CN113106137A - Protein-inorganic hybrid nano flower and preparation method thereof - Google Patents
Protein-inorganic hybrid nano flower and preparation method thereof Download PDFInfo
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- 239000002057 nanoflower Substances 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 81
- 235000018102 proteins Nutrition 0.000 claims abstract description 58
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 58
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 229910001868 water Inorganic materials 0.000 claims abstract description 29
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 239000004365 Protease Substances 0.000 claims abstract description 17
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 10
- 238000007865 diluting Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims description 51
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 46
- 239000011259 mixed solution Substances 0.000 claims description 37
- 239000001103 potassium chloride Substances 0.000 claims description 23
- 235000011164 potassium chloride Nutrition 0.000 claims description 23
- 239000011780 sodium chloride Substances 0.000 claims description 23
- 235000002639 sodium chloride Nutrition 0.000 claims description 23
- 229940088598 enzyme Drugs 0.000 claims description 18
- 230000009849 deactivation Effects 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 11
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 10
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- 238000010438 heat treatment Methods 0.000 claims description 9
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- 229910000365 copper sulfate Inorganic materials 0.000 claims description 8
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 8
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 8
- 239000012460 protein solution Substances 0.000 claims description 8
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- 238000005406 washing Methods 0.000 claims description 8
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 7
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- 235000018262 Arachis monticola Nutrition 0.000 claims description 7
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 7
- 235000020232 peanut Nutrition 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 6
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 239000011565 manganese chloride Substances 0.000 claims description 6
- 235000002867 manganese chloride Nutrition 0.000 claims description 6
- 229940099607 manganese chloride Drugs 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000011592 zinc chloride Substances 0.000 claims description 5
- 235000005074 zinc chloride Nutrition 0.000 claims description 5
- 108090000526 Papain Proteins 0.000 claims description 4
- 108010073771 Soybean Proteins Proteins 0.000 claims description 4
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 4
- 239000005018 casein Substances 0.000 claims description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 4
- 235000021240 caseins Nutrition 0.000 claims description 4
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 4
- 229940055729 papain Drugs 0.000 claims description 4
- 235000019834 papain Nutrition 0.000 claims description 4
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 102000057297 Pepsin A Human genes 0.000 claims description 3
- 108090000284 Pepsin A Proteins 0.000 claims description 3
- 102000005158 Subtilisins Human genes 0.000 claims description 3
- 108010056079 Subtilisins Proteins 0.000 claims description 3
- 108010007119 flavourzyme Proteins 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 229940111202 pepsin Drugs 0.000 claims description 3
- 108090000145 Bacillolysin Proteins 0.000 claims description 2
- 108010004032 Bromelains 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
- 108010084695 Pea Proteins Proteins 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 102000007544 Whey Proteins Human genes 0.000 claims description 2
- 108010046377 Whey Proteins Proteins 0.000 claims description 2
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 2
- 235000019835 bromelain Nutrition 0.000 claims description 2
- 235000021329 brown rice Nutrition 0.000 claims description 2
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 2
- 229960002089 ferrous chloride Drugs 0.000 claims description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 2
- MIVBAHRSNUNMPP-UHFFFAOYSA-N manganese(2+);dinitrate Chemical compound [Mn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MIVBAHRSNUNMPP-UHFFFAOYSA-N 0.000 claims description 2
- 235000019707 mung bean protein Nutrition 0.000 claims description 2
- 235000019702 pea protein Nutrition 0.000 claims description 2
- 235000021119 whey protein Nutrition 0.000 claims description 2
- 239000004246 zinc acetate Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 4
- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 229940001941 soy protein Drugs 0.000 claims 1
- 235000021120 animal protein Nutrition 0.000 abstract description 4
- 239000013543 active substance Substances 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000011068 loading method Methods 0.000 abstract description 2
