Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
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polymers Article Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization Dongyue Wang, Xu-Hong Yang *, Ren-Cheng Tang * ID and Fan Yao National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, 199 Renai Road, Suzhou 215123, China; firstname.lastname@example.org (D.W.); email@example.com (F.Y.) * Correspondence: firstname.lastname@example.org (X.-H.Y.); email@example.com (R.-C.T.) Received: 8 August 2018; Accepted: 3 September 2018; Published: 6 September 2018 Abstract: Keratin from a variety of sources is one of the most abundant biopolymers. In livestock and textile industries, a large amount of rabbit hair waste is produced every year, and therefore it is of great significance to extract keratin from waste rabbit hair in terms of the treatment and utilization of wastes. In this study, a novel, eco-friendly and benign choline chloride/oxalic acid deep eutectic solvent at a molar ratio of 1:2 was applied to dissolve waste rabbit hair, and after dissolution keratin was separated by dialysis, filtration, and freeze-drying. The dissolution temperature effect was discussed, and the resulting keratin powder was characterized by X-ray diffraction, scanning electron microscope, Fourier transform infrared spectroscopy, protein electrophoresis, thermogravimetry and differential scanning calorimetry, and amino acid analysis. During the dissolution process, the α-helix structure of rabbit hair was deconstructed, and the disulfide bond linkages were broken. The solubility of rabbit hair was significantly enhanced by increasing dissolution temperature, and reached 88% at 120 ◦ C. The keratin produced by dissolving at 120 ◦ C displayed flaky powders after freeze-drying, and had a molecular weight ranging from 3.8 to 5.8 kDa with a high proportion of serine, glutamic acid, cysteine, leucine, and arginine. Such features of molecular weight and amino acid distribution provide more choices for the diverse applications of keratin materials. Keywords: keratin; rabbit hair; deep eutectic solvent; dissolution; extraction; biopolymers 1. Introduction Keratin is one of the most abundant biopolymers, and is widely distributed in animal hairs and feathers . In agricultural and industrial production, a large amount of hair and feather wastes are produced every year, resulting in the loss of keratin resources and the aggravation of environmental pollution. Therefore, the recovery and utilization of keratin from animal hairs and feathers are of great significance and have become a research hotspot. As a high value-added and environmentally compatible substance, the extracted keratin is widely studied in biological materials , and has found its applications in many fields. The wool-based keratin hydrolyzates obtained by superheated water and alkaline hydrolysis, and with low molecular weight, were used as an amendment fertilizer to improve plant growth and soil condition [3,4]. The polypeptides of chicken feather keratin hydrolyzed by calcium hydroxide at a high temperature were used as an animal feed supplement . The application of keratin peptide and protein to human hair had a restoring ability in chemically damaged hair . The blends of keratin with other polymers were frequently electrospun to nanofiber mats for diverse applications . Keratin was also used as an additive in natural and synthetic polymers to fabricate composite fibers and materials for improved properties [8,9]. In textile industry, the keratin hydrolyzate modified with a cationic agent was an alternative to sodium sulfate for enhancing the uptake of reactive dyes by cotton fabrics ; the superheated water hydrolyzed keratin Polymers 2018, 10, 993; doi:10.3390/polym10090993 www.mdpi.com/journal/polymers
Polymers 2018, 10, 993 2 of 11 was employed as a foaming agent in the foam dyeing of cotton and wool fabrics ; the keratin extracted by the reduction method could enhance the hydrophilic property of aminolyzed polyester fabric . The different applications of keratin mentioned above have individual requirements. Usually, as an additive in composite materials, keratin with high molecular weight should be used due to the strength requirement, whereas as regarding dyeing and finishing agents, a low molecular weight keratin would be suitable because the water solubility of keratin and the softness of textiles must be taken into account. The different dissolution and extraction approaches of animal hairs and feathers produce different molecular weight keratins which can provide more choices for the diverse applications of keratin materials. The keratin fiber has a stable three-dimensional conformation maintained by a range of noncovalent (hydrogen bonds, ionic bonds, and hydrophobic interactions) and covalent bonds (disulfide bonds) [1,13]. Keratin is distinct from other fibrous proteins in its high sulfur content, high stability of physical and chemical structures, and strong resistance to chemical attacks mainly due to inter- and intrachain cysteine disulfide bond cross-links [1,13]. As far as the crystalline structure is concerned, keratin has three structural forms: α, β, and γ keratin. α-Keratin is tightly coiled, leading to the formation of a rod-like structure, whereas β-keratin consists of a high proportion of parallel sheets of molecular chains . The high stability of the keratin fiber presents a challenge for the dissolution and extraction of keratin substances. Therefore, the breakage of peptide bonds by strong acids and alkalis, the disruption of disulfide bonds by oxidizing and reducing agents, and the deconstruction of physical structures by hydrogen bond disrupters as well as their combinations must be used to dissolve the keratin fiber . Of these approaches, some are limited because of the use of long time, high temperature, and harsh reaction conditions, whereas others have the shortcomings of the use of toxic and harmful reagents. In order to solve the existing problems in the current methods, novel solvents deserve to