Food Chemistry 152 (2014) 56–65

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Optimisation of soy flour fermentation parameters to produce

b-glucosidase for bioconversion into aglycones
C.L. Handa a, U.R. Couto a, A.H. Vicensoti a, S.R. Georgetti b, E.I. Ida a,⇑
a
Departamento de Ciência e Tecnologia de Alimentos, Universidade Estadual de Londrina, 86057-970 Londrina, Paraná, Brazil
b
Departamento de Ciências Farmacêuticas, Universidade Estadual de Londrina, 86057-970 Londrina, Paraná, Brazil

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

Article history: The solid state fermentation (SSF) parameters of defatted soybean flour (DSF) with Aspergillus oryzae IOC
Received 3 June 2013 3999/1998 or Monascus purpureus NRRL 1992 was evaluated using a rotational central composite exper-
Received in revised form 16 October 2013 imental design to optimise the production of b-glucosidase and convert glycosidic isoflavones in agly-
Accepted 19 November 2013
cones. Variables investigated were initial pH of DSF, volume of water added to 10 g of DSF and
Available online 27 November 2013
incubation temperature. b-Glucosidase activity was measured using the synthetic substrate, p-nitro-
phenyl-b-D-glucoside. The content of isoflavones was determinate by ultra performance liquid chroma-
Keywords:
tography. The highest production of b-glucosidase for both strains occurred when adding 10 mL of
Soybean flour
Solid state fermentation
water to the DSF, incubating at 30 °C and using 6.0 as the initial DSF pH. A. oryzae IOC 3999/1998
b-Glucosidase expressed b-glucosidase activity at 10.7 times higher than M. purpureus NRRL 1992. The DSF fermentation
Isoflavones was more efficient in converting isoflavones with M. purpureus NRRL 1992.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction and type of processing (heat treating, baking, enzyme hydrolysis

Soybeans contain several compounds of biological interest,
including phytosterols, protease inhibitors, saponins and isoflav-
ones. Soybean isoflavones have attracted attention because of their
ability to reduce cardiovascular disease risk, inhibit cancer cell
growth, prevent several diseases, such as osteoporosis, and allevi-
ate the symptoms of menopause. Isoflavone effects are strongly
influenced by their chemical structures (Chen et al., 2012).
Isoflavones occur in four distinct chemical forms, namely agly-
cones (daidzein, genistein and glycitein), b-glycosides (daidzin,
genistin and glycitin), acetyl glycosides (600 -O-acetyl dadzin, 600 -
O-acetyl genistin and 600 -O-acetyl glycitin) and malonyl glycoside
conjugates (600 -O-malonyl daidzin, 600 -O-malonyl genistin and 600 -
O-malonyl glycitin), for a total of twelve different forms. b-glyco-
sidic forms have a glucose molecule bound to position seven of
the benzene ring, and the conjugated forms are esterified at carbon
6 of the glucose molecule (Liu, 1997). Among these different forms,
the glycosidic forms are predominant in soybeans, constituting
50–90% of the isoflavones. However, the aglycone forms, which
are present in lower amounts, have higher biological activity
(Izumi, Piskula, Osawa, Obata, & Tob, 2000). The content and com-
position of isoflavones in soy products depends on the condition
⇑ Corresponding author. Address: Km 380 Celso Garcia Cid Road, Campus
Universitário, CCA, DCTA, post box 10.011, CEP 86057-970 Londrina, Paraná, Brazil.
Tel./fax: +55 (43) 3371 4080.
E-mail address: elida@uel.br (E.I. Ida).
or fermentation) (Wang & Murphy, 1994).
Aglycones are potentially more bioactive and absorbable in the
intestine than their glycosidic forms. Therefore, many soy products
have been enriched with aglycones that were obtained by different
processes to convert glycosidic into aglycone forms (Izumi et al.,
2000). These processes can occur through the action of enzymes
obtained from sources such as microorganisms (Pham & Shah,
2009), plants or animals. b-Glucosidase produced by fermentation
has advantages over the others in relation to its low cost and easy
large-scale production (Langston, Sheehy, & Xu, 2006). However,
the conversion of different isoflavone chemical forms depends on
the type of microorganism and its hydrolysis potential (Otieno,
Ashton, & Shah, 2006). Furthermore, glycosidic bond hydrolysis
of isoflavones by enzymes also depends on the time and tempera-
ture of fermentation (Matsuura & Obata, 1993).
During the process of semi-solid or solid state fermentation
(SSF), microorganisms are grown on the surface of solid materials
with limited amounts of water and can be used to obtain enzymes.
The culture media should be favourable for growth and contain all
the nutrients necessary for cell synthesis and desired product for-
mation (Ward, 1991). The production of fungal b-glucosidase by
SSF depends on the pH and initial substrate moisture, temperature
and time of incubation (Qian, Fu, Zhou, Sun, & Weng, 2012). There-
fore, to increase the SSF process yield, it is important to optimise
the physical and chemical parameters (Francis et al., 2003).
As a consequence of their physiological and biochemical prop-
erties, filamentous fungi are better adapted to grow during the
SSF process. Aspergillus oryzae is a fungus used largely in Japan,

