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Eco-friendly enzymatic dehairing of skins and hides by C. brefeldianus

protease

Article in Clean Technologies and Environmental Policy · February 2014

DOI: 10.1007/s10098-014-0791-y

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Clean Techn Environ Policy (2015) 17:393–405
DOI 10.1007/s10098-014-0791-y

ORIGINAL PAPER

Eco-friendly enzymatic dehairing of skins and hides

by C. brefeldianus protease
Harish B. Khandelwal • Snehal V. More •

K. M. Kalal • R. Seeta Laxman

Received: 22 July 2013 / Accepted: 14 May 2014 / Published online: 6 July 2014
Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Alkaline protease from Conidiobolus brefeldi- Introduction

anus was efficient in unhairing various types of skins and
hides. The crude protease preparation was active toward Global trade in leather and leather goods has grown several
keratin-azure, elastin-orcin, azocasein, and azocoll, but did folds during past few decades and constructively affected
not show true collagenase activity. In addition, the crude the socio-economic status of several developing countries
enzyme exhibited other enzyme activities such as chon- like India. Leather sector of India also established itself as
droitinase, laminarase, and chitinase. Complete hair a large employment generator during its growth. It is
removal of skin/hide by the protease achieved in 16–18 h. providing jobs to about 2.5 million people and most of
The dehaired pelt showed smooth and white appearance them are from weaker sections of society. The leather
due to hair removal along with epidermal layer. In addition, industry also holds a prominent position in the Indian
the grain was clean and without damage in enzymatically economy ([Link]
dehaired pelts. The microscopic observation of the cross- [Link]). Leather making involves series of oper-
section of dehaired goat skin and cow hide showed absence ations collectively termed as ‘‘tanning’’ and dehairing is the
of epidermis, hair shaft with empty follicles. Enzymatic major step of tanning operation wherein the hair, epider-
dehairing resulted in complete and uniform fiber opening in mis, noncollagenous proteins, and other cementing mate-
the dermis and corium region. Physical properties viz. rials are removed from the skin (Sivasubramanian et al.
tensile strength, elongation, and tear strength of dyed crust 2008a). Generally, conventional method of dehairing
of enzymatically and conventionally dehaired pelts were involves applying lime and sodium sulfide mixture paste on
comparable. Results were also validated on large scale with the flesh side of skin/hide. Hair removal by this method
goat skins and cow hides. involves chemical reaction of lime and sulfide on hair root,
which is mainly composed of cysteine rich fibrous keratin,
Keywords Conidiobolus brefeldianus
Alkaline a protein which is prone to alkaline hydrolysis (Choudhary
protease
Enzymatic dehairing
Sheep skins
Cow hides
et al. 2004). Conventional dehairing is considered to be one
Dehaired pelts of the most polluting process and has severe impact on
water sources and soil. Apart from this, there are number of
negative factors associated with conventional dehairing
process viz. damage of skin due to overexposure to sulfide,
H. B. Khandelwal
S. V. More
R. S. Laxman (&)
Division of Biochemical Sciences, National Chemical difficulty in precise process control, loss of hair and wool
Laboratory, Pune 411 008, India due to sulfide damage, and costly effluent treatment. The
e-mail: rseetalaxman@[Link] most important among the various factors that are likely to
S. V. More affect the growth of the leather industry is environmental-
e-mail: [Link]@[Link] related concerns. Therefore, the challenge today is to find
environmental-friendly alternatives to chemical processing
K. M. Kalal
Division of Organic Chemistry, National Chemical Laboratory, of hides and leather. Enzymes as alternatives to some of the
Pune 411 008, India chemical-based processing are gaining attention in recent

123
394 H. B. Khandelwal et al.

