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73
Sheep wool-derived hydrolyzed........................................................... (Prayitno et al.)
Majalah Kulit, Karet, dan Plastik, 33(2), 73-78, 2017
Author(s), https://doi.org/10.20543/mkkp.v33i2.3336
Sheep wool-derived hydrolyzed keratin from tannery waste of
the tanning industry using perhydrol
Prayitno*, Dona Rahmawati, Gresy Griyanitasari
Center for Leather, Rubber, and Plastics. Jl. Sokonandi No. 9 Yogyakarta 55166, Indonesia
*Corresponding author. Tel.:+62 274 512929, 563939; Fax.:+62 274 563655
E-mail: prayitno_bbkkp@yahoo.com
Received: 06 September 2017 Revised: 25 September 2017 Accepted: 16 October 2017
ABSTRACT
Sheep wool waste discharged from leather tanning industry recently has posed a problem in relation to its
treatment because of its sizeable quantity and its diculty to degrade. Wool is composed mainly of keratin. It is a
protein with a high content of disulde bonds which cause the protein keratin cannot dissolve in water and resist
of diluted acids and alkalis. Keratin can be hydrolyzed to produce keratin hydrolysates which have many benets
such as for cosmetic additives. Research into the use of waste wool of sheep originated from the sheep leather
tanning industry had been performed by using a hydrolyzed system to produce protein keratin. The waste wool used
came from unhairing by painting and conventional unhairing. Hydrolysis was done using hydrogen peroxide 50%
amounting to 70 ml for every 40 gr of wool. Hydrogen peroxide was added to wool immersed in the 0.5 M NaOH
solution for three hours. The length of hydrolysis ranged from 4, 5, to 6 hours and the mix was stirred shortly every
1 hour followed by ltration using a coarse sieve. To precipitate the hydrolyzed keratin, the pH was decreased to
4-5 using the 2 M HCl solution and after separation of the precipitation, it was dried in the oven at a temperature
not more than 50 oC for 2 days. The research ndings showed that a maximum of 69.19% of keratin hydrolysates
was generated using the raw material of waste wool through a conventional process with a total of hydrolysis time
by 6 hours, whereas the maximum protein generated was 66.99% using waste wool through a conventional process
with a total of hydrolysis time by 4 hours. The FTIR test showed the presence of groups of amides, cysteic acids,
and cystine-S-monoxide.
Keywords: Hydrolyzed keratin, hydrogen peroxide, unhairing by painting, conventional unhairing, waste wool.
INTRODUCTION
The waste wool which resulted from the tan-
ning process reaches 20-30% by weight against
the skin resulted. This raise problems in relation
to the treatment of this waste, because waste wool
cannot be degraded immediately by microorgan-
isms. Currently, waste wool is controlled using
the landll system. Some problems arise because
the diculty in degradation causes problems to
soil fertility and other soil functions. According
to Bayramoglu et al. (2014), wool is essentially
an epidermis structure that forms the outer cov-
erings of the main structures of the body in the
form of the protein keratin, it is a protein brous
which is rich content of sulfur. Protein keratin is a
type of structural proteins that are chemically non-
reactive, have strong mechanical properties, and
are insoluble in water and organic compounds.
It is chemically characterized by a cysteine con-
tent in the sequence of keratin amino (Gupta
et al., 2012). According to Cardamone (2010),
wool contains 95% w/w pure keratin and kera-
tin derived from wool is clay, hard, and insoluble.
According to Sarkar (1995), the amount of kera-
tin in the skin varies according to species and age
of the animal. Keratin is classied into two types,
those are α-keratin and β-keratin. The α-keratin is
mainly the keratin in the hair and weaves, where-
as the β-keratin is the keratin mostly found in
nails, horns, reptile claws, and bird beak (Kannahi
& Ancy, 2012). Whereas Bragulla & Homberger
(2009) classies keratin into two types accord-
ing to their isoelectric point (pI), namely keratin
type I (acidic) and keratin type II (basic). Bovine
keratin type I has a pI < 5.6, while keratin type
II has a pI > 6.0. Keratin is brous which can be
74 MAJALAH KULIT, KARET, DAN PLASTIK Vol. 33 No. 2 Desember Tahun 2017: 73-78
isolated from the wool into a protein hydrolysate
used for a variety of additives in some types of
cosmetics. Studies have been performed to extract
keratin from several sources of raw materials.
Villa et al. (2013) extracted keratin from chicken
feathers using enzyme-producing microbial ker-
atinase Bacillus subtilis sp whereas Kannahi &
Ancy (2012) extracted keratin from chicken feath-
ers using enzymes produced by Aspergillus avus
sp and can hydrolyze protein keratinase until 90-
95%. Mokrejs et al. (2011) state that keratin hy-
drolysates can be extracted using proteolytic en-
zymes. The principle for extracting keratin is to
breakdown the sulfur bonds in cysteine into a hy-
drolysate which can easily dissolve protein. A pre-
vious experiment has been done by Bayramoglu
et al. (2014) to hydrolyze wool keratin for creat-
ing skin emulsion using hydrogen peroxide with
the following conditions: hydrolysis time lasted
for 5 (ve) hours and keratin was precipitated by
0.5 M HCl solution. To improve the resulting ker-
atin and to get a greener process, research into ex-
traction of keratin using hydrogen peroxide needs
to be performed to nd out the optimum hydroly-
sis time and reduce the volume of the HCl solu-
tion used.
