Process Biochemistry 40 (2005) 1199–1205

Soymilk oligosaccharide hydrolysis by Aspergillus oryzae

␣-galactosidase immobilized in calcium alginate
S.J. Prashanth, V.H. Mulimani∗
Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India

Received 4 August 2003; received in revised form 3 April 2004; accepted 11 April 2004

Abstract

␣-Galactosidase was immobilized in calcium alginate in the presence of glutaraldehyde. The optimum pH of the soluble enzyme was 4.8,
whereas that of the immobilized enzyme was 4.5. The optimum temperature of the soluble enzyme was 50 ◦ C and that of the immobilized
enzyme was increased to 57 ◦ C. The immobilized enzyme retained its activity for a longer period. The immobilized enzyme exhibited higher Km
and lower Vmax compared to the soluble enzyme. Immobilized ␣-galactosidase was used in batch, repeated batch and in continuous mode. After
12 h, soluble and immobilized enzyme resulted in 93 and 81% reduction in raffinose family oligosaccharide content in soymilk. Immobilized
calcium alginate beads were used for repeated hydrolysis at 50 ◦ C and showed good operational stability. The performance of immobilized
␣-galactosidase was also tested in a fluidized bed reactor at different flow rates and 90% reduction of raffinose family oligosaccharides in
soymilk was obtained at 40 ml h−1 flow rate.
© 2004 Elsevier Ltd. All rights reserved.

Keywords: ␣-Galactosidase; Soymilk; Oligosaccharides; Aspergillus oryzae; Calcium alginate; Fluidized reactor

1. Introduction legumes. Thus, ␣-galactosides (raffinose and stachyose)

Soy-based foods are a promising supplements to over-
come existing protein-calorie malnutrition problems [1].
Soymilk is especially seen as a low-cost substitute for dairy
milk for the poor in the developing countries and as a nu-
tritive supplement for the lactose-intolerant population as
soybeans do not contain lactose [2]. Soymilk is an economi-
cal raw material, which serves as base material for a variety
of beverages and for the production of proteinacious foods
[3]. Soy usage has been limited because of anti-nutritional
compounds, which not only diminish its nutritive value, but
also restrict its wider acceptance. Flatus-causing oligosac-
charides are one such anti-nutritional factor [4]. Soybeans
typically contain 9–12% total sugars including 4–5%
sucrose, 1–2% raffinose, 3.5–4.5% stachyose along with
melibiose and verbascose in smaller quantities [1]. Mono-
gastric animals, including humans lack the ability to synthe-
size sufficient ␣-galactosidase in their intestinal systems to
hydrolyze the ␣-galactosides present in soybeans and other
∗ Corresponding author. Tel.: +91-8472-448819;
fax: +91-8472-445632.
E-mail address: v h mulimani@rediffmail.com (V.H. Mulimani).
pass into the large intestine, where the resident microflora
act on them, causing flatulence and gastrointestinal (GI)
disturbance. These disturbances reduce both feed efficiency
in monogastric animals and general consumer acceptance
of soy foods [5]. As a result, there is a high demand for
␣-galactoside-free soybean product.
␣-d-Galactosidase (␣-d-galactoside galactohydrolase EC
3.2.1.22) is widely distributed in microorganisms, plants
and animals. Generally, ␣-galactosidase hydrolyzes a va-
riety of simple ␣-d-galactosides as well as more complex
molecules, such as oligosaccharides and polysaccharides
[6]. There are several reports in the literature of the use
of ␣-galactosidase from plant [7], bacteria [8] and fungal
sources [9–11] for the removal of raffinose family sugars
from soymilk. Although satisfactory hydrolysis is obtain-
able by such processing, a single usage of free enzyme
appears uneconomical and the processed milk has the pos-
sibility to contain foreign proteins.
A literature survey indicates that there are few reports
on immobilization of ␣-galactosidase [12,13] for reducing
oligosaccharides in soymilk. Some papers [12,13] report the
immobilization of ␣-galactosidase in polyacrylamide for re-
ducing oligosaccharides in soymilk but polyacrylamide sup-

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.04.011
1200 S.J. Prashanth, V.H. Mulimani / Process Biochemistry 40 (2005) 1199–1205
ports are not useful in food processing because of toxicity
[14]. Alginate is a non-toxic polysaccharide consisting of
guluronic acid and mannuronic acid residues and complexes
with metal ions via its carboxyl groups. Alginate entrapment
was chosen since alginate is widely used in food, pharma-
ceutical, textile and paper products [15], and immobilization
can be carried out in a single-step under mild conditions [16].