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.11.101
 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65
mainly for making fermented products, such as soy sauce, sake,
vinegar and spices. Because of its potential to produce various
enzymes, this fungus is widely used in modern biotechnology
(Machida, Yamada, & Gomi, 2008). Aspergillus fungi species are also
among the most promising for b-glucosidase production
(Solovyeva, Ananjin, Boev, & Okunev, 1997). Solid state fermenta-
tion with A. oryzae in soya flour for 48 h yielded significantly
increased isoflavone aglycones, from 6.94% to 75.51% (Silva,
Celeghini, & Chang, 2011). Monascus purpureus is a fungus tradi-
tionally used in East Asia to produce fermented food and products
that are used as food pigments or biological agents (Lee, Yang, &
Mau, 2008). Its use as a b-glucosidase producer had only been
evaluated by Daroit, Silveira, Hertz, and Brandelli (2007) with a
submerged fermentation with grape waste and peptone as
nitrogen sources.
In considering potential b-glucosidase production for hydroly-
sing the b-glycosidic linkages of isoflavones, this study aimed to
optimise the process parameters of solid state fermentation (SSF)
with A. oryzae IOC 3999/1998 or M. purpureus NRRL 1992 in defat-
ted soy flour (DSF), by using a rotation central composite design
(RCCD) to produce b-glucosidase and to evaluate the bioconversion
of glycosidic isoflavones into aglycones.
2. Material and methods
2.1. Materials
The DSF substrate for the SSF was purchased from Industry
Foods Brazil (Curitiba, Brazil). The chemical composition of DSF
was 8.95% moisture, 1.07% fat, 48.96% protein (N  6.25), 5.98%
ash and 35.04% carbohydrates determined by the difference from
the other constituents. A. oryzae IOC 3999/1998 fungus was used
for fermentation, and it originated from the Oswaldo Cruz Founda-
tion (Fiocruz, RJ), and the M. purpureus NRRL 1992 fungus
(GenBank: JQ614061.1) came from the Laboratory of Biochemistry
and Microbiology at the Institute of Applied Science and Food
Technology at the Federal University of Rio Grande do Sul, Porto
Alegre, RS, Brazil.
The ultra performance liquid chromatography (UPLC) included
standard isoflavones (malonyl conjugates and acetyl conjugates)
from Wako Pure Chemical Industries (Osaka, Japan). Isoflavones
(b-glucosides and aglycones), the synthetic substrate p-nitro-
phenyl-b-D-glucopyranoside (p-NPG) and 4-methylumbellipheryl
b-D-glucopyranoside (4-MUG) were used to determine b-glucosi-
dase activity, both of which were of chromatographic grade and
were obtained from Sigma Aldrich (St. Louis, USA). The other re-
agents were of analytical grade from different sources.
2.2. Reactivation of microorganisms and obtaining spore suspensions
A. oryzae IOC 3999/1998 or frozen M. purpureus NRRL 1992 fun-
gi were left at room temperature, activated in Sabouraud broth and
incubated at 30 °C for seven days. They were then transferred to in-
clined potato dextrose agar (PDA) medium and incubated at 30 °C
for seven days and then used for fermentation. To count the spores,
5 mL of 2% Tween 20 (v/v) was added to the inoculant. The spores
from the surface of the culture medium were suspended with the
aid of a glass rod. The suspension was diluted by 1:10 (v/v) and
the spore count was performed in a Neubauer chamber.
2.3. Rotation central composite design (RCCD) and solid state
fermentation of DSF
To evaluate the DSF fermentation parameters with A. oryzae IOC
3999/1998 or M. purpureus NRRL 1992 in terms of b-glucosidase
57
production, RCCD was applied with three variables and replicates
at the central point (Table 2). The coded independent variables
