Production of Fungal Galactosidase and I

Production of Fungal wGalactosidase and Its Application
to the Hydrolysis of Galactooligosaccharides
in Soybean Milk
RUBEN CRUZ and YONG K. PARK

ABSTRACT
Production of extracellular or-galactosidaseof Aspergillusoryzae
was induced remarkably well by the addition of soybean carbohydrate to culture medium. Addition of stachyoseor raffinose induced
slight production. Soybean carbohydrate, stachyose, and raffinose
also served well for the synthesis of invertase. Maximum hydrolysis
of p-nitrophenyliu-D-galactopyranoside (PNPG) by the enzyme
occurred at pH 4.0, and optimum temperature for hydrolysis of
PNPG was 50°C. The enzymeseemed to be stable from 30-50°C.
The mixture of o-galactosidase and invertase totally hydrolyzed
raffinose and stachyose at 50°C pH 4.0 in 2 hr. The mixed preparation of the enzyme substantially hydrolyzed galactooligosaccharides
in soybean milk at 50°C pH 6.3 in 2 hr.

INTRODUCTION
a-GALACTOSIDASE
(a-D-galactoside
galactohydrolase,

EC. 3.2.1.22) is an important enzyme in the processing of
beet sugar (Kobayashi and Suzuki, 1972). This enzyme is
also potentially important for hydrolysis of galactooligosaccharides, especially stachyose and raffinose in soybean
because these are responsible for intestinal discomfort
and flatulence (steggerda et al., 1966; Cristofaro et al.,
1974).
c+Galactosidase activity has been reported in various
microorganisms, animal tissues, and plant seeds (Suzuki et
al., 1970). It was reported previously by other laboratories
that addition of enzyme preparation from Aspergillus
saitoi (Sugimoto and Van Buren, 1970), Aspergillus awamori (Smiley et al., 1976; McGhee et al., 1978), MortiereZla
vinacea (Thananunkul
et al., 1976), and Cladosporium
cladosporioides (Cruz et al., 198 l), to soybean milk resulted
in hydrolysis of galactooligosaccharides.
In preliminary studies on production of various enzymes
for food processing with a strain of Aspergillus oryzae, it
was found that the strain of fungus produces good yields
of a-galactosidase and invertase (P-D-fructofuranoside
fructohydrolase,

EC. 3.2.1.26) on addition of soybean
carbohydrate as an inducer to a culture medium. The combination of a-galactosidase and invertase efficiently hydrolyzed galactooligosaccharides. This paper demonstrates that
the enzyme preparation of A. oryzae hydrolyzed raffinose
and stachyose in soybean milk.

MATERIALS & METHODS
Microorganism
The culture employed was a strain of Aspergillus oryzae, which
is used for commercial or-amylasein the baking industry. The culture
was maintained on Sabouraud dextrose agar. The spore suspension
of the strain was prepared by incubating the microorganism at 30°C
for 1 wk on Sabouraud dextrose agar slant. Ten ml of sterilized
water were added to the slant culture and the surface gently rubbed
with a sterilized wire loop to give a suspensionof the spores.
Production of the enzyme. Both solid state fermentation and the
submerged culture method were employed for production of the
Authors
Cruz and Park are with Univarsidade
Estadual de Campinas,
Faculdade de Engenharia de Alimentos

(UNICAMP),
Campinas, SP.
Brazil.

enzyme. The composition of the submerged culture medium was as
follows: peptone 1.5%, yeast extract 0.5%, NaNOa 0.2%, KHzP04
OS%, MgS04 O.OS%,KC1 0.05%, and carbohydrate 2%. Soluble
starch, stachyose, raffinose, sucrose, glucose, maltose, lactose,
melibiose, and soybean carbohydrate were used as carbohydrates.
Soybean carbohydrate was prepared by the method described
previously (Cruz et al., 1981). The composition of soybean carbohydrate was mainly manninotriose and free of protein. The pH of
the culture medium was adjusted to 6.0. A volume of 100 ml of the
medium was employed in 500 ml Erlenmeyer flasks. After sterilization of the culture medium, it was Inoculated with 1 ml of the spore
suspensionand then incubated at 30°C on a rotary shaker with agitation of 250 rpm for 5 days. After incubation, the medium was
filtered and activities of cu-galactosidase
and invertase determined in
the filtrate. The solid state fermentation medium consisted of 1OOg
of wheat bran and 100 ml of water or 1OOgof wheat bran and 100
ml of soybean carbohydrate solution (3.7% dry matter). Twenty
grams of the mixture were placed in 500 ml Erlenmeyer flasks and