years due to stringent pollution norms. Currently many precipitation (90 % saturation) and used for dehairing
commercial enzyme preparations as well as most of the studies.
reported proteases used for dehairing require presence of
small amount of sulfide with or without lime and termed as Protease assay
enzyme-assisted dehairing (Sivasubramanian et al. 2008b;
Thanikaivelan et al. 2005; Kandasamy et al. 2012). How- Protease as caseinolytic activity was estimated at 50 °C,
ever, few reports have appeared recently on lime and sul- pH 9 as described earlier (Laxman et al. 2005). Amount of
fide-free dehairing with alkaline proteases (Rajkumar et al. tyrosine released is calculated from a pre-calibrated graph
2011; Sundararajan et al. 2011; Verma et al. 2011). of absorbance at 280 nm against tyrosine concentration.
Majority of the proteases studied for dehairing are of 1 U of protease activity is defined as the amount of enzyme
bacterial origin and are produced by Bacillus strains, while required to produce 1 lmol of tyrosine/min.
reports on fungal proteases used for dehairing are limited
(Madhavi et al. 2011; Archana and Pillai 2012). Determination of various activities in crude enzyme
Enzymatic dehairing is carried out with crude enzyme
preparations. If the crude protease preparation contains Azocaseinase
collagenase activity, then it attacks the collagen of the
grain layer leading to damage of grain structure and its Protease activity was estimated with azocasein as substrate
destruction, which has a major impact on the final quality at 50 °C, pH 9 by the method described by Singh et al.
of leather (Choudhary et al. 2004; Macedo et al. 2005). (1999). 1 U of azocaseinase activity is defined as the
Therefore, it is important that the crude protease prepara- amount of enzyme required to increase the absorbance by
tion is devoid of collagenase activity. In contrast, presence 1 U at 420 nm in 1 min.
of other associated enzymes like chondroitinase, elastase
keratinase, etc., may have added advantage for dehairing. Elastase
Present paper describes lime and sulfide-free enzymatic
dehairing of goat/sheep skins and cow/buffalo hides. To estimate elastase activity, an aliquot of suitably diluted
Enzymatic dehairing of goat skins was optimized with enzyme was added to and 20-mg elastin-orcin in 0.1 M
respect to enzyme concentration and time. Dehairing trials sodium carbonate buffer pH 9 in a total volume of 3 ml.
were validated on large scale for its commercialization. Heat-inactivated enzyme (by boiling for 15 min) was taken
as blank. After incubation at 50 °C for 30 min, the reaction
was terminated by addition of 2-ml 0.7 M phosphate buffer
Materials and methods pH 6. Contents were centrifuged and absorbance of the
supernatant was measured at 578 nm. 1 U of enzyme
Materials activity is defined as the amount of enzyme required to
cause an increase in absorbance by 1 U at 578 nm in
Various substrates viz. elastin-orcin, azocasein, azocoll, 1 min.
laminarin, chondroitin-A, and chitin were procured from
M/s. Sigma Chemicals, USA. Casein was procured from Keratinase
M/s. Sisco Research Laboratory, India. Wet-salted skins
(goat and sheep) and hides (cow and buffalo) were pur- The keratinase activity was estimated at 50 °C, pH 8 using
chased from local market. Lime, sodium sulfide, ammo- keratin-azure as substrate by the method described by
nium sulfate, salt (NaCl), H2SO4, basic chromium sulfate Bressollier et al. (1999). 1 U is defined as the amount of
(Cr(OH)SO4), sodium formate, sodium bicarbonate, dyes, enzyme required to increase the absorbance at 595 nm by
fat liquors, etc., used for conventional dehairing and tan- 0.01, under assay conditions.
ning operations were of commercial grade. All other
chemicals used were of analytical grade. Collagenase

Microorganism and protease production True collagenase activity was estimated at 28 °C, pH 8 using
synthetic collagenase substrate. Change in fluorescence of
Crude protease from newly isolated Conidiobolus bre- collagenase substrate was measured (Laxman et al. 2011a).
feldianus MTCC 5185 was used in dehairing trials. Prote- Azocollagenase activity was estimated with azocoll as sub-
ase production was carried out as described earlier and cell- strate at 37 °C, pH 8 (Laxman et al. 2011a). 1 U of enzyme
free broth was used as source of enzyme (Laxman et al. activity was defined as the amount of enzyme required to
2011a). The broth was concentrated by ammonium sulfate cause an increase in absorbance by 1 U at 580 nm/min.