To our knowledge this is the first report of the usage of algi-
nate as a matrix for the immobilization of ␣-galactosidase.
Aspergillus oryzae has been shown to be a potential source
of ␣-galactosidase [17]. Cruz and Park [17] have success-
fully demonstrated significant oligosaccharide removal by
treatment of soymilk with crude extract of ␣-galactosidase
obtained from A. oryzae culture. Their results indicate the
possibility of using ␣-galactosidase in immobilized form,
which may lead to a more viable process with the added
advantages of reusability and cost-effectiveness. In the
present investigation, we report the application of A. oryzae
␣-galactosidase immobilized in calcium alginate for the
treatment of soymilk and to study the extent of oligosaccha-
ride removal by batch, repeated batch and continuous mode.
2. Materials and methods
2.1. Chemicals
Raffinose, stachyose and PNPG (p-nitrophenyl-␣-d-
galactopyranoside) were procured from Sigma chemicals,
USA. Sodium alginate and calcium chloride were from S.D.
Fine-Chem Ltd., India. All other chemicals used were of an-
alytical grade. Soybean seeds were bought at a local market
in Gulbarga, India, and cleaned from foreign particles.
2.2. Microorganism
Aspergillus oryzae capable of producing extracellular
␣-galactosidase was isolated from a soil sample. It was
identified by the Indian type culture collection (ITCC),
Indian agricultural research institute, New Delhi, India. It
was maintained on potato-dextrose-agar (PDA) slants and
stored at 4 ◦ C.
2.3. Preparation of acetone precipitated α-galactosidase
from A. oryzae
The following medium was used. Basal media with
guar gum (1.5%) as carbon source and peptone, 6.0 g 1−1 ;
(NH4 )2 SO4 , 1.5 g 1−1 ; KH2 PO4 , 6 g 1−1 was used for sub-
merged fermentation. The pH of the medium was adjusted
to 5.5. After autoclaving, the flasks were inoculated with
spores (2 × 106 ) of A. oryzae. Submerged fermentation was
carried out in 250 ml Erlenmeyer flasks each containing
50 ml culture medium. The flasks were incubated at 37 ◦ C
for 5 days on an orbital shaker at 120 rpm. On the 5th
day, mycelia were separated from culture broth by filtration
through Whatman No. 1 filter paper and the filtrate was
used as crude ␣-galactosidase (1.68 U ml−1 ). To the crude
␣-galactosidase, acetone (1:1 (v/v)) was added and the
mixture was allowed to stand for 2 h at 0 ◦ C and then the
precipitate which contained ␣-galactosidase was recovered
by centrifugation at 15,000 rpm for 20 min at 4 ◦ C. The pre-
cipitate thus obtained was dissolved in a minimal amount
of 0.1 M acetate buffer (pH 4.8) and then dialyzed against
0.05 M acetate buffer (pH 4.8) for 12 h at 4 ◦ C. This enzyme
preparation (5.21 U ml−1 ) was used for further studies.
2.4. α-Galactosidase assay
␣-Galactosidase assay was carried out in test tubes by the
method of Dey and Pridham [6]. One milliliter of reaction
mixture contains 0.1 ml of suitably diluted enzyme + 0.8 ml
of 0.2 M acetate buffer (pH 4.8) + 0.1 ml of 2.0 mM PNPG
was added and incubated at 37 ◦ C for 15 min. The reaction
was arrested by adding 3 ml of 0.2 M Na2 CO3 solution and
the absorbance was read at 405 nm in a spectrophotometer
(Elico Ltd., India). The immobilized enzyme was assayed in
the same manner as that of soluble enzyme except that ap-
propriate quantity of immobilized beads were used in place
of soluble enzyme for the assay.
2.4.1. Determination of kinetic parameters Km and Vmax
The soluble and immobilized ␣-galactosidase was assayed
at increasing concentrations of artificial substrate PNPG
as well as natural substrate raffinose in the correspond-
ing activity buffer. Fresh beads were used for each sub-
strate concentration. Km and Vmax was determined using the
Lineweaver–Burk plot method.
2.4.2. Units of α-galactosidase
One unit of enzyme activity is defined as the amount of en-
zyme preparation required to liberate 1 ␮M of p-nitrophenol
from PNPG per minute under the standard assay conditions.
