Hydrolysis of soybean isoflavonoid glycosides by Dalbergia β-glucosidases

Article abstract of DOI:10.1021/jf062885p

Two β-glucosidases from the legumes Dalbergia cochinchinensis and Dalbergia nigrescens were compared for their ability to hydrolyze isoflavonoid glycosides from soybean. Both D. nigrescens and D. cochinchinensis β-glucosidases could hydrolyze conjugated soybean glycosides, but D. nigrescens β-glucosidase hydrolyzed both conjugated and nonconjugated glycosides in crude soybean extract more rapidly. The kinetic properties K _m, k_cat, and k_cat/K_m of the Dalbergia β-glucosidases toward conjugated isoflavonoid glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of the D. nigrescens β-glucosidase in hydrolyzing these substrates. The D. nigrescens β-glucosidase could also efficiently hydrolyze isoflavone glycosides in soy flour suspensions, suggesting its application to increase free isoflavones in soy products.

Full text of DOI:10.1021/jf062885p

J. Agric. Food Chem. 2007, 55, 2407−2412 2407
Hydrolysis of Soybean Isoflavonoid Glycosides by
Dalbergia
â
-Glucosidases
†
†
PHIMONPHAN CHUANKHAYAN, THIPWARIN RIMLUMDUAN,
JISNUSON SVASTI,§ AND JAMES R. KETUDAT CAIRNS*
,†
Schools of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology,
Nakhon Ratchasima 30000, Thailand, and Department of Biochemistry and Center for Protein
Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Two â-glucosidases from the legumes Dalbergia cochinchinensis and Dalbergia nigrescens were
compared for their ability to hydrolyze isoflavonoid glycosides from soybean. Both D. nigrescens
and D. cochinchinensis â-glucosidases could hydrolyze conjugated soybean glycosides, but
D. nigrescens â-glucosidase hydrolyzed both conjugated and nonconjugated glycosides in crude
m m
soybean extract more rapidly. The kinetic properties K , kcat, and kcat/K of the Dalbergia
â-glucosidases toward conjugated isoflavonoid glycosides, determined using high-performance liquid
chromatography, confirmed the higher efficiency of the D. nigrescens â-glucosidase in hydrolyzing
these substrates. The D. nigrescens â-glucosidase could also efficiently hydrolyze isoflavone
glycosides in soy flour suspensions, suggesting its application to increase free isoflavones in soy
products.
KEYWORDS: Isoflavones; â-glycosidases; Dalbergia; â-glucosidase; soy
INTRODUCTION
to prevent coronary heart disease (9). Because of these health
benefits, there is interest in increasing the amounts of free
isoflavones in soy products.
Isoflavones are a group of diphenolic secondary metabolites
produced in a very limited distribution of higher plants, most
frequently in the Leguminosae (1). Among the common dietary
legumes, soybean contains the highest level of isoflavones. From
the natural food sources, soybeans and soy foods contain the
most dietary isoflavones, which provide many health benefits.
The major types of isoflavones in soybean are daidzein,
genistein, and glycitein. Most of these isoflavones in soybean
seeds are conjugated with glucose or malonylglucose (Figure
Hessler et al. (10) reported that â-glucosidases from Saccha-
ropolyspora erythraea could hydrolyze genistin during fermen-
tation of soy-based media, and â-glucosidase from Bifidobac-
teria in soy milk was capable of converting glucosides to their
aglycones (11). Pandjaitan et al. (12) treated soy protein isolate
with almond â-glucosidase to convert most of its isoflavone
glucosides to their aglycones. However, it has been shown that
Escherichia coli â-glucosidase was more effective than almond
emulsin â-glucosidase, and neither of these enzymes could
effectively hydrolyze malonylglucosyl isoflavone conjugates,
even at high concentrations and extended times (13). In 2001,
Hsieh et al. (14) partially purified and characterized â-glucosi-
dase from soybean that could hydrolyze isoflavone conjugates.