123
Eco-friendly enzymatic dehairing of skins and hides 395

Chondroitinase Histological analysis

Chondroitinase assay was performed as per manufacture’s A small piece (2 9 2 cm) of dehaired skin and hide was
protocol at 37 °C, pH 8 (Sigma Chemicals, USA). 1 U cut and washed with water and fixed in 10 % formaldehyde
chondroitinase activity is defined as the amount of enzyme solution. Sample pieces were dehydrated by series of eth-
required to liberate 1 lmol of 2-acetamide-2-deoxy-3-O- anol treatment and embedded in paraffin blocks. Sections
(b-D-gluc-4-ene-pyranosyluronic acid)-6-O-sulfo-D-galact- of three micron thickness of the embedded samples were
ose from chondroitin sulfate A/min under assay conditions. cut using ASCO make microtome and stained with hema-
toxylin and eosin to examine the histological features.
These sections were examined under light microscope and
Chitinase
photomicrographs were taken.
Chitinase activity was estimated at 50 °C, pH 7 as
SEM studies
described by Kulkarni et al. (2008). 1 U is defined as the
amount of enzyme required to liberate 1 lmol of N-acetyl
Morphological characterization of dehaired pelts and dyed
glucosamine/min under assay conditions.
crust leather of goat skin was performed by means of
scanning electron microscope (SEM). The surface of de-
Laminarase haired pelts and cross-sections of dyed crust leather were
scanned on SEM Model Sterioscan-440 Model from LE-
The reaction mixture contained 0.5-ml 1 % laminarin, pre- ICA, Cambridge, UK.
pared in 50 mM phosphate buffer pH 7, and 0.5 ml of suit-
ably diluted enzyme. The reaction mixture was incubated at
50 °C for 30 min. The reducing sugar liberated was mea- Results and discussion
sured using di-nitro salicylic acid method (Bernfeld 1955).
Laminarase activity was expressed as lmol of reducing sugar Various enzyme activities in crude protease preparation
liberated/min under assay conditions.
Crude protease preparation exhibited various enzyme
activities other than caseinolytic activity (Table 1). The
Soaking of skins/hides
enzyme preparation has azocaseinase, elastase, keratinase,
azocollagenase, chondroitinase, chitinase, laminarase
Prior to dehairing, skins/hides were soaked with three
activities, but was devoid of true collagenase activity. This
changes of water containing wetting agent and preservative
is very important for its application in leather processing
(300 % v/w of salted skin/hide) until they are free from
because collagen forms the chief constituent of leather and
dirt, dung, and blood stains. After soaking, the skins/hides
its destruction has adverse impact on the leather quality.
were piled for about 1 h before application to drain excess
On the other hand, activities like elastase, keratinase,
water. The soaked weight of the skins/hides was noted. The
chondroitinase, and azocollagenase are desirable in leather
lime and sulfide or enzyme used in dehairing experiments
processing. Foroughi et al. (2006) conducted systematic
was based on the soaked weight of skin/hide.
study of different dehairing preparations from commer-
cially available sources as well as those produced from
Dehairing of skins/hides newly isolated cultures. Most of the efficient dehairing
preparations showed activity toward azocoll and the action
Dehairing was performed by paint method. A paste of of these enzymes did not affect the grain structure. Elastin
lime (10 %) and sulfide (3 %) or protease with 10 % is insoluble protein, which is responsible for maintaining
water was applied uniformly on the flesh side of the elasticity of skin, however in view of leather manufactur-
skin/hide and left at ambient temperature (28–32 °C) for ing, its elimination becomes essential to render adequate
different periods. Hair removal was performed either by softness to the garment leather, and consequently presence
gently scraping the hair with hand or by blunt knife of elastase in dehairing preparation is advantageous.
depending on the skin type. Dehairing was expressed as Although destruction of hard keratin is difficult and unre-
percentage removal where complete hair removal was lated to dehairing, the breakdown of soft prekeratin, which
treated as 100 %. Enzymatic dehairing was performed contains much fewer di-sulfide bonds, is more likely to be
with 0.2–3 % crude concentrated protease. The dehaired beneficial and can be facilitated by keratinase. Archana and
pelts were processed further to dyed crusts by conven- Pillai (2012) summarized characteristics of several dehai-
tional methods. ring proteases from different sources and found that

123
396 H. B. Khandelwal et al.

