cannot realize industrial production.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a large-scale preparation method of marine source type I collagen subunits.
The technical scheme of the invention is as follows:
a large-scale preparation method of marine source type I collagen subunits comprises the following steps:
(1) extracting the pretreated marine organism processing by-product with acetic acid solution, centrifuging to remove solid impurities, and purifying the obtained clear liquid by salting out with sodium chloride and electrodialysis to obtain type I collagen solution;
(2) adding collagenase into the type I collagen solution for normal-temperature enzymolysis, and then centrifuging to remove impurities; removing the non-enzymatic hydrolysis type I collagen from the obtained clear liquid by using a nanofiltration membrane with the relative molecular mass of 200kD intercepted, so as to obtain a permeate; removing inorganic salt from the permeate by using a nanofiltration membrane with the relative molecular mass cut-off of 50kD to obtain a mixed solution of type I collagen subunits alpha 1 and alpha 2;
(3) salting out the mixed solution of the type I collagen subunit alpha 1 and the alpha 2 by sodium chloride, and centrifuging to obtain clear liquid and precipitate; carrying out electrodialysis desalination on the clear solution, and then carrying out freeze drying to obtain type I collagen subunit alpha 1 shown as SEQ ID NO. 01; dissolving the precipitate with acetic acid solution, desalting by electrodialysis and freeze drying to obtain type I collagen subunit alpha 2 shown in SEQ ID NO. 02.
In a preferred embodiment of the invention, the marine bioprocess by-product comprises fish scales, fish skins and swim bladders.
Further preferably, the pretreatment is: and putting the marine biological processing by-product into a cleaning tank, adding a sodium bicarbonate solution with the mass volume concentration of 2-4%, stirring at room temperature for 4-6h, and then washing with water, wherein the ratio of the sodium bicarbonate solution to the marine biological processing by-product is 8-12L: 1 kg.
In a preferred embodiment of the present invention, the concentration of the acetic acid solution in the step (1) is 0.4 to 0.6M.
In a preferred embodiment of the present invention, the salting-out in step (1) comprises adding 2-4% by mass volume of sodium chloride to the clear solution.
In a preferred embodiment of the present invention, the polar water for electrodialysis in step (1) is a sodium chloride solution with a mass volume percentage of 4-6%.
In a preferred embodiment of the present invention, the flow rate of the electrodialysis in step (1) is 1m or less3/h。
In a preferred embodiment of the present invention, the polar water for electrodialysis in step (3) is a sodium chloride solution with a mass volume percentage of 1.5-2.5%.
In a preferred embodiment of the present invention, the flow rate of the electrodialysis in the step (3) is 1m or less3/h。
In a preferred embodiment of the invention, the electrodialysis membranes used for the electrodialysis in steps (1) and (3) are polyethylene heterogeneous ion exchange membranes, and the current for the electrodialysis in step (1) is 14-16A and the current for the electrodialysis in step (3) is 10-12A.
The invention has the beneficial effects that:
1. aiming at the technical bottleneck that the existing preparation process of the collagen subunits can only prepare milligram-level and microgram-level samples and cannot realize large-scale production, the invention constructs a technical system integrating a collagenase enzymolysis synergistic multi-level membrane separation linkage technology and a salting-out synergistic electrodialysis technology, and prepares the type I collagen subunits alpha 1 and alpha 2 in a large scale.
2. The invention takes the low-value marine organism processing by-products as raw materials, has low production cost and easily controlled process, and is suitable for large-scale production.
3. The type I subunit alpha 1 and alpha 2 produced by the invention have definite amino acid sequences and purity of more than 90 percent, and can be applied to the fields of medicines and health-care foods.
Drawings
FIG. 1 is a process flow diagram of the present invention.
FIG. 2 is a high performance gel chromatogram of a standard sample of example 1 of the present invention.
FIG. 3 is a standard curve of standard high performance gel chromatography of example 1 of the present invention.
FIG. 4 is a gel chromatogram of subunit type I collagen α 1 of example 1.
FIG. 5 is a chromatogram of type I collagen subunit α 2 gel of example 1 of the present invention.
FIG. 6 is a peptide fingerprint of type I collagen subunit α 1 of example 1 of the present invention.
FIG. 7 is a peptide fingerprint of type I collagen subunit α 2 of example 1 of the present invention.
Detailed Description
The technical solution of the present invention is further illustrated and described by the following detailed description.
