Process Biochemistry 47 (2012) 896899

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Short communication

Extracting keratin from chicken feathers by using a hydrophobic ionic liquid

Yun-Xian Wang, Xue-Jun Cao
State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e i n f o a b s t r a c t

Article history: Keratin was extracted from chicken feathers by using a hydrophobic ionic liquid (IL), 1-hydroxyethyl-3-
Received 4 September 2011 methylimidazolium bis(triuoromethanesulfonyl)amide ([HOEMIm][NTf2 ]). Extracted keratin has good
Received in revised form 5 January 2012 solubility in water while the ionic liquid is immiscible with water, and therefore the extracted keratin
Accepted 14 February 2012
could be easily separated from the reaction system by water. The effects of ionic liquid, NaHSO3 , reaction
Available online 22 February 2012
temperature and time were investigated and extracting conditions were optimized. The maximum yield
of keratin was up to about 21% with mass ratio of feathers to NaHSO3 1:1 and mass ratio of feathers to
Keywords:
ionic liquid 1:40 at 80 C for 4 h. Moreover, there was no obvious loss in the yield after ionic liquid was
Ionic liquid
1-hydroxyethyl-3-methylimidazolium
reused for ve batches under optimized conditions. In addition, the recovery of ionic liquid was about
bis(triuoromethanesulfonyl)amide 95% each time. The results indicated that [HOEMIm][NTf2 ] was very efcient as catalyst and solvent for
Keratin dissolving feathers and could be easily recovered due to its hydrophobicity.
Chicken feathers 2012 Elsevier Ltd. All rights reserved.
Separation

1. Introduction However, one of the most serious shortcomings concerning these

The poultry industry produces a great amount of waste feathers
each year, e.g. 1.8 million tons in US [1], which causes an envi-
ronmentally difcult disposal problem. Therefore, from both an
economic and environmental point of view, it is quite desirable
to develop effective and protable process to use these resources.
However, feathers are mainly used as low nutritional value animal
feed. Currently, researchers have been searching for new appli-
cations of feathers and many publications and patents proposing
applications of feathers have been issued. Polyethylene-based com-
posites can be prepared using keratin bers obtained from chicken
feathers [2]. Keratin bers from chicken feathers were used as a
short-ber reinforcement for a poly(methyl methacrylate) matrix
[3]. Feather keratin and polyurethane were combined to synthe-
size hybrid synthetic-natural membranes which could be applied
to separation process [4].
Keratin is the major component of feathers, which is a structural
protein characterized by a high cystine content and a signicant
amount of hydroxyl amino acids, especially serine (about 15%) [5,6].
It contains a range of noncovalent interactions (electrostatic forces,
hydrogen bonds, hydrophobic forces) and covalent interactions
(disulde bonds), which must be destroyed in terms of dissolu-
tion of feathers. Keratin is insoluble in polar solvents like water,
weak acids and bases, as well as in apolar solvents while it is active
because cystine can be reduced, oxidized and hydrolyzed [711].
Corresponding author. Tel.: +86 21 6425 2695; fax: +86 21 6425 2695.
E-mail address: caoxj@ecust.edu.cn (X.-J. Cao).
methods for extracting keratin is that a large quantity of reagents
such as acids or reductants is consumed and cannot be recycled.
Thus, researchers have focused on nding simple and eco-friendly
processing methods to dissolve feather keratin.
Recently, ionic liquids (ILs) have received recognition as green
and promising materials for potential applications in various elds
because they are typically non-volatile, non-ammable, chemi-
cal and thermal stability and remarkable solubility [1216]. They
have also been termed as designer solvents as their proper-
ties can be manipulated by a careful choice of cation/anion
according to the requirements. Ionic liquids exhibit excellent
solubility characteristics because of their special structures com-
pared to the traditional molecular solvents. Some recent works
have been reported regarding the dissolution and regeneration
of keratin bers in ionic liquids. Xie et al. [17] reported the
dissolution and regeneration of wool keratin bers in 1-butyl-3-
methylimidazolium chloride (BMIMCl) ionic liquid. Hameed et al.
[18] prepared natural wool/cellulose blends in BMIMCl and the
lms were formed subsequently from the coagulated solutions. In
fact, the costs of ionic liquids are obviously higher than that of inor-
ganic reagents, but they can be reused and improve the efciency of
whole process, leading to lower the overall cost. Separation of ker-
atin from hydrophilic ionic liquids after reaction is an inconvenient
and troublesome problem.
In this study, a hydrophobic ionic liquid ([HOEMIm][NTf2 ])
was used to dissolve chicken feathers to obtain keratin. The aim
of our research work was to extract keratin with hydrophobic
ionic liquids instead of acids and bases. The effects of mass ratio
of feathers and ionic liquid, mass ratio of feathers and NaHSO3 ,

