Separation and Purification Technology 89 (2012) 282–287

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Enzymatic hydrolysis of bovine hide and recovery of collagen hydrolysate

in aqueous two-phase systems
Pitchaivelu Selvakumar a,b,⇑, Tau Chuan Ling c, Anthony D. Covington d, Andrew Lyddiatt a,e
a
Biochemical Recovery Group, Department of Chemical Engineering, School of Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b
FUJIFILM Diosynth Biotechnologies UK Ltd., P.O. Box 2, Belasis Avenue, Billingham, Cleveland TS23 1YN, UK
c
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
d
British School of Leather Technology, University College Northampton, Park Campus, Northampton NN2 6JD, UK
e
Lyddallan Consultancy Ltd., Wood Manor Wood Street, Shotley Bridge, Consett, County Durham DH8 0JE, UK

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

Article history: The present study focused on the recovery of collagen and its hydrolysate from a homogenate obtained
Received 26 October 2011 by enzymatic hydrolysis of bovine hide. The influence of Bat enzyme, protease and pepsin on the hydro-
Received in revised form 19 January 2012 lysis of hide material is investigated. Aqueous two-phase systems (ATPS) having a composition of 14%
Accepted 29 January 2012
polyethylene glycol (PEG) 600 and 15% potassium phosphate was loaded with 20% w/w of collagen
Available online 6 February 2012
homogenate. Such loaded systems yielded a two phase system, a top phase (97% recovery yield of colla-
gen) and a bottom phase (94% recovery yield of pepsin enzyme). The study indicated that it is possible to
Keywords:
selectively obtain collagen and its hydrolysed product in one phase and the enzyme used for the hydro-
Recovery
Collagen
lysis in another phase in ATPS partition.
Aqueous two-phase systems (ATPS) Ó 2012 Elsevier B.V. All rights reserved.
Enzymatic hydrolysis
Bovine hide

1. Introduction
Collagen is the major component that provides structure,
strength and shape for the extracellular connective tissue. About
14 types of collagen have been reported, however, Type I collagen
is the principal component of the tissue [1]. Collagen and its hydro-
lysate (obtained by hydrolysis of collagen) are generally obtained
from an animal and human origin. Nevertheless, bovine origin is
considered as primary source, which finds importance in medicine
[2], drug delivery, in cosmetic and as nutrition. For example, colla-
gen is successfully used in wound dressings, drug delivery, and bone
repair. In addition to this, recent awareness on nanoscience created
a unique advantage for collagen molecule, since collagen is an active
biomolecule having a nanometer size (about 285 nm); hence, it
could be effectively used as a model for the construction of artificial
biomaterial that can provide a similar property of native tissue.
Collagen and its hydrolysates from animal skin and hides are pro-
duced by controlled thermal and enzymatic hydrolysis [3] as shown
in Fig. 1. However, enzymatic hydrolysis is preferred, because it pro-
duces the material in its native state. For examples, treatment with
protease enzyme is generally employed for obtaining soluble colla-
gen (at elevated temperature) whereas pepsin treatment is adopted
for the production of native collagen (at 4 °C) [4–7]. The major prob-
⇑ Corresponding author at: FUJIFILM Diosynth Biotechnologies UK Ltd., P.O. Box 2,
Belasis Avenue, Billingham, Cleveland TS23 1YN, UK.
E-mail address: selvakumar.pitchaivelu@fujifilmdb.com (P. Selvakumar).
lem associated with enzyme treated collagen is the recovery of colla-
gen free from the enzyme, because the enzyme used for the
treatment co-precipitates with the product during the usual recov-
ery procedure, such as salt precipitation. There is no reliable method
available to recover collagen free of enzyme.
Application of aqueous two-phase system (ATPS) partitioning
technique has been widely reported for the recovery of bioprod-
ucts from biological feedstock [8–12]. The ATPS result from the
mixing of two hydrophilic solutes display incompatibility above
critical concentration. The solutes commonly considered are either
two polymers such as poly (ethylene glycol, PEG) and dextran, or
one polymer and one salt such as PEG and potassium phosphate.
Since it offers mild environment (e.g. less interfacial tension be-
tween phases, [13]), it’s widely used for the recovery and separa-
tion of proteins from the biological feedstock, fermentation
broth, etc. ATPS offers a unique possibility of recovery and separa-
tion of two proteins having an identical molecular weight as re-
ported in the case of the separation of BSA (bovine serum
albumin) and haemoglobin from bovine blood, the G3PDH from
bakers’ yeast, etc. [11,12]. The present study was thus focused on
the recovery of collagen and its hydrolysate from a homogenate
obtained by enzymatic hydrolysis of bovine hide. The results indi-
cate that it is possible to selectively obtain collagen and its hydro-
lysed product in one phase and the enzyme used for the hydrolysis
in another phase in ATPS partition. The experiments related to the
enzymatic hydrolysis of collagen and recoveries are discussed here.