Recently, Suzuki et al. (15) reported the purification and
characterization a â-glucosidase (GmICHG) from the roots of
soybean seedling with high specificity toward conjugated
isoflavones.
1
). In addition, acetylglucose conjugates are also detected in
small amounts in soy products, but it appears that acetyldaidzin
and acetylgenistin may be generated from daidzin and genistin
during their heat-induced decompositions (2).
There are many reports that soybean isoflavones may have
potential benefits for reducing the occurrences of diseases
afflicting humans, such as certain types of breast, prostate, and
colon cancer (3, 4). Besides being anticarcinogenic, they exhibit
antiatherosclerotic, blood glucose lowering, antibacterial (5), and
antioxidative (6) properties. With the discovery of increased
cancer risks associated with estrogen-based hormone replace-
ment therapy, the use of isoflavones as an alternative for
menopausal women has received much public and scientific
interest (7, 8). In addition, isoflavones may reduce low-density
lipoproteins and increase high-density lipoproteins, which help
Dalbergia is a large genus of small- to medium-size
leguminous trees native to the tropical regions of Central and
South America, Africa, and southern Asia (16). Isoflavonoid
â-glycosidases have been described from the seeds of Dalbergia
cochinchinensis Pierre (Thai rosewood) and Dalbergia nigre-
scens Kurz (Thai blackwood) (17-19). These enzymes had high
hydrolytic activity on isoflavonoid glycosides from the same
seeds. D. cochinchinensis â-glucosidase had high substrate
specificity toward dalcochinin-8′-O-â-glucoside (Figure 1),
*
Author to whom correspondence should be addressed (telephone +66
4
4 224304; fax +66 44 224185; e-mail cairns@sut.ac.th).
†
Suranaree University of Technology.
Mahidol University.
§
1
0.1021/jf062885p CCC: $37.00
© 2007 American Chemical Society
Published on Web 02/21/2007

2408 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Chuankhayan et al.
Figure 1. Structures of soybean isoflavonoid glycoside natural substrates of D. nigrescens and D. cochinchinensis
â-glucosidase.
which has an aglycone structure similar to that of rotenone, a
natural insecticide and pesticide (18). D. nigrescens â-glucosi-
dase efficiently hydrolyzed its diglycoside natural substrates
dalpatein 7-O-â-D-apiofuranosyl-(1,6)-â-D-glucopyranoside and
dalnigrein 7-O-â-D-apiofuranosyl-(1,6)-â-D-glucopyranoside (Fig-
ure 1) to release a â-1,6-apiosylglucose (acuminose) disaccha-
ride unit and the aglycones (19). It could also hydrolyze genistin
and daidzin, which are isoflavonoid 7-O-â-D-glucosides. In this
study, we evaluate the potential of Dalbergia â-glucosidases
for release of isoflavones in soy products by comparing their
ability to hydrolyze conjugated and nonconjugated isoflavonoid
glycosides in soybean extracts.
extracts treated with the Dalbergia â-glucosidases. Separation and
quantification of isoflavonoids were achieved with an Eclipse XDB-
C18 (4.6 × 250 mm, 5 µm) reverse phase column on an HP series
1
100 HPLC (Agilent Corp., Palo Alto, CA) with the UV detector set
at a wavelength of 260 nm, in a manner similar to that previously
described for the separation of soy isoflavones (20). Solvent A was
0
.1% phosphoric acid in water, and solvent B was acetonitrile. The
sample was injected in 10% solvent B, which was held for 5 min and
then increased in a linear gradient from 10 to 35% B over 45 min. The
flow rate was 0.8 mL/min.
Peaks of soy isoflavone glucosides and aglycones were identified
by matching retention times with isoflavonoid standards. Malonyldaid-
zin, acetyldaidzin, and acetylgenistin were tentatively identified by
comparison of the relative retention times of the peaks with those in
published HPLC methods (20). Relative amounts were calculated from
relative peak areas, because all peaks were well within the linear range
of the instrument.