1359-5113/$ see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2012.02.013
 Y.-X. Wang, X.-J. Cao / Process Biochemistry 47 (2012) 896899
Table 1
Water content of equilibrated and dried IL; solubility in water of IL.
Ionic liquid Water equilibrateda (g/100 ml)
[HOEMIm][NTf2 ] 4.890
a
Water equilibrated refers to IL that has been stored in contact with water, which incorporates vigorous agitation and mixing.
b
Dried IL is water equilibrated IL that has been dried at 70 C for 6 h on a vacuum line.
reaction temperature and time on the extraction yield of keratin
were investigated and the reusability of ionic liquid was also stud-
ied. Furthermore, the extracted keratin was analyzed by infrared
spectrometry (IR) to conrm its structure and determined by gel
permeation chromatography (GPC) to get its molecular weight.
2. Materials and methods
2.1. Water content and solubility in water
The water content of [HOEMIm][NTf2 ] was determined using a volumetric
Aquastar Karl Fischer titrator (Super Scientech, Shanghai, China) with composite
5 solution as the titrant and anhydrous methanol as the solvent. Each sample was
at least 0.5 g and duplicate measurements were performed.
The absorption spectrum of [HOEMIm][NTf2 ] was measured by full wave-
length scan with UVvis spectrophotometer (PerkinElmer Lamda 900). In a 10 ml
polypropylene centrifuge tube, 1.0 g of the ionic liquid and 2.5 ml of deionized water
were shaken on a vortex mixer for 30 min and centrifuged for 15 min at 4000 rpm.
A 2 l aliquot of the aqueous phase was taken with a microsyringe and diluted to
5 ml with deionized water. The absorbance of this solution at maximum absorption
peak was measured and compared with that obtained from dissolving a weighed
amount (0.10.6 mg) of the ionic liquid in 5 ml of deionized water [19].
2.2. Preparation of feather keratin solution
Before dissolution, chicken feathers were washed with water, ltered, dried and
cut into small pieces or milled. Reactions were performed in a round ask containing
1.0 g chicken feathers, 40 g ionic liquid, 1.0 g NaHSO3 and 500 l water. The mixture
was agitated at 80 C for 4 h. After the process was nalized, water (20 g, 50% wt,
based on the weight of ionic liquid) was added to the mixture and tri-phase sys-
tems containing ionic liquid/parts of insolubilized feathers/keratin solution were
formed after centrifugation. Then the system of keratin solution was ltered, and
the ltrate was transferred to dialysis bag (MWCO 35005000 Da) and extensively
dialyzed against distilled water for 48 h. The protein concentration of the extracts
was measured by the Bradford protein assay method (BioRad), using bovine serum
albumin as standard. The amount of protein dissolved was expressed as a percent-
age of the total weight of dried feathers used. In addition, the other two systems
were vacuumized for 6 h (70 C) and introduced into the fresh substrates for the
next batch. The recovery of ionic liquid was expressed as a percentage of the total
weight of it used.
2.3. Molecular weight of feather keratin
The molecular weight of extracted keratin was determined by gel permeation
chromatography. It was carried out on a PerkinElmer Series 200 system at 35 C
(TSKgel PWXL 10 m, 7.8 300 mm column). 0.05 M NaNO3 was used as the eluent
and the ow rate was 0.8 ml/min.
3. Results and discussion
3.1. Properties of [HOEMIm][NTf2 ]
According to full wavelength scan, [HOEMIm][NTf2 ] existed
maximum absorption peak at 217.5 nm, which mainly attributes
to imidazole ring in the ionic liquid. As shown in Table 1, the water
content of equilibrated [HOEMIm][NTf2 ] was 4.89%(w/v) and the
solubility of [HOEMIm][NTf2 ] in water was 7.941%(w/v), which
indicated that [HOEMIm][NTf2 ] was slightly soluble in water and