- 108010082495 Dietary Plant Proteins Proteins 0.000 abstract 1
- 238000006555 catalytic reaction Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 239000007788 liquid Substances 0.000 description 18
- 238000001878 scanning electron micrograph Methods 0.000 description 11
- 238000002156 mixing Methods 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 241000209094 Oryza Species 0.000 description 8
- 238000004108 freeze drying Methods 0.000 description 6
- 239000011521 glass Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 3
- 108010064851 Plant Proteins Proteins 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 235000021118 plant-derived protein Nutrition 0.000 description 3
- 235000019710 soybean protein Nutrition 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 238000010979 pH adjustment Methods 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 108010089934 carbohydrase Proteins 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002431 foraging effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000007769 metal material Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- -1 rare earth nitrate Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000003746 surface roughness Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—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
Abstract
The invention discloses a protein-inorganic hybrid nano flower and a preparation method thereof. The preparation method comprises the following steps: protein and water are mixed according to the mass-volume ratio of 1-10:100g/mL, adding protease for enzymolysis for 0.2-24h, diluting the enzymolysis solution with phosphate buffer solution until the protein concentration is 0.005-5mg/mL, adding metal ion solution, and incubating to synthesize the protein-inorganic hybrid nano flower. The invention combines the enzymolysis technology to successfully apply various large vegetable proteins and animal proteins to the preparation of the protein-inorganic hybrid nanoflower, can reduce the cost of producing the protein-inorganic hybrid nanoflower by taking biological enzyme as a protein raw material conventionally, has mild preparation conditions and simple method, and is expected to further promote the application of the protein-inorganic hybrid nanoflower in the fields of biosensor design, reaction catalysis, high-efficiency loading of active substances, adsorption separation and the like.
Description
Technical Field
The invention belongs to the field of nano materials, and particularly relates to a protein-inorganic hybrid nano flower and a preparation method thereof.
Background
The nanoflower, which is a kind of art in the scientific community, is not only pleasant, but also exhibits many excellent characteristics due to its great specific surface area and high surface roughness, and has been successfully applied to the fields of biocatalysis, biosensing, medicine, environment, energy, and the like. Early-stage nanoflowers are mostly prepared by using inorganic metal materials, and Ge et al occasionally find that the nanoflowers can be formed by mixing bovine serum albumin dissolved in phosphate buffer and copper sulfate in 2012, so that organic-inorganic hybrid nanoflowers are rapidly developed. Up to now, various types of hybrid nanoflowers have been prepared using various organic materials, such as proteins, DNA, RNA, peptides, amino acids, polysaccharides, etc., wherein studies on the utilization of proteins have been particularly focused since proteins have numerous binding sites and easily form hybrid nanoflowers with inorganic materials. Among many kinds of proteins, biological enzymes (including proteases, carbohydrases, peroxidases, lipases, etc.) have been receiving considerable attention and research because of their small molecular weight and catalytic activity. CN108130321B prepares the protease-inorganic hybrid nano flower by mixing papain, trypsin, alkaline protease or acid protease with different copper ion solutions and self-assembling. CN110669755A mixes alpha-amylase, horseradish peroxidase or laccase with the rare earth nitrate aqueous solution, and further adds ammonium nitrate and ammonia water for aging to prepare the organic-inorganic hybrid nano flower. CN107586770A is prepared by mixing and incubating porcine pancreatic lipase with a copper sulfate solution, and the porcine pancreatic lipase-inorganic hybrid nano flower is applied to the conversion of biodiesel. Although various functional nanoflower materials are prepared by using the biological enzyme at present, the biological enzyme is expensive and high in application cost, and has certain limitations in large-scale production and large-scale use of hybrid nanoflowers. Therefore, widening the protein sources for preparing the hybrid nanoflowers has become an urgent problem to be solved by researchers.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a protein-inorganic hybrid nano flower and a preparation method thereof.
The invention aims to provide a protein-inorganic hybrid nano flower.
The invention also aims to provide a preparation method of the protein-inorganic hybrid nano flower.