autoclaved. After sterilization, the flasks were inoculated with 1 ml
of spore suspension and then incubated at 30°C for 5 days. After
incubation, 100 ml of deionized water was added to the flasks and
thoroughly mixed to extract the enzymes. After 1 hr, the mixture
was filtered and activities of the enzymes in the filtrate determined.
Assaysof enzyme activities
The activity of cu-galactosidasewas determined by using pnitrophenyl-cu-D-galactopyranoside (PNPG) and milibiose as substrates. The reaction mixture for PNPG hydrolyzing activity consisted
of 1 ml of 0.002M PNPG in 0.05M McIlvaine buffer of pH 4.0 and
0.05 ml of adequately diluted enzyme solution. It was Incubated at
50°C for 10 min. After incubation, the reaction was then stopped
by addition of 3 ml of O.lM boric acid-borax buffer, pH 10.7, and
p-nitrophenol was estimated with a spectrophotometer at 400 nm.
One unit of cu-galactosidaseactivity is defined as the amount of
enzyme which wiIl liberate 1 &mole of p-nitrophenol per min under
the conditions described above.
Melibiose hydrolyzing activity was determined by incubating
the mixture of 0.5 ml of enzyme solution, 0.5 ml of 0.15M McIlvaine buffer, pH 5 .O, and 0.5 ml of 0.6M melibiose solution at
50°C for 40 min. After incubation, 0.25 ml of 0.3M Ba(OH)a and
0.25 ml of ZnS04 0.18M were added and the mixture centrifuged
to separate the precipitate. The amounts of glucose formed in the

supernatant were measured by the glucose oxidase-peroxidasechromogen method. One unit of melibiose hydrolyzing activity is
defined as the amount of enzyme which wilI liberate 1 nmole of
glucose per min under the conditions described above.
Invertase activity was measured by incubating a mixture of 4.5
ml of 8% sucrose in 0.05M acetate buffer pH 5.0 and 0.5 ml of
enzyme solution at 50°C for 15 min. After incubation, the glucose
liberated was measuredby theglucose oxidase-peroxidase-chromogen
method. The total reducing substanceswere also measured by the
method of Nelson-Somogyi (Nelson, 1944; Somogyi, 1945). One
unit of invertase activity is defined asa potency of the enzyme
which will form 1 nmole of glucose per min under these conditions.
Enzymatic hydrolysis of raffinose and stachyose
A mixture of 1 ml of enzyme extract which contained 0.26 unit
of ol-galactosidaseactivity and 1 ml of raffinose or stachyose (6%)
in O.lM McIlvaine buffer pH 4.0, was incubated at 50°C for 2 hr.
After incubation, hydrolysates of sugars were immediately tested
by thin-layer chromatography.
Enzymatic hydrolysis of galactooligosaccharides
in soybean milk
Commercial soybean milk powder was reconstituted with deVolume

47 (7982)~-JOURNAL

OF FOOD SCIENCE-1973

PRODUCTlON/At=‘PLICATlON
Table l-induction
submerged culture

of ol-galactosidase

or-Galactosidase
PNPGa
hydrolyzing
activity

Addition
of
carbohydrates

OF FUNGAL a-GALACTOSIDASE.
by various

carbohydrates

activity
Melibiose
hydrolyzing
activityb

I nvertase
activitvb

Starch
Stachyose
Raffinose
Sucrose
Glucose
Maltose

0
0.27
0.02
0
0
0

0
0.14
0.05
0
0
0

Lactose

0

0

Melibiose
Soybean
carbohydrate

0

0

0.80
4.00
2.44
0.70
0.16
0.12
0.17
0.31

1.65

0.2

4.10

E PNPG represents
Enzyme activity

p-nitrophenyl-(Y-D-galactopyranoside
represents unit/min/ml
of culture

Table 24nduction
of or-galactosidase
solid state fermenlation

by soybean

cu-galactosidase
Composition
of solid
culture medium

PNPGa
hydrolyzing
activity

Wheat bran;
50 g
Water;
40 ml
Soybean carbohydrateC
solution;
IOml

in

RESULTS 8c DISCUSSION
Production of the enzyme

on

activity
Melibiose
hydrolyzing
activityb

0.93

I nvertase
activityb

0.25

6.8
/

Wheat bran;
50 g
Water;
25 ml
Soybean carbohydrate
solution;
25 ml
Wheat bran;
50 g
Soybean carbohydrate
solution;
50 ml

