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Keratin based bioplastic film from chicken feathers and its characterization

Article  in  International Journal of Biological Macromolecules · January 2018

DOI: 10.1016/j.ijbiomac.2018.01.037

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 International Journal of Biological Macromolecules 111 (2018) 352–358

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: https://www.journals.elsevier.com/biomac

Keratin based bioplastic film from chicken feathers and

its characterization
Navina Ramakrishnan 1, Swati Sharma ⁎,1, Arun Gupta ⁎, Basma Yahya Alashwal
Faculty of Chemical Engineering and Natural Resources, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia

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

Article history: Plastics have been one of the highly valued materials and it plays an significant role in human's life such as in food
Received 10 November 2017 packaging and biomedical applications. Bioplastic materials can gradually work as a substitute for various mate-
Received in revised form 4 January 2018 rials based on fossil oil. The issue like sustainability and environmental challenges which occur due to
Accepted 6 January 2018
manufacturing and disposal of synthetic plastics can be conquering by bio-based plastics. Feathers are among
Available online 07 January 2018
the most inexpensive abundant, and renewable protein sources. Feathers disposal to the landfills leads to envi-
Keywords:
ronmental pollutions and it results into wastage of 90% of protein raw material. Keratin is non-burning hydro-
Bioplastic philic, and biodegradable due to which it can be applicable in various ways via chemical processing. Main
Biodegradable objective of this research is to synthesis bioplastic using keratin from chicken feathers. Extracted keratin solution
Chicken feather mixed with different concentration of glycerol (2 to 10%) to produce plastic films. The mixture was stirred under
Keratin constant magnetic stirring at 60 °C for 5 h. The mixtures are then poured into aluminum weighing boat and dried
Glycerol in an oven at 60 °C for 24 h. The mechanical properties of the samples were tested and the physic-chemical prop-
Protein erties of the bioplastic were studied. According to the results, Scanning Electron Microscopy test showed good
compatible morphologies without holes, cavity and edge. The difference in chemical composition was analyzed
using Fourier transform infrared spectroscopy (FTIR). The samples were also characterized by thermo gravimet-
ric analysis (TGA), differential scanning calorimetry (DSC), X-Ray diffraction (XRD) to check the thermal and
crystallinity properties. Other than that, bioplastic made up from keratin with 2% of glycerol has the best mechan-
ical and thermal properties. According to biodegradability test, all bioplastic produced are proven biodegradable.
Therefore, the results showed possible application of the film as an alternative to fossil oil based materials which
are harmful to the environment.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction
From late 1970s, after introduction of plastic carrier bags they turn
into a common element in daily life [1]. But the different causing envi-
ronmental pollution by synthetic polymers leads to research on biode-
gradable materials formed from renewable materials such as
polysaccharides [2, 3], proteins [4, 5] and lipids [6, 7]. Bioplastic said to
be important because of tremendous increase in price of petroleum oil
and its stock will end in future. Most of the plastics derived from fossil
resources are largely non-biodegradable and harmful to the environ-
ment and marine life. Majority of the thermoplastics available on the
market are derived from petroleum based resources. Packaging is one
of the main applications of synthetic thermoplastics and about 28.9 mil-
lion tons of plastics were consumed by the packaging industry in the
⁎ Corresponding authors.
E-mail addresses: sspandit.89@gmail.com (S. Sharma), arungupta10@gmail.com
(A. Gupta).
1
Both the authors have equal contribution.
United States in 2005 [8, 9]. Only about 5.7% of the plastics are recycled
after use and most of the packaging is disposed in landfills [10].
By-products from various animal sources (legs, heads, viscera, bones
and feather) are presently being used for valuable purposes [11] such as
fertilizers, livestock feed and pet foods. Also, large amount of it are
dumped, incinerated or disposed into landfills which causes disease,
economic issues, environmental pollution and also wastage of renew-
able materials [12]. In particular, feather is among the most significant
potential resources to manufacture biodegradable materials. More
than 4 billion pounds of feathers generated as co products every year
in the USA have limited use and are mostly disposed in landfills [13].
Disposal of feathers in landfills leads to environmental pollutions and
also results in discarding a valuable raw material containing more
than 90% proteins. Feathers are inevitably generated during poultry pro-
duction and are therefore a sustained and indigenous source. Utilizing
feathers to develop bio-products will add value to feathers and also pro-
vide inexpensive and renewable raw material.
Feather contains 90% of protein named as keratin which is abun-
dantly produced by the poultry industry all over the world [14, 15].
Keratin is naturally non-burning, hydrophilic, biocompatible,