2.5. Immobilization of α-galactosidase
The enzyme was immobilized in calcium alginate beads
according to the method of Ates and Mehmetoglu [18]. Four
milliliters of sodium alginate solution (3.75%) was mixed
with 1 ml enzyme solution (7.5 mg ml−1 ) to a homogenous
final alginate concentration of 3%. The mixture was extruded
drop by drop using a sterile hypodermic syringe needle into
0.2 M CaCl2 solution maintained at 4 ◦ C to form beads. The
beads were allowed to harden in the CaCl2 solution for 2 h.
The resulting spherical beads were washed with sterile dis-
tilled water. The beads were stored in 0.2 M acetate buffer
(pH 4.8) at 4 ◦ C. Glutaraldehyde was used as crosslinking
agent according to the method of Ates and Mehmetoglu
[18]. Calcium alginate beads were treated for 3 min in 5 ml
of 1% glutaraldehyde. The beads were filtered off, washed
with sterile water and stored at 4 ◦ C until used.
 S.J. Prashanth, V.H. Mulimani / Process Biochemistry 40 (2005) 1199–1205
2.6. Preparation of soymilk
Soymilk was prepared according to the method of Muli-
mani and Ramalingam [11]. Soybean seeds were ground
to flour. The soybean flour was defatted with hexane (1:1
(w/v)). The fat free soybean flour was suspended in 10 vol-
ume of distilled water and heated to boiling. Undissolved
residue was separated from soymilk by centrifugation for
5 min at 5000 rpm. The supernatant containing soymilk was
stored at 4 ◦ C for a short period till further use.
2.7. Estimation of oligosaccharides in soymilk
Soymilk (15 ml) was poured into 35 ml of absolute ethyl
alcohol and centrifuged at 6000 rpm for 15 min at 37 ◦ C. The
centrifugate was concentrated and dissolved in 15 ml of dis-
tilled water. The amount of sucrose, raffinose and stachyose
was estimated by the method of Tanaka et al. [19].
2.8. Treatment of soymilk by enzyme
2.8.1. Batch reaction
Batch reactions were performed for both soluble and im-
mobilized enzymes at different incubation periods. For the
reaction involving soluble enzyme around 10 ml enzyme
(5.21 U ml−l ) was added to 60 ml of soymilk in Erlenmeyer
flaks (250 ml). For the reaction involving immobilized en-
zyme around 220 mg (0.24 U mg−1 ) immobilized enzyme
was added to 60 ml of soymilk. The hydrolysis reaction was
carried out at 50 ◦ C in an incubator shaker (200 rpm) at dif-
ferent incubation periods, i.e. 2, 4, 8, and 12 h. After the in-
cubation period an aliquot of the reaction mixture was taken
out. For the reaction involving soluble enzyme, reaction mix-
ture was kept in boiling water bath for 10 min to arrest the
enzyme reaction. Afterwards the sample was analyzed for
degradation of oligosaccharides. Control experiments were
performed in the same manner with 0.2 M acetate buffer (pH
4.8) replacing the enzyme.
2.8.2. Repeated batch
Soymilk (60 ml) and 25 ml of immobilized enzyme beads
were taken in 250 ml Erlenmeyer flasks and were kept in an
incubator shaker (200 rpm) maintained at 50 ◦ C. After every
incubation period, i.e. 4 h, an aliquot of soymilk sample was
taken and its oligosaccharide concentration was determined.
The beads were separated by filtration on the sinter, washed
with sterile distilled water and transferred into fresh batch
of soymilk (60 ml) for another 4 h incubation.
2.8.3. Design of fluidized reactor for the continuous
degradation of raffinose family oligosaccharide sugars in
soymilk
Fluidized bed reactor studies were carried out in jacketed
glass column of 75 cm in length and 1.5 cm in diameter,
with bed volume of 130 ml. The jacket temperature was
maintained at 50 ◦ C (water bath from Julabo, Germany).
1201
The soymilk feed solution (containing 10 mM CaCl2 ) pre-
heated in a water bath (50 ◦ C) was introduced from the
bottom of the column through a peristaltic pump (Amer-
sham Pharmacia Biotech, Sweden) and the product was
withdrawn from the top of column. The alginate beads
containing ␣-galactosidase from A. oryzae were packed
in the column. The upward substrate stream fluidized the
beads filled up in the column. Different flow rates of 30,
60, 90 and 120 ml h−1 were used for the degradation of
oligosaccharides in soymilk. The outlet stream was contin-
uously collected from the frontal end of the column in a
container. The effluents were analyzed for the degradation
of oligosaccharides raffinose and stachyose.