MATERIALS AND METHODS
Materials. High-performance liquid chromatography (HPLC) grade
methanol, acetonitrile, and water were purchased from Fisher Scientific
Hydrolysis of Crude Soy Flour Extract. To compare the hydrolysis
efficiencies of D. nigrescens â-glucosidase, D. cochinchinensis â-glu-
cosidase, and almond â-glucosidase toward the isoflavonoid glycosides
in crude soy flour extract, 10 µL aliquots of crude soybean extract
were hydrolyzed with 0.001 unit of each â-glucosidase in 100 µL of
0.1 M sodium acetate, pH 5.5. The reaction mixtures were incubated
at 37 °C for 10 min or 16 h, and the reaction was stopped by boiling
for 5 min. The stopped reactions were dried by speed vacuum and
resuspended in 100 µL of 10% acetronitrile in 0.1% phosphoric acid/
water. A control reaction of crude extract without enzyme was set up
in the same manner.
(Hanover Park, IL). Isoflavone standards of daidzin, daidzein, genistin,
genistein, malonylgenistin, and glycitin were purchased from LC
Laboratories (Woburn, MA). Glycitein standard was produced by
digestion of glycitin with D. nigrescens â-glucosidase. All other
chemicals and reagents were of analytical grade.
Crude Soy Flour Extraction. Ten grams of defatted soybean flour
from ADM Protein Specialties (Decatur, IL) was extracted with 40
mL of 80% methanol by stirring overnight at room temperature. The
solid was removed from the extract supernatant by centrifugation at
1
2000 rpm for 15 min.
â-Glucosidase Enzymes. Dalbergia â-glucosidases were purified
Hydrolysis of Soy Flour Suspension. The defatted soybean flour
(0.15 g/mL) was suspended in 0.1 M sodium acetate, pH 5.5, and 200
µL aliquots of the suspension were incubated with and without 0.01
unit of D. nigrescens â-glucosidase. The reaction mixtures were
incubated at 37 °C for 10 min, and the reaction was stopped by boiling
for 5 min. The reactions were centrifuged at 12000 rpm for 5 min to
remove the supernatant, the solid was extracted with 80% methanol,
and the methanol extract was removed by centrifugation at 12000 rpm
for 5 min. The reaction supernatant and methanol extract were dried
by speed vacuum and resuspended in 20 µL of 10% acetronitrile in
0.1% phosphoric acid/water. The hydrolysis of daidzin and genistin in
the soy flour suspension supernatant and particles was evaluated by
thin-layer chromatography on analytical silica gel 60 F254 aluminum
plates (Merck, Darmstadt, Germany) with ethyl acetate/methanol/acetic
from D. cochinchinensis Pierre and D. nigrescens Kurz and seeds as
previously described (17, 19). Almond â-glucosidase was purchased
from Sigma Fine Chemicals (St. Louis, MO). Quantification of protein
was by Coomassie brilliant blue staining (Bio-Rad Protein Assay, Bio-
Rad Laboratories, Hercules, CA) with bovine serum albumin (BSA,
frac 5, GE Healthcare, Uppsala, Sweden) as a reference, and enzyme
activity was quantified by p-nitrophenol release from p-nitrophenyl â-D-
glucoside (pNPG), as previously described (19). One unit was defined
as the amount of enzyme that hydrolyzes 1 µmol of pNPG per minute
at 1 mM pNPG and 30 °C in 0.1 M sodium acetate, pH 5.5.