https://doi.org/10.1016/j.ijbiomac.2018.01.037
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 N. Ramakrishnan et al. / International Journal of Biological Macromolecules 111 (2018) 352–358
biodegradable and can be utilized in different ways via chemical pro-
cessing. The keratin protein extracted from chicken feathers can be
used into different forms for biotechnological application, such as
sponges, films, fibers, alone or blended with other natural or synthetic
polymers [2, 15–19]. Furthermore, to solve the feather waste pollution
and use it in various applications numbers of researchers are working
to manufacture potential materials from feather and keratin [20–22].
Among all, bioplastic might be an application for feather [9, 12, 23]
and keratin [4, 24] which is biodegradable or bio-based thus, harmless
to the environment [25]. There are various methods of extraction of
functional keratin protein from chicken feather such as chemical
[26–34], enzymatic [13, 35–37] and with ionic solutions [38–40]. The
properties of the keratin depend on the extraction method from
which it is extracted. In a recent study, the keratin microparticles
were synthesized from chicken feather using chemical method and
their antioxidant and anticancer activities were studied [28].
In the present research biodegradability and mechanical properties
of bioplastic film formed from dissolution of chicken feathers followed
by mixing of different concentration of glycerol are studied. The
bioplastic film was characterized using Scanning Electron Microscopy
(SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffrac-
tion Analysis (XRD), Thermo gravimetric Analysis (TGA), Differential
Scanning Calorimetry (DSC). The properties studied, with a vision of
producing bioplastic film which can be commercialized in future.
2. Materials and methods
2.1. Chemicals
Chicken feathers were collected wet and fresh from a chicken pro-
cessing plant at Jaya Gading, Kuantan, Malaysia. Sodium sulfide
(Na2S), sodium hydroxide (NaOH), hydrochloric acid (HCl),
cetrimonium bromide (CTAB) and petroleum ether were purchased
from Sigma Aldrich (Selangore, Malaysia). Sodium chloride (NaCl), po-
tassium chloride (KCl), disodium phosphate (Na2HPO4),
monopotassium phosphate (KH2PO4) used to prepare phosphate buffer
and were acquired from Sigma Aldrich Sdn Bhd. Milli-Q water was used
for solutions except washing and soaking in which filtered laboratory
water was used. Another material used was glycerol ACS reagent, anhy-
drous, (99.5%) supplied from Chemmart Asia Sdn. Bhd.
2.2. Extraction of keratin
Keratin solution used as base material and its extraction was carried
out according to the process described previously [28]. Chicken feathers
were washed and dried in a ventilated oven at 40 °C for 72 h. The wash-
ing process of feathers includes degreasing with petroleum ether, con-
ditioning at 20 °C with relative humidity (RH) 65% for 24 h, treatment
Fig. 1. SEM graph show the (a) 2 wt% glycerol bioplastic film (b) 5 wt% glycerol bioplastic film (c) 10 wt% glycerol bioplastic film.
353
with CTAB (1 g/L) for 3 h to remove microorganisms. Cleaned defatted
feather were dried under sunlight for 1–2 days and then chopped into
small pieces (2–25 cm) using grinder and stored at 4 °C for further
usage in sealed bags.
The feathers dissolved by mixing 25 g of the chopped feathers into
Na2S (0.5 M in1 L) as optimized and used by previous studies [27, 28],
digested at 50 °C using IKA RW 20 overhead mechanical stirrer for 6 h.
The prepared mixture was filtered twice by using Whatman filter
paper of 110 mm diameter and centrifuged using Eppendorf Centrifuge
5810 R at 10,000 rpm for 15 min to separate the supernatant from un-
dissolved feathers. The pH of the solution was adjusted to 3.5 to precip-
itate the protein using FE20 Mettler Toledo pH meter [27]. The keratin
protein sediments were collected by centrifugation at 10,000 rpm for
10 min, washed with the double distilled water (ddH2O), freeze dried
to obtain keratin particles. The total extracted yield of keratin powder
was 79.6%. The keratin powder was dissolved in NaOH (1 mg/2 ml 2 N
of NaOH) to make it solution form.
2.3. Preparation of bioplastic film
60 ml of keratin solution mixed with different concentration of (2, 5,
10 wt%) glycerol. Then, the mixture was mixed under constant mag-
netic stirring at 60 °C for 5 h. The aliquot were spread over circular alu-
minum weighing boat with top diameter of 43 mm which was placed in
oven at 60 °C for 24 h. After 24 h the mixture checked to make sure it is
fully dried and let it cool. Then, aluminum weighing boat was torn to de-
tach bioplastic film from the weighing boat. Bioplastic was then labeled
and stored for analysis. This process was repeated with all the concen-
tration of glycerol.
2.4. Characterization
2.4.1. Scanning electron microscope (SEM)
The surfaces of synthesized bioplastic films were studied under a
Hitachi's Tabletop Electron Microscope TM3030 Plus at an accelerating
voltage of 15 kV. SEM is used to identify the molecular structure of the
sample to check if the mixtures are well-mixed during the polymeriza-
tion process.
2.4.2. Fourier transform infrared spectroscopy (FTIR)
The FTIR spectra of bioplastic and keratin were analyzed using a
Perkin-Elmer Model 1000 Series FTIR instrument equipped with a at-
tenuated total reflectance (ATR) accessory. The spectra were collected
from 4000 cm−1 to 500 cm−1 frequency ranges. The data were analyzed
for each film using the program FTIR Spectrum Software.
354 N. Ramakrishnan et al. / International Journal of Biological Macromolecules 111 (2018) 352–358

