Extraction of Keratin from Rabbit Hair by a Deep Eutectic Solvent and Its Characterization - MDPI
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polymers
Article
Extraction of Keratin from Rabbit Hair by a Deep
Eutectic Solvent and Its Characterization
Dongyue Wang, Xu-Hong Yang *, Ren-Cheng Tang * ID
and Fan Yao
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering,
Soochow University, 199 Renai Road, Suzhou 215123, China; 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
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,
Polymers 2018, 10, 993 3 of 11
citric acid, and oxalic acid at 1:1, 1:2, and 1:3 molar ratios of choline chloride to other additives as DESs
to dissolve waste rabbit hair at 98 ◦ C for 2 h, and found that the solubility of rabbit hair was 19–22% in
choline chloride-urea, 15–20% in choline chloride-ethylene glycol, 20–27% in choline chloride-citric
acid, and 36–71% in choline chloride-oxalic acid, and the choline chloride-oxalic acid mixture at a molar
ratio of 1:2 or 1:3 showed a better dissolution effect on rabbit hair. Additionally, prolonging time could
increase the solubility of rabbit hair at 98 ◦ C. In this work, the combination of choline chloride and oxalic
acid at a molar ratio of 1:2 was used as DES to dissolve rabbit hair, and the effect of temperature on the
dissolution efficiency of rabbit hair was discussed. Afterwards, the keratins obtained by dissolving at
different temperatures were separated by dialysis, filtration, and freeze-drying. The resulting keratin
powders were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction
(XRD), thermogravimetry (TG)/differential scanning calorimetry (DSC), protein electrophoresis,
scanning electron microscope (SEM), and amino acid analysis.
2. Materials and Methods
2.1. Materials
Waste rabbit hair used in this paper was collected from a slaughterhouse (Shiyan, China). It was
scoured in a 2 g/L sodium carbonate solution at 50 ◦ C for 30 min, rinsed thoroughly in tap water,
and then dried in open air. The resulting rabbit hair was used in the dissolution test.
Choline chloride and oxalic acid were of analytical reagent grade, and purchased from 9-Ding
Chemistry Co. Ltd. (Shanghai, China) and Titanchem Co. Ltd. (Shanghai, China), respectively.
The dialysis bag with a theoretical molecular weight cut-off of 3 kDa was provided by Biosharp Co.
Ltd. (Hefei, China).
2.2. Extraction of Keratin from Rabbit Hair with DES
Choline chloride and oxalic acid were mixed at a molar ratio of 1:2 in the conical flask, and then
the flask was placed in the XW–ZDR low-noise oscillated dyeing machine (Jingjiang Xinwang Dyeing
and Finishing Machinery Factory, Jingjiang, China) and oscillated at 70 ◦ C for 20 min. The resulting
homogeneous and colorless solution was called DES.
Rabbit hair (0.2 g) was thoroughly immersed in DES (20 g) present in the conical flask which
was placed and heated in a glycerol bath. The mixture of rabbit hair and DES was heated for
2 h under a stirring condition at various temperatures ranging from 80 to 120 ◦ C. After cooling,
the solution was dialyzed in a water bath for 2 days using the dialysis bag with a theoretical molecular
weight cut-off of 3 kDa. After dialysis, the mixture was filtered to remove undissolved rabbit hair.
Afterwards, the resulting undissolved rabbit hair was dried, and the solubility of rabbit hair was
calculated using the following equation:
W0 − W1
Solubility (%) = × 100 (1)
W0
where W 0 and W 1 represent the weight of rabbit hair before and after dissolution, respectively.
In order to obtain the keratin powders for further characterization, the above filtrate solution was
freeze-dried using the VirTis freeze mobile 25EL lyophilizer freeze dryer (SP Scientific Inc., New York,
NY, USA).
2.3. Measurements
The UV-Vis absorption spectra of rabbit hair keratin solutions were recorded by the Shimadzu
UV-1800 UV-Vis spectrophotometer (Shimadzu Co., Kyoto, Japan). The FT-IR spectra of rabbit hair
and keratin powder were recorded by the Nicolet 5700 FTIR spectrometer (Thermo Fisher Scientific
Inc., Waltham, MA, USA) using KBr pellets. The amino acid analysis of rabbit hair and keratin powder
was carried out using the L-8900 automatic amino acid analyzer (Hitachi High-Technologies Co.,
Polymers 2018, 10, 993 4 of 11
Tokyo, Japan); the sample (10 mg) was hydrolyzed with 6 mol/L HCl at 110 ◦ C for 24 h in nitrogen
atmosphere, and then the diluent (0.2 mg/mL) was used for the free amino acid analysis; the amino
acid composition was determined by calibration with the external standard amino acid solutions,
and each sample was analyzed in duplicate.
The molecular weight distribution of keratin was measured on the PowerPac universal
Polymers 2018, 10, x FOR PEER REVIEW 4 of 11
type protein electrophoresis (Bio-Rad Laboratories Inc., Hercules, CA, USA) by sodium dodecyl
sulphate-polyacrylamide
the amino acid composition gel electrophoresis
was determined (SDS-PAGE). According
by calibration with to standard
the external the method
aminodepicted
acid by
Laemmlisolutions,
[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).
Polymers 2018, 10, 993 5 of 11
Polymers 2018, 10, x FOR PEER REVIEW 5 of 11
3.2.
