CN101168763A - Method for preparing nano natural silk protein powder - Google Patents
Method for preparing nano natural silk protein powder Download PDFInfo
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- CN101168763A CN101168763A CNA2007100929696A CN200710092969A CN101168763A CN 101168763 A CN101168763 A CN 101168763A CN A2007100929696 A CNA2007100929696 A CN A2007100929696A CN 200710092969 A CN200710092969 A CN 200710092969A CN 101168763 A CN101168763 A CN 101168763A
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- 239000000843 powder Substances 0.000 title claims abstract description 46
- 102000004169 proteins and genes Human genes 0.000 title claims description 15
- 108090000623 proteins and genes Proteins 0.000 title claims description 15
- 238000000034 method Methods 0.000 title abstract description 25
- 108010022355 Fibroins Proteins 0.000 claims abstract description 66
- 238000002360 preparation method Methods 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims abstract description 10
- VGTPCRGMBIAPIM-UHFFFAOYSA-M sodium thiocyanate Chemical compound [Na+].[S-]C#N VGTPCRGMBIAPIM-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000012046 mixed solvent Substances 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 18
- 108091005804 Peptidases Proteins 0.000 claims description 15
- 102000035195 Peptidases Human genes 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 108090000145 Bacillolysin Proteins 0.000 claims description 11
- 102000035092 Neutral proteases Human genes 0.000 claims description 11
- 108091005507 Neutral proteases Proteins 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 9
- 230000007062 hydrolysis Effects 0.000 claims description 9
- 238000006460 hydrolysis reaction Methods 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- 230000009849 deactivation Effects 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 3
- -1 ether compound Chemical class 0.000 claims description 3
- 239000002245 particle Substances 0.000 abstract description 9
- 239000002105 nanoparticle Substances 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004108 freeze drying Methods 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract 1
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 abstract 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 31
- 239000000463 material Substances 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 9
- 239000003960 organic solvent Substances 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 239000012620 biological material Substances 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000002086 nanomaterial Substances 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000008204 material by function Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000012730 carminic acid Nutrition 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 229920000867 polyelectrolyte Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000005549 size reduction Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
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- Cosmetics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing nanometer natural silk fibroin albumen powder. Silk fibroin albumen is dissolved in mixed solvent of 50 to 60 wt percent of NaSCN or LiBr: CH3OH: H2O (the weight ratio is 40 to 60: 22 to 42: 10 to 30), dialyzed and concentrated by water, enzymatically hydrolyzed by Alpha chymotrypsin or neutral proteinase, and finally evaporated, dried and collected through a freeze drying method or centrifugation. The size of the nano-particles obtained in the method is 80 to 500 nm. The method has the advantages that the preparation process is simple, the mass and industrialized production can be performed, and simultaneously the obtained nanometer natural silk fibroin albumen powder particles are uniform, have appropriate sizes, and are favorable for application.
Description
Technical field
The present invention relates to a kind of method of utilizing natural silk to prepare nano level silk fibroin powder, belong to function nano material technology field.
Background technology
The function nano material is field the most dynamic in the nanometer material science, and it can be applied to high-tech areas such as chemical industry, biology, the energy, environment, and the generation far-reaching influence also has broad application prospects.Can be applied in fields such as organizational project, regenerative medicine material and physiologically acceptable boundary material as nano meter biomaterial.When the application of physiologically acceptable boundary material, it mainly is to pass through laminated assembling technology, realization comprises that protein, DNA and polyelectrolyte etc. are in the assembling on numerous base materials under the condition of gentleness, not only realized structural simulation to natural biologic material, and can obtain surperficial stratiform biomaterial, solve the biocompatibility key issue of some materials, can be applied to as cell growth support, medicament slow release material, enzyme and scopes such as imitative enzyme carrier and bio-sensing modulator material.And the nanometer chemical materials, can add by chemical reaction or physics give chemical materials such as degraded, catalysis, smelly function such as antibiotic disappears.