3. Results and discussion
Crude ␣-galactosidase was subjected to acetone precipi-
tation. The precipitate was dialyzed against water to remove
salts and other lower molecular weight substances. Entrap-
ment of ␣-galactosidase in 3% calcium alginate resulted
in 54.5% loss of ␣-galactosidase activity. Glucose oxidase
entrapped in 2% calcium alginate exhibited only 35% of
the activity of free preparation [20]. ␣-Galactosidase from
Pycnoporus cinnabarinus when immobilized on chitin and
chitosan exhibited 27.5% and 23.4% activity of free prepa-
ration [21]. A loss of 40% activity was observed when
␣-galactosidase from Mortierella vinacea was entrapped
in polyacrylamide [12]. Compared to the other reports
[20,21,12] the range of recovery of enzyme activity is rather
satisfactory.
An inherent problem of using alginate is the leakage of
enzyme from the beads, even at high concentrations, which
gradually leads to loss of the enzyme activity [15]. In order
to avoid leakage glutaraldehyde solution (1%) was used as
a hardener in the present study. Ates and Mehmetoglu [18]
have reported that on treatment with glutaraldehyde solution
(1%) the activity of ␤-galactosidase in the alginate beads
was stable.
3.1. Effect of pH
The effect of pH on soluble and immobilized enzyme is
depicted in Fig. 1. The soluble form had an optimum pH of
4.8, which is in agreement with the value reported earlier
[17]. ␣-Galactosidase produced from A. awamori and A.
saitoi that were used for the hydrolysis of oligosaccharides
in soymilk had an optimum pH of 5.0 and 5.5, respectively
[9,10]. The immobilized enzyme showed optimum pH at
4.5. Immobilized ␣-galactosidase was slightly more stable
at acidic pH. Ates and Mehmetoglu [18] have also made a
similar observation. Adami et al. [22] have reported that the
pH-activity profile shift of the immobilized enzyme activity
towards more acidic pH values, consequently the displace-
ment of pH optimum and pK-values may be caused by the
micro environmental effects of the calcium alginate ma-
1202 S.J. Prashanth, V.H. Mulimani / Process Biochemistry 40 (2005) 1199–1205
Fig. 1. Effect of pH on: (䉬) soluble and (䊏) immobilized ␣-galactosidase
from A oryzae.
trix, particularly by the presence of positive charges on the
carrier.
3.2. Effect of temperature
The temperature optima of the soluble and alginate en-
trapped enzyme preparation was determined by assaying
the enzyme activity at indicated temperatures. Optimum
temperature for the activity of alginate entrapped enzyme
was shifted to a higher value than that of the soluble en-
zyme (Fig. 2). The maximum activity of alginate entrapped
enzyme was obtained at 57 ◦ C compared to 50 ◦ C for the
soluble enzyme. The soluble enzyme exhibited 30% activ-
ity at 65 ◦ C, whereas immobilized enzyme had around 69%
activity. The shift in temperature optimum for Cu-alginate
entrapped tyrosinase and Cu-alginate entrapped naringinase
towards higher side is also reported [23,24]. Arica et al.
[25] speculated that hydrophobic interactions and other
secondary interactions of the immobilized enzyme might
impair conformational flexibility necessitating higher tem-
peratures for the enzyme molecules to reorganize and attain
a proper conformation for its functioning and binding of
the substrate. The calcium alginate immobilization system
could protect the enzyme of thermal effects allowing activ-
Fig. 2. Effect of temperature on: (䉬) soluble and (䊏) immobilized
␣-galactosidase from A. oryzae.
Fig. 3. Thermostability of: (䉬) soluble and (䊏) immobilized ␣-gala-
ctosidase from A. oryzae at 50 ◦ C, pH 4.7.
ity at even 57 ◦ C. This is suitable since high temperature
diminishes microbial contamination during treatment of
soymilk.