1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2012.01.046
 P. Selvakumar et al. / Separation and Purification Technology 89 (2012) 282–287 283

Enzyme treatment
Thermal treatment

Hide material

Temp. < 60 o C

Temp. > 100 o C

Long chain

Collagen
hydhydrolysate

Short chain

Amino acids
Amino acids

Fig. 1. Hydrolysis of hide material by thermal and enzymatic methods. The above figure shows two different possibilities of the hydrolysis of the hide material. Thermal
hydrolysis: here, the temperature adopted for hydrolysis is normally very high, hence most of the collagen present in the hide is converted into amino acids. Enzymatic
hydrolysis: here, the hide material can be processed at a low temperature. By choosing suitable enzymes the material can be processed into native, soluble collagen, peptides
and amino acids.

2. Materials and methods tions. One portion was directly used as a feedstock for ATPS exper-
iment and another portion was dialysed against 0.02 M di-sodium
2.1. Enzymatic treatment of hide powder hydrogen phosphate (pH 8.5) then used for ATPS experiments as
another feedstock.
2.1.1. Hydrolysis using protease-based enzyme
Bovine hide powder, kindly donated by the British School of
2.2. Construction of ATPS using pre-dissolved (liquid) phase forming
Leather Technology (BSLT, Northampton) that is sourced from the
chemicals
British Leather Consortium (BLC, Northampton) was used for most
of the experimental studies (which is relatively free from fat hav-
ATPS were constructed by subsequent addition from 50% and
ing undergone certain conventional leather processing steps) as a
40% (w/w) stock solutions, respectively of poly(ethylene glycol)
model substrate. The hide powder was suspended to replicate
and a mixture of potassium di-hydrogen orthophosphate and di-
the wet hide in deionised water in a conical flask. The pH of the
potassium hydrogen orthophosphate to yield the appropriate
medium was maintained at 8.5 by the addition of Na2CO3. A known
weight percent system at the desired pH. Whole bovine blood
quantity of protease enzyme [Bat enzyme (BASF, Germany) and
was then loaded into the system (ranged from 5% to 20%, % w/w)
Protease (Sigma, sabtilisin based)] was then introduced in different
and the variation in the measured pH of these systems was negli-
experiments at the required concentration. The flasks were kept in
gible. All batch experiments were of a constant 10 g mass in a
a rotating (at 80 rpm) constant water bath/shaker (Grant W8, Ger-
14 ml volume centrifuge tube and the system pH was maintained
many) at the required temperature for up to 180 min. The samples
at 7.5 by the buffering action of a mixture of phosphates (e.g.
were drawn at a regular interval of time and were frozen at –20 °C
18:7; K2HPO4:KH2PO4,% w/w). The centrifuge tubes were mixed
to arrest the activity of the added enzyme and subsequently ana-
for 30 min using a laboratory blood mixer (to achieve effective
lysed for dry cell weight, protein content and hydroxy proline.
mixing of phase forming chemicals and proteins) and then centri-
fuged (Jouan K442) at 1000g for 3 min to accelerate the phase sep-
2.1.2. Enzymatic activity aration. Samples were drawn from the appropriate phases and
The enzymatic activities are expressed as: one unit will hydro- suitably diluted before the estimation of protein content.
lyse Casein to produce colour equivalent to 1.0 lmole (181 lg) of
tyrosine per minute at 37 °C.
2.3. Construction of ATPS using solid phase forming chemicals

