CN115368432B - Preparation method of in-situ interface glycosylation modified oil body membrane protein - Google Patents
Preparation method of in-situ interface glycosylation modified oil body membrane protein Download PDFInfo
- Publication number
- CN115368432B CN115368432B CN202211076604.5A CN202211076604A CN115368432B CN 115368432 B CN115368432 B CN 115368432B CN 202211076604 A CN202211076604 A CN 202211076604A CN 115368432 B CN115368432 B CN 115368432B
- Authority
- CN
- China
- Prior art keywords
- glycosylated
- centrifuging
- solution
- membrane protein
- precipitate
- 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.)
- Active
Links
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 68
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 238000006206 glycosylation reaction Methods 0.000 title claims abstract description 24
- 230000013595 glycosylation Effects 0.000 title claims abstract description 20
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 135
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 82
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 82
- 238000006243 chemical reaction Methods 0.000 claims abstract description 74
- 239000004519 grease Substances 0.000 claims abstract description 63
- 239000011259 mixed solution Substances 0.000 claims abstract description 54
- 239000002244 precipitate Substances 0.000 claims abstract description 47
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims abstract description 43
- 239000003208 petroleum Substances 0.000 claims abstract description 41
- 238000002156 mixing Methods 0.000 claims abstract description 25
- 238000001035 drying Methods 0.000 claims abstract description 22
- 238000010438 heat treatment Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 17
- 150000002632 lipids Chemical class 0.000 claims abstract description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 93
- 210000002468 fat body Anatomy 0.000 claims description 48
- 238000003756 stirring Methods 0.000 claims description 32
- 238000005119 centrifugation Methods 0.000 claims description 28
- 229920001503 Glucan Polymers 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 12
- 238000001556 precipitation Methods 0.000 claims description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 8
- 239000000376 reactant Substances 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 5
- 238000002715 modification method Methods 0.000 abstract description 2
- 239000003921 oil Substances 0.000 description 86
- 108090000623 proteins and genes Proteins 0.000 description 29
- 102000004169 proteins and genes Human genes 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 28
- 229920002307 Dextran Polymers 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 125000003147 glycosyl group Chemical group 0.000 description 16
- 238000001816 cooling Methods 0.000 description 13
- 238000007789 sealing Methods 0.000 description 13
- 239000003517 fume Substances 0.000 description 12
- 101710089395 Oleosin Proteins 0.000 description 9
- 240000001548 Camellia japonica Species 0.000 description 8
- 230000002209 hydrophobic effect Effects 0.000 description 8
- 235000018597 common camellia Nutrition 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 238000004140 cleaning Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101000839489 Pinus massoniana 11-beta-hydroxysteroid dehydrogenase Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 239000010495 camellia oil Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000003084 food emulsifier Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012994 industrial processing Methods 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000009145 protein modification Effects 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 235000020238 sunflower seed Nutrition 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 230000000051 modifying effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001184 polypeptide Chemical group 0.000 description 1
- 108090000765 processed proteins & peptides Chemical group 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of in-situ interface glycosylation modified oil body membrane protein; the preparation method of the in-situ interface glycosylation modified oil body membrane protein comprises the following steps: extracting an oil body; mixing the lipid body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain an upper glycosylated lipid body; adding cold acetone into the upper glycosylated grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain precipitate, and drying the precipitate to obtain glycosylated modified oil body membrane protein. The invention aims to solve the problems of high cost, complex operation process and low yield of the existing modification method of the grease body.
Description
Technical Field
The invention relates to the technical field of protein modification, in particular to a preparation method of in-situ interface glycosylation modified oil body membrane protein.
Background
Grease bodies are important oil storage organelles in oil crop seeds. The grease body structure is similar to emulsion, triacylglycerol is arranged in the grease body structure, and the interface layer consists of single-layer phospholipid and grease body membrane protein. The oil body membrane proteins mainly include oil body proteins (oleosin) and small amounts of calcein and steroleosin. The middle of the oil body protein molecule is a very long conserved central hydrophobic region which consists of 68-74 amino acids, forms a hairpin structure and is inserted into an oil phase, and two ends are respectively an amphipathic N-terminal and an amphipathic C-terminal. Due to the specificity of the oil body membrane protein structure, the oil body membrane protein is promoted to be a protein with stable efficient oil-water interface, and has the potential of becoming a novel food emulsifier. However, the hydrophobic segment is long, which results in limited solubility in the aqueous phase, which greatly limits industrial applications. In addition, the method also provides a general strategy for green hydrophilic modification of hydrophobic proteins.