Example 1
As shown in fig. 1, a large-scale preparation method of marine source type I collagen subunit comprises the following steps:
(1) 100kg of tilapia scales/fish skins/swim bladders are put into a cleaning tank, 1000L of sodium bicarbonate solution is added, the mass volume concentration of the solution is 3%, the solution is stirred for 5 hours at room temperature, after being cleaned by water, the solution is added into a reaction kettle, 0.5M acetic acid solution is added for stirring and extraction, after the extraction solution is subjected to impurity removal by a centrifuge with 10000rpm, the obtained clear solution is the type I collagen crude extract solution. Adding 3% (W/V) sodium chloride into the I type collagen crude extract solution for salting out, and centrifuging by a centrifuge with the rotation speed of 10000rpm to obtain a precipitate. Adding 0.5M acetic acid solution to dissolve precipitate, removing acid and inorganic salt by electrodialysis with polyethylene heterogeneous ion exchange membrane at 15A current and 5% (W/V) sodium chloride solution in water tank, and electrodialysis flow not more than 1M3And h, obtaining the type I collagen solution.
(2) Adding the I-type collagen solution into a reaction kettle, adding 100g of collagenase, and carrying out enzymolysis for 4 hours at normal temperature. Removing impurities from the enzymolysis liquid by a 10000rpm centrifugal machine, and removing the non-enzymolysis type I collagen from the clear liquid by using a nanofiltration membrane with the intercepted relative molecular mass of 200kD to obtain a permeate. And removing inorganic salt in the solution by using a nanofiltration membrane with the relative molecular mass of 50kD intercepted, wherein the pressure is 2Mpa, and the temperature is 25 ℃, so as to obtain the mixed solution of the type I collagen subunits alpha 1 and alpha 2.
(3) 5% (W/V) sodium chloride is added into the mixed solution of the subunit alpha 1 type I collagen and the alpha 2 type collagen for salting out. Centrifuging at 10000rpm, desalting the clear solution by electrodialysis with polyethylene heterogeneous ion exchange membrane at 11A current in 2% (W/V) sodium chloride solution in polar water tank at electrodialysis flow rate of 1m or less3And h, freeze-drying the electrodialysis material solution to obtain the I type collagen subunit alpha 1 freeze-dried sample. In addition, 0.1M acetic acid solution is used for dissolving the precipitate after centrifugation by a centrifuge, and desalination is carried out by electrodialysis technology, wherein the electrodialysis membrane is a polyethylene heterogeneous ion exchange membrane, the current is 13A, the polar water tank is 3% sodium chloride solution, and the electrodialysis flow is less than or equal to 1M3And h, freeze-drying the electrodialysis material solution to obtain the I type collagen subunit alpha 2 freeze-dried sample.
The purity and molecular weight of the type I collagen subunit alpha 1 and the type I collagen subunit alpha 2 obtained by the invention are detected by high performance gel chromatography. The detection conditions are as follows: superdex (TM) Peptide 10/300GL column, mobile phase: 50mmol/L phosphate, 0.15mpl/L sodium chloride and pH7.0 buffer solution; the flow rate is 0.5ml/min, the detection wavelength is 210nm, and the sample injection amount is as follows: 50 uL. The Mark samples are respectively as follows: thyroglobulin, molecular weight 669000Da, retention time 8.04; ferritin, molecular weight 440000Da, retention time 14.30; ③ bovine serum albumin, the molecular weight is 67000Da, the retention time is 16.02; beta-lactoglobulin, the molecular weight is 35000Da, and the retention time is 16.02; cytochrome with 13600Da molecular weight and 18.06 retention time; sixthly, aprotinin with molecular weight of 6512Da and retention time of 19.37 (figure 2). The standard curve obtained for the standard is lgMw ═ 0.1726x +7.2402 (R)20.9951) (fig. 3). As can be seen from FIGS. 4 and 5, the collagen subunit alpha prepared in this example1And alpha2。α1Purity 93.2%, alpha2The purity was 90.3%. Calculated by standard curve (FIG. 2) control, collagen subunit alpha1Has a molecular weight of 131KD and a collagen subunit alpha2Has a molecular weight of 125 KD.