2.1.3. Pepsin treatment
The hide powder (2 g) was first suspended in 50 ml of 0.5 M
acetic acid (pH 2.8 at 4 °C) and a known quantity of Pepsin (Sigma)
was added and extracted for 48 h. The medium yielded colloidal
solution containing collagen extracts was divided into two por-
The ATPS were also constructed by adding solid phase forming
chemicals to feedstock. Here, ATPS were constructed by the subse-
quent addition of solid poly(ethylene glycol); potassium di-hydro-
gen orthophosphate, di-potassium hydrogen orthophosphate,
citrated bovine blood and water to yield the appropriate weight
284 P. Selvakumar et al. / Separation and Purification Technology 89 (2012) 282–287
percent system. The other conditions were maintained as given in
Section 2.2. The effect of using solid phase forming chemicals in-
stead of liquid phase forming chemicals had a minimal impact
and did not alter the partition performance.
2.4. SDS–PAGE analysis
Most of the protein concentrations reported here for BSA and
haemoglobin were recorded from gel analysis with duplicate or
triplicate experimentations. The SDS–PAGE analysis of samples
recovered from the phases was conducted according to the discon-
tinuous method [8]. Samples obtained from the phases were suit-
ably diluted 20–40 times (to ensure the band intensity of stained
protein fell within the standard limit of 2 mg/ml to minimise the
error in the quantitation) before denaturation in reducing buffer
(3.25% SDS, 20% sucrose and 5% b-mercaptoethanol in 62.5 mM
Tris–HCl, pH 6.8). Mixtures were boiled at 100 °C for 10 min prior
to being loaded into electrophoretic analysis on 12% T-2.65% C
poly(acrylamide) separating gel and a 4% stacking gel using a Mini
Protean vertical electrophoresis cell (Bio-Rad Labs.). The gel was
stained with 0.1% Coomassie Brilliant Blue R-250, 40% methanol,
10% acetic acid. The gels were subsequently developed in a
destaining solution (comprising 8% v/v acetic acid and 25% metha-
nol in deionised water), and dried using a gel drying kit (Promega).
2.5. Protein assay
The total protein content of the samples from the ATPS was esti-
mated using the Bicinchoninic Acid assay (BCA; Pierce, Rockford, IL,
USA) and the results were expressed relative to a calibration plot
derived from the assay using known concentrations of BSA. The
percentage deviation was within an average error of less than 5%.
2.6. Determination of hydroxyproline
The samples obtained at time intervals were diluted to the re-
quired level and hydrolysed at 120 °C for 20 min as described by
Reddy and Enwemeka [14]. The concentration of hydroxyproline
was determined spectrophotometrically (at 550 nm) against a
standard plot within a working range of 0–20 lg ml 1. The value
of hydroxyproline thus obtained was multiplied by 13 to arrive
at the amount of collagen present in the sample.
3. Results and discussion
3.1. Comparison of enzymatic hydrolysis of hide powder
3.1.1. Bat enzyme treatment of hide material
The effects of temperature and pH are important factors for en-
zyme hydrolysis, since the enzymatic activity is greatly influenced
by the rate of reaction. Here, the hydrolysis of hide powder was
studied at varying concentrations (1–10%, w/w of wet weight of
the hide powder) of bat enzyme at 55 °C (pH 8.5). Bat enzyme is
basically a protease-based product, normally used in the batting
of hide during leather manufacturing processes to remove the un-
even surface of the hide material. The optimal working tempera-
ture and pH for the bat enzyme are 55 °C and 8.5, respectively
(BASF, Germany). Fig. 2 depicts the time course of hydrolysis and
release of soluble collagen in the medium. The results show that
the release of soluble collagen is in proportion to the quantity of
bat enzyme used for the process. Guerard et al. [4] investigated
the hydrolysis of tuna waste using a commercial available neutral
protease. The authors revealed that hydrolysis degree increased
with increasing of enzyme/substrate ratio and reaction time. The
soluble collagen release increased from 20 to 121 mg within
160
Collage n, m g(hydr oxy pr o bas is )
140
120
100
80
60
Bat 1%
40 Bat 2%
Bat 5%
20
Bat 10%
0
0 20 40 60 80 100
Time, min
Fig. 2. Influence of Bat enzyme on the hydrolysis of hide material. The hydrolysis of
hide material was carried out as described in Section 2.1 using Bat enzyme at a
concentration ranging from 1% to 10% w/w (wet weight of hide powder). Samples
were drawn at different time intervals and analysed for the presence of hydroxy-
proline using the method described in Section 2.5. This value was multiplied by 13
to attain collagen concentration in the medium.
15 min of hydrolysis with 10% w/w bat enzyme, and afterwards,
the value reached a plateau (Fig. 2). The hide materials rate of
hydrolysis was substantially lower at an enzyme concentration
<10% w/w. However, the quantity of soluble collagen recovered
was much lower than quantity of collagen present in the hide
materials. This was evidenced by the insoluble hide material seen
in the medium, indicating that a >10% w/w of bat enzyme is
needed to enhance a complete hydrolysis of the hide material.
Moreover, the quantities of different additives present in the bat
enzyme have shown that use of bat enzyme is not a viable eco-
nomic option, neither for the purity of the material produced nor
because of the presence of high concentrations of additives. Hence,
alternate enzymatic hydrolysis options have been investigated.
3.1.2. Pure protease treatment of hide material
In order to improve the efficiency of hydrolysis, a pure protease
enzyme (substilisin based) was used for further experimentation.
The effect of temperature is an important factor for enzyme hydro-
lysis, since the enzymatic activity is greatly influenced by the rate
of reaction. Hence, the experiments were carried out at tempera-
tures ranging from 40 to 55 °C. The enzyme activity introduced
in the medium was maintained at a concentration of 2.3 IU/mg of
dry hide. Fig. 3 shows the variation in the soluble collagen released
estimated on the basis of hydroxyproline. The results indicate that
the temperature has a marked influence in the hydrolysis of the
hide material. At 40 °C, the quantity of soluble protein released
in the medium was only about 12% than the quantity hydrolysed
at 55 °C. Within 5 min of hydrolysis, the experiment conducted
with 55 °C showed at dramatic change in hydrolysis than the
experiment conducted at <55 °C. Yung and Deng [5] studied the ef-
fect of temperature on enzyme digestion of bird feet collagen. The
study indicated that collagen yield increased with increasing of
temperature (4–18 °C). In any biological process such as fermenta-
tion, enzyme hydrolysis undergoes a different phases during the
time course of the reaction including a lag phase (here, the sub-
strate and biologically active component undergoes an adoption
process to adjust to the local environment), an exponential phase
(a rapid conversion process of substrate into product) and a sta-
tionary phase (declining process of product formation due to lack
 P. Selvakumar et al. / Separation and Purification Technology 89 (2012) 282–287 285