According to the invention, biological enzymolysis is utilized to treat various plant proteins and animal proteins, the plant proteins and the animal proteins are modified into assembly units with smaller molecular weights, and the assembly units can form protein-inorganic hybrid nano flowers with regular and uniform appearance with various metal ions through coordination, so that the protein source for preparing the hybrid nano flowers is widened, the preparation conditions are mild and simple, and the amplification is easy.
The purpose of the invention is realized by at least one of the following technical solutions.
The preparation method of the protein-inorganic hybrid nano flower provided by the invention comprises the following steps:
(1) adding protein into water (deionized water or purified water), and stirring to obtain protein solution;
(2) adjusting the pH value of the protein solution in the step (1) to 1.5-8.5 (hydrochloric acid solution or sodium hydroxide solution can be added for adjustment), adding protease, and performing heating treatment (enzymolysis treatment) under a stirring state to obtain an enzymolysis solution;
(3) centrifuging the solution obtained after enzymolysis in the step (2), taking supernatant, and diluting the supernatant with phosphate buffer solution to obtain mixed solution;
(4) and (4) adding a metal ion solution into the mixed solution obtained in the step (3), carrying out incubation treatment, centrifuging to obtain a precipitate, washing with water, and drying to obtain the protein-inorganic hybrid nano flower.
Further, the protein in the step (1) is one of rice protein, pea protein, potato protein, mung bean protein, soybean protein, brown rice protein, oat protein, peanut protein, chlorella protein, whey protein and casein; the volume ratio of the mass of the protein to the volume of the water is 1-10:100 g/mL; the stirring time is 0.5-2.5 h.
Preferably, the stirring time of the step (1) is 1-2 h.
Further preferably, the stirring time of step (1) is 2 h. The stirring mode comprises glass rod stirring, stirring paddle stirring or magnetic stirring, and preferably magnetic stirring.
Preferably, the ratio of the mass of the protein in the step (1) to the volume of the water is 3-6:100 g/mL.
Further preferably, the ratio of the mass of the protein in step (1) to the volume of water is 4:100 g/mL.
Further, the protease in the step (2) is one of pancreatin, Alcalase, pepsin, papain, neutral protease, flavourzyme, compound protease and bromelain; the addition amount of the protease in the step (2) is 0.05-2% of the mass of the protein in the step (1).
Preferably, the protease in the step (2) is added in an amount of 0.1-0.5% by mass based on the protein in the step (1).
Further, the stirring speed in the stirring state in the step (2) is 120-180 rpm; the temperature of the heating treatment in the step (2) is 35-60 ℃, and the time of the heating treatment is 0.2-24 h.
Preferably, the temperature of the heat treatment in the step (2) is 37-55 ℃, and the time of the heat treatment is 0.5-12 h. The heat treatment may be carried out in an incubator, or any device that provides for incubation and agitation, preferably an incubator.
Further, the pH value of the protein solution in the step (2) can be adjusted by adopting a hydrochloric acid solution or a sodium hydroxide solution; the concentration of the hydrochloric acid solution or the sodium hydroxide solution is 1-4mol/L, and 2mol/L is preferred.
Preferably, the pH value of the protein liquid in the step (2) is adjusted, so that the pH value of the adjusted protein liquid is 2.0-8.0.
Further, the stirring in step (2) may preferably be magnetic stirrer stirring.
Further, the speed of the centrifugal treatment in the step (3) is 6000-10000rpm, the time of the centrifugal treatment is 5-20min, and the temperature of the centrifugal treatment is 4-25 ℃.
Preferably, the speed of the centrifugal treatment in the step (3) is 7000-9000rpm, the time of the centrifugal treatment is 10-18min, and the temperature of the centrifugal treatment is 10-22 ℃; further preferably, the rotation speed of the centrifugation is 8000rpm, the time of the centrifugation is 15min, and the temperature of the centrifugation is 20 ℃.
