Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization

Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
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; 18896500672@163.com (D.W.);
 lucky940829@163.com (F.Y.)
 * Correspondence: yangxuhong@suda.edu.cn (X.-H.Y.); tangrencheng@suda.edu.cn (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 [1]. 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 [2], 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 [5].
The application of keratin peptide and protein to human hair had a restoring ability in chemically
damaged hair [6]. The blends of keratin with other polymers were frequently electrospun to nanofiber
mats for diverse applications [7]. 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 [10]; the superheated water hydrolyzed keratin


Polymers 2018, 10, 993; doi:10.3390/polym10090993                            www.mdpi.com/journal/polymers
Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
Polymers 2018, 10, 993                                                                               2 of 11



was employed as a foaming agent in the foam dyeing of cotton and wool fabrics [11]; the keratin
extracted by the reduction method could enhance the hydrophilic property of aminolyzed polyester
fabric [12]. 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 [1]. 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 [14]. 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) [18]. HBA and HBD interact with each other to produce a lower freezing point,
allowing them to form a stable solvent at a low temperature [19]. 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 [18]. DES integrates the advantages
of good biocompatibility, environmental protection, economy, innocuity, and harmlessness [20],
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 [25], and a combination of choline chloride and oxalic acid was
successfully used to extract collagen peptides from cod skins at 65 ◦ C [26].
     China is an important producer of rabbit hair and Chinese rabbit hair production makes up a large
proportion of world output [27]. 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 [29].
Recently, we preliminarily explored the combinations of choline chloride with urea, ethylene glycol,
Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
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.,
Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
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,
           [30], 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 [30], 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
                                                                                      [25]. Moreover,        is also
                                                                                                            only     found amount
                                                                                                                  a small    in      of
         the dissolution of wool by the DES of choline chloride and urea [25]. 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).
Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
Polymers 2018, 10, 993                                                                                                            5 of 11
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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 [31].
                                                                          acidThus,    the absorbance
                                                                                sequences     [31]. 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 [26].  Additionally,     the swelling
effect between DES and rabbit hair [26]. 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 [26],
  interactions between protein molecules [26], 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 [26]. The choline chloride and urea solvent at a molar ratio of 2:1 can extract keratin    peptides      from cod
skins
  fromatwool65 ◦ Cfiber
                    [26].atThe
                            170 choline
                                  °C [25]. 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 [25].
  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 [32]. structure makes the
regenerated keratin easier to be applied in various fields [32].
Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization
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              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 [16]. 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     [16]. 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 [16]. 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   [29].
                               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   [29]. 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 [29].




              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   [36],
                                                                                                     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     [36], and   it is  coincident    with
                                                                                                                                        to the
lowerthe loss of crystallinity
       molecular    weight and indicated by XRD.structure of regenerated keratin [36], 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       [1].respectively   [1]. 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 [1].of α-helical   crystallites
                                                       Additionally,  three novel[1]. 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.
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