900 900
Soluble collage n, m g (dry w t. bas is )

800 800

Collagen, m g (hydroxy pro * 13)

700 700

600 600

500
500
400
400 .27 IU/mg
300 .53 IU/mg
300 1.12 IU/mg
200
2.3 IU/mg
200
100 4.6 IU/mg
11.7 IU/mg
100
0 23.4 IU/mg
0 15 30 45 60 75
0
Tim e, m in 0 20 40 60 80 100
Tim e, m in
40 Deg C 45 Deg C 50 Deg C 55 Deg C
Fig. 4. Influence of protease on the hydrolysis of hide material. The hydrolysis of
Fig. 3. Influence of temperature on the hydrolysis of hide material. The hydrolysis the hide material was carried out using protease (Sigma) at an enzyme activity
of hide material was carried out as described in Section 3.2.1 using protease (Sigma) ranging from 0.27 to 23.4 IU. Samples were drawn at different time intervals and
at an enzyme activity of 2.3 IU. Experiments were carried out at temperatures analysed for the presence of hydroxyproline using the method described in Section
varying from 40 to 55 °C. Samples were drawn at different time intervals and 2.6. This value was multiplied by 13 to attain the collagen concentration in the
analysed for the presence of hydroxyproline using the method described in Section medium.
2.6. The values were multiplied by 13 in order to attain the collagen concentration
in the homogenate.
Lane 1 2 3 4 5 6 7

of substrate available). In the present case, at 55 °C, the process Phosphorylase b 94.0
must have reached an exponential phase quickly because right Albumin (Bovine) 67.0
temperature needed for the hydrolysis process was available. The Ovalbumin 43.0
result showed, at this temperature, about 70% of the hide was
Carbonic anhydrase 30.0
hydrolysed into soluble collagen in 15 min reaching about 85%
conversion within an hour. Hence, further experiments were car-
ried out at 55 °C. Tripsin 20.1