2.4.3. X-ray diffraction (XRD)

The crystal structure of bioplastics was examined by using Rigaku
Miniflex II X-ray diffractometer with CuKa radiation (X = 1.542 A)
worked at 30 kV and 15 mA. Data were recorded within the scattering
angles range of 5° to 50° at the rate of 0.02°/min. The basal spacing
was resulted from the peak position in the XRD diffractogram which
was according to the Bragg eq. (X = 2d sin 9).

2.4.4. Thermo gravimetric analysis (TGA)

TGA analysis was carried out using a Mettler Toledo thermal ana-
lyzer instrument under nitrogen atmosphere. Sample was heated from
room temperature to 900 °C at a rate of 10 °C/min in air with a flow
rate of in general 50 ml/min. The weight change of sample was recorded
as a function of temperature. Then mass loss and differential thermal
analysis profiles were recorded. The whole process was repeated for
all samples.

2.4.5. Differential scanning calorimetry (DSC)

DSC analysis was performed by TA-Instrument DSC/Q 1000 to study
the non-isothermal crystallization kinetics. The specimen weight was in
general in the range of 3–5 mg and the heating rate employed 10 °C per Fig. 3. Comparison of XRD analysis of keratin with bioplastic made with different
concentrations of glycerol.
minute from room temperature to 250 °C.

