CN105088540A - Method for preparing nano fibroin material on basis of tyrosinase/polyphenol medium - Google Patents
Method for preparing nano fibroin material on basis of tyrosinase/polyphenol medium Download PDFInfo
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Abstract
The invention discloses a method for preparing a nano fibroin material on the basis of a tyrosinase/polyphenol medium. Catalytic oxidation is carried out on tyrosine in fibroin and phenolic hydroxyl groups in the polyphenol medium through tyrosinase, so that reactive active quinone structure is generated, reaction between the fibroin and an exogenous functional amino compound is promoted and the functional nano fibroin material is prepared. The method specifically comprises the following steps: (1), preparing a fibroin solution; (2), carrying out catalytic oxidation on fibroin by tyrosinase to enable the fibroin to be crosslinked with the amino compound, and after reaction is carried out for a period of time, adding the polyphenol medium into the system for promoting reaction; (3), forming the nano fibroin material: spreading the solution obtained after the fibroin reacts with the amino compound in a tape casting manner, air-drying the spread solution into a film at room temperature or freeze-drying the spread solution into a film, preparing a spinning solution with formic acid and preparing the nano fibroin material by adopting an electrostatic spinning technology. Compared with the conventional chemical cross-linking method for preparing the functional fibroin material, the method provided by the invention has the advantages that enzyme catalysis efficiency is high, enzyme treatment conditions are mild and the performance of the nano fibroin material can be improved obviously.
Description
Technical field
The present invention relates to a kind of method preparing nano silk cellulosic material based on tyrosinase/Polyphenols amboceptor, particularly one utilizes tyrosinase catalysis to be oxidized fibroin and Polyphenols amboceptor, promote that fibroin albumen is combined with outer source functional amino-compound, prepare the method for functional nano silk fibroin material, belong to field of textile biotechnology.
Background technology
Fibroin has good biocompatibility, and the fibroin protein film being Raw material processing with it has purposes more widely in tissue engineering material builds.Natural silk fibroin toughness of material is poor, and water dissolve-loss ratio is high, for improving the mechanical property of regenerated silk membrane material and functional, has carried out correlative study both at home and abroad in fibroin albumen and composite modification thereof.Wherein chemical method application is more, generally carries out crosslinked or and external source functional compounds grafting mainly with glutaraldehyde, polybasic carboxylic acid etc. to fibroin albumen.Also there is certain deficiency in these class methods, comprise and easily produce the problems such as chemical noxious material remains while improving silk fibroin material performance.Biological method has safe, efficient, single-minded and advantages of environment protection, with enzyme process, modification is carried out to fibroin material, not only maintain the original good characteristic of fibroin albumen, and give fibroin material new application performance, also less to ambient influnence, therefore there is certain application prospect.
Biology enzyme to modification effect of fibroin material and fibroin albumen structure, enzyme selective etc. relevant.Hydrophobicity glycine in fibroin albumen, that alanine remains base content is higher, is secondly serine (containing methylol) and tyrosine (phenolic hydroxy group) residue of polar functionalities.Tyrosinase is the polyphenol oxidase with catalytic oxidation activity; the tyrosine of energy catalytic oxidation phenolic hydroxy group remains base and in succession generates adjacent phenol and DOPA quinone; and can react, for the functional modification of fibroin albumen provides method further with containing amino functional compound.Result of study shows, because tyrosine residue in fibroin albumen is distributed in the macromolecular chain be made up of the stronger alanine of hydrophobicity and glycine, create certain influence to the accessibility of enzymatic reaction, therefore, the fibroin albumen functional modification efficiency based on tyrosine enzyme process has much room for improvement.