The current methods for improving the solubility of oleosin are mainly based on biosynthesis, e.g. "Self-assembly of tunable protein superstructures from recombinant oleosin (Proceedings of the National Academy of Sciences of the United States of America,2012,109 (29): 11657-11662") ", recording that Kevin et al have modified and modified the sunflower seed oleosin gene sequence, knocking out 22 amino acids at the hydrophobic-hydrophilic junction, reducing the length of the hydrophobic segment by 25%, expressing the variant protein using E.coli, resulting in a water-soluble oleosin; "Protease-Triggered, interin-Targeted Cellular Uptake of Recombinant Protein Micelles (Macromolecular Bioscience,2016,16 (9): 1398-1406.)" states that the soluble expression of oleosin is achieved by knocking out 57 amino acids from the hydrophobic core of sunflower seed oleosin, substituting glycine for the more hydrophobic amino acid, and constructing an oleosin mutant so that the hydrophilic arm is only negatively charged. The biosynthesis method has high cost, complex operation process and low yield, and cannot meet the industrial processing requirement of food.
Disclosure of Invention
The invention mainly aims to provide a preparation method of in-situ interface glycosylation modified oil body membrane protein, and aims to solve the problems of high cost, complex operation process and low yield of the existing modification method of the oil body membrane protein.
In order to achieve the above purpose, the preparation method of the in-situ interface glycosylation modified oil body membrane protein provided by the invention comprises the following steps:
extracting an oil body;
mixing the lipid body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain an upper glycosylated lipid body;
adding cold acetone into the upper glycosylated grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain precipitate, and drying the precipitate to obtain glycosylated modified oil membrane protein.
Optionally, mixing the fat body with sugar solution, adding pH regulator, heating for reaction, centrifuging to obtain upper glycosylated fat body,
the mass ratio of the sugar solution to the grease body is 1: (0.2-5).
Optionally, mixing the fat body with sugar solution, adding pH regulator, heating for reaction, centrifuging to obtain upper glycosylated fat body,
the temperature of the heating reaction is 60-100 ℃.
Optionally, mixing the fat body with sugar solution, adding pH regulator, heating for reaction, centrifuging to obtain upper glycosylated fat body,
the heating reaction time is 30-90 min.
Optionally, mixing the fat body with sugar solution, adding pH regulator, heating for reaction, centrifuging to obtain upper glycosylated fat body,
the sugar solution comprises any one of dextran 5k solution, dextran 10k solution and dextran 20k solution.
Optionally, the step of mixing the fat body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain an upper glycosylated fat body comprises the steps of:
mixing the grease body with a sugar solution to obtain a mixed solution;
adding a sodium hydroxide solution into the mixed solution until the pH value is not lower than 9.0, and heating until the reaction is finished to obtain a reactant;
centrifuging the reactant for 10-15 min under the condition of 10000-12000 rpm to obtain an upper glycosylated grease body.
Optionally, adding cold acetone into the upper glycosylated oil, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixture, centrifuging to obtain precipitate, drying the precipitate to obtain glycosylated modified oil membrane protein,
the volume ratio of the upper glycosylated grease body to the cold acetone is 1: (2.5-3); and/or the number of the groups of groups,
the volume ratio of the upper glycosylated grease body to the petroleum ether is 1: (2.5-3); and/or the number of the groups of groups,
the volume ratio of the upper glycosylated grease body to the chloroform-methanol mixed solution is 1: (2.5-3).
Optionally, adding cold acetone into the upper glycosylated grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain precipitate, and drying the precipitate to obtain glycosylated modified oil membrane protein, wherein the step of obtaining the glycosylated modified oil membrane protein comprises the following steps:
adding cold acetone into the upper glycosylated grease body, and centrifuging after vortex to obtain a first precipitate;
adding petroleum ether into the first precipitate, and centrifuging after vortex to obtain a second precipitate;
and adding chloroform-methanol mixed solution into the second precipitation product, performing vortex centrifugation to obtain a third precipitation product, and drying the third precipitation product to obtain the glycosylation modified oil body membrane protein.