be further attempted. In this regard, the extraction of keratin by superheated water and ionic liquids has been successfully carried out, and considered as a more environmentally friendly method [13,15–17]. On the other hand, deep eutectic solvent (DES) as a novel and eco-friendly solvent has received great attention. DES is formed by the complexation of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) . HBA and HBD interact with each other to produce a lower freezing point, allowing them to form a stable solvent at a low temperature . For instance, whereas the melting points of choline chloride and urea are 303 and 133 ◦ C, respectively, the freezing point of their mixture at a urea to choline chloride molar ratio of 2:1 is 12 ◦ C . DES integrates the advantages of good biocompatibility, environmental protection, economy, innocuity, and harmlessness , and its research and application have permeated in many chemical fields, such as extraction and separation, synthesis media, metal processing, catalytic reactions, polymer dissolution, etc. [20–24]. Recently, the application of DES in the extraction and separation of natural proteins has also attracted interest from researchers. The DES consisting of choline chloride and urea was employed to deconstruct wool and extract keratin at 170 ◦ C , and a combination of choline chloride and oxalic acid was successfully used to extract collagen peptides from cod skins at 65 ◦ C . China is an important producer of rabbit hair and Chinese rabbit hair production makes up a large proportion of world output . In recent years, the application of rabbit hair in textile industry has attracted great attention. However, because of the low friction coefficient and cohesion force between rabbit hairs, rabbit hair and its mixture with other fibers have low spinnability, and the resulting yarns suffer from a shortcoming of low yielding rate [27,28], thereby producing waste rabbit hair in the spinning process. Additionally, in farms and slaughterhouses, an amount of waste rabbit hair is produced. Like other animal hairs, rabbit hair is rich in keratin which is a valuable natural protein. Thus, how to recycle the waste rabbit hair has also become a research subject. In the previous research, a L-cystein and urea mixture was used to extract keratin from rabbit hair at pH 10.5 and 75 ◦ C for 5 h; the resulting keratin had a molecular weight of about 11 kDa . Recently, we preliminarily explored the combinations of choline chloride with urea, ethylene glycol,
Polymers 2018, 10, 993 3 of 11 citric acid, and oxalic acid at 1:1, 1:2, and 1:3 molar ratios of choline chloride to other additives as DESs to dissolve waste rabbit hair at 98 ◦ C for 2 h, and found that the solubility of rabbit hair was 19–22% in choline chloride-urea, 15–20% in choline chloride-ethylene glycol, 20–27% in choline chloride-citric acid, and 36–71% in choline chloride-oxalic acid, and the choline chloride-oxalic acid mixture at a molar ratio of 1:2 or 1:3 showed a better dissolution effect on rabbit hair. Additionally, prolonging time could increase the solubility of rabbit hair at 98 ◦ C. In this work, the combination of choline chloride and oxalic acid at a molar ratio of 1:2 was used as DES to dissolve rabbit hair, and the effect of temperature on the dissolution efficiency of rabbit hair was discussed. Afterwards, the keratins obtained by dissolving at different temperatures were separated by dialysis, filtration, and freeze-drying. The resulting keratin powders were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), thermogravimetry (TG)/differential scanning calorimetry (DSC), protein electrophoresis, scanning electron microscope (SEM), and amino acid analysis. 2. Materials and Methods 2.1. Materials Waste rabbit hair used in this paper was collected from a slaughterhouse (Shiyan, China). It was scoured in a 2 g/L sodium carbonate solution at 50 ◦ C for 30 min, rinsed thoroughly in tap water, and then dried in open air. The resulting rabbit hair was used in the dissolution test. Choline chloride and oxalic acid were of analytical reagent grade, and purchased from 9-Ding Chemistry Co. Ltd. (Shanghai, China) and Titanchem Co. Ltd. (Shanghai, China), respectively. The dialysis bag with a theoretical molecular weight cut-off of 3 kDa was provided by Biosharp Co. Ltd. (Hefei, China). 2.2. Extraction of Keratin from Rabbit Hair with DES Choline chloride and oxalic acid were mixed at a molar ratio of 1:2 in the conical flask, and then the flask was placed in the XW–ZDR low-noise oscillated dyeing machine (Jingjiang Xinwang Dyeing and Finishing Machinery Factory, Jingjiang, China) and oscillated at 70 ◦ C for 20 min. The resulting homogeneous and colorless solution was called DES. Rabbit hair (0.2 g) was thoroughly immersed in DES (20 g) present in the conical flask which was placed and heated in a glycerol bath. The mixture of rabbit hair and DES was heated for 2 h under a stirring condition at various temperatures ranging from 80 to 120 ◦ C. After cooling, the solution was dialyzed in a water bath for 2 days using the dialysis bag with a theoretical molecular weight cut-off of 3 kDa. After dialysis, the mixture was filtered to remove undissolved rabbit hair. Afterwards, the resulting undissolved rabbit hair was dried, and the solubility of rabbit hair was calculated using the following equation: W0 − W1 Solubility (%) = × 100 (1) W0 where W 0 and W 1 represent the weight of rabbit hair before and after dissolution, respectively. In order to obtain the keratin powders for further characterization, the above filtrate solution was freeze-dried using the VirTis freeze mobile 25EL lyophilizer freeze dryer (SP Scientific Inc., New York, NY, USA). 2.3. Measurements The UV-Vis absorption spectra of rabbit hair keratin solutions were recorded by the Shimadzu UV-1800 UV-Vis spectrophotometer (Shimadzu Co., Kyoto, Japan). The FT-IR spectra of rabbit hair and keratin powder were recorded by the Nicolet 5700 FTIR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) using KBr pellets. The amino acid analysis of rabbit hair and keratin powder was carried out using the L-8900 automatic amino acid analyzer (Hitachi High-Technologies Co.,
