J. Biol. Macromol., 8(2), 48-55 (2008)

ArticlePreparation and properties of protein films and particles from chicken feather
Toshihiro Fujii*1 and Daguang Li2
1

Bioengineering Course, Division of Applied Biology, Faculty of Textile Science and Technology,
Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan,
2
Shinshu University Graduate School of Science and Technology, 3-15-1 Tokida, Ueda,
Nagano 386-8567, Japan

Received May 11, 2008; Accepted June 21, 2008
A convenient procedure for protein extraction from hair, nail, and wool were applied for chickenfeathers. The velocities of protein extraction by the Shindai method were 1.1−1.8 fold faster than thoseby the conventional method. The protein components mainly consisted of β-keratin. Protein films
with white color were formed from β-keratin solution by Pre-cast, Post-cast, and soft Post-cast methods,
which were effectively applicable for the conversion of α-keratin into protein films. The protein
recovery from solution to solid was 60−80%. Interestingly, films were formed under the conditions athigh pH and low concentrations of MgCl2 and KCl where no film formation occurred in the case of hair,nail, and wool proteins. Scanning electron microscopy (SEM) showed that the surface of thetranslucent protein films was smooth, while that of white films was rough with porous structure and wascomposed of aggregated small particles. The protein particles were easily prepared from the films.
The distribution of the particles was mono-disperse with the average diameter of 0.5−3 μm. These
results suggested that the procedures for protein extraction and preparations of film and particle fromhair α-keratin could be also effective for chicken β-keratin.
Keywords: feather keratin, protein extraction, film formation, protein particle
Introduction
Keratin is a member of intracellular protein
filaments contained in hair, nail, wool, skin, andfeather.
The analyses of the amino acidcomponents indicated that the cysteine content of
keratins was remarkably high (6−17%), comparedwith the other general proteins. Keratin wasclassified into α- and β-keratins. Feather is mainly
made from β-keratin with molecular mass of 10 kDa.
On the other hand, α-keratin consists of two
subfamilies, which are acidic keratins with 40−50
kDa (type I) and neutral/basic ones with 55−65 kDa(type II) [1]. The cysteine contents of chickenfeather and human hair were approximately 7−8%
and 17−18%, respectively [1,2].
Thus, thebiomaterial properties of α- and β-keratins werequite different.
*Corresponding author: Dr. Toshihiro Fujii
Tel: +81-(0)268-21-5518, Fax：+81-(0)268-21-5511
E-mail: fujiit1@shinshu-u.ac.jp

In Japan, chickens have been consumed several
hundred million per year and most of their feather
was abandoned as garbage. Therefore, recycling of
the feather is desirable as a valuable biomaterial
resource. Martelli et al. [3] reported that β-keratin
from chicken feather was extracted using urea,
2-mercaptoethanol, and SDS and that the protein
films were prepared by casting the solutions after
dialysis against distilled water. Schrooyen et al. [4]showed that the mechanical and physiologicalproperties such as tensile strength, elongation,
humidity, and crystallinity of feather keratin films
depended on the degrees of carboxymethylated
cysteine. We developed a rapid and convenientmethod for extracting protein from hair, nail, andwool (Shindai method) [5] and three kinds ofmethods (Pre-cast, Post-cast, and soft Post-cast
methods) to form films from the protein solution
containing hard α-keratin without chemical
modification [6,7].
Recently, we reported theChicken feather protein film and particle

preparation of hair protein particles from the films[8,9].
In this study, we applied these procedures tochicken feather to prepare protein solution, films andparticles. The properties of the films and particles
were also examined.

mM KCl). The protein particles were preparedfrom the aggregates by mechanical stress using asonicator as described previously [8,9].
The protein concentrations were determinedaccording to Bradford using bovine serum albuminas the standard [10].

Materials and Methods
Preparation of chicken feather proteins
Feather proteins were extracted fromethanol-treated chicken feathers by Shindai method
at 50°C for 24 h employing 20 mM Tris-HCl (pH8.5), 2.6 M thiourea, 5 M urea, and 5%(v/v)
2-mercaptoethanol. The extraction was also carriedout by the conventional method employing 20 mM
Tris-HCl (pH 8.5), 8 M urea, and 5%(v/v)
2-mercaptoethanol [5].
After filtration andcentrifugation of the extracted solution, the obtained
protein solution was used to form films. Human
hair protein solution was also prepared by the
Shindai method for comparison.