An appropriate enzyme concentration is essential for any enzy-

matic process. The influence of enzyme concentration (based on
initial activity) was studied ranging from 0.27 to 23.4 IU mg 1 of
dry hide material whilst other conditions were maintained as de-
scribed in the previous section. Fig. 4 depicts the variation in the
soluble collagen released during the hydrolysis of hide material
on the basis of hydroxyproline estimation. The results show that
the initial enzyme activity has a remarkable influence on the
hydrolysis of the hide material. At a lower activity of enzyme
(0.27 and 0.53 IU mg 1), the conversion showed a lag phase (dis-
cussed in the previous section) until after 5 min when the hydroly-
sis then progressed to the exponential phase, Herein, the process
has not seen to approach a stationary phase. At higher activities
of enzyme (0.53 IU), the hydrolysis was quite rapid. This indicates
that the process requires more enzymes for complete conversion.
An initial activity of 2.3 IU mg 1 was found to be optimal for effi-
cient conversion within 60 min. Any reduction in the enzyme em-
ployed promotes a longer duration of the hydrolysis process. The
result obtained in this study is in agreement with the Michaelis–
Menten enzyme reaction kinetics model [15].
Fig. 5 depicts 12% SDS–PAGE the analysis of samples obtained at
15 min of the hydrolysis process. This indicates that at higher con-
centrations of enzyme (24.3 IU mg 1; lane 2 and 11.7 IU mg 1;
lane 3), the hide material is hydrolysed into a lower molecular
weight peptide (about 25,000 Dalton) immediately. But, at inter-
mittent concentrations of enzyme (4.6 IU mg 1; lane 4 and
2.3 IU mg 1; lane 5), the hide material is hydrolysed into a lower
molecular weight peptide (about 25,000 Dalton) and collagen sub-
units [a doublet around 100,000 Dalton corresponds to a1(I) and
a2(I)]. This, however, was not seen at lower concentrations of
α-Lactalbumin 14.4
Fig. 5. SDS–PAGE analyses of samples from enzyme hydrolysis of hide. The
hydrolysis of the hide material was carried out as described in Fig. 5. Lane 1: low
molecular weight (LMW) marker. Lanes 2–7: Homogenate samples obtained ranged
from 0.27 to 23.4 IU.
enzyme, wherein the molecular distribution of peptides varied (a
ranges of bands from 25,000 to 75,000 Dalton, lanes 6 and 7) indi-
cating that the mode of enzyme action differs in different condi-
tions. Similar observations are being made in the case of
hydrolysis of chrome tanned leather shaving by Taylor et al. [16].
3.1.3. Pepsin treatment of hide material
The hide material was treated with pepsin basically to produce
native collagen. In addition to this the telopeptide of type I collagen
is the major component causing immunogenic responses in the
hosts, which hinders the usage of collagen for biomedical applica-
tions. The hide powder suspended in 0.5 M acetic acid was treated
with pepsin ranging from 25 to 100 mg for 10 g of dry hide mate-
rial. The content was digested at 4 °C with continuous stirring for
48 h. Samples were drawn at regular intervals of time and the den-
sity of the homogenate was measured in relation to initial suspen-
sion. The variation in density indicates the level of solubilisation in
the hide material. Fig. 6 shows the variation in the relative density
for pepsin treated homogenate (with different proportion of pepsin
and hide). The results indicate that the collagen solubility in-
creased with increasing of time [17] and the complete solubilisa-
286 P. Selvakumar et al. / Separation and Purification Technology 89 (2012) 282–287

tion of hide material reached a plateau value within the 48 h of 10

digestion in all the cases. However, the time of reaching the pla-
9
teau value mainly depended upon the concentration of pepsin.
For example, the hide treated with 25 mg of pepsin required about 8
42 h whilst the hide treated with 100 mg of pepsin only required
7
24 h to reach the plateau value. Bajza and Vrcek [15] investigated