The type I collagen subunit alpha 1 and the type I collagen subunit alpha 2 obtained by the invention are identified by a high performance liquid chromatography-mass spectrometry combined technology. Collagen subunit alpha1The peptide fingerprint identification (figure 6) shows that the collagen subunit alpha1Has a MASCOT score of 1137, coverage was 32%. Collagen subunit alpha1For collagen, type I, alpha 1 OS ═ oreochromics niloticus GN ═ COL1a1 PE ═ 2 SV ═ 1, the protein code is tr | G9M6I5| G9M6I5_ ORENI, the molecular weight is 137283Da, and the isoelectric Point (PI) value is 5.64. Collagen subunit alpha1The amino acid sequence of (A) is shown in SEQ ID NO. 01. Collagen subunit alpha2The peptide fingerprint identification (FIG. 7) shows that the collagen subunit alpha2The MASCOT score of (a) was 1344, coverage was 36%. Collagen subunit alpha2The protein is encoded as tr | G9M616| G9M6I6_ ORENI, molecular weight 126477Da, isoelectric Point (PI) value 9.18, for a Collagen type I alpha 2 OS ═ oreochromics niloticus GN ═ COL1A2PE ═ 2 SV ═ 1. Collagen subunit alpha2The amino acid sequence of (A) is shown in SEQ ID NO. 02.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
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Pro Lys Gly Glu Lys Gly Glu Pro Gly His Lys Gly Pro Asp Gly Asn
645 650 655
Pro Gly Arg Asp Gly Pro Arg Gly Leu Ala Gly Pro Ala Gly Pro Pro
660 665 670
Gly Pro Thr Gly Ala Asn Gly Asp Lys Gly Glu Gly Gly Ser Phe Gly
675 680 685
Pro Ala Gly Pro Ala Gly Pro Arg Gly Pro Ser Gly Glu Arg Gly Glu
690 695 700
Val Gly Pro Ala Gly Ala Pro Gly Phe Ala Gly Pro Pro Gly Ala Asp
705 710 715 720
Gly Gln Ala Gly Ala Arg Gly Glu Arg Gly Pro Ser Gly Ala Lys Gly
725 730 735
Glu Val Gly Pro Ser Gly Leu Ala Gly Pro Ala Gly Gln Ser Gly Pro
740 745 750
Ala Gly Pro Ala Gly Pro Gly Gly Pro Pro Gly Ala Arg Gly Asp Asn
755 760 765
Gly Pro Pro Gly Leu Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly
770 775 780
Ala Ala Gly Pro Ala Gly Ile Val Gly Pro Pro Gly Pro Ala Gly Pro
785 790 795 800
Ser Gly Lys Asp Gly Pro Arg Gly Pro Arg Gly Asp Pro Gly Pro Ser
805 810 815
Gly Pro Ser Gly Glu Pro Gly Ile Ile Gly Pro Pro Gly Leu Ala Gly
820 825 830
Glu Lys Gly Pro Ser Gly Glu Ser Gly Pro Pro Gly Ser Pro Gly Ala
835 840 845
Pro Gly Thr Ser Gly Pro Leu Gly Leu Gln Gly Phe Val Gly Leu Pro
850 855 860
Gly Ser Arg Gly Asp Arg Gly Ala Pro Gly Gly Ala Gly Gly Val Gly
865 870 875 880
Glu Pro Gly Arg Leu Gly Pro Ala Gly Pro Pro Gly Ala Arg Gly Ala
885 890 895
Pro Gly Asn Ile Gly Leu Pro Gly Met Thr Gly Pro Gln Gly Glu Ala
900 905 910
Gly Arg Glu Gly Ser Pro Gly Asn Asp Gly Pro Pro Gly Arg Pro Gly
915 920 925
Ala Ala Gly Leu Lys Gly Asp Arg Gly Glu Pro Gly Ser Ala Gly Thr
930 935 940
Thr Gly Leu Ala Gly Ala Pro Gly Pro Ala Gly Pro Thr Gly Ala Ala
945 950 955 960
Gly Arg Pro Gly Asn Arg Gly Glu Ala Gly Pro Ser Gly Pro Ser Gly
965 970 975
Ala Val Gly Pro Ala Gly Ala Arg Gly Ala Ser Gly Pro Ala Gly Pro
980 985 990
Arg Gly Glu Lys Gly Val Ala Gly Asp Lys Gly Glu Arg Gly Met Lys
995 1000 1005
Gly Leu Arg Gly His Pro Gly Leu Gln Gly Met Pro Gly Pro Ser Gly
1010 1015 1020
Pro Pro Gly Asp Thr Gly Ala Ala Gly Ala His Gly Pro Ser Gly Pro
1025 1030 1035 1040
Arg Gly Pro Ala Gly Pro His Gly Pro Val Gly Lys Asp Gly Arg Pro
1045 1050 1055
Gly Ala His Gly Thr Met Gly Ala Pro Gly Ala Arg Gly Pro Asn Gly
1060 1065 1070
Tyr Ser Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Leu Pro Gly Pro
1075 1080 1085
Pro Gly Pro Ala Gly Gly Gly Tyr Asp Val Ser Gly Gly Tyr Asp Glu
1090 1095 1100
Tyr Arg Ala Asp Gln Pro Ala Leu Arg Ala Lys Asp Tyr Glu Val Asp
1105 1110 1115 1120
Ala Thr Ile Lys Ser Leu Asn Thr Gln Ile Glu Asn Leu Leu Thr Pro
1125 1130 1135
Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Ile Lys Leu
1140 1145 1150
Ser His Pro Asp Trp Ser Ser Gly Phe Tyr Trp Ile Asp Pro Asn Gln
1155 1160 1165
Gly Cys Thr Asn Asp Ala Ile Lys Val Phe Cys Asp Phe Thr Thr Arg
1170 1175 1180
Glu Thr Cys Ile Tyr Ala His Pro Glu Ser Ile Ala Arg Lys Asn Trp
1185 1190 1195 1200
Tyr Arg Ser Thr Glu Asn Lys Lys His Val Trp Phe Gly Glu Thr Ile
1205 1210 1215
Asn Gly Gly Thr Glu Phe Thr Tyr Asn Asp Glu Thr Leu Ser Pro Gln
1220 1225 1230
Ser Met Ala Thr Gln Leu Ala Phe Met Arg Leu Leu Ser Asn Gln Ala
1235 1240 1245
Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp
1250 1255 1260