Table 1 Enzyme activities in crude enzyme preparation Dehairing of goat and sheep skins
Enzyme activity U/ml
The dehairing potential of crude protease from C. bre-
Protease 200 feldianus toward goat and sheep skins was initially inves-
Elastase 6.50 tigated on small pieces (12 9 15–12 9 21 cm) weighing
True collagenase Nil approximately 50–90 g. Two percent protease was applied
Azocollagenase 94.74 on the flesh side and skin pieces were piled. After 16 h,
Keratinase 4,661 hair was removed manually by gentle scraping with scal-
Chondroitinase 0.15 pel. Dehairing by conventional method was also performed
Chitinase 0.11 in similar manner for comparison. Hair could be easily
b-1,3 glucanase 7.30 removed by both conventional and enzymatic method from
Azocaseinase 200 goat as well as sheep skins (Figs. 1, 2). The dehairing
efficacy of the enzyme was assessed in comparison with
that of the lime and sulfide method. In the case of the
majority of them were showed keratinase activity and enzyme dehairing, the hair was removed with the epidermis
lacked collagenase activity. Role of elastase and chondro- whereas in the case of the chemical method, short hairs in
itinase activities in leather processing and their impact on some places and incomplete removal of epidermis were
leather quality is not well understood because their effect is seen. Enzymatically dehaired pelts were cleaner, more
not directly visible. However, Sivasubramanian et al. whitish and had smooth feel due to epidermis removal and
(2008a) observed marked reduction of proteoglycan con- hair obtained was intact (Fig. 3).
stituents during enzymatic dehairing of goat skins which Majority of the proteases used for dehairing of goat
play important role in the opening of fiber bundles. In light skins were from the strains belonging to the genus Bacillus
of this observation, it can be concluded that the enzyme viz. B. subtilis (Sivasubramanian et al. 2008b; Senthilvelan
preparation containing keratinase, elastase, and chondro- et al. 2012; Pillai and Archana 2008), B. megaterium
itinase along with protease may improve dehairing and (Rajkumar et al. 2011), B. licheniformis (Nadeem et al.
fiber opening due to the complementary action of these 2010; Haddar et al. 2011), B. halodurans (Prakash et al.
enzyme activities. 2010), B. cereus (Sundararajan et al. 2011; Saleem et al.
The enzyme used in this study is produced using com- 2012), and Bacillus sp. (Raju et al. 1996; Arunachalam and
mercial grade media chemicals and not analytical grade. Saritha 2009). Some of these studies were conducted as
The media constituents are not cost incurring as media used dehairing assays using very small pieces of skins ranging
contain only glucose (1 %) and yeast extract (0.3 %) with from 2 9 2 to 5 9 5 cm by dipping the skin pieces in
an agricultural byproduct, Soyabean meal, as an inducer. enzyme solutions without lime and sulfide held in alkaline
The enzyme production has been scaled up to 1,000 L range and temperatures ranging from 30 to 40 °C, which
fermentor. Being a fungal sp. downstream processing is are optimum for the enzyme action (Pillai and Archana
much easier. The fermentation time required is also shorter 2008; Nadeem et al. 2010; Haddar et al. 2011; Prakash
(2–3 days), hence energy input is also saved so the whole et al. 2010; Saleem et al. 2012). Haddar et al. (2011)
process is economically feasible. reported dehairing of goat skin pieces with B. licheniformis
protease (7,000 U/ml) and found no hair removal at 25 °C,
Dehairing of skins and hides while incomplete dehairing was seen at 30 °C, and com-
plete hair removal was achieved only at 37 °C after incu-
Normally, hides and skins of domestic live stock mainly cow bating for 24 h under shaking conditions. Excessive
and buffalo, goat and sheep are used in tanning industry. For damage to the skin can occur when dehairing has to be
broad utility of the C. brefeldianus protease and its com- carried out at elevated temperatures (Edmonds 2008).
mercialization, it is necessary that the enzyme is useful in Dehairing of goat skins by keratinase from Streptomyces
dehairing wide range of skins and hides varying in fat con- fradiae in presence of alkali is reported by Robison and
tent, thickness, etc. Therefore, dehairing efficiency of C. Roselle Nickerson (1961). Dehairing of goat skins with S.
brefeldianus protease was evaluated with goat/sheep skins nogalator protease was studied where dehairing capacity of
and cow/buffalo hides. Dehairing was initially carried out for wild and the mutant strains was correlated to the levels of
16 h with fixed enzyme concentrations of 2 and 3 % having protease secreted by them (Mitra and Chakrabartty 2005).
specific activity of 48.30 for skins and hides, respectively. Verma et al. (2011) reported an alkaline protease active at
Since hides are thicker than skins, higher enzyme concen- 80 °C and pH 8 from Thermoactinomyces sp. RM4 which
trations were used. Since hides are thicker than skins, higher showed good in dehairing of goat skin in 24 h with intact
enzyme concentrations were used. hair and clean pelt.