Preferably, in the step (3), the solution after enzymolysis is subjected to enzyme deactivation treatment under a stirring state and then subjected to centrifugal treatment, wherein the temperature of the enzyme deactivation treatment is 80-100 ℃, the time of the enzyme deactivation treatment is 10-40min, and the stirring speed under the stirring state is 120-180 rpm.
Further preferably, during the enzyme deactivation treatment, the stirring may preferably be magnetic stirrer stirring.
Further preferably, during the enzyme deactivation treatment, the stirring speed in the stirring state is 140-160 rpm.
The enzyme deactivation treatment may be carried out in an incubator, or any device that provides for incubation and agitation, preferably an incubator.
Preferably, the temperature of the enzyme deactivation treatment is 90-100 ℃, and the enzyme deactivation time is 25-35 min; further preferably, the temperature of the enzyme deactivation treatment is 95 ℃, and the time of the enzyme deactivation treatment is 30 min.
Further, the pH value of the phosphate buffer solution in the step (3) is 7.2-9.0; in the mixed solution in the step (3), the concentration of the protein is 0.005-5 mg/mL.
Preferably, in the mixed solution in the step (3), the concentration of the protein is 0.01-2 mg/mL.
More preferably, in the mixed solution of step (3), the concentration of the protein is 0.1-0.5 mg/mL.
Preferably, the pH value of the phosphate buffer solution in the step (3) is 7.3-8.7. Further preferably, the phosphate buffer of step (3) has a phosphate buffer pH of 7.4.
Preferably, the phosphate buffer solution is one of a mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, a mixed solution of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, a mixed solution of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water, and a mixed solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water; in the phosphate buffer solution, the concentration of phosphate ions is 5-20mmol/L, the concentration of sodium chloride is 100-150mmol/L, and the concentration of potassium chloride is 2-10 mmol/L.
Preferably, in the phosphate buffer solution, the concentration of phosphate ions is 10mmol/L, the concentration of sodium chloride is 125mmol/L, and the concentration of potassium chloride is 5 mmol/L.
Further, the metal ion solution in the step (4) is more than one of copper sulfate solution, copper chloride solution, copper nitrate solution, manganese sulfate solution, manganese chloride solution, manganese nitrate solution, calcium chloride solution, calcium nitrate solution, ferrous chloride solution, ferrous sulfate solution, zinc chloride solution, zinc nitrate solution and zinc acetate solution; the concentration of the metal ion solution is 100-200 mmol/L; the volume ratio of the metal ion solution to the mixed solution is 1: 15-150.
Preferably, the metal ion solution in step (4) is more than one of copper sulfate solution, copper chloride solution, manganese sulfate solution, manganese chloride solution, calcium chloride solution, ferrous sulfate solution, zinc sulfate solution and zinc chloride solution.
Further preferably, the metal ion solution in step (4) is one or more of a copper sulfate solution, a copper chloride solution, a manganese chloride solution, a calcium chloride solution, a ferrous sulfate solution, and a zinc sulfate solution.
Preferably, the concentration of the metal ion solution in the step (4) is 120-160 mmol/L.
Further preferably, the concentration of the metal ion solution in the step (4) is 150 mmol/L.
Preferably, the volume ratio of the metal ion solution to the mixed solution in the step (4) is 1: 30-80.
Further preferably, the volume ratio of the metal ion solution to the mixed solution in the step (4) is 1: 60.
further, the temperature of the incubation treatment in the step (4) is 0-40 ℃, and the time of the incubation treatment is 4-72 hours.
Preferably, the incubation treatment of step (4) is performed at rest or under stirring.
Further preferably, when the incubation treatment in step (4) is performed under stirring, the stirring rate is 30-90 rpm. The stirring manner may preferably be magnetic stirrer stirring. The rate of stirring is preferably 40-70 rpm.
Preferably, the incubation period in step (4) is 12-30 h.
Further preferably, the incubation period in step (4) is 24 h.
Preferably, the temperature of the incubation treatment in the step (4) is 4-37 ℃.