Electrophoresis
Tricine/sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Tricine/ SDS-PAGE) was carriedout on 10/20% discontinuous polyacrylamide gel
according to the method of Schagger and Jagow [11].
Films were incubated with the Shindai solution for 1
day at 50℃ and the components were analyzed by
Tricine/SDS-PAGE.
Proteins in the gel were
stained with 0.1% Coomassie brilliant blue R-250,
10% acetic acid and 40% ethanol for 1 h anddestained in 10% acetic acid and 40% ethanol.

Preparation of chicken feather protein films andparticles
Pre-cast, Post-cast, and soft Post-cast methods
were used for the formation of protein films [6,7].
As for Pre-cast method, the proteins in Shindai
solution were premixed with guanidine-HCl (GHA)or trichloroacetic acid (TCA) or perchloric acid
(PCA), and then they were poured into tissue culture
dishes containing distilled water to form films. For
Post-cast method, the protein solution was directly
cast in tissue culture dishes containing 10 ml
solution of GHA, TCA, PCA, and/or 100 mM
acetate buffer (pH 4−6). For soft Post-cast method,
the solution of chloride salt such as KCl, NaCl,
MgCl2, and CaCl2 was used instead of those for
Post-cast method. After standing for 0.5−1 h atroom temperature, a membrane-like protein
aggregate was formed and washed by rinsing withwater for over 24 h and then replaced with distilledwater for 6 h. The films were thoroughly dried in asilicagel box and the protein recovery as the film
was calculated by weighing using an electronicbalance.
The protein aggregates were recovered fromwet films prepared by Pre-cast method (5% GHA),
Post-cast method (5% GHA and 100 mM acetate
buffer, pH 5), and soft Post-cast method (40 and 500

Scanning electron microscopy (SEM)
The morphology of the protein films was
examined with Hitachi S-2380N scanning electron
microscope. The samples on brass stubs using
double-sided adhesive tape were made electricallyconductive by coating in a vacuum with a thin layerof gold. The pictures were taken at an excitation
voltage of 10 kV and a magnification of 1500× [6].
Particle size analysis
The diameters of the protein particles in thesuspension and their distribution were measured
using dynamic light scattering system (Zetasizer
Nano-ZS, Sysmex). The mechanical stimuli weredone at 8 mg/ml in distilled water, and the protein
particle solution was diluted to 0.4 mg/ml in distilledwater. The average particle size was expressed asboth the number average diameter and the volume
average one.
Results and Discussion
Preparation of protein solution and protein
compositions
Figure 1A shows time courses of the protein
extraction from feather vane and shaft using theShindai and conventional methods. The protein
extraction by the Shindai method almost reached tothe saturation level within 1−2 h, while it required
more than 8 h for the conventional method.
Especially for feather shaft, the extraction efficiency

T. Fujii and D. Li

(A)

(A)
Film recovery (%)

Protein conc. (mg/ml)

5
4
3
2
1

00

0 1 2 3 4 5 6 7 8 24
Time (h)

(B)
94
67
43

1

2 3 4 5Hairkeratin
TypeⅡ
TypeⅠ

30
20
14

0

50

100 150 200
KCl (mM)

500(B)

Feather
keratins

Figure 1. Time course of the extraction of proteinfrom chicken feather vane and shaft andelectrophoretical analyses.
(A) The protein was
extracted from feather vane (○,△) and shaft (●,▲) at
50°C using the Shindai method ( ○ , ● ) or theconventional method ( △ , ▲ ) and aliquots were
measured for protein concentration. (B) The chickenfeather vane and shaft proteins extracted were
subjected to 10% Tricine/SDS-PAGE.
Lane 1,
Shindai method, feather vane; lane 2, Shindai method,feather shaft; lane 3, the conventional method, feather
vane; lane 4, the conventional method, feather shaft;lane 5, Shindai method; human hair.