123
Eco-friendly enzymatic dehairing of skins and hides 397

Fig. 1 Dehairing of goat skins: untreated (above), after dehairing (below)

Reports on dehairing with fungal proteases are very few flesh side of skin pieces on small pieces (100–250 g). After
compared to bacterial proteases and mostly confined to the enzyme application, skin pieces were piled and incubated
genera of Aspergillus (Malathi and Chakraborty 1999; for 16 h and dehairing was performed with blunt knife. A
Dayanandan et al. 2003; Madhavi et al. 2011), with few control with conventional lime and sulfide method was also
reports on dehairing with proteases from C. coronatus, carried out for comparison. The protease was effective for
Beauveria sp., Rhizopus oryzae and Penicillium gris- dehairing of both cow and buffalo hides. The dehaired pelts
eofulvum (Pal et al. 1996; Laxman et al. 2007, 2011b; showed smooth and white appearance and were more
Madhavi et al. 2011). cleaner and whiter compared to lime and sulfide-dehaired
Although reports on enzymatic dehairing of goat skins pelts (Figs. 4, 5).
are many, reports on dehairing of sheep skins are very few. Reports on dehairing of cow and buffalo hide are
Pal et al. (1996) reported dehairing of goat and sheep skins limited and mainly restricted to bacterial proteases (Sa-
with R. oryzae protease (5 U/cm2) after soaking and leem et al. 2012; Macedo et al. 2005; Zambare et al.
equilibrating the skins to pH 8 in buffer. Complete hair 2007, 2011). Reports on dehairing of cow hides are more
removal was reported after 11–12 h. compared to that of buffalo hides which are mostly from
India fewer compared to that of cow hides. Dehairing of
calf skins by proteases from S. fradiae in presence of
Dehairing of cow and buffalo hides lime is reported by Robison and Roselle Nickerson
(1961). Anderson (1998) reported dehairing of bovine
The structural features and thickness of skin and hide vary hides for 24 h with a commercial protease in presence of
greatly, and dehairing efficiency of enzyme may vary lime and protein disulfide redox agent. Laxman et al.
accordingly. Therefore, dehairing of hides is considerably (2007) reported dehairing of cow hides using alkaline
difficult than that of skins (Sivasubramanian et al. 2008b). protease from C. coronatus devoid of lime, but in pre-
Dehairing efficiency of the protease toward cow and buf- sence of small amounts of sodium sulfide. Dehairing of
falo hide was investigated by applying 3 % protease on the cow hides with protease from Beauveria sp. in absence

123
398 H. B. Khandelwal et al.

Fig. 2 Dehairing of sheep skins: untreated (above), after dehairing (below)

Physical properties of dyed crust

It is essential that there is no damage to the quality of

leather in terms of strength, elongation, etc., while
complete removal of hair is achieved. Over exposure
should be avoided to get good quality leather. Therefore,
the dehaired pelts were processed further with conven-
tional methods to obtain dyed crust leather to study the
physical properties of the leather. Evaluation of physical
properties of dyed crust leather obtained from goat skin
and cow hide were performed and compared with cor-
responding control samples dehaired by lime and sulfide
Fig. 3 Hair removal from goat skin by protease along with epidermis method (Table 2). The values for tensile strength, elon-
and its enlarged view
gation, and tear strength were slightly higher than that of
control samples which is desirable. This is in agreement
with some of the earlier reports, wherein enzymatic de-
of any added chemicals is also reported recently (Lax- hairing improved tensile properties and elongation of
man et al. 2011b). Rose et al. (2007) reported lime and crust leather (Pal et al. 1996; Dayanandan et al. 2003;
sulfide-free unhairing of skins/hides using animal and/or Sivasubramanian et al. 2008b).
plant enzymes. Saravananbhavan et al. (2005) reported
enzymatic unhairing of cow and buffalo hides for 18 h Histology of dehaired pelts
with commercial bacterial proteases. Four among the five
commercial proteases tested were found promising for Reported literature indicates that diffusion of protease to
dehairing of hide by dip method but resulted in incom- proteolytic sites is important. Once enzyme is pene-
plete removal of epidermis with dark coloration on the trated, broad spectrum protease activity can bring about
pelt (Dettmer et al. 2011). the destruction of various proteins located around the

