CN103819707B - A kind of antioxidation fibroin membrane preparation method based on enzymatic grafting - Google Patents
A kind of antioxidation fibroin membrane preparation method based on enzymatic grafting Download PDFInfo
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- CN103819707B CN103819707B CN201410056821.7A CN201410056821A CN103819707B CN 103819707 B CN103819707 B CN 103819707B CN 201410056821 A CN201410056821 A CN 201410056821A CN 103819707 B CN103819707 B CN 103819707B
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Abstract
The invention discloses a kind of antioxidation fibroin membrane preparation method based on enzymatic grafting, belong to field of textile biotechnology。The present invention by means of tryrosinase, is grafted to fibroin albumen surface after having the flavone compound catalytic oxidation of non-oxidizability；Tyrosine and flavone compound crosslinking in enzymatic fibroin simultaneously, promotes the antioxygenic property of fibroin membrane。Concrete technology flow process includes: silk fibroin solution prepare, fibroin membrane prepare, enzymatic fibroin grafting flavone compound, Modified silk fibroin film post processing。Fibroin protein film not only antioxidation and the anti-microbial property prepared by the present invention are improved, and membrane material mechanical property is also improved。Compared with chemically based crosslinking fibroin protein film method of modifying, the present invention addresses that enzymatic modification method energy consumption is low, efficiency is high, pollution is few, is conducive to environmental conservation。
Description
Technical field
The present invention relates to a kind of antioxidation fibroin membrane preparation method based on enzymatic grafting, a kind of method that particularly flavone compound utilizing tyrosinase catalysis to have antioxidant activity prepares antioxidation fibroin membrane at fibroin albumen surface grafting, belongs to field of textile biotechnology。
Background technology
The fibroin albumen coming from mulberry silk has good biocompatibility, has wide purposes with its fibroin protein film being Raw material processing in medical tissue engineering material builds。For improving the application performance of fibroin albumen membrane material, by means of chemical or biological method, silk fibroin material need to be modified and functionalization processing。
The method of modifying that fibroin protein film is conventional includes high temperature wet heat treatment method, chemical crosslinking modified method and other polymer blended methods and modifiies。In different fibroin membrane method of modifying, chemic modified method application is wide, its ultimate principle is the feature utilizing and containing relatively polyamino, hydroxyl isopolarity reactive group in fibroin albumen, make to cross-link between fibroin macromole by means of chemical cross-linking agent (such as glutaraldehyde, polybasic carboxylic acid etc.), or with external source functional compound generation grafting。This kind of method there is also certain deficiency while improving fibroin membrane performance, and the cross-linking agent as used is easily generated harmful substance residual, produces environmental pollution etc.。In recent years, along with biotechnology research in polymer modification is increasingly deep, the research carrying out silk fibroin material functional modification with biotechnology is increasingly subject to pay attention to。
In fibroin material bioanalysis is modified, the enzyme preparation with latent effect effect includes protease, transglutaminase, oxidoreductase (such as laccase, tryrosinase, horseradish peroxidase) etc.。Wherein, protease is used for the degraded of silk fibroin material；Transglutaminase can catalysis fibroin Glutamic Acid and adjacent silk polypeplide or external source amino-compound grafting, but owing to fibroin Glutamic Acid content is less, therefore this enzyme is applied less in fibroin material bio-modification。Tryrosinase is as a kind of cupric polyphenol oxidase, energy catalytic oxidation list phenol, the adjacent multiple compounds such as diphenol, triphenol, the o-quinone scaffold intermediate that generation of reactive is more active, can react with primary amine further, it is achieved the synthesis or macromolecular grafted modified of new compound。Amino and to have the tyrosine content of phenolic hydroxyl structure higher in fibroin albumen, can according to this feature, by means of the catalysed oxidn of tryrosinase, by catalysis polyphenolic substance and fibroin albumen grafting, it is achieved fibroin albumen functional modification。