HPLC Analysis of Soy Isoflavones. HPLC analysis was used to
measure the amounts of daidzein, genistein, daidzin, genistin, and
malonylgenistin to quantify changes in isoflavone content of crude

Soybean Isoflavonoid Glycosides
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2409
Table 1. Comparison of the Effects of 10 min and Prolonged
Incubation of Dalbergia
Soy Flour Extract Isoflavones
â
-Glucosidases and Almond
â
-Glucosidase on
a
%
hydrolysis activity of â-glucosidases
control
D. nigrescens
D. cochinchinensis
almond
16 h
soybean
glycoside
10
10
10
min 16 h
min
16 h
min
16 h
daidzin
glycitin
genistin
malonyldaidzin
acetyldaidzin
malonylgenistin
acetylgenistin
1
9
1
12
0
0
9
10
8
12
3
100
100
100
8
100
63
100
100
100
60
100
96
52
5
62
0
0
0
100
67
98
10
44
12
47
74
33
70
12
14
12
18
14
7
4
100
100
0
a
The crude soy flour methanol extract was hydrolyzed with 0.001 unit of the
C in 0.1 M sodium acetate, and the
â
-glucosidases for the indicated times at 37
°
amounts of isoflavones were quantified by analytical reverse phase HPLC, as
described under Materials and Methods.
RESULTS AND DISCUSSION
Figure 2. HPLC separation of isoflavonoids in crude soybean flour
methanol extract: (A) crude soybean flour methanol extract (peaks: 1,
daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin;* 5, acetyldaidzin;* 6,
malonylgenistin; 7, daidzein; 8, acetylgenistin;* 9, genistein; 10, glycitein);
Hydrolysis of Crude Soy Flour Extract. To evaluate the
usefulness of D. cochinchinensis and D. nigrescens â-glucosi-
dases for the hydrolysis of soy isoflavone glycosides, the
enzymes were compared for digestion of the glycosides in soy
flour extracts. Defatted soy flour was extracted with methanol,
and the crude methanol extract was separated and analyzed by
HPLC, as shown in Figure 2A. The peaks of daidzin, genistin,
malonylgenistin, glycitin, daidzein, and genistein were identified
in the crude extract by comparison with commercial standards,
whereas glycitein aglycone was identified by digestion of
glycitin with D. nigrescens â-glucosidase, which gave complete
digestion (data not shown). Three additional isoflavonoid
glycosides (malonyldaidzin, acetyldaidzin, and acetylgenistin)
could be provisionally identified by comparison of their relative
elution positions with those in a previous paper (20). Genistin
and daidzin were the most predominant isoflavonoids in the
crude soy flour extract, whereas the peaks for their aglycones
were very small. Actually, malonylglucosides are expected as
the predominant isoflavone in unprocessed soybean, but in the
processed flour evaluated here, the glucosides were present in
higher amounts. The content and composition of isoflavones
can be changed by various processing conditions, and previous
studies suggest that malonylglucosides can easily be converted
to less conjugated forms depending on the thermal conditions
of processing and preparation (22, 23).
(
B, C) crude soybean flour methanol extract after 10 min of hydrolysis
reaction with 0.001 unit of D. nigrescens -glucosidase (B) or
D. cochinchinensis -glucosidase (C) in 0.1 M sodium acetate, pH 5.5.
â
â
The digested and undigested methanol extracts were separated on
analytical C18 reverse phase HPLC as describedunder Materials and
Methods. * These peaks were provisionally identified on the basis of their
relative elution times in comparison to a previous study that used a similar
solvent system and HPLC column (20).
acid/water (15:1:2:2) (v/v) as solvent. Isoflavonoids were visualized
by absorbance under UV light and identified by comparison to
commercial standards.