Further preferably, the temperature of the incubation treatment in the step (4) is 25 ℃.
Preferably, the drying manner in the step (4) comprises freeze drying and drying in a forced air drying oven; the temperature of the freeze drying is-50 to-40 ℃, and the drying is carried out for 24 to 36 hours; and the drying temperature of the air blowing drying box is 30-80 ℃, and the drying is carried out for 4-24 h.
The invention provides a protein-inorganic hybrid nano flower prepared by the preparation method.
The protein-inorganic hybrid nanoflower provided by the invention can be applied to biocatalysis, biosensor design, active substance loading and food.
Compared with the prior art, the invention has the following advantages and beneficial effects:
according to the preparation method of the protein-inorganic hybrid nano flower, provided by the invention, various plant proteins and animal proteins are reformed into assembly units with smaller molecular weight by utilizing biological enzymolysis, and the assembly units can form the protein-inorganic hybrid nano flower with regular and uniform appearance with various metal ions through coordination, so that the application range of the protein in the conventional preparation of the protein-inorganic hybrid nano flower is widened, meanwhile, the preparation condition is mild, the process is simple, the industrial production prospect is good, and the application of the protein-inorganic hybrid nano flower in the fields of medicine, environment, food and the like can be further promoted.
Drawings
Fig. 1 is a Scanning Electron Microscope (SEM) image of the peanut protein-inorganic hybrid nanoflower prepared in example 1.
Fig. 2 is an SEM image of the soy protein-inorganic hybrid nanoflower prepared in example 2.
Fig. 3 is an SEM image of the rice protein-inorganic hybrid nano-flowers prepared in example 3.
Fig. 4 is an SEM image of the casein-inorganic hybrid nanoflower prepared in example 4.
Fig. 5 is an SEM image of chlorella protein-inorganic hybrid nanoflower prepared in example 5.
Fig. 6 is an SEM image of the inorganic nanoflower prepared in comparative example 1.
Detailed Description
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
The preparation method of the protein-inorganic hybrid nano flower provided in embodiment 1 comprises the following steps:
mixing 0.2g of peanut protein with 20mL of deionized water, stirring with a glass rod for 0.5h to obtain protein liquid, adjusting the pH of the protein liquid to 7.0 by using 1mol/L sodium hydroxide solution, transferring the protein liquid after the pH adjustment to a constant temperature oscillator, adjusting the temperature of the constant temperature oscillator to 60 ℃, adjusting the rotating speed of the constant temperature oscillator to 120rpm, adding papain for enzymolysis according to 0.05% of the peanut protein under the stirring of a magnetic stirrer, and keeping the enzymolysis time to be 2 h. After enzymolysis is finished, the enzymolysis liquid is centrifuged for 20min at 6000rpm and 4 ℃, supernatant is taken and diluted to 5mg/mL protein concentration by phosphate buffer solution (mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water; the concentration of phosphate ions is 5mmol/L, the concentration of sodium chloride is 100mmol/L, the concentration of potassium chloride is 2mmol/L, pH is 7.2) to obtain mixed solution, 100mmol/L copper sulfate solution is added into the mixed solution, and the volume ratio of the copper sulfate solution to the mixed solution is 1: and 15, standing and incubating for 4h at the temperature of 0 ℃, centrifuging, taking precipitate, washing with water, and freeze-drying for 24h at the temperature of-50 ℃ to prepare the peanut protein-inorganic hybrid nano flower.
An SEM image of the peanut protein-inorganic hybrid nanoflower prepared in this example is shown in fig. 1, and it can be seen from fig. 1 that the prepared hybrid nanoflower has regular and uniform morphology, is in a flower ball shape, has many pores, and has a very high specific surface area.