Polymers 2018, 10, 993 4 of 11 Tokyo, Japan); the sample (10 mg) was hydrolyzed with 6 mol/L HCl at 110 ◦ C for 24 h in nitrogen atmosphere, and then the diluent (0.2 mg/mL) was used for the free amino acid analysis; the amino acid composition was determined by calibration with the external standard amino acid solutions, and each sample was analyzed in duplicate. The molecular weight distribution of keratin was measured on the PowerPac universal Polymers 2018, 10, x FOR PEER REVIEW 4 of 11 type protein electrophoresis (Bio-Rad Laboratories Inc., Hercules, CA, USA) by sodium dodecyl sulphate-polyacrylamide the amino acid composition gel electrophoresis was determined (SDS-PAGE). According by calibration with to standard the external the method aminodepicted acid by Laemmlisolutions, , 5%and concentration each sample was gelanalyzed and 12% separation gel were chosen as electrophoresis plastics, in duplicate. The molecular weight and the low-molecular-weight distribution standard of keratin protein was measured (3.8–20.1 kDa) wason the as used PowerPac a marker universal type for electrophoresis. protein electrophoresis (Bio-Rad Laboratories Inc., Hercules, CA, USA) by sodium dodecyl The surface morphologies of rabbit hair and its residues in DES solution as well as keratin powders sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). According to the method depicted by were observed by the TM3030 tabletop SEM (Hitachi High Technologies America, Inc., Schaumburg, Laemmli , 5% concentration gel and 12% separation gel were chosen as electrophoresis plastics, IL, USA)andwith an acceleration voltage the low-molecular-weight of 15 kV. standard The(3.8–20.1 protein XRD patterns kDa) wasof rabbit used ashair and keratin a marker for were obtainedelectrophoresis. by X’Pert-Pro MPD X-ray diffraction (PANalytical B.V., Almelo, The Netherlands). The TG The surface and DSC thermal analyses morphologies of rabbit of rabbit hair andhair and its keratin residues powder in DES were solutionbyasthe obtained well as keratin SDT Q600 thermal powders were observed by the TM3030 tabletop SEM (Hitachi High analyzer (TA Instruments Inc., New Castle, DE, USA) in nitrogen with the temperature range Technologies America, Inc., from 40 Schaumburg, IL, USA) with an acceleration voltage of 15 kV. The XRD patterns of rabbit hair and to 600 ◦ C at the heating rate 10 ◦ C/min. keratin were obtained by X’Pert-Pro MPD X-ray diffraction (PANalytical B.V., Almelo, The Netherlands). The TG and DSC thermal analyses of rabbit hair and keratin powder were obtained by 3. Results and Discussion the SDT Q600 thermal analyzer (TA Instruments Inc., New Castle, DE, USA) in nitrogen with the temperature range from 40 °C to 600 °C at the heating rate 10 °C/min. 3.1. Dissolution of Rabbit Hair in DES 3. Results and Discussion The solubility of rabbit hair in DES at various temperatures was observed from the quantity of residual3.1. rabbit hair in Dissolution of the solvent, Rabbit the transparency of the solvent, and the morphological structure Hair in DES of residual rabbit hair. Figure 1 shows the appearance of the rabbit hair and DES mixture as well The solubility of rabbit hair in DES at various temperatures was observed from the quantity of as the SEM images residual rabbitofhair residual rabbit the in the solvent, hairs. At roomoftemperature, transparency the solvent, andrabbit hair was insoluble the morphological structure in DES, and the ofshape of rabbit residual hairFigure rabbit hair. was clearly 1 shows visible with the the appearance clean of the scales rabbit on its hair and DES surface. mixture At 80 ◦as as well C, a large number the SEM images of rabbit hairsofwere residual rabbit hairs. dispersed At room in DES, andtemperature, most of the rabbit hairon scales wasthe insoluble rabbitinhair DES, and surface were the shape still present of rabbit but some hairshairand wasscales clearlywere visibledamaged, with the clean scales onthe indicating its low surface. At 80 °C, solubility ofarabbit large hair at number of rabbit hairs were dispersed in DES, and most of the scales on the rabbit hair surface were this temperature. At 98 ◦ C, most of rabbit hairs were broken into pieces by DES, and lost their clean still present but some hairs and scales were damaged, indicating the low solubility of rabbit hair at fibrous shapes. At 120 ◦At this temperature. C,98almost °C, most noofrabbit hair fiber rabbit hairs was found were broken in solution, into pieces andlost by DES, and thetheir solution clean turned out to be viscous and dark brown. The increased viscosity is due to the high fibrous shapes. At 120 °C, almost no rabbit hair fiber was found in solution, and the solution turnedsolubility of keratin, while theoutcolor to bechange viscous originates and dark brown. fromThetheincreased release of melanin viscosity in cortical is due to the high cells, whichofiskeratin, solubility also found in while theofcolor the dissolution wool change by the originates DES of from cholinethe release of melanin chloride and urea in cortical cells, which . Moreover, is also only found amount a small in of the dissolution of wool by the DES of choline chloride and urea . Moreover, only a small amount residues with a sheet structure were collected. The above observations indicate that with the increase of residues with a sheet structure were collected. The above observations indicate ◦ rabbitthat with the of dissolving increase temperature, of dissolving the solubility temperature, theofsolubility rabbit hair increases of rabbit and at 120 hair increases and atC120 hair approaches °C rabbit hair to complete dissolution. approaches to complete dissolution. Figure 1. Appearance of the rabbit hair and DES mixture (A) and SEM images of residual rabbit hairs Figure 1.atAppearance of the rabbit hair and DES mixture (A) and SEM images of residual rabbit hairs various temperatures (B). at various temperatures (B).