In different fibroin protein film functional modifications processing, there is antioxidation, the fibroin membrane of antibacterial and anti-inflammation functions has higher medical value and application prospect。Containing multiple phenolic hydroxyl structures similar to tyrosine in flavone compound (such as flavonol, flavanone etc.), there is the multiple efficacies such as anti-inflammatory, antioxidation。If by means of the catalysed oxidn of tryrosinase, above-mentioned flavone compound is aoxidized, can react with amino in fibroin albumen further；Meanwhile, in fibroin albumen, tyrosine also can be aoxidized by tryrosinase, then reacts with flavone compound, it may be achieved based on the preparation of the antioxidation fibroin protein film of enzymatic grafting。
Summary of the invention
The technical problem to be solved is to provide a kind of antioxidation fibroin membrane preparation method based on enzymatic grafting, and gained fibroin protein film has good antioxidation and antibiotic effect, has expanded the fibroin membrane performance as medical engineering material。
For solving above-mentioned technical problem, the present invention utilizes the catalysed oxidn of tryrosinase, and catalysis has flavone compound and the fibroin albumen generation graft reaction of phenolic hydroxyl group, improves the application performance of fibroin protein film。
Concrete technology is as follows with step:
(1) prepared by silk fibroin solution: dissolves fibroin fiber with lithium bromide or calcium chloride, prepares silk fibroin solution。
Processing technology preparation and condition: joined by silkworm silk in the water solution system of lithium bromide or calcium chloride, dissolving prepared concentration at about 30～80 DEG C is the silk fibroin protein solution of 10～50g/L；Silk fibroin solution is loaded bag filter, filters after dialysis in deionized water, prepare silk fibroin aqueous solution。
(2) prepared by fibroin membrane: taking silk fibroin solution and make it smooth sprawl by prolonging stream, under-50～-20 DEG C of conditions, abundant lyophilization, obtains fibroin protein film。
(3) enzymatic fibroin grafting flavone compound: add appropriate tryrosinase in the flavone compound solution of phenolic hydroxy group, by fibroin protein film impregnation process in above-mentioned solution under aerobic conditions, carries out enzymatic graft modification。
Process technology preparation and condition: tryrosinase is 1～200U/g fibroin protein film relative to the consumption of fibroin membrane, fibroin protein film 50～200g/L, flavone compound 0.5～10g/L, temperature 20～50 DEG C, pH scope 6.0～8.0, processes 1～24 hour。Preferred 6-12 hour of described process time。
(4) Modified silk fibroin film post processing: after enzymatic grafting in vain, fibroin membrane is through 2～3 secondary chamber Warm Wash, and-50～-20 DEG C obtain antioxidation fibroin protein film after drying。
The preparation of described silk fibroin solution also can be selected for the lithium-bromide solution of variable concentrations, lithium bromide-ethanol water or calcium chloride-ethanol water。
Described tryrosinase includes the enzyme kind of the separate sources such as animal, plant and microorganism。
Described flavone compound all contains the phenolic hydroxyl group of varying number, specifically includes the compounds such as flavonol, flavanone, flavanonol, isoflavone and isoflavanone。
The present invention utilizes the catalysed oxidn of tryrosinase, makes flavone compound be grafted on fibroin albumen surface, compared with preparing functional type fibroin membrane with traditional chemical cross-linking method, the invention have the advantages that
(1) enzyme catalysis efficiency is high, utilizes tyrosinase catalysis flavone compound high with catalytic efficiency in fibroin albumen graft reaction, and enzyme preparation consumption is less。
(2) ferment treatment condition relaxes, the preparation of antioxidation fibroin membrane is carried out under low temperature and near-neutral sulfite deinking, there is the advantage that energy consumption is low, process process safety, it is to avoid chemical crosslink technique reaction easily causes all many-sided defects such as environmental pollution, fibroin membrane biological safety are low。