Optionally, the step of extracting the fat body comprises:
crushing a sample, adding ultrapure water, stirring uniformly, homogenizing, and filtering to obtain a filtrate;
adding sucrose into the filtrate, regulating the pH value to be not less than 9, stirring and centrifuging to obtain an upper paste;
and (3) cleaning the upper paste by using 8M urea solution, centrifuging, and repeating for 2-3 times to obtain the grease body.
According to the technical scheme provided by the invention, the lipid body and the sugar solution are mixed for glycosylation reaction, so that the hydrophilicity of the lipid body membrane protein is increased, meanwhile, the steric hindrance between lipid body membrane protein molecules is increased, the aggregation of protein molecules is prevented, and the solubility of the lipid body is improved; in addition, the preparation method provided by the invention has the advantages of simple operation steps, low cost and rapid reaction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of an embodiment of a method for preparing an in situ interfacial glycosylation modified oil body membrane protein according to the present invention;
FIG. 2 is a schematic diagram of glycosyl modified oil body membrane proteins prepared in examples 1 to 12 and oil body proteins provided in comparative examples;
FIG. 3 is a square chart showing the solubility of the glycosyl modified oil body membrane proteins prepared in examples 1 to 9 of the present invention and the oil body proteins provided in comparative examples;
FIG. 4 is an electrophoresis chart of glycosyl modified oil body membrane proteins prepared in examples 1 to 15 of the present invention, and oil body proteins provided in comparative examples.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Grease bodies are important oil storage organelles in oil crop seeds. The grease body structure is similar to emulsion, triacylglycerol is arranged in the grease body structure, and the interface layer consists of single-layer phospholipid and grease body membrane protein. The oil body membrane proteins mainly include oil body proteins (oleosin) and small amounts of calcein and steroleosin. The middle of the oil body protein molecule is a very long conserved central hydrophobic region which consists of 68-74 amino acids, forms a hairpin structure and is inserted into an oil phase, and two ends are respectively an amphipathic N-terminal and an amphipathic C-terminal. Due to the specificity of the oil body membrane protein structure, the oil body membrane protein is promoted to be a protein with stable efficient oil-water interface, and has the potential of becoming a novel food emulsifier. However, the hydrophobic segment is long, so that the solubility in the water phase is limited, and the industrial application is greatly limited; the prior art mostly adopts biosynthesis, and the method has high cost, complex operation process and low yield, and cannot meet the industrial processing requirement of food.
In view of the above, the invention provides a preparation method of in-situ interface glycosylation modified oil body membrane protein, and the glycosylation modified oil body membrane protein prepared by the preparation method has hydrophilicity, high solubility, simple preparation method and low cost; in combination with the schematic flow chart of an embodiment of the method for preparing an in-situ interface glycosylation modified oil body membrane protein shown in fig. 1, the method for preparing an in-situ interface glycosylation modified oil body membrane protein comprises the following steps:
s10, extracting an oil body;
before extracting the grease body, selecting a sample, and selecting a sample with good quality and uniform texture when selecting the sample, wherein in the embodiment, the camellia seeds are preferably selected as the sample; specifically, in performing step S10, the following steps may be performed:
s101, crushing a sample, adding ultrapure water, uniformly stirring, homogenizing, and filtering to obtain filtrate;
step S102, adding sucrose into the filtrate, adjusting the pH value to be not less than 9, stirring and centrifuging to obtain an upper paste;
step S103, cleaning the upper paste by 8M urea solution, centrifuging, and repeating for 2-3 times to obtain the grease body.
Specifically, in actual operation, the following steps may be performed: weighing a certain amount of camellia seeds, putting the camellia seeds into a refiner, adding ultrapure water into the refiner according to a proportion (namely, 9-10L of ultrapure water is needed for every 1kg of camellia seeds), filtering the mixture by adopting 200-300 meshes of gauze after refining to obtain a filtered grease suspension, adding sucrose into the grease suspension, adjusting the pH value of the grease suspension until the pH value of the grease suspension is not less than 9.0, stirring the grease suspension at 25-30 ℃ for 30-40 min, centrifuging the grease suspension for 30-40 min under the condition of 6000-6500 rpm, layering, taking an upper paste, and cleaning the upper paste for 2-3 times by adopting 8M urea solution, wherein the condition that the upper paste is cleaned by adopting 8M urea solution each time is required to be centrifuged for 10-15 min under the condition of 10000-10500 rpm, and finally obtaining the grease.