Example 2
The preparation method of the protein-inorganic hybrid nano flower provided in embodiment 2 comprises the following steps:
mixing 1g of soybean protein with 50mL of deionized water, stirring for 1h by using a magnetic stirrer to obtain protein liquid, adjusting the pH of the protein liquid to 1.5 by using 1.5mol/L hydrochloric acid solution, transferring the protein liquid after the pH is adjusted to a constant-temperature oscillator, adjusting the temperature of the constant-temperature oscillator to 35 ℃, adjusting the rotating speed of the constant-temperature oscillator to 130rpm, adding pepsin accounting for 0.1 percent of the mass of the soybean protein under the stirring of the magnetic stirrer for enzymolysis, and keeping the enzymolysis time to be 0.2 h. After enzymolysis, centrifuging the enzymolysis liquid at 7000rpm and 10 ℃ for 15min, taking supernate, diluting the supernate with phosphate buffer solution (mixed solution of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water; the concentration of phosphate ions is 10mmol/L, the concentration of sodium chloride is 110mmol/L, the concentration of potassium chloride is 4mmol/L, and the pH value is 7.4) to the concentration of protein is 2mg/mL to obtain mixed solution, adding 120mmol/L calcium chloride solution into the mixed solution, wherein the volume ratio of the calcium chloride solution to the mixed solution is 1: 30, standing and incubating for 12h at 4 ℃, centrifuging to obtain precipitate, washing with water, and freeze-drying for 32h at-45 ℃ to obtain the soybean protein-inorganic hybrid nano flower.
The SEM image of the soybean protein-inorganic hybrid nano flower prepared in this example is shown in fig. 2, and it can be seen from fig. 2 that the prepared hybrid nano flower has regular and uniform appearance, is flower-like, has small size, and has a high specific surface area.
Example 3
The preparation method of the protein-inorganic hybrid nano flower provided in embodiment 3 includes the following steps:
mixing 3.2g rice protein and 80mL purified water, stirring with a glass rod for 1.5h to obtain protein liquid, adjusting the pH of the protein liquid to 8.5 by using 2mol/L sodium hydroxide solution, transferring the protein liquid after the pH is adjusted to a constant-temperature oscillator, adjusting the temperature of a constant-temperature incubator to 50 ℃, adjusting the rotating speed of the constant-temperature incubator to 150rpm, adding Alcalase according to 0.5% of the mass of the rice protein under the stirring of a magnetic stirrer for enzymolysis, wherein the enzymolysis time is 6 h. After enzymolysis, centrifuging the enzymolysis liquid for 10min at 8000rpm and 15 ℃, taking supernate, diluting the supernate with phosphate buffer solution (mixed solution of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water; the concentration of phosphate ions is 15mmol/L, the concentration of sodium chloride is 125mmol/L, the concentration of potassium chloride is 5mmol/L, and the pH value is 8.0) to the protein concentration of 0.2mg/mL to obtain mixed solution, adding 150mmol/L zinc chloride solution into the mixed solution, wherein the volume ratio of the zinc chloride solution to the mixed solution is 1: and 60, stirring and incubating the rice protein-inorganic hybrid nano flower for 24 hours by using a magnetic stirrer at the temperature of 20 ℃, wherein the stirring speed is 30rpm, centrifuging the rice protein-inorganic hybrid nano flower after the incubation is finished, taking the precipitate, washing the precipitate with water, and freeze-drying the precipitate for 36 hours at the temperature of-40 ℃ to obtain the rice protein-inorganic hybrid nano flower.
The SEM image of the rice protein-inorganic hybrid nano flower prepared in this example is shown in fig. 3, and it can be seen from fig. 3 that the prepared hybrid nano flower has regular and uniform appearance, is flower-like, has a large size, and has a high specific surface area.
