Quantitative determination of enzymatic and chemical dehairing of skins by an electronic force sensor

Abstract

The dehairing effect of 2 keratinolytic enzyme preparations from liquid cultures of Bacillus cereus IZ-06b and B. cereus IZ-06r and 3 commercial proteolytic enzyme formulations used in leather manufacturing for soaking, dehairing, and bating processes were quantified by a computerized force sensor. Measurements of the force needed to remove individual hairs from skin, showed that the keratinolytic enzymes did loosen hairs from sheepskins. These enzyme preparations may therefore be useful alternatives to present days' beamhouse chemicals, although the traditional dehairing procedure with lime and sulphide still exhibited the strongest effect in hair loosening.

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quality ..................... 360
by O. Smemvo, E. Gr;rnrsRA-SCrn, [. Kannxa, Mus-rcH awo S. MusARAr
Quantitative Determination of Enzymatic and Chemical
Dchairing of Skins by an Electronic Force
Sensor ................ 367
Dy A. Zelvcnv, B. Btrusu enn N. EnrxsEr.l
Eramination of Gram Positive Bacteria on Salt-Pack Cured Hidcs .........372
by
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367
QueNurATIvE DETEnMINATToN
oF ENzynaATrc
AND CHEuTcAL
DEHITRTNG
or Srws By eu ETECTRoNTc
Foncn SEr*rson
by
A. C" AorcuzEr
ZrNcnr', B. O- Brrrrsur, N. T- EnrrsEN2j
t
Department of'
Leather Engineering, Engineer ing Faculty, Ege (Jniversity
Bonuov.l
35100
Izurn, Tt;nxEy
2Department
of'Biotechnology,
Chemistry and Environmental Engineering,
Aalhorg (Jniversity
Sonuca-q.nnsuolMsvcJ
49, DK-9000 A_{r_BoRG,
Drnlr,qnx
Asffnlcr
The dehairing effect of 2 keratinolytic enzyme preparations
from liquid cultures of Bacillus cereus IZ-O6b and B. tereus
12-O6r and 3 commercial proreolytic enzyme formularions
used in leather manufacturing for soaking, dehairing, and
bating processes were quantified by a computerized force
sensor.
Measurements of the force needed to remove individual
hairs from skin, showed thar the keratinolytic enzymes did
loosen hairs from sheepskins. These enzyme prepiuarions may
therefirre be useful alternatives to present days' beamhouse
chemicals, although the traditional dehairing procedure rvith
lime and sulphide still exhibited rhe srrongesr effect in hair
loosening.
Rpsuunx
El efecto depilante de dos preparaciones queratinolfricas
enzimdticas producidas por cultivos liquidos d,el Bacillus
cereus IZ-O6b y B" cereus 12-O6I, asf crimo las de tres
formulaciones proteoliticas ctlmerciales empleadas en
curtiembres para los procesos de remojo, depilado, y rendido
fueron cuantificadas por medio de un medidor de fuerza
computarizado. Determinaciones de la fuerza requerida para
remover pelos individualmente de la piel demostraron que las
enzimas queratinoliticas si aflojaron los pelos de pieles ovinas.
Estas preparaciones podrian ser alternativas ftiles a los
productos quimicos del pelambre, aunque los procedimientos
tradicionales con cal y sulfuro exhiben todavia los efectos
depilantes mds fuertes.
InrnonucrroN
Microbial keratinases
are becoming
important enzymes
for
the lea&er industry, rvhere
they can be used
as environmenully
friendly dehairing agents. Traditional dehairing processes
using lime and sulphide solutions are some of the rnost
pollutant operational
steps in leather
manufacturing"
Alkaline
proteases,
including keratinase,
collagenase,
and elastase can
be used to minimize the need
for sulphide
and reduce the
organic waste
load of the dehairing process.'
Keratinolyric
proteases
selectively
degrade
the keratin rissue
in the follicles
in the hides and skinsa and keratinolyric acriviry combined
rvith mild collageno\nic and elastolytic
activities
can
resulr in
a proteolytic dehairing process,
rvhich is genrle
rorvards rhe
proteins
forming the leather.3
Different protease formulations are presently available for
different learher manufacruring sreps,
including soaking,
dehairing
and bating.
The proreases
in commercial
soaking
enzyme formulations (e.g- Buzyme 148, Buckman
Laboratories,
Belgium)
remove
blood,
albumin,
and
mucous
from green
hides, and contribute to a uniform rehydration
of
brine cured and wet-salred
hides.
Unhairing enzyme (e.g.
Buzyme 7705,
Buckman Laborarories,
Belgium) is used in rhe
dehairing step
of the liming process,
before
or afier an inirial
treatment
rvith lime. The dehairing
process
can be modified to
rvork either
as a hair save
or hair burn process
and can reduce
BOD and COD in rhe effluent rvaqre.
Baring enzyme (e-g.
Pellucit
1000,
Pulcra
Chemicals,
Germany) is used after the
deliming process
for bating in order to break dolvn non-
structural proteins.
The dehairing effect of microbial proteolytic enzymes
has
been documented
in several studies,
mainly by qualitative
comparisons
of the ease by rvhich hair is removed from
enzymatically treated skins relative to skins not treared by
enzymes,
rvhen hairs are pulled our by gentle scraping
rvirh
fingers, or indirectly from analysis
of color, smoothness,
and
silkiness
of the pelt-2'a-'2
In this study,
we have
used direct
measurements
of the force needed
to remove
individual hairs
*Corresponding Author e-mail address: nte@bio.aau-dk
Manuscript received March 30,2011, accepted for publication July 3,2011-
IALCA,VOL 106,2ot1