3.3. Thermal stability
Thermal stability of the soluble and immobilized
␣-galactosidase was examined by incubating these prepa-
rations without any stabilizers at 50 ◦ C. Fig. 3 depicts the
stability of ␣-galactosidase at 50 ◦ C. After 24 h, soluble and
immobilized enzyme retained 44 and 71% activity respec-
tively. Soluble and immobilized enzyme retained 88 and
92% activity after 12 h incubation. The soluble form retained
92 and 88% activity after 8 and 12 h, whereas immobilized
enzyme retained 94 and 92% after the same incubation
period, respectively. In the initial stages, i.e. up to 12 h in-
cubation activity was retained to greater extent (91%) after
which the residual activity decreased to 70%. Enhanced
thermal stability of naringinase in Ca-alginate gel [24],
Cu-alginate entrapped tyrosinase preparation [23] and phe-
nol oxidase in Cu-alginate gels [26] has been demonstrated
earlier. The greater stability of immobilized enzyme may be
subscribed to the stabilizing effects of immobilization [27].
3.4. Effect on Michaelis constant (Km ) maximal reaction
rate (Vmax )
The kinetic parameters determined for soluble and immo-
bilized enzyme are depicted in the Table 1. Lineweaver–Burk
plot revealed that Km and Vmax for immobilized enzyme
with PNPG as substrate were 1.42 mM and 3.33 U ml−1 ,
Table 1
Kinetic parameters for soluble and immobilized ␣-galactosidase
PNPG Raffinose
Km Vmax Km Vmax
(mM) (U ml−1 ) (mM) (U ml−1 )
Soluble 0.83 2.94 5.5 0.15
Immobilized 1.42 3.33 8.69 0.06
 S.J. Prashanth, V.H. Mulimani / Process Biochemistry 40 (2005) 1199–1205
Fig. 4. Percent hydrolysis of raffinose family sugars after treatment with:
(䉬) soluble and (䊏) immobilized ␣-galactosidase from A. oryzae in
soymilk at different incubation periods.
respectively (the soluble enzyme showed a Km and Vmax of
0.83 mM and 2.94 U ml−1 , respectively). The observed Km
and Vmax for soluble enzyme with raffinose were 5.5 mM
and 0.15 U ml−1 for the immobilized enzyme; 8.69 mM and
0.06 U ml−1 for the soluble enzyme. For soluble enzyme
the Km -values suggests that the ␣-galactosidase prefers for
the synthetic PNPG than for natural substrate raffinose,
which is larger and more bulky trisaccharide. The increase
in Km -values after immobilization may be partially due to
mass transfer resistance of the substrate into the immobi-
lization medium. Low diffusion is characteristic shown by
alginate immobilization system as the alginate network,
due to its high degree of crosslinking limits the permeation
rate of substrate and product [28]. Bodalo et al. [29] have
similarly reported that Km value increased from 0.28 to
0.44 mM and Vmax decreased from 295 to 18.58 U mg−1
upon immobilization of ␤-galactosidase in alginate.
3.5. Enzymic treatment of soymilk
3.5.1. Batch reaction
The hydrolysis of raffinose family sugars by soluble
and immobilized ␣-galactosidase by batch experiment is
displayed in Figs. 4 and 5. The soymilk sample initially
Fig. 5. Repeated degradation of raffinose family sugars in soymilk by
immobilized ␣-galactosidase from A. oryzae.
1203
contained 574 mg of total oligosaccharide per 100 ml, out
of which 464 mg stachyose and 118 mg raffinose were the
main constituents. In the batch experiments, the soluble
and immobilized enzyme was incubated with soymilk at
different incubation periods, i.e. 2, 4, 8, and 12 h. The 2
and 4 h incubation with soluble enzyme resulted in 85 and
87% and immobilized enzyme resulted in 74 and 78% hy-
drolysis, respectively. After 8 and 12 h incubation soluble
␣-galactosidase led to 91 and 93% degradation, whereas
immobilized ␣-galactosidase resulted in 79 and 81% reduc-
tion in raffinose family oligosaccharides in soymilk. The
soluble enzyme showed better degradation. The immobi-
lized enzyme showed lesser degradation, which could be
due to diffusional limitation (i.e. resistance of substrate to
diffuse into the immobilization matrix and resistance of the
products to diffuse out) [30]. The 92% hydrolysis of raffi-
nose family sugars in soymilk by immobilized enzyme for
the incubation period of 12 h is the highest reduction com-
pared to other reports on immobilization of ␣-galactosidase
[12,13]. But interestingly 85% oligosaccharide hydrolysis
occurred within 2 h incubation for soluble enzyme implied
that prolonged incubation does not significantly increase
the percent hydrolysis (Fig. 1). Hydrolysis after 2 h was
very slow probably due to substrate depletion; it could also
be due to product inhibition [9].