Phase volume, in ml
the enzymatic treatment of collagen proteins from leather waste 6
and the study reported that the solubility rate of collagen proteins
increased with increasing of enzyme concentration. 5

4
3.2. Aqueous two-phase partition for the recovery of collagen and its
3 Top phase
hydrolysate
2 Bottom phase
The scouting studies conducted for the recovery of collagen and
1
its hydrolysate from the homogenate indicated that an ATPS con-
sisting of 12.5% PEG average molecular weight 1000 and 13.5% 0
potassium phosphate was the most suitable system. blank loaded

Fig. 7. Distribution of collagen in ATPS. ATPS having a composition of 14% PEG 600
3.2.1. Partition behaviour of homogenate obtained from enzymatic and 15% potassium phosphates was constructed and loaded with 20% w/w of
hydrolysis of hide using protease collagen homogenate. Such loaded systems yielded a two phase system, a top phase
and a bottom phase.
Fig. 7 depicts the visual appearance of the system loaded prote-
ase hydrolysed homogenate. The homogenate contained soluble
collagen and insoluble hide material, the enzyme used for the
100
treatment. The soluble collagen was partitioned to the top phase
whilst the enzyme was partitioned to bottom phase. Fig. 8 shows 90
the recovery of collagen and enzyme in the top and bottom phases
for 20% loaded system. This was evidenced by the soluble protein 80 Collagen
assay and protease assay carried out. The potassium phosphate 70 Enzyme
present in the ATPS actually precipitated the soluble collagen pres-
ent in the homogenate. Due to the salting out effect of phosphate it
% Recovery

60
was excluded from the bottom phase and partitioned to the top
50
phase.
However, when the ATPS was centrifuged at 1000g for 3 min, 40
most of the collagen was seen at the interface of the system. Collagen
is a nanometer-sized protein and its partition behaviour is similar to 30
that of many nanoparticles. It has been reported that, nanoparticles,
20
in general, favour interfacial partitioning [18]. In the present case,
when the system was left overnight under gravity, soluble collagen 10
was seen between the top and bottom phases. This indicates that
soluble collagen was in fact, distributed within the top phase as 0
Top phase Bottom phase

Fig. 8. Recovery of collagen and enzyme from homogenate. ATPS having a

120
composition of 14% PEG 600 and 15% potassium phosphates was constructed and
loaded with 20% w/w of collagen homogenate. Such loaded systems yielded a two
phase system, a top phase and a bottom phase.
100
% Viscosity variation