Polyphenol compound comprises Tea Polyphenols, caffeic acid, forulic acid etc., and this compounds also has the phenolic hydroxyl structure similar to tyrosine.Polyphenol compound also can be oxidized by tyrosinase catalysis, the active quinone structure (A) that generation of reactive is stronger, active quinone molecule not only can be polymerized (A-A-A) mutually, also can react with two molecules of ammonia based compounds (B) simultaneously, form amino-compound intermolecular crosslinked (B-A-B).According to this characteristic, tyrosinase catalysis oxidation fibroin (SF) reaction in, fibroin not only can directly and external source amino-compound react, both formation be directly cross-linked (SF-B); Also can add appropriate Polyphenols medium system in system, generate on the basis of quinones active group at oxidation polyphenol amboceptor, form the indirect grafting (SF-A-B) of amino-compound on fibroin surface being bridge with active quinone structure, thus promote the efficiency of fibroin enzyme process functional modification, prepare functional nano silk fibroin material on this basis.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method preparing nano silk cellulosic material based on tyrosinase/Polyphenols amboceptor, be intended to improve the graft reaction efficiency of fibroin and external source amino-compound under tyrosinase catalysis condition, prepare functional silk fibroin material on this basis.
For solving the problems of the technologies described above, the present invention utilizes tyrosinase catalysis to be oxidized fibroin and Polyphenols amboceptor, what promote between fibroin albumen and outer source functional amino-compound is crosslinked, prepares the method for functional nano silk fibroin material in conjunction with electrostatic spinning, concrete technology and step as follows:
(1) silk fibroin solution preparation: dissolve mulberry silk with lithium bromide or calcium chloride, prepare silk fibroin solution;
Treatment process prescription and condition: mulberry silk is joined in the water solution system of lithium bromide or calcium chloride, after dissolving at 30 ~ 80 DEG C, silk fibroin solution is loaded bag filter, filter after dialysing 8 ~ 36 hours in deionized water, obtained concentration is 10 ~ 50g/L silk fibroin solution;
(2) tyrosinase/Polyphenols amboceptor catalysis fibroin and amino-compound are cross-linked: in step (1) silk fibroin solution, add tyrosinase and amino-compound, react and add Polyphenols amboceptor after 0.5 ~ 8 hour, under identical temperature conditions, continue process 0.5 ~ 24 hour;
Treatment process prescription and condition: tyrosinase 1 ~ 100U/ml, amino-compound 1 ~ 50g/L, Polyphenols amboceptor 1 ~ 10g/L, temperature 20 ~ 60 DEG C, pH scope 6.0 ~ 8.0;
(3) nano silk cellulosic material is shaping: by step (2) reacted silk fibroin solution, makes it smoothly in formation mould sprawl by prolonging stream, at room temperature air-dry film forming or under-50 DEG C ~-20 DEG C conditions freeze drying film forming; Carry out fibroin membrane dissolving with 98% formic acid, preparation spinning solution, adopt Static Spinning technology spinning nano silk cellulosic material;
Spinning technique and condition: in spinning solution, fibroin concentration is 10 ~ 45%, spinning voltage is 10 ~ 40kV, and spinning speed is 0.3 ~ 2ml/h, and the spinning spacing of syringe needle point and metal roller surface is 5 ~ 20cm.
Prepare a method for nano silk cellulosic material based on tyrosinase/Polyphenols amboceptor, time prepared by described silk fibroin solution, select lithium-bromide solution, lithium bromide-ethanol water or calcium chloride-ethanol water; Described tyrosinase cultivar origin is in animal, plant or microorganism; Described Polyphenols amboceptor comprises catechin, epicatechin, caffeic acid, forulic acid and chlorogenic acid; Described amino-compound comprises containing amino chemical finishing agent, functional polypeptide, shitosan, polylysine, elastin laminin and gelatin.
The present invention utilizes tyrosinase catalysis to be oxidized fibroin and external source amino-compound reacts, and generates on the basis of quinones active group, promote the efficiency of fibroin enzyme modification, constructing function nano silk cellulosic material at oxidation polyphenol amboceptor.Compared with preparing silk fibroin composite membrane coating with traditional chemical cross-linking method, the present invention has the following advantages:
(1) enzymatic efficiency is high, utilizes tyrosinase catalysis fibroin and the oxidation of Polyphenols amboceptor, and promote that in the reaction that fibroin and amino-compound are cross-linked, enzymatic efficiency is high, enzyme preparation consumption is less;
(2) ferment treatment condition relaxes, fibroin albumen functional modification is carried out under low temperature and near-neutral sulfite deinking, there is the advantage that energy consumption is low, treatment process is safe, avoid all many-sided defects such as chemical crosslink technique reaction easily causes environmental pollution, silk fibroin material security low;
(3) nano silk cellulosic material performance improvement is obvious, and based on tyrosinase/Polyphenols amboceptor method modified fibroin albumen, not only obtained fibroin nano material is water-soluble low, and have specifically functional, the biocompatibility of product is also better.