Example 4
Embodiment 4 provides a method for preparing protein-inorganic hybrid nanoflowers, comprising the following steps: mixing 8g of casein with 100mL of purified water, stirring for 2h by using a magnetic stirrer to obtain protein liquid, adjusting the pH of the protein liquid to 8.0 by using 2.5mol/L sodium hydroxide solution, adjusting the temperature of a constant-temperature oscillator to 40 ℃, adjusting the rotating speed of the constant-temperature oscillator to 160rpm, adding pancreatin according to 1% of the casein by mass under the stirring of the magnetic stirrer for enzymolysis, wherein the enzymolysis time is 12 h. After enzymolysis, adjusting the temperature to 80 ℃, keeping the temperature of a glass rod for 40min under stirring at the speed of 120rpm for enzyme deactivation, centrifuging the enzymolysis liquid for 8min at the conditions of 9000rpm and 20 ℃, taking supernate, diluting the supernate with phosphate buffer solution (mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, wherein the concentration of phosphate ions is 20mmol/L, the concentration of sodium chloride is 135mmol/L, the concentration of potassium chloride is 8mmol/L, and the pH value is 8.5) until the concentration of protein is 0.01mg/mL to obtain mixed solution, adding 180mmol/L ferrous sulfate solution into the mixed solution, wherein the volume ratio of the ferrous sulfate solution to the mixed solution is 1: 100, incubating for 48 hours under the condition of stirring by a magnetic stirrer at 37 ℃, wherein the stirring speed is 90rpm, centrifuging, taking the precipitate, washing with water, and drying for 24 hours by an air blast drying oven at 30 ℃ to prepare the casein-inorganic hybrid nano flower.
An SEM image of the casein-inorganic hybrid nanoflower prepared in this example is shown in fig. 4, and it can be seen from fig. 4 that the prepared hybrid nanoflower has regular and uniform morphology, is in a flower ball shape, has many pores, and has a very high specific surface area.
Example 5
The preparation method of the protein-inorganic hybrid nano flower provided in embodiment 5 comprises the following steps: mixing 20g of chlorella protein with 200mL of purified water, stirring for 2.5h by a stirring paddle to obtain a protein solution, adjusting the pH of the protein solution to 7.0 by using 4mol/L sodium hydroxide solution, transferring the protein solution after pH adjustment to a constant-temperature oscillator, adjusting the temperature of a constant-temperature incubator to 50 ℃, adjusting the rotating speed of the constant-temperature incubator to 180rpm, adding flavourzyme according to 2% of the mass of the chlorella protein under stirring by a magnetic stirrer for enzymolysis, wherein the enzymolysis time is 24 h. After enzymolysis, adjusting the temperature to 100 ℃, keeping the temperature of a magnetic stirrer for 10min under stirring at the speed of 180rpm for enzyme deactivation, centrifuging the enzymolysis liquid for 5min under the conditions of 10000rpm and 25 ℃, taking supernate, diluting the supernate with phosphate buffer solution (mixed solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water, wherein the concentration of phosphate ions is 10mmol/L, the concentration of sodium chloride is 150mmol/L, the concentration of potassium chloride is 10mmol/L, and the pH value is 9.0) until the concentration of protein is 0.005mg/mL to obtain mixed solution, adding 200mmol/L manganese chloride solution into the mixed solution, wherein the volume ratio of the manganese chloride solution to the mixed solution is 1: 150, standing and incubating for 72h at 40 ℃, centrifuging to obtain a precipitate, washing with water, and drying in an air drying oven at 80 ℃ for 4h to obtain the chlorella protein-inorganic hybrid nano flower.
An SEM image of the chlorella protein-inorganic hybrid nanoflower prepared in this example is shown in fig. 5, and it can be seen from fig. 5 that the prepared hybrid nanoflower has regular and uniform morphology, is in a shape of a bud, has many pores, and has a very high specific surface area.
Comparative example 1
Adding 100mmol/L CuSO4Directly mixing the solution with phosphate buffer solution (mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, wherein the concentration of phosphate ions is 5mmol/L, the concentration of sodium chloride is 100mmol/L, the concentration of potassium chloride is 2mmol/L, and the pH value is 7.2) according to the weight ratio of 1: 15, standing and incubating for 4h at 0 ℃, centrifuging, washing with water, and freeze-drying for 24h at-50 ℃ to obtain the inorganic nanoflower.