Polymers 2018, 10, 993 5 of 11 Polymers 2018, 10, x FOR PEER REVIEW 5 of 11 3.2. 3.2.Solubility SolubilityofofRabbit RabbitHair Hairand and Absorbance Absorbance ofof Regenerated RegeneratedKeratin KeratinSolution Solution InIn order to investigate order the dissolving to investigate property the dissolving of rabbitofhair property at various rabbit hair at temperatures quantitatively, various temperatures the solubility of rabbit hair and the absorbance of keratin solution were quantitatively, the solubility of rabbit hair and the absorbance of keratin solution were measured measured and shown in Figure 2. The in and shown keratin Figuresolution had a maximum 2. The keratin solution had absorption a maximum peakabsorption at about 272 peaknm, at corresponding about 272 nm, to the absorption oftothe corresponding thearomatic absorption amino acid of the sequences aromatic amino . acidThus, the absorbance sequences . Thus,of keratin the solution absorbance of at 272 nm could characterize the quantity of keratin. Figure 2 shows that both keratin solution at 272 nm could characterize the quantity of keratin. Figure 2 shows that both the the solubility of rabbit hair and theofabsorbance solubility rabbit hair and of keratin solution the absorbance ofalmost keratin increased linearly solution almost with the increased rise in linearly temperature. with the rise in temperature. This This reveals reveals the enhanced the enhanced soluble behavior soluble of rabbitbehavior hair with of rising rabbit temperature. hair with rising Attemperature. 80, 98, and 120 At ◦ C, 80,solubility the 98, and 120 of°C, the solubility rabbit hair was of rabbit65.7%, 28.9%, hair wasand28.9%, 65.7%, 88.7%, and 88.7%,Rabbit respectively. respectively. Rabbit hair hair exhibited high exhibited high ◦ solubility at 120 °C. The increased solubility of rabbit hair at high solubility at 120 C. The increased solubility of rabbit hair at high temperatures can be explained by temperatures can be explained the following. by (a) theAsfollowing. (a) As therises, the temperature temperature rises, the the viscosity viscosity of DES of DES reduces, reduces, the promoting promoting the mass transfer mass transfer effect between DES and rabbit hair . Additionally, the swelling effect between DES and rabbit hair . Additionally, the swelling extent of rabbit hair increases extent of rabbit hair atincreases at high temperatures. high temperatures. The two factorsThe twocanfactors can accelerate accelerate the diffusion the of diffusion DES into of the DEShairintointerior the hairand interior and subsequently enhance the dissolution of rabbit hair. (b) With subsequently enhance the dissolution of rabbit hair; (b) With the increase in temperature, the ionization the increase in temperature, the ionization of oxalic acid increases, causing an increase in the acidity of the of oxalic acid increases, causing an increase in the acidity of the dissolution system. Thus the increased dissolution system. Thus the increased ionization of basic amino acid sequences weakens the ionization of basic amino acid sequences weakens the interactions between protein molecules , interactions between protein molecules , and at the same time the strong acidity increases the and at the same time the strong acidity increases the hydrolysis extent of protein, resulting in the hydrolysis extent of protein, resulting in the increased solubility of rabbit hair. increased solubility of rabbit hair. From the results of Figures 1 and 2, rabbit hair has a high solubility at 120 °C in the choline From the results of Figures 1 and 2, rabbit hair has a high solubility at 120 ◦ C in the choline chloride/oxalic acid solvent at a molar ratio of 1:2. The use of the same solvent at a choline chloride to chloride/oxalic oxalic acid molar acidratio solvent of 1:1at provides a molar ratio of 1:2. a high The use extraction of the same efficiency solventpeptides of collagen at a cholinefromchloride cod toskins oxalicatacid molar ratio of 1:1 provides a high extraction efficiency of collagen 65 °C . The choline chloride and urea solvent at a molar ratio of 2:1 can extract keratin peptides from cod skins fromatwool65 ◦ Cfiber .atThe 170 choline °C . chloride andindicate These facts urea solvent that the at dissolution a molar ratio of of 2:1 can keratin extract fibers withkeratin the from wool fiberinatDESs ◦ 170 must C . cuticle layers beThese carriedfacts outindicate that the dissolution at high temperatures becauseofofkeratin fibers their tight with the cuticle structures. layers in DESs must be carried out at high temperatures because of their tight structures. Figure2. Figure 2. Solubility Solubility of of rabbit rabbithair hairininDES DESatatvarious varioustemperatures. temperatures. 3.3.Characterization 3.3. CharacterizationofofRegenerated Regenerated Keratin Keratin 3.3.1.Morphological 3.3.1. MorphologicalStructure Structure Figure3 3shows Figure showsthe themorphologies morphologies of of rabbit rabbit hair hairand andregenerated regeneratedkeratin keratinpowders powdersbyby dissolving dissolving in DES at various temperatures. With regard to the raw rabbit hair, the scales were in DES at various temperatures. With regard to the raw rabbit hair, the scales were clearly clearly visible, visible, and the cuticle scale edges were at large angles to the axial direction as shown in the enlarged image and the cuticle scale edges were at large angles to the axial direction as shown in the enlarged image in in Figure 3a. Compared with the raw rabbit hair, the keratin powders underwent dramatic changes Figure 3a. Compared with the raw rabbit hair, the keratin powders underwent dramatic changes in in shape, and did not display the residue of rabbit hair, indicating that they lost the compact shape, and did not display the residue of rabbit hair, indicating that they lost the compact structure structure of rabbit hair. The keratin obtained by dissolving at 80 °C was in the form of lumpy of rabbit hair. The keratin obtained by dissolving at 80 ◦ C was in the form of lumpy powders, powders, whereas the samples produced at 98 and 120 °C became flaky powders and had a loose whereas the samples produced at 98 and 120 ◦ C became flaky powders and had a loose structure as structure as high temperatures greatly enhanced the rupture extent of rabbit hair. Such a loose high temperatures structure greatly makes the enhanced regenerated the rupture keratin easier toextent of rabbit be applied hair. Such in various a loose fields . structure makes the regenerated keratin easier to be applied in various fields .