Kinetic Studies. The kinetic properties of both Dalbergia â-glu-
cosidases toward soybean isoflavonoid glycosides were determined by
incubating genistin, malonylgenistin, and daidzin at five or six
concentrations in the range of 0.035-1 mM, depending on the K seen
m
in preliminary experiments and whether substrate inhibition was seen
at higher concentrations, in 50 µL reactions containing 5% DMSO in
0
.1 M sodium acetate, pH 5.5, with 0.45 ng of D. nigrescens or 10 ng
of D. cochinchinensis â-glucosidase at 30 °C. These enzyme concentra-
tions were determined to convert <10% of the substrates to products,
in order to ensure initial reaction rates were observed. All reactions
were done in duplicate. After 10 min, the reactions were stopped by
boiling for 5 min. The reactions were dried and resuspended in 50 µL
of 0.1% phosphoric acid in water (solvent A), and then 20 µL of each
sample was injected for HPLC analysis. The column was equilibrated
with 85% solvent A. After the sample was injected, ACN was increased
from 15 to 35% as a linear gradient over 45 min. The aglycone product
of each soybean isoflavonoid glycoside substrate was detected by
measuring the absorbance at 260 nm. The amounts of liberated
aglycones were calculated from standard curves of aglycone (genistein
and daidzein) peak areas over the 0.7-10 mmol range. Because
substrate inhibition was seen at some higher concentrations and
detection limits prevented the use of very low concentrations, nonlinear
regression of the Michaelis-Menten equation curves could not be used
As shown in Figure 2B, 0.001 unit of D. nigrescens
â-glucosidase hydrolyzed nearly all soybean isoflavonoid gly-
cosides that appeared in the crude extract within 10 min.
Daidzin, glycitin, genistin, acetyldaidzin, and acetylgenistin were
completely hydrolyzed by D. nigrescens â-glucosidase, although
only approximately 8% of malonyldaidzin and 60% of malo-
nylgenistin could be hydrolyzed in this time. D. cochinchinensis
â-glucosidase could also hydrolyze 52% of the daidzin, 5% of
the glycitin, and 60% of the genistin within the 10 min, but it
showed less hydrolysis of the conjugated forms of genistin and
daidzin (Figure 2C). When the reactions were extended up to
16 h to see whether the enzymes could hydrolyze the remaining
glycosides, D. nigrescens â-glucosidase hydrolyzed 60% of the
malonyldaidzin and achieved nearly complete hydrolysis of
malonylgenistin. By 16 h, D. cochinchinensis â-glucosidase had
hydrolyzed essentially all of the daidzin and genistin, but it still
showed little hydrolysis of the malonylated forms of daidzin
and genistin (Table 1). Essentially no hydrolysis was seen in
control reactions without added enzymes, demonstrating no
m
to estimate the K and kcat, so calculation of kinetic parameters with
standard errors were obtained from linear regression of the double-
reciprocal (Lineweaver-Burk) plots with the Enzfitter 1.05 program
(Elsevier Biosoft, Cambridge, U.K.).

2
410 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Table 2. Kinetic Properties of Purified D. nigrescens
D. nigrescens
Chuankhayan et al.
â
-Glucosidasea
â-glucosidase
D. cochinchinensis â-glucosidase
1
cat (s-1)
(M- s-1)
1
substrate
K
m
(mM)
0.3)
10-
0.11)
1.1)
k
cat (s-1)
k
cat/K
m
(M- s-1)
K
m
(mM)
k
k
cat/K
m
2
7
6
10-
1
106
genistin
daidzin
malonylgenistin
(6.7
(1.44
(5.2
±
±
±
×
700
300
21
±
±
±
15
13
2
(1.05
(2.08
(4.1
±
±
±
0.03)
0.03)
×
×
×
10
10
(1.26
1.57
(5.10
±
0.69)
×
×
208
50
1.88
±
99
(1.7
(3.22
(3.7
±
±
±
0.8)
0.18)
0.4)
×
-
1
4
×
10
10
10
±
±
0.09
1.09)
±
3
×
10
10
-2
5
10-
2
4
×
0.4)
±
0.18
×
a
Reactions were done in the standard reaction at 37 °C in 0.1 M acetate and stopped at 10 min when <10% of the substrate had been hydrolyzed. The isoflavone
aglycones were quantified by HPLC, as described under Materials and Methods, and the rates of product formation were calculated. Kinetic parameters and standard errors
were calculated from the linear regression of the Lineweaver Burk plots with the Enzfitter program.