Just because of the function nano material has great using value, how to prepare and just Cheng Liao expert, scholar's research focus of application function nano material.Natural silk is to be made of special amino acid, and it has possessed the very big using value as biology, weaving, fine chemistry industry material, and is very active to the preparation technology and the method research of its nano level silk fibroin powder.The present industrial nano level silk fibroin powder, preparation method thereof that still do not have, the industrial now situation that has the employing mechanical crushing method to prepare silk fibroin powder, but be subjected to the restriction of size reduction machinery technology, can only make micron particles, and micron particles and nano-scale particle, its range of application and effect are inconsistent.For the preparation of silk fibroin powder nano-scale particle, all be in laboratory stage at present.Such as application number is " 200610039191.8 "; denomination of invention discloses so a kind of method for the Chinese invention patent application of " silk nano granular of immobilized enzyme and preparation method thereof "; it has adopted water-soluble fibroin solution and can mix with miscible proton type organic solvent or the aprotic, polar type organic solvent of water; forming milky spherical particle is dispersed in the organic solvent system; obtain nano thread crude granule mixed solution or suspension; remove organic solvent; obtain silk nano granular suspension or fibroin nanometer powder, the nano particle that makes is about 35～125nm.In this method, insoluble or be insoluble in water as fibrinous silk fibroin, and do not point out the preparation method of water-soluble fibroin solution in this patent, simultaneously water-soluble fibroin solution with can with water miscible proton type organic solvent or aprotic, polar type organic solvent mixing process in, the fibroin stripping is low, loss is big, and the preparation difficulty is big, yield is low, be difficult to industrialized deficiency so exist.Also has a Chinese invention patent (patent No. is that " 200410016856.4 ", denomination of invention are " fibroin Nano microsphere and preparation method thereof ") utilization biological nano technology, make silk protein that self-assembly take place under proper condition and form Nano microsphere, the nano particle that makes is about 80～3000nm.This method also exists the preparation difficulty big, is difficult to industrialized deficiency, and also there is big, the uneven problem of nano particle difference in size in this patent simultaneously.
The patent No. is that " 02138016.3 ", denomination of invention also disclose a kind of method for preparing silk fibroin for the Chinese invention patent of " a kind of preparation method of bioactive peptide-silk fibroin peptide ", it is a raw material with leftover bits and pieces silk cocoon or useless silk, through the Na of 0.3-1.0% (w/v)
2CO
3After solution is concise, with the CaCl of 20-60% (w/w)
2The solution heating for dissolving gets molten silk liquid, at pH2.5-12.0, with proteolytic enzyme molten silk liquid is carried out enzymatic hydrolysis reaction under 30-80 ℃, removes the CaCl of enzymolysis solution middle and high concentration again with nanofiltration
2Salt, to the concentrated solution spraying drying promptly.Though this method can make the silk fibroin powder, particle is big, can't reach nano level, and this is because CaCl
2This metal-salt, the Ca ion can be wrapped in the fibroin by complexing when the dissolving fibroin, also the Ca ion can't be removed even if carry out aqueous solution dialysis, and this technology is not remove CaCl
2Situation under the enzyme digestion reaction that carries out, except the formation of fibroin-Ca ionic complex has influenced enzyme digestion reaction, has reduced the hydrolysis result CaCl
2Existence in whole enzymolysis system also will make its Ca ion and proteolytic enzyme form chelation structure, make enzyme digestion reaction be difficult to normally carry out thereby cause enzyme to lose activity.Be to remove CaCl behind the first enzymolysis
2Technical process, be the purpose that can't reach preparation nano level silk fibroin powder.
Summary of the invention
At the prior art above shortcomings, the purpose of this invention is to provide a kind of method of utilizing natural silk to prepare nano level silk fibroin powder, present method is simple for process, be fit to industrialization, production in enormous quantities, gained protein powder uniform particles is of moderate size, and is beneficial to application.