was remarkably improved by the Shindai method incomparison with the conventional method. Thus,
Shindai method is effectively applicable for protein
extraction from hard tissues. About 80−90% offeather vane and shaft were converted into protein
solutions.
The obtained proteins were analyzed by
Tricine/SDS-PAGE (Fig. 1B).
Proteins fromfeather vane and shaft by the two methods weremainly composed of β-keratin with a molecular mass

Film recovery (%)

M.W.
（kDa）

90
80
70
60
50
40
30
20
10
0

90
80
70
60
50
40
30
20
10
0

0

10 20 30 40 50 60
Time (min)Figure 2. Effect of KCl concentration on the film
recovery and time course of film formation. (A)The films were prepared by soft Post-cast method.
Feather ( ○ ) and hair ( ● ) protein solutions weredirectly exposed to various concentrations of KCl as
indicated. (B) Feather protein solution was exposedto the developing solutions containing 40 mM KCl(○), 500 mM KCl (●), and 100 mM acetate buffer,
pH 5 (△). After storing for 1 h (A) and various
times as indicated (B), the resulting protein films were
washed and the quantities were calculated byweighing.

of 10 kDa, as reported previously [12]. We could
not find any significant difference among them.
Furthermore, the composition of amino acids wasalmost same between in vane and shaft proteins (datanot shown)
Chicken feather keratin consists of 97 aminoacid residues [2]. From the amino acid sequence,pI value was calculated to be 7.8, while the values ofhuman keratin type I and II were 4.8−6.0 and
5.9−8.5, respectively. When the sequences werecompared using CLUSTALW, the identity were 13%

Chicken feather protein film and particle

Chicken feather

Human
Hair

MethodSolvent

Pre-cast

5% GHA
5% TCA
5% PCA

75 ± 1 80 ± 1 76 ± 5
69 ± 2 73 ± 1 86 ± 1
70 ± 3 80 ± 1 79 ± 2

Post-cast

5% GHA
5% TCA
5% PCA
100 mM Acetate buffer, pH 4.0
100 mM Acetate buffer, pH 5.0
100 mM Acetate buffer, pH 5.5
100 mM Acetate buffer, pH 6.0

55 ± 585 ± 385 ± 260 ± 268 ± 465 ± 265 ± 160 ± 181 ± 182 ± 068 ± 570 ± 468 ± 268 ± 191 ± 582 ± 274 ± 182 ± 481 ± 60±0
0±0soft Post-cast

40 mM KCl
500 mM KCl
40 mM NaCl
500 mM NaCl
40 mM MgCl2
40 mM CaCl238 ± 165 ± 140 ± 060 ± 165 ± 277 ± 040 ± 271 ± 151 ± 067 ± 170 ± 383 ± 30±0
73 ± 50±0
74 ± 792 ± 498 ± 2

Vane
Shaft
Recovery (%)

Table 1. Recoveries of proteins in films from chicken feather
Chicken feather (10 mg) and human hair protein (6 mg) films were prepared by Pre-cast, Post-cast, and soft
Post-cast methods and the quantities of protein recovered as the films were calculated with their weights.
The ”recovery %” represents the ratio of the protein amount in a formed solid film to that in a cast solution.

(hHa4 of type I) and 16% (hHb6 of type II) [13,14],
respectively, indicating that both keratins were quite
different in the primary structure. Our results
suggest that the Shindai method is effective for the
extraction of proteins irrespective of primarystructure of keratins. In addition, it was founduseful even for the proteins with high contents ofdisulfide bond.
Preparation of protein film
Based on the conventional method, wedeveloped the method to prepare films from theprotein solution of hair, nail, and wool mainly
consisting of α-keratin. Then, we established themethods named Pre-cast, Post-cast, and soft
Post-cast methods [5,6]. Table 1 shows the amountof protein recovered as film consisting of feather andhuman hair proteins using these methods. The
film-like aggregates were formed from the feather
protein solutions like hair protein solution. The
recoveries of feather proteins as films were 40−85%
and somewhat lower than the values for hair proteins.
No significant difference was found between thevane and the shaft [5,6], so that the subsequent
experiments were undertaken by using the mixtureof feather vane and shaft. Interestingly, feather
proteins formed the film at slightly acidic pH andlower concentrations of KCl or NaCl. Under these
conditions hair protein solution could not be
converted to the film.
The film formation of feather and hair proteins
was examined as a function of KCl concentrations(Fig. 2A). Feather protein films were generated at
the developing solution more than 20 mM KCl,
whereas higher concentrations of KCl more than 200
mM were required for film formation from hairproteins. These two proteins were quite different in
the sensitivity for KCl in applying soft Post-cast
method. Recently, we found that hair α-keratin
films prepared by soft Post-cast method (50 mM
CaCl2) significantly interacted with Ca2+ after
washing enough them with distilled water(unpublished data). It can therefore be presumed
that α- and β-keratins contain direct binding sites forK+ and the bindings promote the conversion from thesolution to the film.
Next, we examined the time course of film
formation of the feather proteins when KCl (40 and
500 mM) and acetate buffer (pH 5) were used as thedeveloping solution (Fig. 2B). At 500 mM KCl or