80 a separate entity. Prolonged gravity settling or centrifugation eased

to promote it to separate at the interface. This has an advantage, from
a process operational view point, because the study indicates that
60
soluble collagen can be separated as enriched form at the interphase
whilst other components are partitioned to the bottom phase.
40 The collagen obtained is relatively free from enzymes and other
insoluble hide material, so it could easily harvested and processed
25 mg for further application.
20 50 mg In order to increase the throughput of ATPS, the influence of
100 mg homogenate loading was addressed. This indicated that collagen
always partitioned at the interface, irrespective of the homogenate
0
introduced into the system. Fig. 9 shows the variation in the parti-
0 10 20 30 40 50 60
tion co-efficient of the enzyme at different homogenate loading
Time, in hours ranging from 10 to 70% w/w. Both the partition co-efficient of the
collagen and the enzyme was found to be uniform throughout
Fig. 6. Variation in viscosity during pepsin treatment of hide material. The pepsin
solubilisation of hide material was carried out at a concentration ranging from 25 to
the loading, indicating that the system is robust in handling the
100 mg (per 10 g of hide powder). Samples were drawn at different time intervals homogenate. This is similar to the observation reported in the case
and the viscosity was measured in relation to the initial value. of intracellular protein recovery from bakers’ yeast [19].
 P. Selvakumar et al. / Separation and Purification Technology 89 (2012) 282–287
4 4. Conclusion
3.5
3 hydrolysate from a homogenate obtained by the enzymatic hydro-
partition co-effecient, ln K
2.5
2 Kcollagen
1.5 Kenzyme
1
0.5
0 zyme). The study indicated that it is possible to selectively obtain
0 20 40 60
-0.5
-1
-1.5
% Homogenate loading, (w/w)
Fig. 9. Partition behaviour of collagen and enzyme from homogenate. ATPS having
a composition of 14% PEG 600 and 15% potassium phosphates was constructed and
loaded with 10–70% w/w of collagen homogenate. Such loaded systems yielded a
two phase system, a top phase and a bottom phase.
3.2.2. Partition behaviour of homogenate obtained by pepsin treated
hide material
The homogenate obtained at 24 h and treated with 100 mg of
pepsin was used as feedstock for the ATPS experiments (Section
3.1.3). The pepsin treated homogenate normally has a pH of 2.8.
Hence, for ATPS partition studies the homogenate is actually di-
vided into two parts. One part was dialysed against 0.02 M phos-
phate buffer to neutralise the pH to 7.5, before introducing into
ATPS and the other part was used directly. The dialysed homoge-
nate behaved similarly to the soluble collagen reported for collagen
hydrolysate. This indicates that both collagen and collagen hydroly-
sate partition in the same manner. Although, the collagen and its
hydrolysate have different molecular weights, the surface proper-
ties of both are the same- hence it behaved like a protein. The
partition behaviour of raw pepsin treated homogenate was investi-
gated in order to see whether the homogenate pH influenced the
system performance. The advantage of ATPS is that it actually acts
as a buffered system. Because, the concentration of phosphate used
for the construction ATPS is quite high, it often buffers any feed-
stock’s pH variation. This was seen in the case of the bakers’ yeast
loaded system, where, the feedstock’s pH did not alter the partition
performance of the individual components and the pH of the sys-
tem. However, in the present case, the pH of the feedstock was
about 2.8, so it may have some influence on the partition perfor-
mance of the system. The system was constructed with raw homog-
enate (undialysed) and the variation in the pH was measured in the
system. The variation was observed to be minimal and the partition
behaviour was found to be similar to that of other ATPS at lower
homogenate loadings (up to 20% w/w). The system pH gradually
decreases if the system is constructed with homogenate with an in-
creased loading, especially from 40% to 75% w/w. This, however, has
not changed the interfacial partition of collagen.
The main aim of the present study was to investigate the feasi-
bility of the recovery of collagen and its hydrolysate from a homog-
enate obtained by the enzymatic hydrolysis of bovine hide using
ATPS where the recovery performance was determined by measur-
ing the recovery yield of collagen and enzyme. However, the result
reported here requires further study (e.g. purification factor and
purity of collagen obtained in the ATPS) in order to document