Detailed description of the invention
Adopt tyrosinase and Polyphenols amboceptor catalysis fibroin and external source amino-compound to be cross-linked, prepare functional nano silk fibroin material, specific embodiment is as follows:
Embodiment 1
(1) silk fibroin solution prepares: mulberry silk joins in lithium bromide-alcohol-water (mass ratio 45:45:10) solution, after 60 DEG C of dissolvings, silk fibroin solution is loaded bag filter, dialyse 24 hours in deionized water, period is per hour changes 1 water, filters the silk fibroin aqueous solution obtaining 20g/L after dialysis;
(2) in step (1) silk fibroin solution, add 10U/ml tyrosinase and 5g/L elastin laminin, 30 DEG C, under pH7 condition process add 2g/L catechin after 4 hours, continue process 12 hours;
(3) nano silk cellulosic material is shaping: sprawl smooth in molded polytetrafluoroethylarticle mould for step (3) reacted silk fibroin solution, air-dry film forming under room temperature; Fibroin membrane dissolving is carried out with 98% formic acid, compound concentration is the fibroin spinning solution of 15%, and adopt Static Spinning technology spinning nano silk cellulosic material, wherein spinning voltage is 15kV, spinning speed is 0.6ml/h, and the spinning spacing of syringe needle point and metal roller surface is 10cm.
Fibroin nano material sample 1: add tyrosinase in process, do not add catechin;
Fibroin nano material sample 2: add tyrosinase in process, add catechin;
After above-mentioned PROCESS FOR TREATMENT, evaluate NIH/3T3 cell and flood the survival rate after 24 hours in fibroin/elastin laminin composite membrane culture medium, and measure fracture strength and the elongation at break of nano silk cellulosic material.Result shows, the NIH/3T3 cell survival rate of sample 1 is 80%, fracture strength and elongation at break are respectively 2.1MPa, 1.9%; The cell survival rate of sample 2 is 83%, fracture strength and elongation at break can reach 2.8MPa, 2.8% respectively.
Embodiment 2
(1) silk fibroin solution prepares: mulberry silk joins calcium chloride-alcohol-water (mol ratio 1:1:7), after 70 DEG C of dissolvings, silk fibroin solution is loaded bag filter, dialyse 24 hours in deionized water, period is per hour changes 1 water, filters the silk fibroin aqueous solution obtaining 18g/L after dialysis;
(2) in step (1) silk fibroin solution, add 8U/ml tyrosinase and 2g/L shitosan, 30 DEG C, under pH7 condition process add 1g/L caffeic acid after 3 hours, continue process 15 hours;
(3) nano silk cellulosic material is shaping: after step (3) being reacted, solution is smooth in molded polytetrafluoroethylarticle mould sprawls, air-dry film forming under room temperature; Fibroin membrane dissolving is carried out with 98% formic acid, compound concentration is the fibroin spinning solution of 15%, and adopt Static Spinning technology spinning nano silk cellulosic material, wherein spinning voltage is 20kV, spinning speed is 0.8ml/h, and the spinning spacing of syringe needle point and metal roller surface is 12cm.