T. Fujii and D. Li

a

d

b

e

c

a

b

c

d

f

e

20 mm

10 μm

Figure 3. Photographs of feather vane protein films
by obtained by Pre-cast, Post-cast, and soft Post-cast
methods.
a, Control (tissue culture dish); b,
Pre-cast method (5% GHA); c, Post-cast method (5%GHA); d, Post-cast method (100 mM acetate buffer,pH 5); e, soft Post-cast method (40 mM KCl); f, soft
Post-cast method (500 mM KCl).

Figure 4. Morphological observation of feather
protein films.
a, Pre-cast film (5% GHA); b,
Post-cast film (5% GHA); c, Post-cast film (100 mM
acetate buffer, pH 5); d, soft Post-cast method (40mM KCl); e, soft Post-cast method (500 mM KCl).

acetate buffer (pH 5), the half-times required for the
maximal film formation were almost 10 min,whereas the time was fairly longer at 40 mM KCl.
Similar phenomena were observed when NaCl wasused in place of KCl (data not shown). The curves
also showed the presence of a lag time for film
formation. It may be due to the formation of
nucleation center in a similar way as the assembly of
microtubules.
The addition of plasticizers such as sorbitol,
glycerol, and polyethylene glycol to feather keratin
films prepared by casting method caused theincrease of moisture content and water vapor
permeability [3]. Thus, we examined the effect ofsorbitol and glycerol (10−50 mg/g of feather protein)to the films prepared by Post-cast (acetate buffer, pH5) and soft Post-cast (40 and 500 mM KCl) methods.
These reagents had little effect on the flexibility of
the films (data not shown).

Morphological observations of the protein films
When the films containing approximately sameamount of protein were prepared on tissue culture
dishes, the films by soft Post-cast method using 40
mM KCl was clear enough to show the letter on thepaper after drying, whereas the films made by theother conditions were white and opaque (Fig. 3).
We attempted to image the films using SEM (Fig. 4).
The fine structure of the white films prepared by
Pre-cast method (guanidine-HCl), Post-cast method(GHA and acetate buffer, pH 5), and soft Post-cast
method (500 mM KCl) exhibited aggregates ofparticles with a diameter of 0.5−3 μm and formed
heterogeneous porous surface. On the other hand,the surface of the translucent films prepared by soft
Post-cast method (40 mM KCl) was smooth and theparticles over ca. 0.5 μm in diameter were not found.
Similar images were observed in the human hairfilms [6,7].

Chicken feather protein film and particle

M.W.
（kDa）

(A)

3
Particle diameter (μm)

Preparation and properties of protein particles
Recently, we reported a novel preparation
method of protein particles from human hair protein
films and used the suspension as a blood analog
fluid [8,9].
A particle suspension was easily
prepared from the wet feather protein films bymechanical fracture with a sonicator and the size
distribution of each protein suspension wasmeasured with Zeta-Sizer. The average diameters

were 1.9−2.6 μm without the particles (0.6 μm)
prepared by soft Post-cast method using 20 mM KCl(Table 2). When soft Post-cast method was used infilm formation, the average diameters of the particles
increased with increasing the concentration of KCl(Fig. 6A). This seems to correlate directly with thefine structure of the films as shown in Fig. 4, since
the film prepared at 40 mM KCl consists of smaller
particles than the film obtained at 500 mM KCl. In
the hair protein particles [9], the number-average
size was apparently distributed in a monodisperse
manner, while two peaks were observed in the
distribution of volume-average size. On the other