Polymers 2018, 10, x FOR PEER REVIEW 6 of 11 Polymers 2018, 10, 993 6 of 11 Polymers 2018, 10, x FOR PEER REVIEW 6 of 11 Figure 3. SEM images of rabbit hair (a) and keratin samples produced by dissolving at 80 (b), 98 (c), Figure 3. SEM images of rabbit hair (a) and keratin samples samples produced produced by by dissolving dissolving at at 80 80 (b), (b); 98 98 (c), (c); and ◦ C120 °C (d). and 120 °C (d). 3.3.2. Molecular Weight Distribution 3.3.2. Molecular Weight Distribution The SDS-PAGE analysis was employed to investigate the molecular weight distribution of regenerated The SDS-PAGE keratins produced analysis was by dissolving employed to ininvestigate DES at various temperatures the molecular (Figuredistribution weight 4). The of dissolving regenerated regenerated temperature keratins keratins produced hadby produced an by obvious dissolving influence dissolvingin DES inonat the molecular various DES weight temperatures at various of the keratin.4). (Figure temperatures For keratin The (Figure dissolving 4). The produced at 80 °C, the entire band was stained, indicating that the molecular weight is distributed temperature dissolving had an obvious temperature had an influence obviouson the molecular influence on the weight molecular of the keratin. weight For of the keratinFor keratin. produced keratin throughout the band and some fragments had molecular weight exceeding 20.1 kDa, but the at 80 ◦ C, the produced at entire 80 °C, band was stained, the entire band was indicating stained, that the molecular indicating that theweight molecular is distributed weight is throughout distributed majority had a molecular weight ranging from 5.8 to 10 kDa. The molecular weight of regenerated the band throughout and keratins some the band producedfragments and by some had dissolving atmolecular fragments 98 and 120had weight °C exceeding molecular was between weight 3.8 20.1 and kDa, 7.8exceeding kDa andbut3.8the 20.1 andmajority kDa, 5.8 kDa, had but thea molecular majority weight had respectively. ranging a molecular from weight As the temperature5.8ranging toincreases, 10 kDa. fromThe the5.8 molecular to 10 kDa. decomposition weight degree ofofregenerated The molecular weight rabbit hair iskeratins produced of regenerated aggravated by dissolving keratinsand moreat produced 98byand keratin 120 ◦ C dissolving molecules arewas at 98 between and 120 to disintegrated °C3.8 andbetween was smaller 7.8 kDa pieces, and 3.8 and3.8 resulting in7.8and thekDa 5.8 and increased kDa, 3.8 respectively. and content 5.8 kDa, of the low respectively. As molecular weight As theincreases, temperature keratin. temperature The lower molecular increases, the decomposition the degree weight decomposition of regenerated of rabbit degree keratinshair of rabbit hair is aggravated produced andismore atkeratin aggravated and higher more keratin molecules temperatures is in molecules are are disintegrated agreement with disintegrated to smaller the morphological pieces,toresulting smaller pieces,structure of resulting in in the increased Figure 3, where the increased content the high content of of low molecular temperature dissolution produces a more loose form of keratin. low molecular weight weight keratin. The lowerkeratin. The weight molecular lower molecular of regenerated weight of regenerated keratins produced atkeratins produced at higher temperatures higher temperatures is in agreement is in with the agreement with morphological the morphological structure of Figure 3, where structure the highof Figure 3, where temperature the high dissolution temperature produces dissolution a more loose formproduces a more loose form of keratin. of keratin. Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures. 3.3.3. XRD Pattern The XRD patterns of rabbit hair and keratin obtained using DES at 120 °C are shown in Figure 5. The raw rabbit hair showed a small diffraction peak at 9° and a broad diffraction peak at 20°, Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures. Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures. 3.3.3. XRD Pattern 3.3.3. XRD Pattern The XRD patterns of rabbit hair and keratin obtained using DES at 120 °C are shown in Figure 5. The XRD patterns of rabbit hair and keratin obtained using DES at 120 ◦ C are shown in Figure 5. The raw rabbit hair showed a small diffraction peak at 9°◦ and a broad diffraction peak at 20°, The raw rabbit hair showed a small diffraction peak at 9 and a broad diffraction peak at 20◦ ,
Polymers 2018, 10, x FOR PEER REVIEW 7 of 11 corresponding to the α-helix and β-sheet structures, respectively . In the XRD pattern of the Polymers 2018, 10, x FOR PEER REVIEW 7 of 11 extracted keratin, the diffraction peak at 9° disappeared, indicating that the α-helix structure of rabbit hair2018, corresponding Polymers is to destroyed in the the α-helix 10, 993 anddissolution process [16,29,33]. β-sheet structures, Interestingly, respectively . In thecompared with XRD pattern the 7 of of raw 11 the rabbit hair, extracted the the keratin, extracted keratin diffraction peakdisplayed an increaseindicating at 9° disappeared, in the diffraction intensity that the α-helix of β-sheet structure of structure at 20°, rabbitcorresponding hair is destroyedsuggesting the increased content of β-sheet structure in keratin. in the dissolution process [16,29,33]. Interestingly, compared with the raw These changes to the α-helix and β-sheet structures, respectively . In the XRD pattern of the revealhair, rabbit the disorder the and rearrangement extracted keratinpeak of keratin displayed an crystallites increase induring thethat the dissolution diffraction and regeneration extracted keratin, the diffraction at 9◦ disappeared, indicating the α-helix intensity structure ofofrabbit β-sheet process. structure Similar changes were also found in the keratin