368 MElsunrNc ILcrR REr{ovA,r
FoncE
from skin for quantitative comparisons of the dehairing
activity of commercial proteases on sheep skin, as well as
crude preparations of the proteolytic enzymes secreted by two
keratinolytic strains of Batillus cereus named IZ-06b and
12-O6r. These bacteria produce a mixture of keratinolytic,
collagenolytic, and elastolytic activiries rvhen grorvn in barch
culturer. The kinetics of hair loosening by rhe various
enzymatic preparations were compared in experimental
dehairing processes.
ExprnnnsNrll
Enzymatic preparations end production
Bacillus tereus lZ-O6b and B. cereus 12-O6r, isolated from
rvool, were used for the production of crude proteolytic
enzyme preparations rvith keratinolytic, collagenolyric, and
elastolytic activities-4 The cultures were grorvn in barch
cultures
in liquid media containing 0.5 g l-' NaCl,0.3 g l-'
K,HPO4,0-4 g l-r KH,POo,O.O4 g l-rMgCl,5 g l-' mahose,3
g
l-' meat peptone, pH 7.4 in conical flasks shaken at 150 rev
min-r
on an
orbital shaker
at 30"C. After 20-24 hours rvhen
the
maximal keratinolytic activities in the batch cultures rvere
attaincda
cell-free culture supernatants
were harvested and
used as crude enzymatic preparations
for enzymatic
dehairing
of sheepskins-
Dehairing
of sheepskins by commercially available
enzyme
formulations used in the leather industry rvas investigated
at
conditions recommended
by the manufacturer.
The effect of
the soaking enzyme formulation, Buzyme 148 (Buckman
Laboratories, Belgium) was investigated
ar pH 7 ar a dosage
level of O-l%.The unhairing enzyme
formularion
(Buzyme
7705,
Buckman Laboratories,
Belgium) rvas investigared
at pH
6 at dosage level of O.2%.The bating enzyme
formulation
(Pellucit
l0O0
LVU g-r,
Pulcra
Chemicals,
Emery,
USA) rvas
investigated
at pH 7 at a dosage level of l%.
Enzymetic and chemical heatment of skins
The dehairing effects of the enzymatic prepararions
were
tested using rarv,
dry salted (metis type) sheepskin.
Afrer
soaking of sheepskins for 2 h in water,
pieces of 25 cm2
sheepskins
rvere incubated
for up to 48 h at 30"C in 15-30 ml
B. cereus lZ-O6b or B. cereus 12-O6r crude enzyme
preparatiDns
depending on leather weight, or in solutions of
soaking,
dehairing, or bating enzymes prepared
according
to
recommended
dosage levels
of the manufaclurer
as
described
above,
and shaken
at ll0 rev min-rin an orbital shaker.
Microbial activity rvas inhibited by addition of O l% of the
commercial bactericide Gemacide
LP (Gemsan,
Istanbul,
T[rkey). Sheepskins
were also incubated
in a solution of 3%
lime and 2% sodium sulphide in order to compare the
enzymatic dehairing to the naditional beamhouse
dehairing
process-
JALCA, VOL. 106,2011
Force measurements
The force needed to remove individual hairs from sheepskins
was determined by a computerized PS-2104 force sensor
{Pasco, California). The sheepskins ivere mounted on a hook
on the force sensor and 1-lO hairs were gently pulled out by a
pair of lweezers rvhile the force applied to the hairs rvas
recorded at a frequency of 10 Hz. The force needed to remove
the hairs rvas taken as the maximal force recorded at the time
the hairs got loose. Every 2 hours, the sheepskins were
removed from the incubation baths where they were
enzymatically or chemically treated, mounted on the force
sensor, and hairs rvere repeatedly pulled out for ar leasr lO
times. The number of individual hairs remr)ved each time rvas
counted and compared to the maximal force that had been
recorded by the force sensor each time hairs were pulled out.
By this rvay, the action of the enzymes rvas recorded as a
decrease in the force needed to remove individual hairs from
the skins over time-
Rnsurrs
Figure I shorvs how sheepskins were mounted onto the
PS-2104 force sensor and horv individual hairs rvere pulled
out from the skin by a pair of tleezers. Selection and removal
of single hairs proved impractical and time consuming due to
high densities of hairs on the skin surfaces, and the ttveezers
typically got hold on l-l0 hairs each time. A force berrveen 0
and 2 N rvas needed to remove these numbers of hairs
depending on the skin and horv it had been treated. Figure 2
compares sets of force measurements on hair repeatedly
removed from raw metis type sheepskin soaked for 2 h in
rvater and after additional incubation in soaking enzyme for
48 h.
The maximal force needed to remove hairs increased non-
linearly by the number of hairs removed (Figure 3)^ In
particularly rvhen more than l0 hairs rvere simultaneously
removed, the force per hair decreased, probably because not
all hairs got loose at the same time. The relationship betrveen
fon:e and number of hairs removed follorved a power function
F = a'no (l)
where F is force, n is number of hairs removed, and rr (force
needed to remove I hair) and D are constants specific for a
given piece of skin and treatment. The force needed to remove
I hair rvas calculated from Eq. l, using n = l-
The effects of B. rcreus IZ-O6b and B. cereus 12-06r crude
enzyme preparations on the force needed to remove hairs from
ra\. metis type sheepskin are shorvn in Figure 4 and compared
to skins incubated in rvater- The force measurements have
been normalized relative to the initial force needed in order to
compensate for the variations in strengths of hair-binding to
skin between different pieces of skin- Although incubation ia
waterhad an effect on the loosening ofthe hairs,the effect of