1.73

0.47

5.3

2.58

0.70

4.4

ionized water (10%) and a volume of 50 ml (pH 6.3) poured into a
250 ml Erlenmeyer flask. Thirteen units of a-galactosidasepreparation were added and the mixture incubated at 50°C for 2 hr. Periodically, 2 ml of sample were taken and treated with 0.6 ml of 0.3M
Ba(OH)2 and 0.6 ml of 0.18M ZnSOg and the precipitated protein
separated by centrifugation. The supernatnat was used for the
determination of sugars.
Thin-layer chromatography
Forty 4 of the treated samples were applied on silica gel and
developed in the ascending direction using the solvent system of
ethyl acetate, acetic acid, and water (3:1:1, v/v). Diphenylamine
and aniline in acetone (Ghebregzabher et al., 1979) were applied
to detect sugars.

medium

carbohydrate

..

a PNPG represents p-nitrophenyl-a-D-galactopyranoside
b Enzyme activity
represents unit/min/ml
of enzyme extract
’ Soybean carbohydrate
solution contained
3.7% of solids

As shown in Table 1, a-galactosidase synthesis was
induced remarkably
well by the addition
of soybean
carbohydrate in the submerged culture method. Addition
of stachyose or raffinose induced production slightly. It is
apparent that manninotriose,
the main constituent of soybean carbohydrate, was the best inducer for synthesis of
a-galactosidase.
Soybean carbohydrate,
stachyose, and
raffinose also induced remarkably the synthesis of invertase. Furthermore,
it was found that the strain of A. oryzae
produced both cr-galactosidase and invertase extracellularly.
Such extracellular
enzymes are secreted into the mash
during microbial growth. These may readily be separated
from the microbial
cells by filtration
or centrifugation,
whereas intracellular
enzymes are bound within the cells
and may be released only upon disruption of the cell structure. Therefore,
it is very advantageous for producing
industrial enzymes. Table 2 shows production
of the enzyme by the solid state fermentation
method. It is noted
that synthesis of cu-galactosidase was also induced by addition of soybean carbohydrate to wheat bran. Furthermore,
productivity
of the enzyme by solid state fermentation is
greater than by submerged fermentation.
Therefore, the
enzyme extract from wheat bran culture was used for
hydrolysis
of galactooligosaccharides
in soybean milk.
Although various microorganisms
produce ol-galactosidase
(Sugimoto
and Van Buren, 1970; Smiley et al., 1976;
Thananunkul
et al. (1976; Cruz et al. 1981), the enzymes
100’

loo-

w
>
c
*, 50w
@z
-

r

I
7
6
5
PH
Fig. l-Effect
of pH on ol-galactosidase
activity.
[Enzyme
activities
were determined
at various pH’s as described
in the test. Substrate
(PNPG)
was dissolved in 0.05M Mcllvaine
buffer
of various pH.1
3

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I

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TEMPERATURE
=‘C
Fig. 2-Effect
of temperature
on or-galactosidase
activity.
[Mixtures
of the enzyme solution
and substrate
were incubated
at various
temperatures
for 10 min, and p-nitrophenol
estimated as described
in the text./

1

1

I

,

35

40

45

50

TEMPERATURE

I

55

%

Fig. 3-Thermostability
of or-galactosidase
activity.
enzyme extract in test tubes were incubated
at various
for 10 min, and residual enzyme activities determined
in the text./

[One ml of
temperatures
as described

Fig. 4-Thin-layer
chromatography
tor hydrolysates
ot raffinose
and stachyose by the enzyme. [Method
is described in the text. 1.
Raffinose
before incubation
with enzyme; 2. Raffinose after incobation with enzyme;
3. Stachyose
before incubation
with enzyme;
4. Stachyose after incubation
with enzyme.1

from A. oryzae
are more advantageous for use in food
processing because they appear to be GRAS.