123
Eco-friendly enzymatic dehairing of skins and hides 399

Fig. 4 Dehairing of cow hides: untreated (above), after dehairing (below)

cells of the epidermis and the basal layer (Edmonds SEM analysis of dehaired pelt and dried crust
2008). Complete removal of epidermal layer from skin
and hide was observed when dehairing was performed SEM studies of grain and cross-section of dehaired pelt and
using C. brefeldianus protease. The optical micrograph dyed crust were carried out. Grain structure was clean, and
of the stained cross-section of goat skin and cow hide empty hair follicles were clearly seen in case of enzymatic
confirms the above visible assessment (Figs. 6, 7). The dehairing, however, in conventionally dehaired pelt, hair
microscopic structure of cross-section of dehaired pelt follicles were not clearly visible due to deposits of lime and
clearly distinguishes the enzymatic dehairing from the sulfide and scuds were seen. Cross section of dyed crust
conventional process of dehairing with respect to the shows more visible fiber separation as compared to con-
extent of removal of epidermis, hair shaft, and follicles. ventional (Figs. 8, 9).
Complete absence of these structures was observed in the
sections from enzymatically dehaired pelt of goat skin Effect of enzyme concentration on dehairing
and cow hide. In case of conventionally dehaired pelts, of goat skin
remnants of epidermal layer and hair shaft were
observed. Besides, enzymatic dehairing leads to com- Since C. brefeldianus protease was effective in dehairing of
plete and uniform collagen fiber opening in the dermis all the animal skins/hides, tested optimization of dehairing
and corium region, while in conventional dehaired pelt, conditions such as effect of enzyme concentration and time
the fiber structure appeared to be compact and unevenly was carried out before large scale trials were conducted
opened. Similar observations on differences between with goat skin as a representative of skin type. Dehairing
conventionally and enzymatically dehaired pelts were was performed on goat skin pieces (300–800 g) with pro-
also reported by other researchers (Sivasubramanian tease concentrations ranging from 0.2 to 2.0 % for 16 h.
et al. 2008b; Saleem et al. 2012; Raju et al. 1996; Dehairing was performed with scalpel as described earlier
Zambare et al. 2007). and expressed as percentage hair removal. The enzyme was

123
400 H. B. Khandelwal et al.

Fig. 5 Dehairing of buffalo

hides: untreated (above), after
dehairing (below)

Table 2 Tensile properties of dyed crust

Skin/hide Dehairing method Tensile strength (kg/cm) Elongation at break (%) Tear strength (kg/cm)
Parallel Perpendicular Parallel Perpendicular Parallel Perpendicular

Goat skin Conventional 233.13 186.85 48.11 71.22 45.10 38.11

Enzymatic 239.45 192.36 49.04 73.26 46.66 38.87
Cow hide Conventional 290.01 242.71 48.12 68.77 47.01 41.00
Enzymatic 302.25 250.36 50.23 72.24 47.65 42.04

effective for dehairing of goat skin even at lower enzyme method when used in presence of 0.1 % lime. Rajkumar
concentrations of 0.2–0.5 % with 65–90 % dehairing and et al. (2011) carried out dehairing of goat skin using B.
increasing the concentration to 1 % resulted in 98 % de- megaterium RRM2 protease and required 12 h of treatment
hairing (Fig. 10). time for visible dehairing activity. Dayanandan et al.
(2003) studied dehairing efficiency of A. tamarii alkaline
Time course of dehairing protease (applied in combination with 10 % kaolin) on goat
skin and complete dehairing was observed after 18 and
Time span required for dehairing was investigated on goat 12 h with 1 and 2 % enzyme, respectively. Decreasing the
skin pieces using 1.5 % protease. Extent of hair removal protease concentration to 0.5 % gave moderate dehairing
was examined after regular time interval. No dehairing was even after 24 h, while addition of 1 % sulfide to 0.5 %
observed in 2 h was initiated only after 6 h and increased protease resulted in complete dehairing 24 h. Dehairing of
with time. Around 70–75 % dehairing was seen after 12 h goat skins by protease from Elizabethkingia meningosep-
and was completed within 16 h (Fig. 11). Pillai and Ar- tica resulted in loosening of hair after 12 h, and complete
chana (2008) reported that keratinolytic protease from B. removal was observed only after 18 h (Nagal et al. 2010).
subtilis required 18 h to dehair goat skin piece by dip Saravananbhavan et al. (2005) reported enzymatic