extracted from rabbit hair using the mixture hair isatdestroyed 20°, suggesting the increased in the dissolution processcontent of β-sheet [16,29,33]. structure Interestingly, in keratin. compared with theThese changes raw rabbit of L-cystein reveal thethe hair, and urea disorder extractedand . rearrangement keratin displayed anofincrease keratinincrystallites during the diffraction the dissolution intensity at 20◦ , and regeneration of β-sheet structure process. Similarthe suggesting changes were increased alsooffound content instructure β-sheet the keratin extracted in keratin. from These rabbit changes hairthe reveal using the mixture disorder and rearrangement of L-cystein of keratin and urea . crystallites during the dissolution and regeneration process. Similar changes were also found in the keratin extracted from rabbit hair using the mixture of L-cystein and urea . Figure 5. XRD patterns of rabbit hair and regenerated keratin by dissolving in DES at 120 °C. 3.3.4. Thermal Stability Figure Figure 5. XRD 5. XRD patterns patterns of rabbit of rabbit hairand hair andregenerated regenerated keratin keratinby bydissolving dissolvingin DES at 120 in DES ◦ C. at 120 °C. Figure 6 shows the thermal behaviors of rabbit hair and regenerated keratin obtained using DES3.3.4. 3.3.4. Thermal at 120 Thermal InStability Stability °C. the TG curves of Figure 6a, both rabbit hair and regenerated keratin underwent a three-step Figure 6weight Figure 6 shows shows loss. thethe The thermal firstbehaviors thermal weight loss behaviors step occurred of rabbit of rabbit hair hair and below 150keratin andregenerated regenerated °C, corresponding keratin obtained obtainedusing to the using DES at 120 ◦ C. In the TG curves of Figure 6a, both rabbit hair and regenerated keratin underwent evaporation DES at 120 °C.of Inbound the TGwater. curves The of second Figure 6a,weight bothloss stephair rabbit wasand in the temperature regenerated range keratin of 220 to 375 underwent a a three-step weighthair loss.and The190 first weight loss stepregenerated occurred below 150 ◦respectively, C, corresponding to the three-step weight loss. The first weight loss step occurred below 150 °C, correspondingistoa rapid °C for the raw rabbit to 370 °C for the keratin, which the evaporation weight lossofstep ofcaused bound by water. melting The second weight loss step α-helix was in the temperature range of 220 to evaporation ◦ bound water. the The second and destruction weight ◦ loss stepof was in thestructure, temperature the breakage range of 220 of disulfide to 375 375 C for the raw rabbit hair and 190 to 370 C for the regenerated keratin, respectively, which is bonds, °C fora the the raw thermal rabbitlossdecomposition hair and 190 toby of 370 peptide bridges, chain linkages, and small lateral chains, and rapid weight step caused the°C for theand melting regenerated destruction keratin, respectively, of α-helix structure, the which is a rapid breakage the evolution weight of loss step bonds, gaseous caused products bythermal the melting [34,35]. The last step and destruction was a gradual the of α-helix weigh loss caused by the of disulfide the decomposition of peptide bridges,structure, chain linkages,breakage and smalloflateral disulfide further bonds,chains, pyrolysis the thermal of the degraded decomposition and the evolution products. of peptide of gaseous Compared productsbridges, [34,35]. Thechainwith the lastlinkages, raw step was aand rabbit small gradual hair, the lateral weigh regenerated losschains, caused and keratin the evolutionshowed by the further reduced of gaseous thermal pyrolysis products stability, [34,35]. of the degraded as indicated The last products. by step was Compared its lower witha the initial gradual degradation weigh raw rabbit hair,loss temperature caused by the the regenerated and higher weight loss at the second weigh loss step. This poor further pyrolysis of the degraded products. Compared with the raw rabbit hair, the regenerated keratin showed reduced thermal stability, as indicated by its lower thermal initial stability degradation is mainly temperaturedue to the lower keratin molecular andshowed higher weight weight reduced and lossthermal disordered at the second weigh stability, structure asloss of step. This indicated regenerated bypoor thermal its lower keratin , stability initial and is mainly due degradation it is coincident to the temperature with the lower loss of molecular crystallinity weight and indicated disordered by XRD. structure of regenerated keratin and higher weight loss at the second weigh loss step. This poor thermal stability is mainly due , and it is coincident with to the lowerthe loss of crystallinity molecular weight and indicated by XRD.structure of regenerated keratin , and it is coincident disordered with the loss of crystallinity indicated by XRD. Figure 6.Figure TG (a)6.and DSC TG (a) and(b) DSCcurves of rabbit (b) curves hair of rabbit and hair andregenerated keratin regenerated keratin byby dissolving dissolving in DES in DES ◦ C. at 120at 120 °C. In the DSC curves of Figure 6b, the raw rabbit hair had an endothermic peak at 242 °C, which is Figure 6. TG (a) and DSC (b) curves of rabbit hair and regenerated keratin by dissolving in DES at 120 °C. assigned to the melting and destruction of α-helical crystallites [13,37]. However, the same peak In the DSC curves of Figure 6b, the raw rabbit hair had an endothermic peak at 242 °C, which is assigned to the melting and destruction of α-helical crystallites [13,37]. However, the same peak