The object of the present invention is achieved like this: a kind of preparation method of nano natural silk protein powder is characterized in that it may further comprise the steps:
1., purified fibroin raw material added carries out in the salts solution of capacity that salt is molten to obtain molten silk liquid, described salts solution is 50～60wt%NaSCN or by LiBr, CH
3OH and H
2O three is by weight 40～60: 22～42: 10～30 organic-inorganic mixed solvents that are made into;
2., molten silk liquid that the first step is obtained dialyses, and obtains silk fibroin water solution;
3., second silk fibroin water solution that obtain of step is carried out enzymolysis, enzymolysis proteolytic enzyme is Chymetin or neutral protease;
4., the dry collection of enzymolysis solution promptly got silk fibroin powder.
The preparation method of described fibroin raw material is: will discard cocoon shell or waste silk and soak to remove waxy substance (discarded cocoon shell immersion 48 hours wherein with ether compound, waste silk soaked 24 hours), wash with water to boil with ethanol again after the drying and removed carbohydrate and ash content in 1 hour, after cleaning, in water, boil again, wash the pure fibroin that obtains with water drying for standby to slough silk gum.
Described enzyme solution is: silk fibroin water solution is heated in 70～80 ℃ of water-baths takes out behind the 5min and adjust pH, add the proteolytic enzyme of enzymolysis aequum, stir hydrolysis to the enzymolysis desirable value, through after certain reaction times with the enzyme deactivation stopped reaction.
If enzymolysis proteolytic enzyme is Chymetin, then the pH value transfers to 5.5～7.5 with 25% ammonia solution, add proteolytic enzyme 37 ℃ ± 3 ℃ stir the about 1～1.5h of hydrolysis down after, solution is put into-40 ℃ and is frozen the storehouse and allow the enzyme deactivation stopped reaction.
If enzymolysis proteolytic enzyme is neutral protease, then the pH value transfers to 6.0～7.5 with 25% ammonia solution, add proteolytic enzyme 50～55 ℃ stir the about 1～1.5h of hydrolysis down after, solution is put into-40 ℃ and is frozen the storehouse and allow the enzyme deactivation stopped reaction.
Compare prior preparation method, the present invention has following beneficial effect:
1, because Chymetin or neutral protease have the enzyme of silk fibroin is cut strong, the even advantage of enzyme chopping fibroin of function, so the silk fibroin uniform particles that obtains is of moderate size, be beneficial to application, granular size is 80～500nm.This nano thread fibroin can be used as cosmetics additive less than the human skin pore size.
2, present method technology is simple, is easy to realize that the single preparation amount is big, and is raw materials used for discarded silk, for the leftover bits and pieces raw material utilizes category again, possessed the application prospect in field of functional materials.
3, salts solution and the enzymolysis protein enzyme low price selected for use of present method, preparation cost is low.
4, yield height of the present invention can reach 65～80%.
Based on above-mentioned advantage, so the present invention can carry out in enormous quantities, suitability for industrialized production.
Experiment shows that the nano thread fibroin powder of present method preparation has anti-microbial property, can be used as biomaterial and uses, and also can be used as functional fiber and substance thereof, and the while also can be used as the food interpolation, the fine chemistry industry goods add material and are applied.
Description of drawings
The protein powder particle that Fig. 1-the present invention uses the Chymetin enzymolysis to obtain;
The protein powder particle that Fig. 2-the present invention uses neutral protease enzymolysis to obtain;
Fig. 3-sample is cultivated the fungistatic effect figure behind the staphylococcus aureus on every side.
Embodiment
The present invention is with 50～60wt%NaSCN salts solution or LiBr, CH
3OH and H
2(weight ratio 40～60: 22～42: 10～30) mixed solvent that is made into of three dissolves silk fibroin to O, carry out the enzymolysis refinement with Chymetin or neutral protease again after dialysis concentrates and prepare evengranular nano thread fibroin powder, its nano particle size is 80～500nm.With the nano-grade silk silk fibroin powder uniform particles that present method makes, technology is simple, and is raw materials used for discarded silk, for the leftover bits and pieces raw material utilizes category again, possessed the application prospect in field of functional materials such as biomaterials.