maximum recovery of it could be up to 96% (50% wt water, based on
the weight of ionic liquid). Moreover, [HOEMIm][NTf2 ] possesses
unexpected hyperpolarity close to protic ILs and water [20],
which could greatly enhance the dissolution of feathers. Therefore,
[HOEMIm][NTf2 ] was a proper ionic liquid for extracting keratin
from feathers.
897
Driedb (g/100 ml) Water solubility (g/100 ml)
0.453 7.941
3.2. Solubility of chicken feathers in ionic liquid
3.2.1. Effect of mass ratio of feathers and NaHSO3 on the yield of
keratin
To reduce the disulde bonds, the reaction was carried out
with various mass ratio of feathers to NaHSO3 (1:0, 1:0.3, 1:0.5,
1:0.75, 1:1, 1:1.25 and 1:1.5) under identical conditions. As shown
in Fig. 1a, the extraction yield of keratin increased obviously with
the increasing of mass ratio from 1:0 to 1:1 and then very slowly
from 1:1.25 to 1:1.5.
It suggested that more disulde bonds of feather keratin were
destroyed with higher mass ratio. Nevertheless, it also showed that
the yield did not have signicant increase when the mass ratio sur-
passed 1.0. This effect could be because all the disulde bonds of
feather keratin were reduced when excessive NaHSO3 was sup-
plied. In the reaction system without NaHSO3 , the yield of extracted
keratin was up to 7.86% only relying on the hyperpolarity of ionic
liquid [HOEMIm][NTf2 ]. Therefore, the optimal mass ratio was 1:1
considering both the yield of keratin and the production costs.
3.2.2. Effect of mass ratio of feathers and ionic liquid on the yield
of keratin
The dissolution of feathers can be divided into two stages,
including swelling and solubilization. The rst stage is that solvent
molecules gradually permeate into feathers so that the physi-
cal interactions of feather keratin are taken place by interactions
between solvent molecules and feather keratin. The second stage
is that chains of feather keratin unfold from aggregation due to
salvation effects and reduction of disulde bonds. Therefore, solu-
bility of feathers is signicantly related to polarity of ionic liquid
and the yield of keratin is increased with the increasing polarity
of ionic liquid. Reactions were conducted with different mass ratio
of feathers to ionic liquid (1:20, 1:25, 1:30, 1:40, 1:45 and 1:50)
under identical conditions. As a result (Fig. 1b), although the high-
est yield of 21.75% was obtained when the mass ratio was 1:45, the
yield of keratin was increased slowly with mass ratio from 1:40 to
1:45 and declined with further increase of mass ratio. This effect
could be attributed to that ionic liquid [HOEMIm][NTf2 ] could pro-
duce electrostatic and hydrogen-bond interactions between itself
and feather keratin [20]. It also indicated that part of peptide bond
of feather keratin was disrupted when excessive amounts of ionic
liquid were applied. From these results, the mass ratio of feathers
to ionic liquid 1:40 was adopted in the subsequent experiments
considering the cost of ionic liquid.
3.2.3. Effects of reaction temperature and time on the yield of
keratin
Temperature is an important factor affecting the yield of keratin,
and the rate of dissolution of feather keratin at various tempera-
tures (70100 C) was determined while keeping other conditions
constant. From the Fig. 1c, it can be seen that the yield increased
obviously with temperature from 70 C to 80 C and approached at
stage from 80 C to 90 C. It was because higher reaction tempera-
ture can provide much energy to accelerate physical and chemical
change of feather keratin and thus promote the dissolution of
feathers. However, when the temperature was over 90 C, the
yield decreased markedly. Peptide bond scission of feather keratin
898 Y.-X. Wang, X.-J. Cao / Process Biochemistry 47 (2012) 896899