Thananunkul et al. [12] have reported 50% hydrolysis,
whereas Thippeswamy and Mulimani [13] have reported
71% hydrolysis after 12 h incubation of soymilk with im-
mobilized enzyme. The ␣-galactosidase from A. oryzae im-
mobilized in calcium alginate caused higher percentage of
degradation in less incubation period.
3.5.2. Repeated batch reaction
The operational stability of the immobilized ␣-
galactosidase was evaluated in a repeated batch process.
The results (Fig. 4) indicated that the catalytic activity of
the immobilized enzyme was durable under repeated use.
Thus, the immobilized enzyme was able to maintain good
oligosaccharide reduction up to 57% even after five runs.
Percent hydrolysis was determined to be 78% for calcium
alginate beads on incubation for 4 h. On subsequent uses,
i.e. after 2nd, 3rd and 4th cycles the percent hydrolysis was
found to be 75, 71, 68 and 65%, respectively. There was no
drastic decrease in percent hydrolysis even after five uses,
which could be due to glutaraldehyde treatment of calcium
alginate beads, which prevented the leakage of enzyme.
Ates and Mehmetoglu found that after treatment with glu-
taraldehyde the Cu-alginate immobilized enzyme could be
used 8 times with high activity [18].
3.5.3. Continuous reaction
The use of an immobilized enzyme makes it economi-
cally feasible to operate an enzyme process in a continuous
mode. A fluidized bed reactor was constructed to explore
the practicability of using the calcium alginate entrapped
enzyme in continuous system. Bodalo et al. [29] suggested
1204 S.J. Prashanth, V.H. Mulimani / Process Biochemistry 40 (2005) 1199–1205
that the final choice of derivative for use in fluidized bed re-
actors should be based not only on the enzyme activity but
also on the hydrodynamic behavior of the support. For the
operation in fluidized reactor, Bodalo et al. [29] suggested
calcium alginate beads over other matrices in spite of low
activity because of their satisfactory hydrodynamic behavior
in the reactor. The continuous degradation of oligosaccha-
rides in soymilk was carried out in a fluidized bed reactor.
At 50 ◦ C, oligosaccharide reduction in soymilk was 90, 78,
74, 52 and 35% for 40, 80, 120, 160 and 200 ml h−1 flow
rates, respectively. These results suggest that a low flow rate
(40 ml h−1 ), i.e. a higher retention time leads to higher per-
cent degradation (90%). Optimal flow rate is one of the im-
portant parameter for the effective operation of a fluidized
column reactor [31]. Thananunkul et al. [10] reported that
a fluidized bed containing ␣-galactosidase-gel complex hy-
drolyzed 60% of the raffinose and stachyose in soymilk but
at a flow rate of 30 ml h−1 . Thippeswamy and Mulimani
[13] reported that oligosaccharide degradation of 84% at a
flow rate of 25 ml h−1 . Compared to the earlier reports in
which 25 and 30 ml h−1 resulted in the maximum reduction,
40 ml h−1 flow rate used in the present study is more practi-
cal to operate and resulted in the maximum oligosaccharide
reduction of 90%.
Thus, immobilized ␣-galactosidase from A. oryzae shows
high optimum temperature and better operational stability by
being stable even after being used five times, ␣-galactosidase
has been previously immobilized in polyacrylamide for the
hydrolysis of oligosaccharide in soymilk [12,13]. The us-
age of toxic chemicals is an important issue in the context
of proteins/enzymes, which are used in food processing or
pharmaceutical purposes [32]. The usage of polyacrylamide
in the process raises considerable apprehension because of
the toxicity. Even though polyacrylamide is known not to
be poisonous, it must be presumed that use of acrylamide
gel in producing processes in the food industry will result in
some contamination of the food by acrylamide fragments of
low molecular weight which result from the breakdown of
the gel matrices after long periods of use. The prevention of
such contamination by acrylamide fragments is very diffi-
cult and verification of the security of acrylamide fragments
is also equally difficult [33]. Calcium alginate, on the other
hand, is perfectly safe from this viewpoint. A. oryzae used
is considered as an excellent host for the safe production
of harmless products [34]. The immobilization of A. oryzae
␣-galactosidase in calcium alginate and its subsequent use is
comparatively safe, simple and cheap with durable enzyme
activity.
Acknowledgements
One of the authors Mr. S.J. Prashanth wish to thank the
Council of Scientific and Industrial Research (CSIR), New
Delhi, India, for its financial support in the form senior re-
search fellowship.
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