2.4.6. Mechanical property testing 3. Results and discussion

Tensile properties were tested using Shimadzu AG-X series universal
testing machine. The dumbbell shape samples were made by following
ASTM D638 (type V) standard. A load of 5.0 kN was applied at constant
crosshead speed of 1 mm/min at room temperature. The Tensile
strength and tensile modulus were evaluated from the force displace-
ment data.
2.4.7. Biodegradability testing
Biodegradability of the bioplastic film by incubating in protease en-
zyme solution was investigated. Protease enzyme solution was pre-
pared using 0.1 M phosphate buffer solution with 7.4 pH for 10 h. 25
ml of the stock solution was poured onto each sample of film in petri
dishes. The petri dishes were then sealed using paraffin film. Sealed
petri dishes were placed in an incubator at 30 °C. The change in sample's
appearance was observed and noted for every 2 h.
3.1. SEM
Fig. 1(A–C) shows the results of SEM analysis of 2 wt%, 5 wt% and 10
wt% of glycerol, respectively. Fig. 1(A) shows very good compatible
morphologies without cavity, edge, and holes. Thus, showed good
bonding between the components, which was ascribed to the presence
of chemical interactions in keratin and glycerol [4]. Thus, the glycerol
helped to make homogenous mixture with clear signs of plasticization
in the keratin matrix, with no separation and single phase morphology
was produced which is in agreement with the previous research [41].
SEM analysis on 5 wt% glycerol bioplastic film (Fig. 1B) and 10 wt% glyc-
erol bioplastic film (Fig. 1C) showed the presence of undissolved parti-
cles. As compared to 5 wt% glycerol bioplastic film (Fig. 1B), 10 wt%
glycerol bioplastic film (Fig. 1C) showed more voids and undissolved
particles. Presence of empty micro voids may be due to the degree of
dispersion of the plasticizer in the polymer matrix is better at lower
plasticizer content. According to the study [42], the tendency to form

Fig. 2. FTIR spectra are given for keratin and bioplastic made with different concentration
of glycerol. Fig. 4. TGA graph of keratin and bioplastic made with different concentrations of glycerol.
 N. Ramakrishnan et al. / International Journal of Biological Macromolecules 111 (2018) 352–358
Fig. 5. DSC curves of keratin and bioplastic made with different concentration of glycerol.
empty voids and phase separation is directly proportional to the glyc-
erol content.
3.2. FTIR
FTIR analysis for bioplastic synthesized using keratin as a base mate-
rial shown in Fig. 2. It was found that bioplastic made having different
concentration have similar wave numbers as for keratin extracted
from chicken feather. Therefore, bioplastic formed was totally from ker-
atin with slight amount of glycerol. The presence of glycerol, showed a
shift towards higher wave number, which specified a decreased β-
sheet interaction with glycerol and the promotion towards disordered
structure. In previous investigations also, when glycerol was used in
pea maize, cereal sorghum, and wheat proteins as a plasticizer, showed
the similar results [43].
The results showed Amide A and Amide I-III groups [44]. A broad and
week single absorption band at 3314 cm−1 was for N\\H bond which
showed the presence amide group and named as Amide A. The protein
conformational arrangements were studied by amides I–III. The absorp-
tion bands of\\CH2 at 1633 cm−1 (amide I), C\\N stretching and N\\H
bending at 1480–1580 cm−1 (amide II), and C\\N, C\\O stretching and
N\\H bending at 1230 cm−1 (amide III) [45, 46] were examined in the
spectra of samples [47]. The absorption bands at 1076 cm−1 and 1024
cm−1 were ascribed to the asymmetric and symmetric S\\O stretching
Fig. 6. Graph show the (A) tensile strength and (B) Young's Modulus of bioplastic made with different concentrations of glycerol.
355
vibration respectively. The S\\O stretching vibration was due to
cysteine-S-sulphonated residues which were formed during extraction
process [48–50]. The sharp peak at 1024 cm−1 observed in the FTIR re-
sults, indicating a higher content of cysteine-S-sulfonated residues in
the sample. However, the relative intensity at 1633 cm−1 increased,
and it was more prominent in glycerol presence. Glycerol, having
more hydroxyl groups thus, can form a large number of hydrogen
bonds, which increase the total hydrogen bonded peptides groups ex-
amined at higher wave number (Fig. 2).
3.3. XRD
The Fig. 3 showed peaks which indicated the strong crystalline char-
acteristics. The shift to higher angle indicated a decline in the subse-
quent interlayer spacing, which means that the mixed component had
an arranged structure. The increase of d-spacing showed that the
blend has a less arranged structure, thus leads to more difficult crystal-
lization [51]. The d-spacing is defined as inter-atomic spacing (D value
in Angstrom units − 8–10 cm). It is showed in Fig. 3 that bioplastic
film synthesized with 2 wt% of glycerol has the highest 2θ = 21° values
and the low d-spacing, which means it has the most ordered structure
among the bioplastic film. It may due to the lower glycerol content in
the mixtures and thus dispersed better [42]. Besides, bioplastic film syn-
thesized with 5 wt% and 10 wt% glycerol showed almost similar results.
Since glycerol amount used is higher for 10 wt% bioplastic film, d- spac-
ing is the lowest compared to other glycerol compositions. It can be seen
from the Fig. 3 that strong crystalline characteristics was showed by
bioplastic film having lowest glycerol concentration.
3.4. TGA
Fig. 4 shows the graph of weight percentage across room tempera-
ture to 900 °C. From Fig. 4, it is shown that the bioplastic synthesized
from keratin with different glycerol concentrations have the same
trend of weight loss. These degradation trends shown in Fig. 4 are al-
most identical to the research done where there is two step weight
losses for plasticized film [23]. The degradation of each plasticized
resin consisted of three weight loss steps. The first gradual weight loss
(below 150 °C) is due to the evaporation of moisture, the second (be-
tween 150 and 250 °C) is attributed to the plasticizer evaporation, and
the final weight loss beyond 250 °C is due to decomposition of quill ma-
terial. The increase of glycerol concentration significantly increases the
thermal degradation rate at constant temperature [52, 53]. However,
in the present study, increase glycerol concentration has an significant
effect on the thermal stability thermal degradation rate as well as ther-
mal stability which is similar to previous research [54].
356 N. Ramakrishnan et al. / International Journal of Biological Macromolecules 111 (2018) 352–358