The SEM image of the inorganic nanoflower prepared in this comparative example is shown in fig. 6, and it can be seen from fig. 6 that the structure of the inorganic nanoflower without protein is significantly loosened, the pores are significantly reduced, and the surface is easily collapsed.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (10)
1. A preparation method of protein-inorganic hybrid nano-flowers is characterized by comprising the following steps:
(1) adding protein into water, and stirring to obtain protein solution;
(2) adjusting the pH value of the protein solution in the step (1) to 1.5-8.5, adding protease, and heating under stirring to obtain a solution after enzymolysis;
(3) centrifuging the solution obtained after enzymolysis in the step (2), taking supernatant, and diluting the supernatant with phosphate buffer solution to obtain mixed solution;
(4) and (4) adding a metal ion solution into the mixed solution obtained in the step (3), carrying out incubation treatment, centrifuging to obtain a precipitate, washing with water, and drying to obtain the protein-inorganic hybrid nano flower.
2. The method for preparing protein-inorganic hybrid nanoflowers according to claim 1, wherein the protein of step (1) is one of rice protein, pea protein, potato protein, mung bean protein, soy protein, brown rice protein, oat protein, peanut protein, chlorella protein, whey protein and casein; the volume ratio of the mass of the protein to the volume of the water is 1-10:100 g/mL; the stirring time is 0.5-2.5 h.
3. The method for preparing protein-inorganic hybrid nanoflowers according to claim 1, wherein the protease in step (2) is one of pancreatin, Alcalase, pepsin, papain, neutral protease, flavourzyme, complex protease and bromelain; the adding amount of the protease in the step (2) is 0.05-2% of the mass of the protein in the step (1); the stirring speed in the stirring state in the step (2) is 120-180 rpm; the temperature of the heating treatment in the step (2) is 35-60 ℃, and the time of the heating treatment is 0.2-24 h.
4. The method as claimed in claim 1, wherein the centrifugation rate of step (3) is 6000-10000rpm, the centrifugation time is 5-20min, and the centrifugation temperature is 4-25 ℃.
5. The method as claimed in claim 1, wherein in step (3), the solution after enzymolysis is processed by enzyme deactivation under stirring and then centrifuged, the temperature of enzyme deactivation is 80-100 ℃, the time of enzyme deactivation is 10-40min, and the stirring speed under stirring is 120-180 rpm.
6. The method for preparing protein-inorganic hybrid nanoflower according to claim 1, wherein the phosphate buffer of step (3) has a pH of 7.2 to 9.0; in the mixed solution in the step (3), the concentration of the protein is 0.005-5 mg/mL.
7. The method of claim 6, wherein the phosphate buffer is one of a mixture of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, a mixture of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride and water, a mixture of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water, and a mixture of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and water; in the phosphate buffer solution, the concentration of phosphate ions is 5-20mmol/L, the concentration of sodium chloride is 100-150mmol/L, and the concentration of potassium chloride is 2-10 mmol/L.
8. The method of preparing protein-inorganic hybrid nanoflowers according to claim 1, wherein the metal ion solution of step (4) is one or more of copper sulfate solution, copper chloride solution, copper nitrate solution, manganese sulfate solution, manganese chloride solution, manganese nitrate solution, calcium chloride solution, calcium nitrate solution, ferrous chloride solution, ferrous sulfate solution, zinc chloride solution, zinc nitrate solution, zinc acetate solution; the concentration of the metal ion solution is 100-200 mmol/L; the volume ratio of the metal ion solution to the mixed solution is 1: 15-150.
9. The method for preparing protein-inorganic hybrid nanoflower according to claim 1, wherein the incubation temperature in step (4) is 0-40 ℃ and the incubation time is 4-72 hours.
10. A protein-inorganic hybrid nano flower obtained by the production method of any one of claims 1 to 9.
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