Mr-csunrNc }Lqrn Rrrraov,c.l FoncE 369
Figure l. Dererminarion
of fonce needed
to remove individual hairs
from raw metis ryp€ sheepskin by PS-2104 force
scnsor.
both of the trvo crude enzyme preparations clearly exceeded
the effect of the rvater.
The dehairing effects of different commercial enzymes and
the traditional dehairing chemicals, lime and sodium sulphide
were also compared by the Pasco PS-2104 force sensor (Figure
5). The lime and sodium sulphide solurions completely
loosened the hairs from the skins in less than 2 h, rvhile the
soaking enzyme, Buzyme 148 rvas the only enzymatic
preparation that also loosened the hairs complerely. Neither
the unhairing enzyme, Buzyrne 7705 nor the bating enzyme,
Pellucit 1000 LVU g-' loosened hairs as efficiently as B. cereus
IZ-O6b or B- cereus 12-O6r crude enzyme prepararions (Figure
a). The effect of the bating enzyme trearmenr rvas similar to
the effect of treatment in only rvater (Figure 5)-
Drscgssrox
The Pasco PS-2104 or similar force senstlrs provide a
quantitative alternative to the qualirative methods that have
usually been used to evaluate the effect of enzymes and
chemicals in dehairing processes-23
r2
The force measurements
are rapidly recorded, and the sensor gives reproducible resuhs
and works well at rhe desired range of forces (0-2 N) needed
to remove hairs from sheepskin. Force measurements can
z_
$ o.e
L
0.9
2
E'0."
o
&
0 50 100 150
Iim6 (s)
Figurc 2. Examples of force measuremenrs during removal of hairs
from raw metis rype sheepskin. A. Hairs rcpeatedly removed I I times
from skin soaked for 2 h in water. The maximum of each spike indicate
the force needed to remove between I and l0 hairs from rhe skin. ts.
Flairs repeatedly removed 16 times from the same skin after addiriorral
incubation in soaking enzyme, Buzyme 148 for 48 h at 30"C. Inser shows
force recordings during the first 5 hair removal trials on expanded soale.
0 20 40 60 80 100
Number
of hdrs
Figure 3. Reluionship bctween maximal fonce recorded and number of
hairs removed from meds qrpe sheep;kin afrer incubxion fu 4h at 30"C in
warer. Force estimated fmm besr fit of Eq. 1 16 dara, a = 0.20 N and D = 0.47.
g 1"2
6
'i
o
E o.e
JALCA,VOL 106,201I