In some embodiments, 9-10L of ultrapure water is required to be added for each 1kg of camellia seeds when the step S10 is performed; every 1m 3 The upper layer paste of the water-based cleaning agent is required to be correspondingly added with 1kg of 8M urea and 4-5L of aqueous solution for cleaning, namely, in the actual cleaning process, firstly adding 1kg of 8M urea into a container, then adding 4-5L of ultrapure water, stirring until the 8M urea is completely melted, and then adding 1M of aqueous solution 3 Cleaning the upper paste of the upper layer; the 8M urea solution is configured according to a conventional configuration method in the art, and will not be described in detail here.
Specifically, as a preferred embodiment of the present embodiment, the pH is preferably 11.
And step S20, mixing the fat body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain the upper glycosylated fat body.
The protein modification is to modify the protein structure by adopting a physical method, a biological method or a chemical method, so that amino acid residues and polypeptide chains of the protein are changed to cause the change of the spatial structure and physicochemical properties of protein molecules so as to obtain better functional characteristics (such as solubility), and the glycosylation modification is one of the protein chemical modifications, and the Maillard reaction is adopted to covalently crosslink food protein and sugar so as to prepare glycosylated protein, thereby improving the solubility of the protein, improving the functional properties and other properties of the protein; specifically, in the present embodiment, by adding a sugar solution to the fat body, the sugar solution is subjected to glycosylation reaction with the fat body interface film protein, thereby achieving improvement in the performance of the fat body film protein; compared with other modes, the mode of adopting glycosylation reaction has lower cost, better effect and quicker reaction.
Specifically, in performing step S20, it may be performed by:
step S201, mixing the grease body with a sugar solution to obtain a mixed solution;
step S202, adding a sodium hydroxide solution into the mixed solution until the pH value is not lower than 9.0, and heating until the reaction is finished to obtain a reactant;
step S203, centrifuging the reactant for 10-15 min under the condition of 10000-12000 rpm to obtain an upper glycosylated grease body.
In some embodiments, the specific operations are as follows: mixing the grease body and the sugar solution according to a proportion to obtain a mixed solution, adding a sodium hydroxide solution into the mixed solution to adjust the pH value of the mixed solution until the pH value of the mixed solution is not lower than 9.0, sealing the mixed solution, heating an oil bath, placing the sealed mixed solution in the heated oil bath, reacting for a certain time to obtain a reactant, placing the reactant in ice water, cooling in a water bath to 25-30 ℃, and centrifuging for 10-15 min under the condition of 10000-15000 rpm to obtain the upper glycosylated grease body.
In this embodiment, the pH of the mixed solution is preferably 11.
In some embodiments, the concentration of the sodium hydroxide solution is 2 to 2.5mol/L, and as a preferred embodiment of the present embodiment, the concentration of the sodium hydroxide solution is 2mol/L.
The solubility is an important index of the applicability of the oil body membrane protein, and the high and low of the solubility directly influences the emulsification and foaming characteristics of the oil body membrane protein, and determines the premise and basis of the applicable range, extraction and processing conditions and the like of the oil body membrane protein; the inventor repeatedly researches and tests to find that the mass ratio of the sugar solution to the grease body, the reaction time, the reaction temperature and the like can influence the solubility.
Specifically, in some embodiments, the mass ratio of the sugar solution to the fat body is 1: (0.2-5). Namely, 0.2 to 5kg of fat body is required to be added to each 1kg of sugar solution, and the mass ratio of the sugar solution to the fat body can be 1:0.2, which may be 1: 1. may be 1: 2. may be 1: 5. may be 1:0.5.
further, in some embodiments, the temperature of the heating reaction is 70-100 ℃. The inventors repeatedly tested that the higher the reaction temperature, the higher the solubility of the finally prepared glycosylated modified fat body membrane protein, and therefore, in practical application, the reaction temperature may be 70 ℃, 80 ℃, 90 ℃ or 100 ℃, and as a preferred embodiment of the present embodiment, the reaction temperature is 80 ℃.