(x1, x2, x3) and uncoded variables (X1 = initial pH of DSF, X2 = mL
water added to 10 g of DSF and X3 = °C incubation) are shown in
Table 1 with their variation levels. The RCCD was conducted in 2
blocks (Table 2), and exploratory modelling block 1 was conducted
with 11 random assays (9 factorial points and 3 central points).
Modelling was conducted in block 2 with 9 random assays (6 axial
points and 3 central points) for optimisation, for a total of 20 assays
in the two blocks.
For each assay, SSF employed 10 g of DSF and the initial pH (X1)
was adjusted according to the levels presented in Table 1. The
amount of water added to DSF (X2) (Table 1) was previously pre-
pared by using distilled water with its pH adjusted by the addition
of aliquots of 1 M hydrochloric acid or 1 M sodium carbonate until
the pH (X1) of the DSF reached the correct levels (Table 1). After
water was added, the DSF (X2) was homogenised and the material
was uniformly distributed into 250 mL Erlenmeyer flasks and ster-
ilised by autoclaving at 121 °C for 15 min. When the medium
reached room temperature, a suspension of 107 spores was spread
evenly over the surface of each sample and incubated (Fanem,
mod. 347F, Sao Paulo, Brazil) for 48 h at different incubation tem-
peratures (X3) according to pre-determined levels (Table 1). After
incubation, the suitably fermented DSF preparations were frozen,
freeze-dried, ground and stored at 5 °C until the time of analysis.
To monitor tests (Table 2) for the production of b-glucosidase, a
control was used in parallel to each assay without an added sus-
pension of fungal spores.
The production of fungal b-glucosidase was evaluated by its re-
sponse functions as follow: YM or YA (YA = AU b-glucosidase from
DSF fermented with A. oryzae IOC 3999/1998. g1 sample or
YM = UA b-glucosidase from DSF fermented with M. purpureus
NRRL 1992.g1 sample). The model equation was as follows:
Y ¼ b0 þ bj þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x21 þ b22 x22 þ b33 x23
þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3 þ e ð1Þ
where Y (response function), x1, x2 and x3 (coded variables), b (esti-
mated coefficients for each term of the response surface model) and
bj (estimated coefficient of the block for the response surface
model).
The response functions (YA or YM) were used to perform regres-
sion analyses and analyses of variance (ANOVA) for the regression.
The equation model was fitted to experimental data to yield the
proposed model. Response surface graphs and desirability param-
eters were generated for each response function (YA or YM). All exe-
cuted analysis, desirability and response surfaces were performed
with STATISTICA 8.0 software (StatSoft Inc., 2007).
After response surface analysis and graphing of the desirability
for b-glucosidase maximum activity, the proposed model was val-
idated by performing new assays in triplicate. The results (Yexp.)
were compared with the estimated response (ŷA or ŷM) by
Student’s t-test (p < 0.05).
2.4. Bioconversion of isoflavone glucosides in aglycones of DSF
fermented with A. oryzae IOC 3999/1998 or M. purpureus NRRL 1992
The bioconversion of isoflavone glucosides into aglycones with
DSF fermented by A. oryzae IOC 3999/1998 or M. purpureus NRRL
1992 under enzyme production conditions with maximum b-glu-
cosidase activity was evaluated by quantifying different isoflavone
forms by ultra performance liquid chromatography (UPLC). A con-
trol was used in parallel to the fermentation without an added
spore suspension. The different isoflavone form concentrations
from fermented DSF in the different fungi and controls were com-
pared by Tukey’s post hoc test.
58 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65