Fig. 1. (a) Effect of mass ratio of feathers and NaHSO3 on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to ionic liquid 1:20, 500 l water, 80 C,
4 h. (b) Effect of mass ratio feathers and ionic liquid on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, 1.0 g NaHSO3 , 500 l water,
80 C, 4 h. (c) Effect of reaction temperature on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic liquid
1:40, 500 l water, 4 h. (d) Effect of reaction time on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic
liquid 1:40, 500 l water, 80 C.

occurred at the higher temperature. Therefore, 80 C was selected

as the optimal reaction temperature.
The effect of reaction time on the dissolution of feathers keratin
was studied and the results were shown in Fig. 1d. It was found that
the optimum reaction time was 4 h and the maximum yield was up
to 21.50%.

3.3. Recycle of ionic liquid

Even though previous works have demonstrated the dissolu-

tion of feathers in ionic liquids, there is yet no effective way to
separate the protein from ionic liquids. Therefore, the reusability
of ionic liquid [HOEMIm][NTf2 ] was investigated under the opti-
mum conditions mentioned above. In our study, extracted keratin
has good solubility in water, while [HOEMIm][NTf2 ] is immiscible
with water. Consequently, keratin could be easily separated from
Fig. 2. The reusability of ionic liquid for dissolving feathers. Reaction conditions:
the reaction mixture by water, and the extracted keratin could mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic liquid 1:40,
be puried by dialysis and precipitated by ethanol according to 500 l water, 80 C, 4 h.
requirement of nal product. The remaining ionic liquid could be
used directly in the next time. As shown in Fig. 2, the ionic liquid
was used for ve batches without essential loss in the process of dis- 3.4. FTIR
solving feathers. It was also found that the recovery of ionic liquid
was about 95% in each cycle. This indicated that [HOEMIm][NTf2 ] The FTIR spectra of extracted keratin in the region
is an effective hydrophobic ionic liquid for extracting keratin from 4004000 cm1 are given in Fig. 3, where the characteristic
chicken feathers. absorption bands are mainly assigned to the peptide bonds
 Y.-X. Wang, X.-J. Cao / Process Biochemistry 47 (2012) 896899
Fig. 3. IR spectrum of extracted keratin.
Fig. 4. The GPC data of extracted keratin.
( CONH ). The vibrations in the peptide bonds originate bands
known as amides IIII [2123]. The amide I band is connected
mainly with the C O stretching vibration and it occurs in the range
of 17001600 cm1 . The amide II band is related to N H bending
and C H stretching vibration and it falls at 1520 cm1 region.
The amide III band occurs in the range of 12201300 cm1 and it
results from in phase combination of C N stretching and in N H
in plane bending, with some contribution from C C stretching and
C O bending rations. It can be seen from Fig. 3 that these bands
exist in extracted keratin.
3.5. Molecular weight
The molecular weight of feather keratin solution was examined
by using GPC. There was only one sharp peak in the molecular
weight distribution of extracted keratin (Fig. 4). The weight-
averaged and number-averaged molecular weights of extracted
899
keratin were 10,240 and 10,000 respectively. This GPC data agrees
with published data that feather keratin consists of 96 amino acid
residues and has a molecular weight of 10,206 [6]. The polydis-
persion degree was at the level of 1.024, which shows that the
extracted keratin was uniform in its molecular weight. However,
the yield of extracted keratin was less 25%, probably because ker-
atin was degraded to amino acids and small peptides and not
separated from reaction mixture completely.
4. Conclusions
The eco-friendly ionic liquid [HOEMIm][NTf2 ] could be a suit-
able solvent and catalyst for dissolving feathers. To our knowledge,
this should be the rst report concerning the dissolution of feathers
in a hydrophobic ionic liquid and an easy separation of extracted
keratin.
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