370 MusunrNc H.qrn Rruovll Foncn
0.8
o
I
"E
o.o
E
,N
6
E 0.4
o
zo.2
o
u
& 0-6
E
o
N
E
o.+
E
=
010?o304050 Time
(h)
Figure 4. Changes
in force needed to remove individual hairs from raw
metis type sheepskin incubated at 3S'C in B. cereus IZ-06b (E; or 8.
cereus V-O6r (O.; crudc enzyme preparations or warer 11.;. Error bars
indicate standard error of mean
of l0 replicue hair removal uials.
Iime(h)
Figure 5.
Changes
in force
needed to remove individual
hairs from raw
metis rype sheepskin incubated at 30"C in 3% limc and 2% sodium
sufphide
(a),O.| oA
soaking enzyme Buzyme
148
(A),0.2
dl unhairing
enzyme Buryme 7705
(O), I ,/ bating enzymc
Pellucir 1000 LVIJ gl
(tr), or warer (O-;. Error bars indicate standard eror of mean of I0
replirate hair removal rials.
therefore be used to determine the kinetics of enzymatic and
chemical dehairing processes. In contrast to qualitative
evaluation methods, the force measurements will also allorv
direct comparisons betrveen different procedures and
dehairing agents.
0.8
0.2
q
3020
10
JALCA,VOL 106,2011
Hairs removed from skins one by one provide direct recordings
of the force needed to remove individual hairs- Horvever,
catching only single hairs by the pair of tweezers was
impractical and time consuming and hair removal trials rvere
conducted more rapidly when simultaneous removal of up to
l0 hairs rvas accepted. The force used to remove hairs rvas
applied manually. No mechanical device was available for this
purpose, and the manual removal of l-10 hairs each time
seems the fastest rvay io conduct the analysis at this moment.
The porver function (Eq-f) provides a suitable description of
the relationship between recorded force maxima and number
of hairs simultaneously removed (Fig- 3), from rvhere the force
used to remr)ve individual hairs can be deducted. Errors on the
frrrce estimates stemming from the manual hair pulling
procedures are minimized when hairs are pulled out
repeatedly a number of times.
The keratinolytic enzyme preparations produced by both B.
ce.reus IZ-O6b and B. ce.re.us 12-O6r reduced the force needed
to remove hairs from the skins. This demonstrates the
dehairing ability of the proteases secreted by these bacteria.
The force needed to remove hairs decreased most rapidly
during the first 8 hours, although the hair loosening process
continued for more than24 hours of incubation.
The trvo keratinolytic enzyme preparations from B. cereus
IZ-O6b and B- cereus 12-O6r were not as efficient dehairing
agents as the traditional lime and sodium sulphide for the
dehairing process but their efficiency rvere comparable to the
commercial soaking and unhairing enzymes, and more
efficient than only water. Holever, soaking rhe skins in only
water also reduced rJte force needed to remove hairs, almost as
efficiently as bating enzyme preparation, rvhich is also not
used for dehairing of skins- The hair loosening effects of the
soaking and unhairing enzymes, as rvell as the B. cereus
supernatants, must therefore be attributed to sper:ific
proteolytic activities in these preparations and supernatants.
Conclusrorrs
A general method for quantitative measurements of
mechanical force to assay the effect of proteases and other
chemicals in hair removal processes has been developed and
successfully employed. Quantitative measurements of the
dehairing rvill provide an objective rvay to compare different
enzymes, chemicals and procedures, something that is not
possible by the qualitative observations that are normally used
to assay the effect of enzymatic dehairing procedures. Force
sensors can be useful also in fur and double-face production to
control hair-loosening defects. In addition, force sensors can
be used to control hair slip in rarv hide and skin, and be useful
supplement to the experience and empirical knorvledge of the
tanners.

Mr.qsunnqc Rc,rR REIraov-qr Foncr 371
Acxuowr,nncunurs
We thank Ege University Research Project Department
Directorate
for financial support
(Project No: O8
MUH 0OB)-
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JALCA,VOL t06,201t