Further, the heating reaction time is 30-90 min. In practical application, the heating reaction time can be 30min, 40min, 50min, 60min, 70min, 80min or 90min; as a preferred embodiment of this embodiment, the heating reaction time was 60 minutes.
In some embodiments, the specific type of sugar solution is not limited, and is preferably selected to be a dextran solution, specifically, may be a dextran 5k solution, may be a dextran 10k solution, and may be a dextran 20k solution.
It should be noted that the dextran 5k solution, the dextran 10k solution and the dextran 20k solution are prepared according to conventional technical means in the art, and are not described in detail herein.
And S30, adding cold acetone into the upper glycosylated grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain a precipitate, and drying the precipitate to obtain the glycosyl modified oil body membrane protein.
It should be noted that in some embodiments, the addition of cold acetone and petroleum ether serves to remove neutral fat from the fat body; the chloroform-methanol mixed solution is added for the purpose of removing phospholipids and the like of the fat body.
Further, in some embodiments, in the chloroform-methanol mixed solution, the volume ratio of chloroform to methanol is 2:1, a step of; the chloroform-methanol mixed solution is prepared by referring to a conventional preparation method in the art, and is not described in detail herein.
Still further, in some embodiments, the volume ratio of the upper glycosylated lipid to the cold acetone is 1:3, a step of; namely 3kg of cold acetone is needed to be added correspondingly to each 1kg of upper glycosylated grease; the volume ratio of the upper glycosylated grease to the petroleum ether is 1:3, a step of; namely, 3kg of petroleum ether is needed to be added correspondingly to each 1kg of upper glycosylated grease; the volume ratio of the upper glycosylated grease to the chloroform-methanol mixed solution is 1:3, a step of; namely, 3kg of chloroform-methanol mixed solution is needed to be added correspondingly to each 1kg of upper glycosylated grease; as a preferred embodiment of the present embodiment, 3kg of cold acetone, 3kg of petroleum ether, and 3kg of chloroform-methanol mixture are added in this order to 1kg of the upper glycosylated fat.
Specifically, in performing step S30, it may be performed by:
step S301, adding cold acetone into the upper glycosylated grease body, and centrifuging after vortexing to obtain a first precipitate;
step S302, adding petroleum ether into the first precipitation product, and centrifuging after vortexing to obtain a second precipitation product;
and step S303, adding chloroform-methanol mixed solution into the second precipitation product, performing vortex centrifugation to obtain a third precipitation product, and drying the third precipitation product to obtain the glycosylation modified oil body membrane protein.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1
(1) Putting 1kg of commercial camellia seeds into a homogenizer, adding 9kg of ultrapure water, homogenizing, filtering with 200-mesh gauze, adding sucrose into the filtrate, adding sodium hydroxide solution until the pH value of the filtrate is 11, stirring for 30min, centrifuging at 6000rpm for 30min to obtain an upper paste, washing with 8M urea solution for 2 times, centrifuging at 10000rpm for 10min after each washing to obtain a grease body;
(2) The mass ratio is 1:1 mixing the fat body with a glucan 5k solution (the concentration of the glucan 5k solution is 0.2 mg/mL), adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil bath pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 2
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with dextran 5k solution (concentration of the dextran 5k solution is 0.2 mg/mL) according to a proportion of 0.2, adding sodium hydroxide solution (concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil bath pot at 100 ℃, stirring at 300rpm for reaction for 30min, cooling in a cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 3
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1:1 mixing the fat body with a glucan 10k solution (the concentration of the glucan 10k solution is 0.2 mg/mL), adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in a cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 4
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with a glucan 10k solution (the concentration of the glucan 10k solution is 0.2 mg/mL) according to the proportion of 0.2, adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil bath pot at 100 ℃, stirring at 300rpm for 30min, cooling in a cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 5
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1:1 mixing the fat body with a glucan 20k solution (the concentration of the glucan 20k solution is 0.2 mg/mL), adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in a cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 6
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with a glucan 20k solution (the concentration of the glucan 20k solution is 0.2 mg/mL) according to the proportion of 0.2, adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil bath pot at 100 ℃, stirring at 300rpm for 30min, cooling in a cold water bath to 28 ℃, and centrifuging at 10000rpm for 10min to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex full reaction, centrifuging to remove redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosylated modified oil body membrane protein.