−
Table 3. Relative Levels of Soy Glycosides and Aglycones before and
after 10 min of Digestion of Suspended Soybean Flour with or without
a
0.01 Unit of D. nigrescens â-Glucosidase
relative amount of isoflavone after reaction
0 min
with D. nigrescens
-glucosidase
1
10 min
control
0
min
â
glycoside or
aglycone
super-
natant
super-
natant
super-
natant
pellet
pellet
pellet
daidzin
glycitin
genistin
malonyldaidzin
acetyldaidzin
malonylgenistin
acetylgenistin
daidzein
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0
0
12
0
3
0
1710
467
1118
0
0
0
54
0
0
90
109
88
109
71
110
77
230
212
229
92
103
93
115
79
124
89
160
143
156
Figure 3. TLC analysis of hydrolysis of soybean flour suspension in 0.1
M sodium acetate buffer with 0.01 unit of D. nigrescens -glucosidase.
Standards and samples were separated on an analytical silica gel 60
254 aluminum plate and isoflavones visualized by absorbance of UV light,
as described under Materials and Methods. Lanes: 1, daidzin; 2, genistin;
, suspension reaction without enzyme at time 0; 4, suspension reaction
without enzyme after 10 min; 5, suspension reaction with 0.01 unit of
enzyme after 10 min; 6, methanol extract of the pellet from the suspension
reaction without enzyme at time 0; 7, methanol extract of the pellet from
the suspension reaction without enzyme after 10 min; 8, methanol extract
of the pellet from the suspension reaction with 0.01 unit of enzyme after
0
â
383
488
670
genistein
glycitein
F
3
a
Reactions were done in 0.1 M sodium acetate buffer with incubation at 37
C, and relative amounts were determined by HPLC, as described under Materials
and Methods. The amounts in the 0 time were set at 100%, and relative levels
°
(
relative HPLC peak areas) at other times are reported.
The kinetic properties of D. nigrescens and D. cochinchin-
10 min.
ensis â-glucosidases toward daidzin, genistin, and malonyl-
genistin were determined using HPLC to quantify the amounts
of products (Table 2). Previously, we had determined provi-
sional kinetic parameters of D. nigrescens â-glucosidase for
daidzin and genistin by the glucose oxidase assay (19). Because
daidzein and genistein are antioxidants that interfere with that
assay, the kinetics were repeated by HPLC. D. nigrescens
â-glucosidase has Km values approximately 6 times lower than
those of D. cochinchinensis â-glucosidase for daidzin and
genistin. Although D. cochinchinensis â-glucosidase has a lower
Km value for malonylgenistin than D. nigrescens â-glucosidase,
its kcat value was 200 times lower than that of D. nigrescens
â-glucosidase. Therefore, D. nigrescens â-glucosidase hydro-
lyzed soybean isoflavonoid glycosides more quickly than
D. cochinchinensis â-glucosidase (Table 2), which correlates
with the results in Figure 2, where Thai rosewood â-glucosidase
hydrolyzed daidzin and genistin more slowly than D. nigrescens
â-glucosidase and had little effect on malonylgenistin.
Hydrolysis of Soy Flour Suspension. The hydrolysis of
isoflavonoid glycoside in methanolic extracts of soy flour by
D. nigrescens â-glucosidase was promising, but it is not directly
relevant to food and feed processing. Therefore, hydrolysis was
evaluated in a suspension of soy flour in aqueous buffer. The
hydrolysis of soy flour extract was analyzed by thin-layer
chromatography as shown in Figure 3. The two most intense
spots, daidzin and genistin, were completely digested with
D. nigrescens â-glucosidase, and daidzin and genistin were not
detected after digestion in either the aqueous solution or the
active endogenous isoflavonoid â-glycosidases were present in
the methanolic extract.