MATERIALS AND METHODS
Materials
The raw material used in this research was
wool obtained from sheepskin tannery waste
using conventional unhairing and unhairing by
painting in beamhouse operations. Chemicals
used to process were Na2CO3, wetting agents,
NaOH, HCl, pH sticks, Na2S, Ca(OH)2, NH4Cl,
degreasing agents, and H2O2 50% solution. The
equipment to hydrolyze keratin consisted of an
oven, glass apparatus, a thermometer, a pH meter
and testing equipments such as Kjeldahl apparatus
and fourier transform infra red (FTIR) IR Prestige-
21 Shimadzu.
Methods
Research design
This experiment was arranged in a random-
ized complete factorial design, which consisted of
two variables of the material used in accordance
with the method of the unhairing process and three
variables in hydrolysis times. Each treatment was
repeated three times so that there were a total of
18 experiments. Each experiment was analyzed
in terms of the weight of the keratin generated in
presentation of the raw material used, total pro-
tein, and structure of protein with FTIR.
Keratin extraction method
The method used in this research was based
on the experiment conducted by Bayramoglu et al.
(2014). Waste wool was washed with water to re-
move Na2S and Ca(OH)2, then it was dried under
the sunlight. 40 grams of waste wool were soaked
in 1 liter of the 0.5 M NaOH solution for 3 hours.
80 ml of H2O2 was added each time, it was stirred
until 4, 5, and 6 hours. Next, it was ltered using a
coarse strainer and the 2 M HCl solution was add-
ed until its pH was equal to 4 to 4.5 to allow pre-
cipitation of keratin. The keratin was dried in the
oven at a temperature of 45 oC for 2 days and pro-
cessed in such a way that it changed into powder.
Testing
Testing was done to determine total protein
content and weight of keratin generated and also
the total protein content and the chemical structure
of protein keratin.
Data analysis
The weight of keratin and the total protein
content obtained from the experiment were an-
alyzed statistically using the method analysis of
variance using SPSS for each treatment contin-
ued with the LSD analysis with a 95% degree of
signicance.
RESULTS AND DISCUSSION
The weight of keratin and the total weight of
the protein content generated in the experiment
using waste wool from conventional unhairing
and using waste wool from unhairing by painting
are presented in Figure 1.
Protein contents
To determine the inuence of the treatment
on the keratin hydrolysate produced, a statistical
analysis, i.e. the analysis of variance was under-
taken as presented in Tables 1 and 2. Results of the
analysis of variance as listed in Table 1 show that
there are signicant dierences viewed from the
factor of raw materials in the weight of the protein
content between the keratin hydrolysates generat-
ed from waste wool processed using the conven-
tional method and the keratin hydrolysates gener-
ated by the painting process (ttable = 48.05 > tcount=
4.96 ), which mean that the raw material greatly
75
Sheep wool-derived hydrolyzed........................................................... (Prayitno et al.)
inuences the protein content of the resulting hy-
drolyzed keratin. Waste wool processed using a
conventional method generated hydrolyzed ker-
atin with a protein content by 63.26% whereas
that produced by the painting process had a pro-
tein content by 54.47%. According to Cardamone
(2010), wool contains up to 95% by weight of
pure keratin. The lower keratin content generated
in this experiment may be caused by the age and
species of the sheep (Sarkar, 1995).
It is likely because the time of wool expo-
sure to basic liquor is much more longer for wool
processed using the conventional unhairing meth-
od than that processed using the painting method
so that the wool processed conventionally became
more swollen, which in turn caused keratin to be
more easily hydrolyzed by hydrogen peroxide. As
for the time factor, according to results of the sta-
tistical analysis of the hydrolyzed keratin using
ANOVA, there was a signicant dierence (tcount
= 4.15 > ttable = 4.10), but the LSD calculation at a
5% degree of signicance performed afterwards
suggested no signicant dierences.
Yield (Percentage of keratin hydrolysate
against the raw material weight)
Results of the ANOVA of the protein kera-
tin extracted are listed in Table 2. The weight per-
centages of the hydrolyzed keratin which was gen-
erated with the raw material of waste wool pro-
duced either using a conventional process or with
a painting process did not indicate signicant dif-
ferences (tcount = 3.77 < t table = 4.96). It means that
the hydrolysis time lasting for 4.5 and 6 hours did
not generate dierent results for the protein kera-
tin extracted (tcount = 2.40 < ttable = 4.10). This is pos-
sibly because the protein content present in kera-
tin of the wool has been extracted completely.