Example 7
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with dextran 5k solution (concentration of dextran 5k solution is 0.2 mg/mL), adding sodium hydroxide solution (concentration of sodium hydroxide solution is 2 mol/L) to adjust pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pan at 80deg.C, stirring at 300rpm for reacting for 60min, cooling in cold water bath to 28deg.C, and centrifuging for 10min under 10000 conditions to obtain upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
Example 8
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1:2, adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) into the mixed solution to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reacting for 60min, cooling in a cold water bath to 28 ℃, and centrifuging for 10min under 10000 conditions to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
Example 9
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with dextran 10k solution (concentration of the dextran 10k solution is 0.2 mg/mL) in a ratio of 0.5, adding sodium hydroxide solution (concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in cold water bath to 28 ℃, and centrifuging for 10min under 10000 conditions to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
Example 10
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1:2, mixing the fat body with a glucan 10k solution (the concentration of the glucan 10k solution is 0.2 mg/mL), adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in a cold water bath to 28 ℃, and centrifuging for 10min under 10000 conditions to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
Example 11
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1: mixing the fat body with dextran 20k solution (concentration of the dextran 20k solution is 0.2 mg/mL) in a ratio of 0.5, adding sodium hydroxide solution (concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in cold water bath to 28 ℃, and centrifuging for 10min under 10000 conditions to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
Example 12
(1) Consistent with the preparation of example 1;
(2) The mass ratio is 1:2, mixing the fat body with a glucan 20k solution (the concentration of the glucan 20k solution is 0.2 mg/mL), adding a sodium hydroxide solution (the concentration of the sodium hydroxide solution is 2 mol/L) to adjust the pH value of the mixed solution to 11, magnetically stirring for 10min, sealing the mixed solution in an oil pot at 80 ℃, stirring at 300rpm for reaction for 60min, cooling in a cold water bath to 28 ℃, and centrifuging for 10min under 10000 conditions to obtain an upper glycosylated fat body;
(3) Adding cold acetone into the upper glycosylated grease body, performing vortex separation and full reaction, removing redundant cold acetone, adding petroleum ether, performing vortex centrifugation to remove redundant petroleum ether after full reaction, adding chloroform-methanol solution, performing vortex centrifugation to remove redundant chloroform-methanol solution after full reaction, obtaining precipitate, and naturally drying the precipitate in a fume hood to obtain glycosyl modified oil membrane protein.
The commercial tea-oil camellia plants used in the above examples 1 to 12 are Zhejiang quzhou white tea-oil camellia.
Comparative example
Unmodified oil body membrane protein (solubility 14.+ -.1. Mu.g/mL).
Performance testing
(1) Solubility test
Taking the glycosyl modified oil body membrane proteins prepared in examples 1 to 9 and the oil body proteins in the comparative example, performing ultrasonic treatment for 10min, performing vortex treatment for 30min, repeating for 5 times, performing centrifugal treatment at 10000rpm for 3min, taking supernatant, measuring protein solubility by using Thermo Scientific Pierce BCA kit, preparing bovine serum albumin with different concentrations according to the operation method thereof, and preparing standard curves (y=0.833x+0.2104, R 2 =0.99), absorbance at 562nm was read using a microplate reader; the samples were diluted to obtain absorbance values within the standard curve range, and then subjected to the sample concentration determination test results are shown in fig. 3.
From fig. 3, it can be derived that: under the same reaction conditions, the higher the reaction temperature is, the higher the solubility of the obtained glycosyl modified oil body membrane protein is, and when the temperature reaches a peak value, the solubility is reduced again; this is because when the sugar content increases in the reaction system, the sugar molecules that each protein molecule can contact per unit volume increases, thus creating conditions for the glycosyl reaction, and improving the production amount and reaction rate of glycoprotein; therefore, the solubility of the glycosyl modified oil body membrane protein can be increased, the whole reaction rate can be improved, and the preparation time can be shortened.
(2) Polyacrylamide gel electrophoresis SDS-PAGE
Taking the glycosyl modified oil body membrane proteins prepared in examples 1 to 9 and the oil body proteins in the comparative examples; centrifuging at 10000rpm for 10min, diluting and diluting for 10 times, collecting 40 μL of the diluted solution in 2mL centrifuge tube, adding 10 μL of loading buffer solution, decocting in boiling water for 10min, and centrifuging at 3000rpm for 60s to obtain sample; preparing separating gel (15%) and concentrated gel (5%) according to the gel preparation formula, taking 5-10 mu L of treated sample and protein marker, loading, running for 90min under constant current mode (60V), taking out gel, dyeing for 30min with Coomassie brilliant blue staining solution, decolorizing, photographing with gel imager, and analyzing to obtain the result shown in figure 4.