Gly Glu Ser Gly Ser Leu Lys Lys Ala Val Val Leu Gln Gly Ser Asn
1265 1270 1275 1280
Asp Val Glu Leu Arg Ala Glu Gly Asn Ser Arg Phe Thr Phe Ser Val
1285 1290 1295
Leu Glu Asp Gly Cys Thr Thr His Thr Gly Glu Trp Ser Lys Thr Val
1300 1305 1310
Ile Glu Tyr Arg Thr Asn Lys Pro Ser Arg Leu Pro Ile Leu Asp Ile
1315 1320 1325
Ala Pro Leu Asp Ile Gly Gly Ala Asp Gln Glu Phe Gly Leu Asp Ile
1330 1335 1340
Gly Pro Val Cys Phe Lys
1345 1350

Claims (10)

1. A large-scale preparation method of marine source type I collagen subunit is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting the pretreated marine organism processing by-product with acetic acid solution, centrifuging to remove solid impurities, and purifying the obtained clear liquid by salting out with sodium chloride and electrodialysis to obtain type I collagen solution;
(2) adding collagenase into the type I collagen solution for normal-temperature enzymolysis, and then centrifuging to remove impurities; removing the non-enzymatic hydrolysis type I collagen from the obtained clear liquid by using a nanofiltration membrane with the relative molecular mass of 200kD intercepted, so as to obtain a permeate; removing inorganic salt from the permeate by using a nanofiltration membrane with the relative molecular mass cut-off of 50kD to obtain a mixed solution of type I collagen subunits alpha 1 and alpha 2;
(3) salting out the mixed solution of the type I collagen subunit alpha 1 and the alpha 2 by sodium chloride, and centrifuging to obtain clear liquid and precipitate; carrying out electrodialysis desalination on the clear solution, and then carrying out freeze drying to obtain type I collagen subunit alpha 1 shown as SEQ ID NO. 01; dissolving the precipitate with acetic acid solution, desalting by electrodialysis and freeze drying to obtain type I collagen subunit alpha 2 shown in SEQ ID NO. 02.
2. The large-scale preparation method according to claim 1, wherein: the marine organism processing byproducts comprise fish scales, fish skins and swim bladders.
3. The large-scale preparation method according to claim 2, characterized in that: the pretreatment comprises the following steps: and putting the marine biological processing by-product into a cleaning tank, adding a sodium bicarbonate solution with the mass volume concentration of 2-4%, stirring at room temperature for 4-6h, and then washing with water, wherein the ratio of the sodium bicarbonate solution to the marine biological processing by-product is 8-12L: 1 kg.
4. The large-scale preparation method according to claim 1, wherein: the concentration of the acetic acid solution in the step (1) is 0.4-0.6M.
5. The large-scale preparation method according to claim 1, wherein: the salting-out in the step (1) comprises adding 2-4% by mass of sodium chloride into the clear liquid.
6. The large-scale preparation method according to claim 1, wherein: the electrodialyzed polar water in the step (1) is a sodium chloride solution with the mass volume percentage of 4-6%.
7. The large-scale preparation method according to claim 1, wherein: the flow rate of electrodialysis in the step (1) is less than or equal to 1m3/h。
8. The large-scale preparation method according to claim 1, wherein: the electrodialyzed polar water in the step (3) is a sodium chloride solution with the mass volume percentage of 1.5-2.5%.
9. The large-scale preparation method according to claim 1, wherein: the flow rate of electrodialysis in the step (3) is less than or equal to 1m3/h。
10. A large-scale preparation method according to any one of claims 1 to 9, characterized in that: the electrodialysis membrane used for electrodialysis in the steps (1) and (3) is a polyethylene heterogeneous ion exchange membrane, the current of electrodialysis in the step (1) is 14-16A, and the current of electrodialysis in the step (3) is 10-12A.
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Publication number Priority date Publication date Assignee Title
CN114805490A (en) * 2022-03-10 2022-07-29 大连海洋大学 Antioxidant peptide prepared from tilapia skin and preparation method thereof
CN114805490B (en) * 2022-03-10 2023-05-23 大连海洋大学 Antioxidant peptide prepared from tilapia skin and preparation method thereof

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