Introduce the present invention in detail below in conjunction with embodiment.
1. at first obtain the fibroin fiber raw material
Discarded cocoon shell or waste silk are soaked to remove waxy substance (discarded cocoon shell immersion 48 hours wherein with ether compound, waste silk soaked 24 hours), wash with water to boil with ethanol again after the drying and removed carbohydrate and ash content in 1 hour, after cleaning, in water, boil again, wash the pure fibroin that obtains with water drying for standby to slough silk gum.After purifying with carmine picric acid solution method check silk gum, wash the pure fibroin that obtains with water drying for standby.
The preparation of fibroin is not limited to aforesaid method, also can adopt traditional other fibroin preparation method to obtain.
2. the preparation of silk fibroin water solution
The salts solution of dissolving fibroin fiber can be by weight 40～60: 22～42: 10～30 (best proportioning is 50: 32: 10) preparation LiBr, CH
3OH and H
2The organic-inorganic mixed solvent that O three forms also can be 50～60wt%NaSCN salts solution.The pure fibroin fiber that obtains in the last step is dissolved in the solvent of preparing above.The amount of salts solution gets final product fully dissolving fibroin fiber.
3. stand-by after dialysing, concentrating.
4. regenerated silk protein powder enzymolysis (enzymolysis can adopt two kinds of proteolytic enzyme, and a kind of is Chymetin, and a kind of is neutral protease)
(1) enzymolysis of Chymetin: through fixed molten and mensuration, the used silk fibroin peptide solution of the present invention concentration is all in 2～5mg/ml scope.Take out after the beaker that silk fibroin water solution is housed is placed in 70～80 ℃ of water-baths heating 5min, solution is adjusted pH to 5.5～7.5 with 25% ammonia solution, according to silk fibroin: the Chymetin weight ratio is (in reality also can adopt other ratio add at 10: 1, as long as the add-on of enzyme can be guaranteed the abundant enzymolysis of silk fibroin powder) ratio add Chymetin, 37 ℃ down stir the about 1.5h of hydrolysis after, solution is directly put into-40 ℃ and is frozen the storehouse and allow the enzyme deactivation stopped reaction.The silk fibroin powder evaporates down at 60 ℃ with the centrifugal back of whizzer and obtained dried powder in 48 hours behind the enzymolysis.Yield about 65～80%.The size of powder is observed and is undertaken by field emission scanning electron microscope, observes it for big or small ball-type powder between the 80nm to 500nm, sees figure one, and a and b are the sectional drawing of different positions.
(2) enzymolysis of neutral protease: through fixed molten and mensuration, the used silk fibroin peptide solution of the present invention concentration is all in 2～5mg/ml scope.Take out after the beaker that silk fibroin water solution is housed is placed in 70～80 ℃ of water-baths heating 5min, solution is adjusted about pH to 6.0～7.5 with 25% ammonia solution, according to silk fibroin: the neutral protease weight ratio is (in reality also can adopt other ratio add at 10: 1, as long as the add-on of enzyme can be guaranteed the abundant enzymolysis of silk fibroin powder) ratio add neutral protease, 50～55 ℃ down stir the about 1.5h of hydrolysis after, solution is directly put into-40 ℃ and is frozen the storehouse and allow the enzyme deactivation stopped reaction.The silk fibroin powder evaporates down at 60 ℃ with the centrifugal back of whizzer and obtained dried powder in 48 hours behind the enzymolysis.Yield about 65～70%.The size of powder is observed and is undertaken by field emission scanning electron microscope, observes it for big or small ball-type powder between the 80nm to 500nm, sees figure two, and a and b are the sectional drawing of different positions.