Fig. 7. Appearance for 2% glycerol bioplastic film during biodegradation in a–f) 0.5% stock solution; g–l) 1% stock solution.

3.5. DSC
The thermal properties like melting temperature and temperature of
binding the molecules (Tg) of keratin bioplastic with different concen-
trations of glycerol were studied using DSC in the temperature range
25 to 250 °C as shown in Fig. 5. The DSC curves showed the melting tem-
perature at 35 °C. According to Reddy et al. [9], unmodified chicken
feathers did not show any melting peak whereas cyanoethyl treated
chicken feathers showed an endothermic melting peak at 167 °C due
to the introduction of cyano group. Tg is defined as the temperature at
which the molecules binding forces binding are relaxed to allow large-
scale molecular movement [3]. The addition of plasticizers into the plas-
tic film reduce the Tg, but according to Sanyang et al. [54], the decrease
of Tg values are insignificant when the plasticizer concentrations in-
creases which is in accordance with present study.
3.6. Mechanical properties
The film formed was 5 mm in thickness. Fig. 6(A) shows the graph of
tensile strength versus concentration of glycerol. According to Sanyang
et al. [54], when the concentration of plasticizer increases, the tensile
strength decreases. As compared to starch-based bioplastic, keratin
bioplastic have relatively lower tensile strength. The 15 wt% of
glycerol-plasticized sugar palm starch film and 10 wt% keratin
bioplastic film have tensile strength of 9.59 MPa and 0.0409 MPa re-
spectively [54]. When compared to citric acid cross-linked films, citric
acid cross-linked feather keratin films have much higher tensile
strength of 230 MPa [9].Tensile strengths of keratin based plastics
films in this work generally have a lower tensile strength values as com-
pared to other biodegradable plastics such as starch-based and citric
acid cross-linked plastics film. Films made from the cyanoethylated
feathers had strength and elongation ranging from 1.6 to 4.2 MPa and
5.8 to 14% respectively depending on the extent of cyanoethylation.
Cyanoethylated feathers had good thermoplasticity and could be useful
to make various thermoplastics [55].
Fig. 6(B) shows the graph of Young's Modulus versus concentration
of glycerol. It is shown that with the increases of glycerol concentration,
the Young's Modulus decreases. As compared to starch-based bioplastic,
keratin bioplastic have lower tensile strength in which the tensile
strength value for 5 wt% of glycerol-plasticized sugar palm starch film
is 9.59 MPa while for keratin based film even for lower glycerol concen-
tration of 10 wt% is 7.5593 MPa [56].When compared to citric acid
cross-linked films, citric acid cross-linked feather keratin films have
much higher tensile strength of 230 MPa [9].Young's Modulus of keratin
based plastics produced in this work films generally has lower Young's
Modulus values as compared to other biodegradable plastics such as
starch-based and citric acid cross-linked plastics film. As the