Polymers 2018, 10, 993 8 of 11 In2018, Polymers the DSC curves 10, x FOR 6b, the raw rabbit hair had an endothermic peak at 242 ◦ C, which PEERofREVIEW Figure is 8 of 11 assigned to the melting and destruction of α-helical crystallites [13,37]. However, the same peak disappeared disappeared for for the the regenerated regenerated keratin, keratin, but but aa weak weak peak peak with with an an endothermic endothermic effect effect appeared appeared at at approximately approximately 196 C. Such changes may be associated with the formation of disordered structures 196 °C. ◦ Such changes may be associated with the formation of disordered structures during during the the dissolution dissolution of of rabbit rabbit hair hair at at high high temperature temperature [13,37]. [13,37]. 3.3.5. 3.3.5. FT-IR FT-IR Spectrum Spectrum The FT-IR spectra The FT-IR spectraofofrabbit rabbit hair hair andand regenerated regenerated keratin keratin obtained obtained usingusing DES atDES 120at◦ C120 are °C are shown shown in Figure 7. The FT-IR spectrum of the raw rabbit hair displayed the characteristic in Figure 7. The FT-IR spectrum of the raw rabbit hair displayed the characteristic absorption bands absorption bands of keratinous of keratinous fibers atfibers at 3420, 3420, 1650, 1543,1650, 1240,1543, 1240, and 685 −1 , which cmand 685 cm −1,assigned are which are assigned to the absorptionto the of absorption of hydrogen hydrogen bonded bonded NH groups NH groups (amide A, N–H(amide A, N–H stretching), amidestretching), amide I (C=O I (C=O stretching), amidestretching), II (N–H amide II (N–H bending), amidebending), III (C–Namide III (C–N stretching), andstretching), and amide IV, amide IV, respectively .respectively . The The regenerated regenerated keratin showed keratin showed no noticeable no noticeable changes changesofin in the positions the positions absorption of indicating bands, absorptionthat bands, indicating the DES that followed dissolution the DES dissolution followed by dialysis can provide by adialysis purified can provide keratin a purified product. keratin with Compared product. Compared the rabbit with hair, the the rabbit regenerated hair, theexhibited keratin regeneratedthe keratin intensifiedexhibited the intensified absorption absorption of the amide I band, of the amide which might Ibe band, which with associated mighta be associated lower quantitywith a lowercrystallites of α-helical quantity .of α-helical crystallites Additionally, three novel. Additionally, weak absorption three bandsnovel weak appeared absorption bands appeared − 1 at 1317, 1170, and 1124 cm −1 , which are related at 1317, 1170, and 1124 cm , which are related to the breakage of disulfide bonds and the formation to the breakage of disulfide bonds and the of sulfur-containing formation derivatives of sulfur-containing derivatives [1,36]. [1,36]. Figure Figure 7. 7. FT-IR FT-IR spectra spectra of of rabbit rabbit hair hair and and regenerated regenerated keratin ◦ C. keratin by dissolving in DES at 120 °C. 3.3.6. 3.3.6. Amino Amino Acid Acid Composition Composition Figure Figure 88shows showsthe theamino aminoacidacidcompositions compositions ofof rabbit rabbithair and hair andregenerated regenerated keratin produced keratin producedby dissolving at 120 °C. ◦ Regardless of rabbit hair or regenerated keratin, by dissolving at 120 C. Regardless of rabbit hair or regenerated keratin, the main amino acids the main amino acids compositions compositions were werealmost almostthethe same, same, andandtheythey werewere serine, serine, glutamicglutamic acid, cysteine, acid, cysteine, leucine, leucine, and and arginine. arginine. In the In the total totalacids amino amino acids analyzed analyzed in the present in the present study, study, the content the content of theoffive the amino five amino acidsacids was was 52.8% for rabbit hair, and 59.8% for regenerated keratin, respectively. Compared 52.8% for rabbit hair, and 59.8% for regenerated keratin, respectively. Compared with the raw rabbit with the raw rabbit hair, hair, the the regenerated regenerated keratinkeratin had obvious had obvious changes changes in amino in amino acidacid compositions. compositions. TheThe contents contents of of aspartic aspartic acid,acid, serine,serine, glycine,glycine, alanine,alanine, tyrosine, tyrosine, phenylalanine, phenylalanine, and histidine anddecreased histidineconsiderably decreased considerably (reduction rate (reduction exceededrate exceeded 15%), whereas 15%), the whereas contentsthe contents of of glutamic glutamic acid, acid, cysteine, cysteine, valine, andvaline, lysine and lysine increased considerably (increment rate exceeded 13%). Glycine and increased considerably (increment rate exceeded 13%). Glycine and cysteine had the highest reduction cysteine had the highest reduction and highest and in increment highest increment content, in content, respectively. respectively. These changes of aminoThese changes acid of amino compositions acid indicate compositions indicate that the different acid amino acid sequences of rabbit hair that the different acid amino acid sequences of rabbit hair are subjected to the different breakages are subjected to the different during the breakages dissolutionduring the dissolution process, thus producingprocess, thus of a series producing polypeptides a series withof different polypeptides with molecular different molecular weights. Because small molecular polypeptides are removed weights. Because small molecular polypeptides are removed during the dialysis with a molecular during the dialysis with weighta molecular cut-off of 3weight kDa, thecut-off of 3difference obvious kDa, the obvious of aminodifference of amino appears acid compositions acid compositions appears between rabbit hair between rabbit hair and regenerated keratin. Additionally, the low-sulfur, and regenerated keratin. Additionally, the low-sulfur, high-sulfur, and high-tyrosine protein fractionshigh-sulfur, and high-tyrosine protein fractions of rabbit hair might be ruptured to different extents. This is also the reason for the changes of amino acid compositions.