The resulting pH of the hydrolyzed kera-
tin accorded with the pH range for settling of
Raw materials Total Average The notation with LSD 5%
t 5% 2,228 P 490.25 54.47 B
LSD5% 3.46 K 569.37 63.26 A
Figure 1. Percentages of the weight and the total protein keratin generated.
Table 2. Analysis of variance (ANOVA) for the protein content.
Sources of Var DF SS MS F. cal F. 5%
Deuteronomy 2 1.06 13, 402.76
Treat 5 408.27
Raw materials 1 347.78 347.78 48.05 4.96
Hydrolysis time 2 60.05 30.03 4.15 4.10
Interaction 2 0.44 0.22 0.03 4.10
Error 10 72.38 7.24
Calculation of LSD (least squared design)
conventional
painting
conventional
painting
Yield (%)
76 MAJALAH KULIT, KARET, DAN PLASTIK Vol. 33 No. 2 Desember Tahun 2017: 73-78
protein keratin, which ranged from 4 to 5. This
is consistent with the pH of the electrostatic pro-
tein containing hydrolyzed keratin. The keratin of
sheep falls into the category of keratin type K25
which has an isoelectric pH of 4.7 (Bragulla &
Homberger, 2009).
FTIR Test
The structure of hydrolyzed keratin analyzed
using FTIR indicated that the spectrum of the hy-
drolyzed keratin generated by Bayramoglu et al.
(2014) and the hydrolyzed protein generated by
the research into keratin extraction as presented
in Figures 2 and 3 had similar spectra. The FTIR
spectrum of the test zone ranging from 1,400 to
4,000 cm-1 is known as the “function group” zone.
The peak spectra of the “function group” zone for
keratin hydrolysates was at wavelengths of 3274,
2962, 1539, and 1450 cm-1 whereas the FTIR spec-
tra of Bayramoglu et al. (2014) absorbs ion peak
to hydrolyze keratin was found at wavelengths of
3279, 2962, 1536, and 1449 cm-1
Research by Bayramoglu et al. (2014) re-
sulted hydrolyzed keratin generate peak at wave-
lengths of 1680-1645 cm-1 for the amide II, 1550-
1515 cm-1 for the amide II, and for the amide III in
1435 cm-1 cystein-S-sulfonate (Cy-S-S)-3) at 1012
cm-1, cysteic acid (Cy-SO3H) at 1045 cm−1 cys-
tine--S-monoxide (Cy-SO-S-Cy) on 1080 cm-1,
cystine-S-dioxide (CySO2-S-Cy) in 1137 cm-1.
The FTIR test of hydrolyzed keratin from this re-
search showed that the zone of amide obtained
at peak amide I was in 1634.81 cm-1, peak amide
II was in 1535.29 cm-1, amide III was in 1448.01
cm-1, cysteic acid was in 1045.02 cm-1, and cys-
tine-S-monoxida was in 1081.10 cm-1. According
to Bayramoglu et al. (2014), amide I with a peak
in 1650 cm-1 and amide II with a peak in 1547
cm-1 have a helix-shaped structure.
CONCLUSIONS
Based on the research ndings, it can be con-
cluded that keratin hydrolysates can be generated
from extraction of waste wool through hydroly-
sis using hydrogen peroxide with hydrolysis time
lasting for 4, 5, and 6 hours. The percentages of
the protein keratin resulting from waste wool orig-
inated from conventional processes were 64.99,
64.30, and 60.50% respectively, while the percent-
ages of the protein keratin resulting from waste
wool originated from the painting process were
56.09, 55.22, and 52.14% respectively. The per-
centages of the hydrolyzed keratin resulting from
the extraction using waste wool as the raw mate-
rial were 58.61, 66.28, and 69.19%, respective-
ly for the conventional process and were 59.24,
52.24, and 59.82%, respectively for painting pro-
cess. The FTIR test results showed the presence
of results of cluster-cluster amide, cysteic acid,
Table 3. Analysis of variance (ANOVA) of the protein keratin extracted.
Sources of Var DF SS MS F. cal F. 5%
Deuteronomy 2 106.16 53.08
Treat 5 676.76
Raw materials 1 158.06 158.06 3.77 4.96
Hydrolysis Time 2 201.68 100.84 2.40 4.10
Interaction 2 317.01 158.50 3.78 4.10
Error 10 419.43 41.94
Figure 2. FTIR spectra of keratin resulting from
the painting process.
Figure 3. FTIR spectra of keratin resulting from
the conventional process.
% Transmittance
105
90
75
60
45
30
15
0
3800 3400 3000 2600 2200 1800 1400 1/cm
% Transmittance
4000 3500 3000 2500 2000 1500 1000 1/cm
105
90
75
60
45
30
15
0
77
Sheep wool-derived hydrolyzed........................................................... (Prayitno et al.)
and cystine-S-monoxide in the hydrolyzed kera-
tin generated.
ACKNOWLEDGEMENT
The authors thank to the Center for Leather,
Rubber and Plastics for the facilities and funding
in this study.
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