Green Modification Of Keratin From Waste Resources And Development Of Regenerated Keratin Fibers

Fiber shortage has become one of the most prominent problems across the globe.On the one hand,availability of natural fibers is largely restricted by land,water and other natural resources required for cultivation and farming,cost of pesticides,fertilizers and labor,and the global economy.In contrast with synthetic fibers,natural fibers such as protein fibers do not show any significant increase in their production volume for nearly a decade.On the other hand,the production volume of synthetic fibers such as polyester could not keep growing due to the limitation of nonrenewable petroleum resources.However,the world annual consumption of textile fibers has exceeded 100 million tons since 2018 and keeps expanding.Therefore,it is of critical importance to find alternative sources for textile fibers.Keratinous wastes,especially poultry feathers,are abundant,safe,cost-effective and readily available materials for fiber production.It is estimated more than 10 million tons of keratin wastes are annually generated in the world.The potential production of protein fibers from such materials is already 2.5 times higher than the current output of both wool and silk.Besides with large availability,keratinous materials have better performances due to the high degrees of crosslinking.Keratins contain cysteine residues of about 7-20%,forming a large number of inter-and intra-chain disulfide bonds.As strong covalent bonds,disulfide crosslinkages are not as sensitive to water molecules as hydrogen bonds and ionic bonds.Yet so far,only a few of the feathers are autoclaved or hydrolyzed and then used as animal feed with low nutritional value,and the rest are disposed through landfills.Developing regenerated keratin fibers（RKFB）could not only provide new sources for the fiber industry to alleviate the fiber shortage but also add value to the poultry industry and address related environmental concerns.However,extraction of densely crosslinked keratin from keratinous wastes has been a long-standing difficulty which hinders the utilization of these abundant and renewable resources.Furthermore,inefficient re-construction of crosslinking bonds within interpolymeric chains prevented the RKFB obtaining similar properties to original materials.To improve the mechanical properties of RKFB suitable for textile application,three green methods for modification of regenerated keratin materials（RKM）according the"filament-matrix"structure of natural keratinous materials（NKM）were developed.Increasing the degree of crosslinking and/or crystallinity of RKM was the aim of the modification methods including crosslinking with non-toxic oligosaccharide derivatives,reinforcement with submicron amino acid particles and chain-extending with dithiol.The mechanical properties of RKM usually increased with the increasing of degree of crosslinking and/or crystallinity.The mechanical properties of RKM showed a significant enhancement via dithiol-extending.Therefore,dithiol-extending was used for development of RKFB.The main research contents and results are as follows:（1）Firstly,to analyze the influence of molecular weight distribution（MWD）on the performance of RKM,a new method for quantitative determination of MWD of keratin by gel permeation chromatography coupled with light-scattering（GPC-LS）was established.It is well known that GPC-LS permits the evaluation of absolute molecular weight and MWD of proteins with a high accuracy.However,a GPC-LS analysis for keratin remains challenging mainly due to the insolubility or degradability of keratin in most solvents which work as a mobile phase of GPC.Herein,we have established a new method（GPC-LS-SDS）to effectively solubilize keratin with the preservation of backbones and to quantificationally determinate keratin’s MWD by GPC-LS.It was proved that the bonding of SDS to protein did not affect the accuracy of GPC-LS-SDS.The GPC-LS-SDS method exhibited pinpoint accuracy（2%）with the verification by lysozyme,ovalbumin and bovine serum albumin,and then applied for feather keratin.The results showed that feather keratin was comprised of polypeptide chains with molecular weight of 9.9 kDa,19.9 kDa,31.5 kDa and 100.3 kDa,and the percentage of each component was about 47.5%,26.1%,11.8%,14.6%,respectively.The quantitative analysis of keratin by this method could provide foundation for further of the structure-function relationship of keratinous materials,bioinspired and biomimetic investigations.（2）Oxidized sucrose（OS）were prepared to crosslink keratin materials to improve the performance properties of RKM.Crosslinking is one of the most common methods to enhance performance properties of RKM.However,most crosslinkers for proteins are either expensive,toxic,or with low efficiencies under mild conditions.In this research,OS,a potent and safe crosslinker,were developed to crosslink RKM.The OS improved both tensile strength and elongation of RKM for 79%and 216%,respectively.Mechanism of the crosslinking reaction between keratin and OS was proposed as forming aminals and Schiff bases,which was verified via ¹³C-NMR and ¹H-NMR.Relationship between degree of crosslinking and tensile properties of keratin films was also quantified.OS crosslinking could be an alternative method to improve performance properties of RKM and promote development of regenerated fibers from keratin wastes.（3）Cystine particles with bio-safety and interfacial compatibility were prepared to effectively reinforce keratin films,leading to high strength and unprecedented pliancy under dry and wet states.Reinforcements for RKM were usually synthetic polymers or polysaccharides,and thus usually had non-ideal interfacial properties due to limited compatibility.In this research,submicron cystine particles were employed to reinforce RKM for their high compatibility with keratin and bio-safety.Transition of primary and secondary structures of keratin due to matrix-reinforcement interaction was analyzed.The RKM showed unprecedented pliancy,good tensile properties under humid conditions and biocompatibility,and thus had good potential for biomedical engineering applications.（4）A method to efficiently restore the secondary structure of keratin via lengthy disulfide crosslinking for the development of high-value products from feather wastes was developed.For decades,good flexibility and wet stability of poultry feathers could not be retained in the regeneration of keratin products due to low recovery of original secondary structures of the protein.We find that extension of intrinsic disulfide linkages in keratin restores not only degree of crosslinking but also secondary structures because lengthy crosslinkages can increase the collision possibility of thiol groups on different protein backbones and help sliding of molecular chains.Using dithiothreitol for disulfide extension,breaking elongation,wet strength and weight retention in water of generated keratin films increased 650%,600%,and 10%,respectively,compared to films without disulfide extension.Disulfide extension did not change the cytocompatibility of keratinous products.The new approach makes industrial applications of keratin products with good performance on a large scale possible.（5）Regenerated keratin filaments with high ductility using a dual-functional dithiol chain extender was produced.Due to the high degrees of intermolecular disulfide crosslinking,natural keratin materials,especially feathers and animal hairs,intrinsically have dry and wet tensile properties better than most protein materials.However,regenerated keratin materials developed by various approaches have tensile properties much lower than their natural counterparts.The disparity was due to damages of backbones and unsuccessful reconstruction of intermolecular disulfide crosslinking.In this work,disulfide crosslinking is efficiently reconstructed using a dithiol reducing agent.The regenerated keratin filaments retained the tenacity of natural feathers,and furthermore,demonstrated much higher stretchability in dry and wet states than natural feathers.The new approach has great industrial potential in regeneration of highly-crosslinked materials with much-improved ductility and high retention of tenacity.In summary,increasing of either crystallinity or crosslinking degree could improve the performance of RKM.However,increasing of both crystallinity and crosslinking degree could leading to a significant enhancement in the mechanical properties of RKM.Dithiol chain-extending was a simple effective method to simultaneously increase crystallinity and crosslinking degree of RKM.The mechanical properties of RKFB prepared by dithiol chain-extending could meet the requirements of textile fibers.Dithiol chain-extending provides a reference for the conversion of keratinous waste into high performance RKFB,showing the direction for the utilization of highly crosslinked materials.