Effects of pH and temperature on enzyme activities
Maximum hydrolysis of PNPG by the enzyme occurred
at pH 4.0, and the enzyme hydrolyzed the substrate substantially at pH 6.5 as shown in Fig. 1. Fig. 2 demonstrates
that optimum temperature for hydrolysis of PNPG by the
enzyme was SO’C. Thermostability
of the enzyme was
examined by incubating a mixture of 0.1 ml of the enzyme
solution and 0.5 ml of 0.1M McIlvaine buffer pH 5.0 at
various temperature for 15 min. After incubation, residual
enzyme activities were examined. As shown in Fig. 3, the
enzyme seemed to be stable from 30 - 5O’C.

Enzymatic hydrolysis of raffinose and stachyose
As shown in Fig. 4, the enzyme extract completely
hydrolyzed raffinose at SO“C, pH 4.0 in 2 hr, while stachyose was substantially hydrolyzed. In the case of soybean
milk, as shown in Fig. 5, the presence of sucrose, raffinose,
and stachyose before treatment of the enzyme was demonstrated, but after enzyme treatment for 2 hr, raffinose and
stachyose were significantly
decreased, whereas sucrose
had totally disappeared.

REFERENCES
Cristofaro,
E.. Mattu, F.. and Wuhrman,
J.J. 1974. Involvement
of
the raffinose
family of oiiaosaccharide
in SatuIence. In “Suears in
Nutrition,”
p. 313..Academic
Press New York.
Cruz. R.. Batistela, J.C., and Wosiacki, G. 1981. Microbial
or-gala&osidase for soy milk processing. J. Food Sci. 46: 1196.
Delente, J. and Ladenburg.
K. 1972. Quantitative
determination
of
ohgosaccharides
in defatted soybean by gas-liquid chromatography.
J. Food Sci. 37: 372.
Ghebregzabher,
M., Rufini.
S., Sapia, G.M.. and Late, M. 1979.
Improved
thin-layer
chromatographic
method for sugar separation.
J. Chromatogr.
180: 1.
Kobayashi,
H. end Suzuki, H. 1972. Studies on the decomposition
of raffinose
by o-galactosidase
of mold. J. Ferment.
Technol. 50:
625.
McGhee,
J.E., SiImsn,
R., and BagIey. E.B. 1978. Production
of
or-galactosidase
from AspergiRus
awamori:
Properties
and action

Fig. B-Thin-layer
chromatography
for hydrolysates
of sucrose,
raffinose,
and stachyose in soybean milk by the enzyme. [Method
is
described
in the text. 1. Before incubation
with enzyme; 2. 1 hr
incubation
with enzyme; 3. 1.5 hr incubation
with enzyme; 4. 2 hr
incubation
with enzyme.

on para-nitrophenyl-c+D-galactopymnoside
and
charides of soymilk.
J. Am. Oil Chem. 55: 244.
Nelson. N. 1944. J. Biol. Chem. 153: 375.

gaIacto-ohgosac-

SmiIey, K.L.. Hensley.
D.E.. and Gesdorf, H.J. 1976. Alpha-galactosidase
production
and use in a Hollow-fiber
reactor.
Appl.
Environ. Microbial.
31: 615.
Somogyi.
M. 1945. A new reagent for the determination
of sugar.
J. Biol. Chem. 160: 61.
Steggerda.
F.R., Richards,
E.A., and Rackis, J.J. 1966. Effects of
various soybean
products
on flatulence
in the adult man. Proc.
Sot. EXP. Biol. Med. 121: 1235.
Sugimoto,
H. and Van Buren,
J.P. 1970. Removal
of oligosaccharides from SOY milk by an enzyme from AsperaiIIus
saitoi. J.
Food Sci. 35: 655.
from
Suzuki.
H.. Li. S.H.. and Li. Y.T. 1970. or-Galactosidase
MortiereIIa
vinacea. J. Biol. Chem. 245: 781.
Thananunkul,
D., Tanaka, M., Chichester,
C.O., and Lee, T.C. 1976.
Dearadation
of raffinose
and stachvose
in soybean
milk bv oga&tosidase
from Mortierella
vinacea. Entrapment
of cu-galactosidase within polyacrylamide
gel. J. Food Sci. 41: 173.

Volume

47 (1982)-JOURNAL

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SCIENCE-1975