From fig. 4, it can be derived that: the molecular mass of the oil body membrane protein is about 14kDa and 27kDa, after glycosylation reaction, the molecular mass of the oil body membrane protein is improved, the reaction temperature is increased along with the increase of sugar adding amount, the reaction time is prolonged, the molecular mass of the oil body membrane protein is gradually increased, the maximum molecular mass of the glycosylated oil body membrane protein can reach 14.7kDa, and the formation of the glucan-oil body membrane protein copolymer is proved.
(3) Appearance test
The modified oil body proteins were observed for the glycosyl modified oil body membrane proteins prepared in examples 1 to 12 and the oil body proteins in comparative examples, and the results are shown in FIG. 2.
From fig. 2, it can be derived that: as the volume ratio of the sugar solution to the fat body decreases, sugar molecules that can be contacted per protein molecule per unit volume decrease, resulting in a gradual decrease in modified protein, and therefore, by increasing the content of the sugar solution, the modifying effect of protein in the fat body can be increased.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (3)
1. The preparation method of the in-situ interface glycosylation modified oil body membrane protein is characterized by comprising the following steps of:
extracting an oil body;
mixing the lipid body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain an upper glycosylated lipid body;
adding cold acetone into the upper glycosylated grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain precipitate, and drying the precipitate to obtain glycosylated modified oil membrane protein;
the sugar solution is any one of a glucan 5k solution, a glucan 10k solution and a glucan 20k solution;
wherein the step of extracting the fat body comprises:
crushing a sample, adding ultrapure water, homogenizing, and filtering to obtain filtrate;
adding sucrose into the filtrate, regulating the pH value to be not less than 9, stirring and centrifuging to obtain an upper paste;
washing the upper paste with 8M urea solution, centrifuging, and repeating for 2-3 times to obtain an oil body;
mixing the lipid body with a sugar solution, adding a pH regulator, heating for reaction, and centrifuging to obtain an upper glycosylated lipid body, wherein the step of obtaining the upper glycosylated lipid body comprises the following steps of:
mixing the grease body with a sugar solution to obtain a mixed solution;
adding a sodium hydroxide solution into the mixed solution until the pH value is not lower than 9.0, and heating until the reaction is finished to obtain a reactant;
centrifuging the reactant for 10-15 min under the condition of 10000-12000 rpm to obtain an upper glycosylated grease body;
wherein the mass ratio of the sugar solution to the grease body is 1: (0.2-5),
the temperature of the heating reaction is 60-100 ℃,
the heating reaction time is 30-90 min.
2. The method for preparing an in-situ interface glycosylated modified oil membrane protein according to claim 1, wherein cold acetone is added into the upper glycosylated oil, the mixture is centrifuged, petroleum ether is added, the mixture is centrifuged, chloroform-methanol mixture is added, the precipitate is obtained after centrifugation, the precipitate is dried, and the glycosylated modified oil membrane protein is obtained,
the volume ratio of the upper glycosylated grease body to the cold acetone is 1: (2.5-3); and/or the number of the groups of groups,
the volume ratio of the upper glycosylated grease body to the petroleum ether is 1: (2.5-3); and/or the number of the groups of groups,
the volume ratio of the upper glycosylated grease body to the chloroform-methanol mixed solution is 1: (2.5-3).