)

m

2.5

∝

2
1.5
1
0.5
0

0

(B)

100

200 300 400
KCl (mM)

500

40
Particle amount (%)

Protein compositions of the films
The protein films by Pre-cast (5% GHA),
Post-cast (100 mM acetate buffer pH 5), and soft
Post-cast (40 and 500 mM KCl) methods werestored in the dry state for 4−6 weeks at room
temperature.
The protein of the films was
re-extracted using the Shindai solution for 1 day at
50 ℃ , and the components were analyzed by
Tricine/SDS-PAGE (Fig. 5).
The extracted
solution of all the films mainly consisted of
β-keratin and no significant difference incomposition was found among the films examined
here. Furthermore, protein hydrolysis was notfound during the film formation nor during thestorage period, indicating that feather keratin was
remarkably stable after forming film-like aggregateslike hair and wool keratins.

)

1

2

3

4

94
67
43
30
20
14
6.5
Figure 5.
Tricine/SDS-PAGE of the extracted
proteins from feather protein films.
Extracted
proteins were analyzed by 10% Tricine/SDS-PAGE.
Lane 1, Pre-cast film (5% guanidine-HCl); lane 2,
Post-cast film (100 mM acetate buffer, pH 5); lane 3,
soft Post-cast film soft Post-cast method (40 mMKCl); lane 4, soft Post-cast method (500 mM KCl).

%

30
20
10
0
0.1

1
Particle diameter (μm)
( m)

10

Figure 6. Effect of KCl on average diameter ofprotein particles and particle diameter distribution.
(A) Feather protein particles were prepared from thefilms formed at various concentrations of KCl asshown in Fig. 2. After treatment of the protein
suspension at 8 mg/ml by sonication, the particle
diameters were measured at the concentration of 0.4
mg/ml. The plots exhibited the number average
diameters (○) and volume ones (●). (B) The size
distributions of the particles from the films formed at
20 mM ( ● , ▲ ) and 500 mM KCl ( ○ , △ ) were
exhibited.
The plots for number-average andvolume-average diameters are shown with circles (○,●) and triangles (△,▲), respectively.

T. Fujii and D. Li

MethodAverage particle diameter (μm)

Solvent

N

V

Pre-cast

5% GHA

1.85 ± 0.02

2.08 ± 0.04

Post-cast

5% GHA

2.39 ± 0.05

2.61 ± 0.09

100mM Acetate buffer, pH 5.0

2.00 ± 0.02

2.11 ± 0.02

soft Post-cast 20mM KCl

0.55 ± 0.05

0.55 ± 0.05

500mM KCl

2.50 ± 0.01

2.53 ± 0.04

Table 2. Average diameter of feather protein particles
Chicken feather protein films prepared by three methods were disintegrated in distilled water using a sonicator at
the 85% intensity of full power for 1.5 min ×8 times. Samples were diluted to 0.4 mg/ml in distilled water andthe particle diameters were measured. The number average diameters and the volume average ones indicated in
the columns denoted with ‘N’ and ‘V’, respectively.

hand, in the feather protein particles, the values ofnumber-average and volume-average diameters werehighly close, indicating that they are monodisperse
protein particles (Table 2 and Fig. 6B).
The
reason for this difference will be caused the distinct
biochemical characteristics of both keratins, that is,
the molecular mass and cysteine content of feather
β-keratins were lower than those of α-keratins in hair,nail, and wool [1,2].
In this report, we applied some processing
methods developed in human hair, nail, and wool tochicken feather. It was found that our methods areconvenient to prepare not only the protein solution
but also the protein materials with the form of films
and/or particles without chemical modification.
Acknowledgments
This study was supported by Grants-in-aid for
the Global COE Program and the CLUSTER (thesecond stage) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
We are also indebted to the Division of Gene
Research Center, Research Center for Human andEnvironmental Science, Shinshu University forallowing the use of their facilities.
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Communicated by Hisayuki Morii