the entire details of separation performance.
287
The present study focused on the recovery of collagen and its
lysis of bovine hide. The influence of Bat enzyme, protease and
pepsin on the hydrolysis of hide material were investigated. Aque-
ous two-phase systems (ATPS) having a composition of 14% poly-
ethylene glycol (PEG) 600 and 15% potassium phosphate were
loaded with 20% w/w of collagen homogenate. Such loaded sys-
tems yielded two phase system, a top phase (97% recovery yield
of collagen) and a bottom phase (94% recovery yield of pepsin en-
80 collagen and its hydrolysed product in one phase and the enzyme
used for the hydrolysis in another phase in ATPS partition.
References
[1] J.P.R.O. Orgel, A. Miller, T.C. Irving, R.F. Fischetti, A.P. Hammersley, T.J. Wess,
The in situ supermolecular structure of type I collagen, Structure 9 (2001)
1061–1069.
[2] R.F. Oliver, H. Barker, A. Cooke, R.A. Grant, Dermal collagen implants,
Biomaterials 3 (1982) 38–40.
[3] L.F. Cabeza, M.M. Taylor, G.L. Dimaio, E.M. Brown, W.N. Marmer, R. Carrió, P.J.
Celma, J. Cot, Processing of leather waste: pilot scale studies on chrome
shavings. Isolation of potentially valuable protein products and chromium,
Waste Manag. 18 (1998) 211–218.
[4] F. Guerard, L. Guimas, A. Binet, Production of tuna waste hydrolysates by a
commercial neutral protease preparation, J. Mol. Catal. B: Enzym. 19–20 (2002)
489–498.
[5] K.L. Yung, C.L. Deng, Effects of pepsin digestion at different temperatures and
times on properties of telopeptide-poor collagen from bird feet, Food Chem. 94
(2006) 621–625.
[6] N. Bhaskar, V.K. Modi, K. Govindaraju, C. Radha, R.G. Lalitha, Utilization of meat
industry by products: protein hydrolysate from sheep visceral mass, Bioresour.
Technol. 98 (2007) 388–394.
[7] S. Nalinanon, S. Benjakul, W. Visessanguan, H. Kishimura, Use of pepsin for
collagen extraction from the skin of bigeye sanpper (Priacanthus tayenus), Food
Chem. 104 (2007) 593–601.
[8] C.W. Ooi, S.L. Hii, S.M.M. Kamal, A. Ariff, T.C. Ling, Extractive fermentation
using aqueous two-phase systems for integrated production and purification
of extracellular lipase derived from Burkholderia pseudomallei, Process
Biochem. 46 (2011) 68–73.
[9] C.W. Ooi, B.T. Tey, S.L. Hii, S.M.M. Kamal, J.C.W. Lan, A. Ariff, T.C. Ling,
Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-
based aqueous two-phase systems, Process Biochem. 44 (2009) 1083–1087.
[10] F. Luechau, T.C. Ling, A. Lyddiatt, Two-step process for initial capture of
plasmid DNA and partial removal of RNA using aqueous two-phase systems,
Process Biochem. 45 (2010) 1432–1436.
[11] P. Selvakumar, T.C. Ling, S. Walker, A. Lyddiatt, Redefinition of working
aqueous two-phase systems: A generic description for prediction of the
effective phase chemical composition for process control and biorecovery, J.
Chromatogr. B: Analy. Technol. Biomed. Life Sci. 878 (2010) 1784–1790.
[12] P. Selvakumar, T.C. Ling, S. Walker, A. Lyddiatt, A practical implementation and
exploitation of ATPS for intensive processing of biological feedstock: a novel
approach for heavily biological feedstock loaded ATPS, Sep. Purif. Technol.
(2010).
[13] P.A. Albertsson, A. Cajarville, D.E. Brooks, F. Tjerneld, Partition of proteins in
aqueous polymer two-phase systems and the effect of molecular weight of the
polymer, BBA – Gen. Subj. 926 (1987) 87–93.
[14] K. Reddy, C.S. Enwemeka, A simplified method for the analysis of
hydroxyproline in biological tissues, Clin. Biochem. 29 (1996) 225–229.
[15] Z. Bajza, V. Vrcek, Thermal and enzymatic recovering of proteins from
untanned leather waste, Waste Manag. 21 (2001) 79–84;
K. Kohler, L. Von Bonsdorff-Lindeberg, S.O. Enfors, Influence of disrupted
biomass on the partitioning of b-galactosidase fused protein A, Enzym.
Microbial. Technol. 11 (1989) 730–735.
[16] M.M. Taylor, E.J. Diefendorf, W.N. Marmer, E.M. Brown, Effect of deionization
on physical properties of gelable protein products recovered from solid
tannery waste, J. Amer. Leather Chem. Assoc. 365 (1995) 90–97.
[17] E. Skierka, M. Sadowska, The influence of different acids and pepsin on the
extractability of collagen from the skin of Baltic cod (Gadus morhua), Food
Chem. 105 (2007) 1302–1306.
[18] P. Jauregi, M.A. Hoeben, R.G.J.M. Van Der Lans, G. Kwant, L.A.M. Van Der
Wielen, Recovery of small bioparticles by interfacial partitioning, Biotechnol.
Bioeng. 78 (2002) 355–364.
[19] J. Huddleston, A. Veide, K. Kohler, J. Flanagan, S.O. Enfors, A. Lyddiatt, The
molecular basis of partitioning in aqueous two-phase systems, Trends
Biotechnol. 9 (1991) 381–388.