(3) fibroin membrane performance improvement is obvious, utilizes the flavone compound of tyrosinase catalysis oxidation phenolic hydroxy group to react with fibroin, not only improves the antioxygenic property of fibroin membrane, enhance fibroin membrane antibiotic property, also improve the mechanical property of membrane material。
Detailed description of the invention
Adopting tyrosinase catalysis flavone compound and fibroin albumen grafting, preparation has the fibroin membrane of better antioxidation and anti-microbial property, and specific embodiment is as follows:
Embodiment 1
(1) silk fibroin solution prepares: joined by the domestic silkworm silk after degumming in lithium bromide-alcohol-water (mass ratio 45:45:10) solution, dissolves at 60 DEG C and prepares 50g/L silk fibroin protein solution。Silk fibroin solution being loaded bag filter, in deionized water dialysis 36 hours, period changes 1 water per hour, is filtrated to get silk fibroin aqueous solution after dialysis。
(2) prepared by fibroin membrane: takes quantitative silk fibroin solution and pours in polytetrafluoroethylene (PTFE) mould, by prolonging stream film forming, and abundant drying and forming-film under-50 DEG C of conditions。
(3) enzymatic fibroin grafting flavone compound: add Mushroom Tyrosinase in epigallo catechin solution, fibroin protein film modification is carried out under persistently logical oxygen condition, wherein, the consumption of fibroin protein film is 100g/L, tryrosinase is 100U/g fibroin protein film relative to the consumption of fibroin membrane, epigallo catechin 2g/L, temperature 30 DEG C, pH scope 6.8, processes 6 hours。
(4) Modified silk fibroin film post processing: after enzymatic graft reaction, fibroin membrane is through 3 secondary chamber Warm Wash, dry under-50 DEG C of conditions, obtains antioxidation fibroin protein film。
Sample 1: only add epigallo catechin during fibroin membrane dipping；
Sample 2: fibroin membrane is through tryrosinase, epigallo catechin combined treatment；
After above-mentioned PROCESS FOR TREATMENT, sample 1 adopts the non-oxidizability of the fibroin membrane that ABTS method records to be 2.4%, and adopting GB15979-2002 to record fibroin membrane to staphylococcus aureus bacteriostasis rate is 8.0%, fibroin membrane fracture strength 15.5N/cm2；The non-oxidizability that sample 2 records is 56.1%, and bacteriostasis rate is 36.6%, fibroin membrane fracture strength 21.0N/cm2。
Embodiment 2
(1) silk fibroin solution prepares: joined by the domestic silkworm silk after degumming in calcium chloride-alcohol-water (mol ratio 1:1:7) solution, dissolves at 70 DEG C and prepares 25g/L silk fibroin protein solution。Silk fibroin solution is loaded bag filter, and dialyse 24h in deionized water, and period changes 1 water per hour, is filtrated to get silk fibroin aqueous solution after dialysis。
(2) prepared by fibroin membrane: takes quantitative silk fibroin solution and pours in polytetrafluoroethylene (PTFE) mould, makes it smooth sprawl by prolonging stream, dry under-50 DEG C of conditions。
(3) enzymatic fibroin grafting flavone compound: add tryrosinase in catechin solution, fibroin protein film modification is carried out under persistently logical oxygen condition, wherein, the consumption of fibroin protein film is 100g/L, tryrosinase is 150U/g fibroin relative to fibroin membrane consumption, catechin 2g/L, temperature 30 DEG C, pH scope 7.0, processes 12 hours。
(4) Modified silk fibroin film post processing: after enzymatic graft reaction, fibroin membrane-50 DEG C of condition cold drying, obtains antibacterial silk fibroin membrane after 2 secondary chamber Warm Wash。
Sample 3: only add catechin during fibroin membrane dipping and process；
Sample 4: fibroin membrane processes through tryrosinase, catechin；
After above-mentioned PROCESS FOR TREATMENT, adopting the performance test methods that sample 1 is addressed, recording sample 3 non-oxidizability is 4.4%, and bacteriostasis rate is 3.8%, fibroin membrane fracture strength 17.0N/cm2；Sample 4 non-oxidizability is 49.0%, and bacteriostasis rate is 37.5%, fibroin membrane fracture strength 22.5N/cm2。
Although the present invention is with preferred embodiment openly as above; but it is not limited to the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can doing various changes and modification, therefore protection scope of the present invention should with being as the criterion that claims define。