The silk fibroin powder can also adopt freeze-drying to collect except above-mentioned centrifugal back evaporation drying collection obtains.
Antibacterial experiment
Use extractum carnis, peptone, sodium-chlor, water to make substratum.Agar powder is the inoculation streptococcus aureus after the dissolving sterilization, maconkey agar inoculation intestinal bacteria.
Cultured intestinal bacteria and streptococcus aureus are taken out, coating method is inoculated into culture dish respectively with two kinds of bacteriums, silk-protein peptide powder, zinc phthalocyanine powder and zinc phthalocyanine-silk fibroin peptide powder is distinguished inoculation culture 18 hours, observe the growing state of bacterium.Entire operation is all carried out at the aseptic technique platform.
Experiment shows, the nano natural silk protein powder of the present invention's preparation possesses stronger anti-microbial property, shown in figure three, wherein scheming a is the silk fibroin peptide powder antibiogram that 50～60wt%NaSCN dissolving obtains, and figure b is the silk fibroin nanometer powder antibiogram behind the enzymolysis.
Claims (5)
1. the preparation method of a nano natural silk protein powder, it is characterized in that: it may further comprise the steps:
1., purified fibroin raw material added carries out salt in the salts solution of capacity and separate and obtain molten silk liquid, described salts solution is 50～60wt%NaSCN or by LiBr, CH
3OH and H
2O three is by weight 40～60: 22～42: 10～30 organic-inorganic mixed solvents that are made into;
2., molten silk liquid that the first step is obtained dialyses, and obtains silk fibroin water solution;
3., second silk fibroin water solution that obtain of step is carried out enzymolysis, enzymolysis proteolytic enzyme is Chymetin or neutral protease;
4., the dry collection of enzymolysis solution promptly got silk fibroin powder.
2. the preparation method of nano natural silk protein powder according to claim 1, it is characterized in that: will discard cocoon shell or waste silk and soak to remove waxy substance wherein with ether compound, discarded cocoon shell soaked 48 hours, waste silk soaked 24 hours, wash with water to boil with ethanol again after the drying and removed carbohydrate and ash content in 1 hour, after cleaning, in water, boil again, wash the pure fibroin that obtains with water drying for standby to slough silk gum.
3. the preparation method of nano natural silk protein powder according to claim 1 and 2, it is characterized in that: described enzyme solution is: take out and adjust pH after heating silk fibroin water solution about 5min to the enzymolysis desirable value in 70～80 ℃ of water-baths, the proteolytic enzyme that adds the enzymolysis aequum makes the enzyme deactivation stopped reaction after the stirring hydrolysis.
4. the preparation method of nano natural silk protein powder according to claim 3, it is characterized in that: described enzymolysis proteolytic enzyme is Chymetin, the pH value transfers to 5.5～7.5 with ammonia solution, add proteolytic enzyme 37 ℃ ± 3 ℃ stir the about 1～1.5h of hydrolysis down after, solution is put into-40 ℃ and is frozen the storehouse and allow the enzyme deactivation stopped reaction.
5. the preparation method of nano natural silk protein powder according to claim 3, it is characterized in that: described enzymolysis proteolytic enzyme is neutral protease, the pH value transfers to 6.0～7.5 with ammonia solution, add proteolytic enzyme and stir hydrolysis down after about 1～1.5 hour, solution is put into-40 ℃ and frozen the storehouse and allow the enzyme deactivation stopped reaction at 50～55 ℃.
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| CN104532365A (en) * | 2015-01-15 | 2015-04-22 | 武汉纺织大学 | Silk nanofiber manufacturing method |
| CN105435300A (en) * | 2015-12-25 | 2016-03-30 | 重庆理工大学 | Nano-silk-fibroin-containing antibacterial medical dressing |
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| CN100427593C (en) * | 2006-03-30 | 2008-10-22 | 苏州大学 | Silk nano granular of immobilized enzyme, and prepn. process thereof |
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