Polymers 2018, 10, 993 9 of 11 ofPolymers rabbit 2018, hair 10, x FOR might bePEER REVIEW ruptured 9 of 11 to different extents. This is also the reason for the changes of amino acid compositions. Figure 8. Amino acid content of rabbit hair and regenerated keratin by dissolving in DES at 120 ◦ C. Figure 8. Amino acid content of rabbit hair and regenerated keratin by dissolving in DES at 120 °C. 4.4.Conclusions Conclusions Considering Consideringthatthatthe thewaste wasterabbit rabbithair hairfrom fromlivestock livestockandandtextile textileindustries industriesisisa avaluable valuablekeratin keratin source, and that keratins obtained by different dissolution methods have source, and that keratins obtained by different dissolution methods have diverse applications, diverse applications,the the present study provided an approach to dissolve and extract keratin from present study provided an approach to dissolve and extract keratin from rabbit hair using the rabbit hair using the mixture mixtureofofcholine cholinechloride chloride andand oxalic acid at oxalic acid ataamolar molarratio ratioofof 1:21:2 as as a DES a DES which which is considered is considered to be tonontoxic be nontoxic and eco-friendly. The instrumental analyses demonstrated that the disorder ofthe and eco-friendly. The instrumental analyses demonstrated that the disorder of the crystalline crystallinestructures structuresofofprotein proteinandandthe thebreakage breakageofofthe thedisulfide disulfidebond bondofofprotein proteinchains chainsoccurred occurred during duringthethedissolution dissolutionprocess. process.The Thesolubility solubilitytest testrevealed revealedthat thatincreasing increasingdissolution dissolutiontemperature temperature remarkably remarkably enhanced the dissolution of rabbit hair. After dissolution, the pure keratin powderwas enhanced the dissolution of rabbit hair. After dissolution, the pure keratin powder was easily easilyobtained obtained byby dialysis, dialysis,filtration, and filtration, freeze-drying. and freeze-drying. TheThe keratin produced keratin produced by dissolving by dissolvingin DES at in DES 120 ◦ C displayed flaky powders after freeze-drying and lower thermal stability than the raw rabbit hair, at 120 °C displayed flaky powders after freeze-drying and lower thermal stability than the raw and hadhair, rabbit a molecular and hadweight ranging a molecular from 3.8 weight to 5.8 from ranging kDa. 3.8 Thetosum5.8 of serine, kDa. Theglutamic acid, cysteine, sum of serine, glutamic leucine, and arginine accounted for 59.8 wt % in the total content of analyzed acid, cysteine, leucine, and arginine accounted for 59.8 wt % in the total content of analyzedamino acids. The keratin amino obtained acids. The keratin obtained by dissolving rabbit hair in DES could be used as the dyeing the by dissolving rabbit hair in DES could be used as the dyeing and finishing agents for and chemical finishingprocessing agents for of the textiles, and may chemical also findofapplication processing in areas textiles, and maywhere a lowapplication also find molecular weight in areas keratin whereisa required. low molecular weight keratin is required. Author Contributions: D.W. carried out the experiments and wrote the manuscript. X.-H.Y. and R.-C.T. guided Author the Contributions: research and revised theD.W. carried out manuscript. the experiments F.Y. participated in theand wrote theanalyses. instrumental manuscript. X.-H.Y. and R.-C.T. guided the research and revised the manuscript. F.Y. participated in the instrumental analyses. Funding: This research was funded by Science and Technology Support Program of Jiangsu Province (grant number BE2015066). Funding: This research was funded by Science and Technology Support Program of Jiangsu Province (grant number BE2015066). Acknowledgments: This study was supported by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions (No. 2014-37). Acknowledgments: This study was supported by the Priority Academic Program Development (PAPD) of Conflicts Jiangsu of Interest: Higher The authors Education declare(No. Institutions no conflict of interest. 2014-37). Conflicts of Interest: The authors declare no conflict of interest. References 1.References Shavandi, A.; Carne, A.; Bekhit, A.A.; Bekhit, A.E.-D.A. An improved method for solubilization of wool keratin using peracetic acid. J. Environ. Chem. Eng. 2017, 5, 1977–1984. [CrossRef] 1. Shavandi, A.; Carne, A.; Bekhit, A.A.; Bekhit, A.E.-D.A. An improved method for solubilization of wool 2. Shavandi, A.; Silva, T.H.; Bekhit, A.A.; Bekhit, A.E.-D.A. 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