123
Eco-friendly enzymatic dehairing of skins and hides 401

Fig. 6 Histology of stained sections of goat skins before and after dehairing (9100)

Fig. 7 Histology of stained sections of cow hides before and after dehairing (9100)

unhairing of cow and buffalo hides for 18 h with com- with whole skins. After optimizing the conditions of de-
mercial bacterial protease. hairing on skin pieces, dehairing of goat skins was vali-
dated at large scale using 10 goat skins.
Dehairing of goat skins at large scale Skins were soaked, washed, and cut into two halves. The
right halves of the skins were applied with the enzyme
The extent of hair integrate varies in different areas of skin. preparations and treated as experimental skins. The left
Hair in the backbone and neck region is tougher to remove halves were the controls and were dehaired by following a
due to presence of excessive proteoglycans. Hence, it is conventional lime sulfide method using 3.0 % sodium
necessary to examine the dehairing potential of protease sulfide and 10 % lime. Two enzyme concentrations (1.5

123
402 H. B. Khandelwal et al.

Fig. 8 SEM showing grain surface of dehaired goat pelts

Fig. 9 SEM showing cross-

section of dyed crust of goat
skins

and 2 %) were used for dehairing. Paste of enzyme and Nadeem et al. (2010) reported complete dehairing in 12 h
10 % water was applied on the flesh side and the skins but on further incubation, considerable grain damage to
were piled and covered with gunny cloth and left for goat skin pieces after 15 h dehairing with B. licheniformis
overnight. Next day (after about 16 h), dehairing was protease by dip method. Pal et al. (1996) reported dehairing
performed with blunt knife on wooden beam in similar of half skins of goat and sheep skin by R. oryzae protease at
manner used in leather industry. Hair could be easily pH 8 and 12 h.
removed from the central area of skin, whereas it required
slight force to unhair the peripheral and backbone part. Dehairing of cow sides
While complete dehairing was observed with 2 % protease,
few hair patches were observed with 1.5 % protease Wet-salted cow hides were soaked, washed, and cut into
(Fig. 12). The dehaired pelts obtained by enzymatic two halves. The right halves of the skins were applied
method showed smooth and white appearance due to with the enzyme preparations and treated as experi-
removal of epidermis and there was no grain damage seen. mental skins. The left halves were the controls and

123
Eco-friendly enzymatic dehairing of skins and hides 403

applied on the grain side and the skins were piled and
covered with gunny cloth and left for overnight. Next
day (after about 18 h), dehairing was performed with
blunt knife on wooden beam in similar manner used in
leather industry. Dehairing was increased with increase
in enzyme concentration, and complete dehairing
(100 %) was observed with 2.5 % protease after 18 h
(Table 3). The resultant dehaired pelt showed white
appearance due to removal of epidermis. In this
experiment, enzyme was applied on grain side instead
of flesh side (as applied in the earlier experiment),
Fig. 10 Effect of enzyme concentration on dehairing of goat skins
taking into account that the thickness of hide is much
more than skin and this might facilitate better pene-
tration of enzyme to reach up to hair follicles. These
were dehaired by following a conventional lime sulfide results suggest that the extent of hair and epidermis
method using 3.0 % sodium sulfide and 10 % lime. removal was not affected by the mode of enzyme
Two enzyme concentrations (1.5, 2, and 2.5 %) were application (enzyme application either on the flesh side
used dehairing. Paste of enzyme and 10 % water was or grain side).

Fig. 11 Time course of dehairing for goat skins

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404 H. B. Khandelwal et al.

Fig. 12 Large scale trials for dehairing of goat skins

Table 3 Effect of protease concentration on dehairing of cow hides Acknowledgments Mr. Harish Khandelwal is grateful to the
Council of Scientific and Industrial Research (CSIR) for financial
Weight of hide (kg) Protease (%) Dehairing (%) assistance. Authors acknowledge the financial support from NMITLI
Project funded by CSIR, Government of India.
7 1.5 70
6 2.0 90
9 2.5 100 References

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