3.2.Solubility
SolubilityofofRabbit
RabbitHair
Hairand
and Absorbance
Absorbance ofof Regenerated
RegeneratedKeratin
KeratinSolution
Solution
InIn
order to investigate
order the dissolving
to investigate property
the dissolving of rabbitofhair
property at various
rabbit hair at temperatures quantitatively,
various temperatures
the solubility of rabbit hair and the absorbance of keratin solution were
quantitatively, the solubility of rabbit hair and the absorbance of keratin solution were measured measured and shown in
Figure 2. The in
and shown keratin
Figuresolution had a maximum
2. The keratin solution had absorption
a maximum peakabsorption
at about 272 peaknm, at corresponding
about 272 nm, to
the absorption oftothe
corresponding thearomatic
absorption amino acid
of the sequences
aromatic amino [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].
Polymers 2018, 10, x FOR PEER REVIEW 6 of 11
Polymers 2018, 10, 993 6 of 11
Polymers 2018, 10, x FOR PEER REVIEW 6 of 11
Figure 3. SEM images of rabbit hair (a) and keratin samples produced by dissolving at 80 (b), 98 (c),
Figure 3. SEM images of rabbit hair (a) and keratin samples
samples produced
produced by
by dissolving
dissolving at
at 80
80 (b),
(b); 98
98 (c),
(c);
and
◦ C120 °C (d).
and 120 °C (d).
3.3.2. Molecular Weight Distribution
3.3.2. Molecular Weight Distribution
The SDS-PAGE analysis was employed to investigate the molecular weight distribution of
regenerated
The SDS-PAGE keratins produced
analysis was by dissolving
employed to ininvestigate
DES at various temperatures
the molecular (Figuredistribution
weight 4). The of
dissolving
regenerated
regenerated temperature
keratins
keratins produced hadby
produced an by
obvious
dissolving influence
dissolvingin DES inonat
the molecular
various
DES weight
temperatures
at various of the keratin.4).
(Figure
temperatures For keratin
The
(Figure dissolving
4). The
produced at 80 °C, the entire band was stained, indicating that the molecular weight is distributed
temperature
dissolving had an obvious
temperature had an influence
obviouson the molecular
influence on the weight
molecular of the keratin.
weight For
of the keratinFor
keratin. produced
keratin
throughout the band and some fragments had molecular weight exceeding 20.1 kDa, but the
at 80 ◦ C, the
produced at entire
80 °C, band was stained,
the entire band was indicating
stained, that the molecular
indicating that theweight
molecular is distributed
weight is throughout
distributed
majority had a molecular weight ranging from 5.8 to 10 kDa. The molecular weight of regenerated
the band
throughout and
keratins some
the band
producedfragments
and
by some had
dissolving atmolecular
fragments
98 and 120had weight
°C exceeding
molecular
was between weight
3.8 20.1
and kDa,
7.8exceeding
kDa andbut3.8the
20.1
andmajority
kDa,
5.8 kDa, had
but thea
molecular
majority weight
had
respectively. ranging
a molecular from
weight
As the temperature5.8ranging
toincreases,
10 kDa. fromThe
the5.8 molecular
to 10 kDa.
decomposition weight
degree ofofregenerated
The molecular weight
rabbit hair iskeratins produced
of regenerated
aggravated
by dissolving
keratinsand moreat
produced 98byand
keratin 120 ◦ C
dissolving
molecules arewas
at 98 between
and 120 to
disintegrated °C3.8 andbetween
was
smaller 7.8 kDa
pieces, and
3.8 and3.8
resulting in7.8and
thekDa 5.8
and
increased kDa,
3.8 respectively.
and
content 5.8 kDa,
of
the low
respectively.
As molecular weight
As theincreases,
temperature keratin.
temperature The lower molecular
increases,
the decomposition the degree weight
decomposition of regenerated
of rabbit degree keratinshair
of rabbit
hair is aggravated produced
andismore atkeratin
aggravated
and higher
more keratin
molecules temperatures is in
molecules are
are disintegrated agreement with
disintegrated
to smaller the morphological
pieces,toresulting
smaller pieces,structure of
resulting in
in the increased Figure 3, where
the increased
content the high
content of
of low molecular
temperature dissolution produces a more loose form of keratin.
low molecular
weight weight
keratin. The lowerkeratin. The weight
molecular lower molecular
of regenerated weight of regenerated
keratins produced atkeratins produced at
higher temperatures
higher temperatures
is in agreement is in
with the agreement with
morphological the morphological
structure of Figure 3, where structure
the highof Figure 3, where
temperature the high
dissolution
temperature
produces dissolution
a more loose formproduces a more loose form of keratin.
of keratin.
Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures.
3.3.3. XRD Pattern
The XRD patterns of rabbit hair and keratin obtained using DES at 120 °C are shown in Figure 5.
The raw rabbit hair showed a small diffraction peak at 9° and a broad diffraction peak at 20°,
Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures.
Figure 4. SDS-PAGE of regenerated keratins by dissolving in DES at various temperatures.
3.3.3. XRD Pattern
3.3.3. XRD Pattern
The XRD patterns of rabbit hair and keratin obtained using DES at 120 °C are shown in Figure 5.
The XRD patterns of rabbit hair and keratin obtained using DES at 120 ◦ C are shown in Figure 5.
The raw rabbit hair showed a small diffraction peak at 9°◦ and a broad diffraction peak at 20°,
The raw rabbit hair showed a small diffraction peak at 9 and a broad diffraction peak at 20◦ ,Polymers 2018, 10, x FOR PEER REVIEW 7 of 11
corresponding to the α-helix and β-sheet structures, respectively [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 peakPolymers 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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