Fibroin nano material sample 3: do not add tyrosinase and caffeic acid in process;
Fibroin nano material sample 4: add tyrosinase in process, do not add caffeic acid;
Fibroin nano material sample 5: add tyrosinase and caffeic acid in process;
After above-mentioned PROCESS FOR TREATMENT, by sample 3,4,5 to flood 6 hours in 30 DEG C of aqueous solution after 50% ethanolic solution process, measure the water dissolve-loss ratio of fibroin membrane material and the bacteriostasis rate to staphylococcus aureus.Wherein, the water dissolve-loss ratio of sample 3 is 12.2%, and bacteriostasis rate is 85%; The water dissolve-loss ratio of sample 4 is 6.2%, and bacteriostasis rate is 91%; The water dissolve-loss ratio of sample 5 is 4.3%, and bacteriostasis rate is 95%.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (2)
1. prepare the method for nano silk cellulosic material based on tyrosinase/Polyphenols amboceptor for one kind, particularly one utilizes tyrosinase catalysis to be oxidized fibroin and Polyphenols amboceptor, promote that fibroin albumen is combined with outer source functional amino-compound, prepare the method for functional nano fibroin membrane material, concrete technology and step as follows:
(1) silk fibroin solution preparation: dissolve mulberry silk with lithium bromide or calcium chloride, prepare silk fibroin solution;
Treatment process prescription and condition: mulberry silk is joined in the water solution system of lithium bromide or calcium chloride, after dissolving at 30 ~ 80 DEG C, silk fibroin solution is loaded bag filter, filter after dialysing 8 ~ 36 hours in deionized water, obtained concentration is 10 ~ 50g/L silk fibroin solution;
(2) tyrosinase/Polyphenols amboceptor catalysis fibroin and amino-compound are cross-linked: in step (1) silk fibroin solution, add tyrosinase and amino-compound, react and add Polyphenols amboceptor after 0.5 ~ 8 hour, under identical temperature conditions, continue process 0.5 ~ 24 hour;
Treatment process prescription and condition: tyrosinase 1 ~ 100U/ml, amino-compound 1 ~ 50g/L, Polyphenols amboceptor 1 ~ 10g/L, temperature 20 ~ 60 DEG C, pH scope 6.0 ~ 8.0;
(3) nano silk cellulosic material is shaping: by step (2) reacted silk fibroin solution, makes it smoothly in formation mould sprawl by prolonging stream, at room temperature air-dry film forming or under-50 DEG C ~-20 DEG C conditions freeze drying film forming; Carry out fibroin membrane dissolving with 98% formic acid, preparation spinning solution, adopt Static Spinning technology spinning nano silk cellulosic material;
Spinning technique and condition: in spinning solution, fibroin concentration is 10 ~ 45%, spinning voltage is 10 ~ 40kV, and spinning speed is 0.3 ~ 2ml/h voltage, and the spinning spacing of syringe needle point and metal roller surface is 5 ~ 20cm.
2. method according to claim 1, is characterized in that: select lithium-bromide solution, lithium bromide-ethanol water or calcium chloride-ethanol water time prepared by described silk fibroin solution; Described tyrosinase cultivar origin is in animal, plant or microorganism; Described Polyphenols amboceptor comprises catechin, epicatechin, caffeic acid, forulic acid and chlorogenic acid; Described amino-compound comprises containing amino chemical finishing agent, functional polypeptide, shitosan, polylysine, elastin laminin and gelatin.
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| CN106149195A (en) * | 2016-08-18 | 2016-11-23 | 华南理工大学 | A kind of embedding fish oil and the nano fibrous membrane of ferulic acid and preparation and application simultaneously |
| CN107050511A (en) * | 2017-04-11 | 2017-08-18 | 江南大学 | A kind of biological enzyme fibroin albumen/calcium phosphate composite materials preparation method |
| CN107099046A (en) * | 2017-04-25 | 2017-08-29 | 江南大学 | A kind of method that fibroin base biomimetic mineralization material is prepared by grafting vinyl monomers |
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| CN108744023A (en) * | 2018-06-15 | 2018-11-06 | 福州大学 | A kind of fibroin albumen medical bio adhesive and preparation method thereof |
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| CN107099046A (en) * | 2017-04-25 | 2017-08-29 | 江南大学 | A kind of method that fibroin base biomimetic mineralization material is prepared by grafting vinyl monomers |
| CN107594068A (en) * | 2017-09-25 | 2018-01-19 | 中国海洋大学 | A kind of be crosslinked using tyrosinase reduces knife volume newly to the method for shrimp allergen tropomyosin allergenicity |
| CN108744023A (en) * | 2018-06-15 | 2018-11-06 | 福州大学 | A kind of fibroin albumen medical bio adhesive and preparation method thereof |
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