Ismail and Hayes (13) reported that high amounts of E. coli
and almond â-glucosidases could hydrolyze genistin and daidzin,
but those enzymes showed little hydrolysis of conjugated
glycosides, even when the amount of the enzyme was increased
up to 6 units and the digestion time to 24 h for E. coli
â-glucosidases and to 30 units and 4 h for almond â-glucosi-
dases. For comparison, we also tested almond â-glucosidase at
the prolonged time and found it could hydrolyze most of the
daidzin and genistin, but hydrolyzed little of the conjugated
forms (Table 1). Although conversion between the conjugated
and nonconjugated forms can occur during processing (24), the
activity of the D. nigrescens â-glucosidase toward conjugated
forms is a useful property. However, both Dalbergia â-glu-
cosidases had highest activity toward daidzin and genistin, as
previously reported for almond, E. coli, and soybean root
â-glucosidases (13, 15). Although both Dalbergia enzymes had
much higher activity than almond â-glucosidase toward both
daidzin and genistin, D. nigrescens â-glucosidase showed higher
activity than D. cochinchinensis â-glucosidase, for which two
small peaks of daidzin and genistin were still observed after
the 10 min digestion (Figure 1C). This experimental result could
be confirmed by the kinetic properties of these enzymes toward
isoflavone glycosides.

Soybean Isoflavonoid Glycosides
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2411
methanol extract of the flour particles left after aqueous
extraction. The hydrolysis of isoflavonoids in the flour particles
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(lanes 8) indicated that D. nigrescens â-glucosidase could
(
(
(
4) Hendrich, S.; Lee, K. W.; Ku, X.; Wang, H. J.; Murphy, P. A.
efficiently hydrolyze these isoflavonoid glycosides inside the
particles or that they are in equilibrium with those free in
solution. The hydrolysis of genistin and daidzin could also be
seen to a lesser extent in controls, which can occur when
soybean flour is soaked in water, probably by an endogenous
soybean â-glucosidase (27). This result was confirmed by HPLC
of the reactions with and without D. nigrescens â-glucosidase
Defining food components as new nutrients. J. Nutr. 1994, 124,
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789S-1792S.
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(Table 3). D. nigrecsens â-glucosidase (0.01 unit) completely
hydrolyzed almost all isoflavonoid glycosides in the suspended
soy flour within 10 min at 37 °C, including both those in solution
and those associated with the insoluble particles. Only malo-
nyldaidzin remained in significant amounts of 12 and 54% of
the initial levels in the solution and particles, respectively. This
hydrolysis resulted in increases of approximately 17-fold in
daidzein, 5-fold in genistein, and 11-fold in glycitein in the
solution and in approximately 4-, 5-, and 6-fold increases of
daidzein, genistein, and glycitein in the particles, respectively.
The soy flour also appeared to have endogenous activity, which
hydrolyzed up to 29% of acetyldaidzin, 23% of acetylgenistin,
(7) Albertazzi, P.; Purdie, D. The nature and utility of the phy-
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2
03.
(
10) Hessler, P. E.; Larsen, P. E.; Constantinou, A. I.; Schram,
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1
2% of genistin, and 10% of daidzin in the solution and lower
3
98-404.
amounts in the particles, but did not affect malonylglucoside
levels. This hydrolysis in the control resulted in increases of
(11) Tsangalis, D.; Ashton, J. F.; McGill, A. E. J.; Shah, N. P.
Enzymatic transformation of isoflavone phytoestrogens in soymilk
by â-glucosidase-producing bifidobacteria. J. Food Sci. 2002,
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genistein in soy protein concentrate with â-glucosidase. J. Food
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<2.5-fold of each isoflavone aglycone in the solution and <2-
fold in the particles. Thus, addition of D. nigrescens â-glucosi-
dase greatly increased the release of free isoflavones in the
suspended soy flour.