3. The method for preparing an in-situ interface glycosylation modified oil body membrane protein according to claim 1, wherein the steps of adding cold acetone into the upper layer glycosylation grease body, centrifuging, adding petroleum ether, centrifuging, adding chloroform-methanol mixed solution, centrifuging to obtain a precipitate, and drying the precipitate to obtain the glycosylation modified oil body membrane protein comprise the steps of:
adding cold acetone into the upper glycosylated grease body, and centrifuging after vortex to obtain a first precipitate;
adding petroleum ether into the first precipitate, and centrifuging after vortex to obtain a second precipitate;
and adding chloroform-methanol mixed solution into the second precipitation product, performing vortex centrifugation to obtain a third precipitation product, and drying the third precipitation product to obtain the glycosylation modified oil body membrane protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211076604.5A CN115368432B (en) | 2022-09-02 | 2022-09-02 | Preparation method of in-situ interface glycosylation modified oil body membrane protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211076604.5A CN115368432B (en) | 2022-09-02 | 2022-09-02 | Preparation method of in-situ interface glycosylation modified oil body membrane protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115368432A CN115368432A (en) | 2022-11-22 |
| CN115368432B true CN115368432B (en) | 2024-04-02 |
Family
ID=84069008
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211076604.5A Active CN115368432B (en) | 2022-09-02 | 2022-09-02 | Preparation method of in-situ interface glycosylation modified oil body membrane protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115368432B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106107014A (en) * | 2016-07-04 | 2016-11-16 | 武汉轻工大学 | The method of compound physical field coordination strengthening rapeseed protein glycosylation modification |
| CN112957266A (en) * | 2021-02-23 | 2021-06-15 | 青岛农业大学 | Peanut oil body membrane protein modified liposome and preparation method thereof |
-
2022
- 2022-09-02 CN CN202211076604.5A patent/CN115368432B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106107014A (en) * | 2016-07-04 | 2016-11-16 | 武汉轻工大学 | The method of compound physical field coordination strengthening rapeseed protein glycosylation modification |
| CN112957266A (en) * | 2021-02-23 | 2021-06-15 | 青岛农业大学 | Peanut oil body membrane protein modified liposome and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 巴氏灭菌对不同油脂体乳液氧化稳定性的影响;武利春等;食品科学;第43卷(第7期);第1.3方法 * |
| 燕麦蛋白质糖基化改性及乳化性研究;张蓓;中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑(第12期) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115368432A (en) | 2022-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3481216B2 (en) | Process for obtaining a rapeseed protein isolate and protein isolate thereby obtained | |
| Petruccelli et al. | Relationship between the method of obtention and the structural and functional properties of soy proteins isolates. 1. Structural and hydration properties | |
| Egelrud et al. | The purification of a lipoprotein lipase from bovine skim milk | |
| Farías et al. | Casein glycomacropeptide pH-dependent self-assembly and cold gelation | |
| Kunz et al. | Human milk proteins: separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion-exchange chromatography | |
| CN111838318A (en) | Process for improving structure and components of homogenized milk fat globule membrane by using phospholipid | |
| DK158587B (en) | AGARO SEAL MATERIALS FOR USE WITH ISO ELECTRIC FOCUS, AIR GELS THEREOF AND PROCEDURE FOR PREPARING SUCH A GEL | |
| CN115368432B (en) | Preparation method of in-situ interface glycosylation modified oil body membrane protein | |
| Poole et al. | Effects of basic proteins on the denaturation and heat-gelation of acidic proteins | |
| CN114246326A (en) | Preparation method and application of modified egg white protein | |
| Fuda et al. | Selective separation of β‐lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB | |
| US20050279636A1 (en) | Gel for electrophoresis and method for preparing the same | |
| CN109444313A (en) | Method based on LC-MS technology analysis protein-PS complex digestibility | |
| Morr et al. | Preparation and properties of an alcohol-precipitated whey protein concentrate | |
| US8716440B2 (en) | Method of liberating and recovering peptide | |
| CN104764788A (en) | Protein electrophoresis method | |
| CN114600969B (en) | Preparation method of solid lipid based on pig plasma protein | |
| CN110791491A (en) | Method for extracting defibrase from snake venom | |
| Boschetti et al. | A Two‐Dimensional Separation of Proteins from Chloroplast Thylakoids and Other Membranes | |
| Bacon et al. | Preparation of transparent pea protein gels: a comparison of isolation procedures | |
| CN110452286B (en) | Iris sepia protein peptide with emulsifying activity and antioxidant activity, and preparation method and application thereof | |
| JP5261732B2 (en) | Method for producing oligosaccharide containing sialic acid | |
| CN105732796A (en) | Cobra venom nerve growth factor purification and preparation method | |
| Layton et al. | Electrophoretic fractionation of soluble antigenic proteins from the seed of Ricinus communis (castor bean) | |
| CN108640986B (en) | Method for separating egg white allergen |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |