The formation of galacto-oligosaccharides (GOS) from lactose by commercially available Biolacta FN5 (β- galactosidase, EC 3.2.1.23) derived from Bacillus circulans was studied under immobilized enzyme condition. The present work utilizes hydrophobic membrane (0.22 m pore size) for immobilization of enzyme. Experiments were conducted in a three compartment cell. The middle compartment (~25 mL) being separated by immobilized membranes was utilized for feed lactose solution; whereas, adjacent compartments were filled with distilled water. The reacted mixture solution was analyzed for tri-, tetra- and penta- forms of GOS which depended on varying amounts of initial lactose (ILC) and enzyme concentrations. Total GOS formation increased from 7 to 28% for ILC from 50 to 200 g/L. However, tri-saccharide was the major (67%) in comparison to tetra (27%) and penta (6%) forms of GOS. There was marginal difference of GOS formations while comparing the result (GOS yield) under both free (~30%) and immobilized (~28%) conditions.

1. Proceedings of the
International Conference on Chemical Engineering 2011
ICChE2011, 29-30 December, Dhaka, Bangladesh
*
Corresponding Author: Prashant K. Bhattacharya,
E-mail: pkbhatta@iitk.ac.in
A study with enzymatic membrane reactor for conversion of lactose in to
galacto-oligosaccharides
Tapas Palai, Pallavi Kumari and Prashant K. Bhattacharya*
Department of Chemical Engineering
Indian Institute of Technology Kanpur
Kanpur-208016, India
The formation of galacto-oligosaccharides (GOS) from lactose by commercially available Biolacta FN5 (β-
galactosidase, EC 3.2.1.23) derived from Bacillus circulans was studied under immobilized enzyme
condition. The present work utilizes hydrophobic membrane (0.22 m pore size) for immobilization of
enzyme. Experiments were conducted in a three compartment cell. The middle compartment (~25 mL) being
separated by immobilized membranes was utilized for feed lactose solution; whereas, adjacent compartments
were filled with distilled water. The reacted mixture solution was analyzed for tri-, tetra- and penta- forms of
GOS which depended on varying amounts of initial lactose (ILC) and enzyme concentrations. Total GOS
formation increased from 7 to 28% for ILC from 50 to 200 g/L. However, tri-saccharide was the major
(67%) in comparison to tetra (27%) and penta (6%) forms of GOS. There was marginal difference of GOS
formations while comparing the result (GOS yield) under both free (~30%) and immobilized (~28%)
conditions.
1. INTRODUCTION
Lactose constitutes over 70% of the total solids in
whey, an abundant byproduct of cheese production.
Considering a 3% increase in cheese production
each year, lactose is a major byproduct of the dairy
industry (Foda and Lopez-Leiva, 2000). Although
there has been extensive research on better ways to
use whey lactose, the dairy industry still is in need
of new technologies for converting lactose into
marketable products. Thus, a process to convert
lactose into a prebiotic food ingredient, which
would be of greater commercial use than lactose,
would find ready application in the food (Albayrak
and Yang, 2002).
Galacto-oligosaccharides (GOS) are complex
mixtures of various sugars, and they are of great
interest as nutraceuticals for food industries
because of their prebiotic properties. The GOS
molecules consist of a galactosyl-galactose chain
with a terminal glucose residue. The general
structure of GOS may be represented as
[galactose]n-glucose, where n is 29 (Czermak et.
al., 2004, Palai, 2010). The GOS can be used in a
variety of products, including fermented milk
products, breads, jams, confectionery, and
beverages (Sako et. al., 1999).
Several studies on GOS synthesis from lactose,
using enzymes from different sources, have been
performed under free enzyme condition. The
enzymes used were well established, owing to their
stronger hydrolytic than transferase activity;
moreover, they result in higher production of
oligosaccharides (Albayrak and Yang, 2002). β-
Galactosidase has been isolated and purified from a
large variety of sources, and the enzyme is well
characterized with regard to its kinetic behavior as
a hydrolase (Mahoney, 1998). The enzymatic
hydrolysis of lactose, catalyzed by β-galactosidase,
mainly yields glucose and galactose, but in the
same biochemical reaction, GOS are also formed
by a trans-galactosylation reaction. In aqueous
systems, trans-galactosylation competes with
hydrolysis. Therefore, GOS mixtures always
contain considerable amounts of unreacted lactose
and monosaccharides (Mahoney, 1998). The
removal of those sugars is essential not only for the
purity of the final product but also for
microbiological studies on the prebiotic effects of
such GOS mixtures.
However, one of the major concerns is the
simultaneous formation of monosaccharides, a
cause for enzyme inhibition, under free enzyme
condition (Boon et. al., 1999). Recently, a concept
is being proposed to immobilize enzyme (Sen et.
al., 2011) and carry out the reaction with
simultaneous removal of mono-saccharides. This
brings the application of enzymatic membrane
reactor. The enzymatic membrane reactor (EMR) is
a flow reactor within which membranes are used
for combined actions of membrane separation and
enzymatic reaction. The membrane aims to

2. separate the cells or enzymes from the feed or
inhibitors of enzymatic reaction product. In the
present work, however, an attempt has been made
to utilize microporous hydrophobic membrane for
the purpose of immobilizing enzymes. The said
membrane is expected to provide high surface area
structure as support. Such reactor when joined with
membrane separator together can function as
immobilized EMR. This work focuses on the
reaction part of the total work on EMR.
EMR offers several other advantages like providing
high surface area of reaction, enzyme reusability, continuous
product formation, increase in reactor stability and
productivity, improvement in product purity and quality
(Albayrak and Yang, 2002), and above all ease of separation
of enzyme after reaction. However, immobilization
also poses certain limitations like membrane
fouling and diffusional problems. Several supports,
such as ion-exchange resins (Mozaffar et. al.,
1986), chitosan beds (Shin and Yang, 1998),
cellulose beads (Kminkova et. al., 1988) and
agarose beds (Berger et. al., 1995) have been used
for the immobilization of enzyme. But, hardly there
attempts to immobilize membranes.
OOBBJJEECCTTIIVVEE
In the present study, main objective is to develop a
new and efficient method for immobilization of
enzyme on hydrophobic polyvinylidene fluoride
(PVDF) membrane for production of galacto-
oligosaccharides (GOS) from a commercially
available enzyme from Bacillus circulans. Further,
the objective was also to investigate the influence
of varying lactose and enzyme concentrations on
GOS formation under immobilized condition in
comparison to free enzyme conditions. In order for
sustainable immobilized reaction, it was also
thought to test the membrane for its reusability.
2. EXPERIMENTAL
2.1 Materials and Chemicals
Commercially available β-galactosidase (Biolacta
FN5), derived from Bacillus circulans, was
provided by Daiwa Kasei K.K., Japan. Lactose GR
(monohydrate), D-galactose (assay~99%) and other
common chemicals were obtained from Loba
Chemie, India and Qualigens Fine Chemicals,
India. Hydrophobic PVDF membrane of pore size
0.22 µm (47 mm diameter) was supplied by
Millipore®
.
2.2 Enzyme Assay
O-nitrophenyl-β-D-galactopyranoside (ONPG) was
used as substrate for activity estimation of β-
Galactosidase (Bahl and Agrawal, 1969). The
reaction was carried out in 0.5 mL of 10 mM
ONPG in citrate buffer (pH 6.0) at 40°C. The
reaction mixture was incubated for exactly 10 min
after the addition of 0.1 mL of β-galactosidase
enzyme solution (0.050.10 U/mL). A blank
sample analysis was similarly performed. After
incubation, 3.0 mL of borate buffer (pH 9.8) was
added for terminating the reaction. The amount of
o-nitrophenol (ONP) released during the reaction
was measured spectrophotometrically (Hitachi,
U2900) by its absorbance at 410 nm. The enzyme
activity was estimated as 1.3 U/mg powder. One
unit (U) of enzyme is defined as the amount of
enzyme required to hydrolyze 1.0 μmol of ONPG
to ONP and D-galactose per minute, at pH 6.0 and
40°C.
2.3 Enzyme Immobilization
The β-galactosidase enzyme was immobilized on
PVDF membrane by cross-linking with
glutaraldehyde (Zhou and Chen, 2001). The two
PVDF membranes were equilibrated in phosphate
buffer solution (pH 6.0) for 1 h. The membranes
were activated for 4 h at 30o
C with 4% v/v aqueous
glutaraldehyde solution to improve covalent
bonding; followed by buffer washing (removal of
excess glutaraldehyde). For completion of the
cross-linking, membranes were immersed in
enzyme solution of desired concentration for 18 h
keeping in an incubator at 20o
C followed by
washing with brine (removal of any excess free
enzyme. Finally, membranes were stored in fresh
buffer solution at 20o
C. The amount of
immobilized enzyme was calculated by difference
of weight.
2.4 HPLC Analysis
The concentrations of lactose, glucose, galactose,
and GOS (tri-, tetra-, penta-saccharides, etc.) were
determined by HPLC. The HPLC system consisted
of: pump (Waters®
515), column (Waters Sugar
Pak I, 300×6.5 mm; packed with calcium-loaded
resin) along with heater, RI detector (Waters®
2414, RID-10A). Empower-2 data processor was
used. The mobile phase consisted of 50 mg EDTA-
calcium disodium/L in deionized water at room
temperature. The mobile phase was filtered through
0.45 μm membrane filter and degassed immediately
before use. The isocratic flow rate of the mobile
phase was maintained at 0.5 mL/min. The
temperatures of the column and detector were
maintained at 75°C and 35°C, respectively.
2.5 Three Compartment Cell
A three compartment test cell was designed and
fabricated as shown in Fig. 1. Middle frames
housed immobilized membranes and filled with
lactose (desired amount), dissolved in phosphate
buffer (50 mM, pH 6.0) to prepare 25 mL solution
at 40o
C. Other two sides were filled with water. At
regular intervals, samples were withdrawn for

3. analysis through HPLC till completion of the
reaction (~ 30 hours). The initial lactose (50-200
g/L) and enzyme concentrations (0.005-0.015
g/mL) were varied. Enzyme was deactivated by
heating the sample in a water bath at 90o
C for 5
min. The samples were stored in the freezer for at
least 1 h to accelerate precipitation of insoluble
material; they were then clarified by centrifugation
at 13,000 rpm for 10 min, and the supernatants
were filtered through a 0.45 μm filter syringe and
diluted as required before HPLC analysis.
Fig. 1: Schematic diagram of reaction cell
3. RESULTS AND DISCUSSION
3.1 Enzyme Loading on PVDF Membrane
Immobilization of enzyme on PVDF membrane
was carried out at three different enzyme
concentrations. A maximum of 51 mg of enzyme
loading (on both the membranes on both sides’)
was noted for 0.01 g/mL enzyme solution (Fig. 2).
Therefore, most of the runs were carried out at 0.01
g/mL enzyme concentration.
Fig. 2: Enzyme loading on PVDF membrane
3.2 GOS Formation Kinetics
The kinetics of lactose conversion under
immobilized enzyme condition was studied at
different initial lactose concentrations (50, 75, 100,
150, and 200 g/L). After the reaction, the individual
saccharide components were analyzed by HPLC. A
typical time-course of lactose hydrolysis in the
batch reaction is shown in Fig. 3. It is clear from
Fig. 3 that after 20 h of reaction, the total GOS
production reached at steady-state condition. The
maximum GOS yield of 28% was observed at 200
g/L ILC, at 40°C, pH 6.0, and 0.01 g/mL enzyme
concentration.
0 5 10 15 20 25 30
0
10
20
30
40
50
60
70
80
90
100
110
120
0 5 10 15 20 25 30
0
5
10
15
20
Tri-saccharide
Tetra-saccharide
Penta-saccharide
Concentration(%)
Time(h)
Lactose
Total GOS
Glucose
Galactose
Concentration(%)
Time (h)
Fig. 3: Time course product composition for lactose
hydrolysis at 200 g/L ILC. GOS formation (inset)
The composition of GOS was mainly tri-
saccharides (~19% of the total reaction mixture),
tetra-saccharides (~7.5%), and a small amount of
penta-saccharides (~1.5%) (Fig. 4a); i.e. tri-
saccharides (67% of total GOS), tetra-saccharides
(27%) and penta-saccharides (6%) (Fig. 4b).
6%
16%
50%
1.5%
7.5% 19%
1.5%
7.5% 19%
6%
Lactose
Tri-
saccharideTetra-saccharide
Penta-saccharide
Glucose
Galactose
a
50%
16%
67% 6%
27%
27%
6%
67%
Tri-saccharide
Tetra-
saccharide
Penta-saccharide
b
Fig. 4: a) Composition of reaction mixture and b)
composition of GOS
3.3 Effect of Initial Lactose Concentration
ILC is an important factor affecting GOS formation
(Albayrak and Yang, 2002). As the ILC increased
from 50 to 200 g/L, the maximum production of
total GOS increased from 7% to 28%, respectively
(Fig. 5). The GOS production was higher at higher
ILCs, because water competes with lactose as an
acceptor for the β-galactosyl group (e.g., hydrolytic
vs. trans-galactosylation reactions). At lower
lactose concentrations, relatively higher amount of
galactose at the active site of the enzyme is
released by reaction with water. At higher lactose
concentrations, lactose can more efficiently act as
the acceptor for the β-galactosyl groups, binding
with the enzyme-galactose complex and forming
GOS (Zhou and Chen, 2001).
With increasing ILC, initially glucose
concentration increases and thereafter attain
plateau. This may be atributed to the fact that
glucose can be formed in both the reactions; trans-
galactosylation and hydrolysis. However, galactose

4. concentration keep-on increases with increasing
ILC.
25 50 75 100 125 150 175 200 225
0
5
10
15
20
25
30
GOS
Glucose
Galactose
Productsconcentration(%)
Initial lactose concentration (g/L)
Fig. 5: Effect of initial lactose concentration on
products formation
3.4 Effect of Enzyme Concentration
Enzyme concentration has lesser effect on GOS
formation than ILC. At higher enzyme
concentration GOS formation quickly reaches a
maximum value
Fig. 6: Effect of enzyme concentration on GOS
formation at 100 g/L ILC
It was observed that equilibrium GOS formation
increases initially and then remains constant with
increase in enzyme concentration (Fig. 6).
3.5 Study of Enzyme Reusability
An investigation of enzyme reusability was carried
out as per the conditions: 100 g/L ILC, 0.015 g/mL
enzyme, and 40o
C. Three runs were taken
consecutively under batch mode and the results are
shown in Fig. 7. The maximum GOS formation
decreases gradually (Fig. 7, inset). This study
clearly indicates that activity of immobilized
enzyme decreases on continuous use of the same
immobilized membrane for different runs.
0 5 10 15 20 25 30 35 40
0
5
10
15
20
25
30
1 2 3
0
5
10
15
20
25
MaximumGOS(%)
1st run
2nd run
Time (h)
3rd run
GOS(%)
Fig. 7: Reusability of immobilized enzyme at 100
g/L ILC and 0.015 g/mL enzyme concentration.
Maximum GOS formation (inset)
Fig. 8: Reuse of membrane for enzyme loading
After each run, the membranes were washed with
water. They were then kept in formalin solution
and shaken for a while. Finally, they were
preaserved in 10% ethanol solution in cold
condition (5-10 o
C) for next run. It was noticed
(Fig. 8) that the amount of enzyme loading
remained more or less same. Further, it was
experienced that membranes could not be used
beyond three runs; observation reveals membrane
damge.
3.6 Comparison of GOS formation between
Free and Immobilized Conditions
The results were compared with our previous study
conducted under free enzyme condition (Palai,
2010). Fig. 9 shows that maximum GOS yield
under immbilized condition (28%) is less than that
of free enzyme condition (31%). This may be due
to diffusional limitation. Encourgingly, there is
hardly any difference of enzyme activity between
free and immobilized conditions.

5. Fig. 9: Comparison of GOS yield between free and
immobilized conditions at different ILC
4. CONCLUSION
Immobilization of enzyme on hydrophobic PVDF
membrane by cross-linking technique for GOS
formation is investigated under batch mode. The
maximum enzyme loading was observed to be 51
mg, for both the membranes on both sides’.
Maximum GOS formation was observed to be
28%. The different forms of GOS obtained were:
tri-saccharides (19%), tetra-saccharides (7.5%) and
penta-saccharides (1.5%). There was marginal
difference of GOS formations while comparing the
result (GOS yield) both under free (~31%) and
immobilized (~28%) conditions. It was observed
that maximum GOS formation increased with
increasing ILC while the same does increase
initially with increasing enzyme concentration and
thereafter attains a plateau.
Acknowledgement
The authors gratefully acknowledge the
Department of Biotechnology, Govt. of India for
partial financial support to carry out this work.
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