Claims (7)
1. the preparation method of an antioxidation fibroin protein film, it is characterised in that utilize tyrosinase catalysis oxidation to have flavone compound and the fibroin albumen generation graft reaction of phenolic hydroxyl group, improve the application performance of fibroin protein film；
Concrete technology step is as follows:
(1) silk fibroin solution is prepared: dissolve fibroin fiber with lithium bromide or calcium chloride, prepare silk fibroin solution；
(2) fibroin membrane is prepared: taking silk fibroin solution and make it smooth sprawl by prolonging stream, abundant lyophilization, obtains fibroin protein film under-50～-20 DEG C of conditions；
(3) enzymatic fibroin grafting flavone compound: add appropriate tryrosinase in the flavone compound solution of phenolic hydroxy group, by fibroin protein film impregnation process in above-mentioned solution under aerobic conditions, carries out enzymatic graft modification；
(4) Modified silk fibroin film post processing: after enzymatic grafting, fibroin membrane is through 2～3 secondary chamber Warm Wash ,-50～-20 DEG C obtain antioxidation fibroin protein film after drying；
Enzymatic reaction condition is: the consumption of tryrosinase is 1～200U/g fibroin protein film, flavone compound 0.5～10g/L, fibroin protein film 50～200g/L, temperature 20～50 DEG C, pH scope 6.0～8.0, processes 1～24 hour。
2. preparation method according to claim 1, it is characterised in that the preparation technology of described silk fibroin solution and condition be: joined by silkworm silk in the water solution system of lithium bromide or calcium chloride, 30～80 DEG C of stirrings to dissolving, prepares 10～50g/L silk fibroin solution；Silk fibroin solution is loaded bag filter, filters after dialysis in deionized water, prepare silk fibroin aqueous solution。
3. preparation method according to claim 1, it is characterised in that the described enzymatic reaction process time is 6-12 hour。
4. preparation method according to claim 1, it is characterised in that lithium bromide-ethanol water or calcium chloride-ethanol water are selected in the preparation of described silk fibroin solution。
5. preparation method according to claim 1, it is characterised in that described tryrosinase is animal, plant or microbe-derived enzyme kind。
6. preparation method according to claim 1, it is characterised in that described flavone compound all contains the phenolic hydroxyl group of varying number。
7. preparation method according to claim 1, it is characterised in that described flavone compound is flavonol, flavanone, flavanonol, isoflavone or isoflavanone。
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| CN105218842B (en) * | 2015-09-25 | 2017-11-03 | 江南大学 | A kind of method that enzyme process prepares fibroin/elastin laminin composite film material |
| CN105088540B (en) * | 2015-09-25 | 2017-03-29 | 江南大学 | A kind of method that nanometer silk fibroin material is prepared based on tryrosinase/polyphenol amboceptoid |
| CN105801771B (en) * | 2016-05-13 | 2018-05-25 | 江南大学 | A kind of method that fibroin base composite absorbent material is prepared based on enzymatic graft copolymerization |
| CN105924596B (en) * | 2016-07-11 | 2018-01-30 | 江南大学 | A kind of method that fibroin base absorbent material is prepared by grafting vinyl monomers |
| CN107099046B (en) * | 2017-04-25 | 2020-03-06 | 江南大学 | Method for preparing fibroin-based biomimetic mineralized material by grafting vinyl monomer |
| CN112679680B (en) * | 2020-12-17 | 2022-12-20 | 苏州经贸职业技术学院 | Bionic piezoresistive material, preparation method thereof and wearable bionic piezoresistive sensor |
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| CN103421201A (en) * | 2012-05-14 | 2013-12-04 | 江南大学 | Preparation method for antibacterial silk fibroin membrane by biological enzyme method |
| CN103526543A (en) * | 2012-08-29 | 2014-01-22 | 江南大学 | Flame-proof treatment method of real silk fabrics by biological enzyme method |
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| CN103421201A (en) * | 2012-05-14 | 2013-12-04 | 江南大学 | Preparation method for antibacterial silk fibroin membrane by biological enzyme method |
| CN103526543A (en) * | 2012-08-29 | 2014-01-22 | 江南大学 | Flame-proof treatment method of real silk fabrics by biological enzyme method |
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