Fig. 8. Appearance for 5% glycerol bioplastic film during biodegradation in a–e) 0.5% stock solution; f–j) 1% stock solution.
 N. Ramakrishnan et al. / International Journal of Biological Macromolecules 111 (2018) 352–358
Fig. 9. Appearance for 10% glycerol bioplastic film during biodegradation in a–d) 0.5% stock solution; e–h) 1% stock solution.
concentration of glycerol increases, tensile strength and Young's Modu-
lus of keratin bioplastic films decreases. These behaviors agree with
other studies on the effect of glycerol concentration on protein-based
films: wheat gluten and soy proteins [57], peanut proteins [58] and
fish muscle proteins [59].
3.7. Biodegradability testing
For 1 wt% stock solution, the plastic film is seen to be degraded faster
compared to 0.5 wt% stock solution as shown in Figs. 7, 8, 9. Thus, it can
be concluded that amount of enzyme present effects the rate of biode-
gradability. Besides, the compositions of plastic film also affect the bio-
degradability rate. When the concentration of glycerol increase, time
taken for the plastic film to degrade decreases. 2 wt% glycerol based
film takes about 10 h to be degraded fully in stock solution (Fig. 7).
This is may be due to high strength and strong bonding between plasti-
cizer and keratin within the film. Whereas for highest composition of
glycerol which is for 10 wt% glycerol, it just took about 6 h for the plastic
film to completely degrade (Fig. 9). This may be due to poor strength
and bonding between keratin and glycerol within the film [23]. Since
total time taken for the plastic film fully degrade is same for all concen-
tration in 1 wt% and 0.5 wt% stock solution, the amount of enzyme does
not give much effect on biodegradability rate. After observation for
about 1 day, the bioplastic film is found to be fully degraded within
10 h time. This proves that the product of this research is totally biode-
gradable and harmless to the environment and living things.
4. Conclusion
By end of this research, bioplastic was successfully synthesized using
keratin from chicken feathers. Glycerol at increasing concentrations de-
creased the maximum tensile strength of chicken feather keratin films.
In this way, the tensile strength and elongation at break can be con-
trolled through glycerol concentration added in the keratin dispersion
used to obtain the samples. The microstructure of keratin films as mod-
ified by the addition of glycerol, which made the film surfaces more ho-
mogeneous, compared with films without a plasticizer. According to the
results, bioplastic made up from keratin with 2 wt% of glycerol has the
best mechanical and thermal properties. Other than that, from SEM
test, it has good compatible morphologies without edge, cavity and
holes and bioplastic produced are proven as a biodegradable. Therefore,
it is a remarkable result, considering the possible application of these
357
films to substitute fossil oil based materials which is harmful to the
environment.
Acknowledgment
Authors are thankful to University Malaysia Pahang (UMP) for prov-
ing facilities and financial support; Author [SS] is thankful to UMP for
providing Doctoral Scholarship Scheme (DSS) scholarship (letter no.
UMP.20.03/13/13.14/1) as financial support.
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