(
(
(
It is noteworthy that D. nigrescens â-glucosidase is able to
hydrolyze almost all isoflavonoid conjugated and nonconjugated
glycoside forms in the crude soy flour extract and suspended
soy flour in a short time. D. cochinchinensis â-glucosidase could
also hydrolyze some of these glycosides, although less rapidly
than D. nigrescens. This may correlate with the differences in
their natural substrates, because D. nigrescens are 7-O-glyco-
sides, as are the soybean isoflavonoids, whereas D. cochinchin-
ensis hydrolyzes a rotenoid glycoside. The hydrolysis of
isoflavonoid glycosides may increase their conversion to isofla-
vones (aglycone), which have bioavailability and health benefits
13) Ismail, B.; Hayes, K. â-Glycosidase activity toward different
glycosidic forms of isoflavones. J. Agric. Food Chem. 2005, 53,
4
918-4924.
14) Hsieh, M. C.; Graham, T. L. Partial purification and characteriza-
tion of soybean â-glucosidase with high specificity towards
isoflavone conjugates. Phytochemistry 2001, 58, 995-1005.
(15) Suzuki, H.; Watanabe, R.; Fukushima, Y.; Fujita, N.; Noguchi,
A.; Yokoyama, R.; Nishitani, K.; Nishino, T.; Nakayama, T. An
isoflavone conjugate-hydrolyzing â-glucosidase from the roots
of soybean (Glycine max) seedlings. J. Biol. Chem. 2006, in
press.
(
(
16) Thai Forest Bulletin (Botany), 2002.
(26-29). D. nigrescens â-glucosidase is a very stable enzyme
17) Srisomsap, C.; Svasti, J.; Surarit, R.; Champattanachai, V.;
Sawangareetrakul, P.; Boonpuan, K.; Subhasitanont, P.; Chokcha-
ichamnankit, D. Isolation and characterization of â-D-glucosidase/
â-D-fucosidase from Dalbergia cochinchinensis Pierre. J. Bio-
chem. 1996, 119, 585-590.
with a temperature optimum of 65 °C, so it should be useful
for industrial processes. Some evidence of endogenous soybean
isoflavonoid â-glucosidase was evident in the processed soy
flour, which suggests that it is also somewhat heat stable, but
this residual activity was much less than that of the small amount
of D. nigrescens enzyme added to the digest. This research
suggests that D. nigrescens â-glucosidase may be useful for
processing of soy foods to enhance their nutritional and
economic value.
(
18) Svasti, J.; Srisomsap, C.; Techasakul, S.; Surarit, R. Dalcochinin-
8′-O-â-D-glucoside and its â-glucosidase enzyme from Dalbergia
cochinchinensis. Phytochemistry 1999, 739-743.
(19) Chuankhayan, P.; Hua, Y.; Svasti, J.; Sakdarat, S.; Sullivan,
P. A.; Ketudat-Cairns, J. R. Purification of an isoflavonoid 7-O-
â-apiosyl-glucoside â-glycosidase and its substrates from Dal-
bergia nigrescens Kurz. Phytochemistry 2005, 66, 1880-1889.
20) Gerhauser, C.; Klimo, K.; Heiss, E.; Neumann, I.; Gamal-Eldeen,
A.; Knauft, J.; Liu, G. Y.; Sitthimonchai, S.; Frank, N. Mech-
anism-based in vitro screening of potential cancer chemopre-
ventive agents. Mutat. Res. 2003, 523-524, 163-172.
ACKNOWLEDGMENT
(
We thank Dr. Sunanta Tongta for providing soy flour and advice
on soy isoflavones and Dr. Prachumporn Toonkool for helpful
discussions.
(
21) Zhang, Y. C.; Lee, J. H.; Vodovotz, Y.; Schwartz, S. J. Changes
in distribution of isoflavones and â-glucosidase activity during
soy bread proofing and baking. Cereal Chem. 2004, 81, 741-
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Received for review October 7, 2006. Revised manuscript received
December 20, 2006. Accepted January 3, 2007. This work was supported
by the grants from the Thailand Research Fund (BRG4780020 and
RTA 4780006) and from Suranaree University of Technology.
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JF062885P