2.5. Extraction and determination of b-glucosidase activity
Extractions of b-glucosidase produced by A. oryzae IOC 3999/
1998 or M. purpureus NRRL 1992 were conducted using the meth-
odology described by Carrão-Panizzi and Bordignon (2000). The
b-glucosidase activity was measured as described by Matsuura
and Obata (1993). One activity unit (AU) of b-glucosidase was de-
fined as the amount of enzyme that freed 1 lmol of p-nitrophenol
(p-NP) per min under assay conditions. b-Glucosidase activity was
expressed as the AU of b-glucosidase from DSF fermented g1 of
fermented DSF or control. The response functions (YA or YM) were
expressed as the differences between the b-glucosidase activity
from fermented DSF and the control.
2.6. Effect of pH on the b-glucosidase activity
The enzyme extracts of DSF fermented by M. purpureus NRRL
1992 or A. oryzae IOC 3999/1998 fungi were obtained as described
in item 2.5. The optimum pH for activity was determined using the
substrate p-NPG, 0.1 M phosphate-citrate buffer, pH between 3.0
and 8.0 and intervals of 0.5 pH units at 30 °C and 30 min incuba-
tion. The activity of b-glucosidase was determined at different
pHs and each respective curve was built.
2.7. Electrophoresis and zymograms of b-glucosidase
To 25 mL of each enzyme extract, 100 mL of acetone at 20 °C
was added, before stirring for 1 h at 4 °C. The precipitate was sep-
arated and re-suspended in 0.1 M phosphate-citrate buffer, pH 5.5.
Samples were diluted in sample buffer (1:1, v/v) and 50–5 lL of
Table 1
Independent variables and levels of variation in RCCD.
Independent variables Levels of variation
1.68 1 0 1
X1 = initial pH of DSF 5.2 5.5 6 6.5
X2 = mL water added to DSF 2 5 10 15
X3 = temperature (°C) incubation 13 20 30 40
enzyme extract of the M. purpureus NRRL 1992 and A. oryzae IOC
3999/1998, respectively, were applied into polyacrylamide gel.
The polyacrylamide gel electrophoresis was performed using the
buffer system of Davis (1964) and as described by Georgetti et al.
(2009). The b-glucosidase activity was detected by zymography
as described by Ferreira, Terra, and Ferreira (2003) using 2 mM
4-MUG in 0.1 M phosphate-citrate buffer, pH 5.5 and incubation
at 30 °C for 30 min. UV light was used to visualise the fluorescent
bands to confirm the activities of b-glucosidase.
2.8. Quantification of isoflavones by UPLC
The fermented DSF and controls stored at 5 °C were defatted
with hexane (1:10 w/v) for 30 min at room temperature by contin-
uous rotary agitation and then vacuum filtered. The isoflavone
extraction was performed using a mixed solvent extractor contain-
ing ultra pure water, acetone and ethanol (1:1:1, v/v/v), as de-
scribed by Yoshiara, Madeira, Delaroza, Silva, and Ida (2012). The
extraction was performed in triplicate with 0.3 g of fermented
and unfermented DSF (control) with 6 ml of organic solvent, and
then stirred each 15 min for 1 h at room temperature. The mixture
was then placed in an ultrasonic bath for 15 min at room temper-
ature, centrifuged (2500g at 4 °C and 15 min) (Centrifuge 5804R -
Eppendorf, Hamburg, GE) and filtered (Millex filter-H (0.22 lm).
Triplicate aliquots of 1.4 lL of filtrate were automatically injected
into the Waters liquid chromatography UPLC (Acquity UPLCÒ
System, Waters, United States). The column was a reversed-phase
type (model ACQUITY-UPLC BEH C18, Waters, United States) with
dimensions of 2.1 mm  50 mm and a particle size of 1.7 lm. Elu-
tion was performed with a non-linear gradient using mobile phase
A, containing acidified water at pH 3.0 and then adjusted with gla-
cial acetic acid, and mobile phase B, containing acetonitrile applied
at a flow of 0.7 mL min1 at 35 °C. The gradient began with 90%
eluent A and 10% eluent B; the 8 min gradient elution ratio ulti-
mately reached 0% A and 100% B. The initial conditions returned
in 9 min with a total run time of 10 min. The detector was a diode
1.68
array (Waters) with a 260 nm wavelength. Standard solutions for
6.8 calibration curve construction (peak area  isoflavone content)
18
were made for daidzin, genistin, glycitin, malonyl daidzin, malonyl
47
genistin, glycitin malonyl, acetyl daidzin, acetyl genistin, acetyl

Table 2
RCCD for DSF fermentation with A. oryzae IOC 3999/1998 or M. purpureus NRRL 1992 and response functions YA and YM.

Assays Independent variables coded and uncoded Response functions

x1 (X1) x2 (X2) x3 (X3) YA YM
1 1(5,5) 1(5) 1(20) 0.000 0.122
2 1(5,5) 1(5) +1(40) 0.015 0.128
3 1(5,5) +1(15) 1(20) 0.000 0.000
4 1(5,5) +1(15) +1(40) 0.152 0.216
5 +1(6,5) 1(5) 1(20) 0.000 0.008
6 +1(6,5) 1(5) +1(40) 0.408 0.007
7 +1(6,5) +1(15) 1(20) 0.021 0.000
8 +1(6,5) +1(15) +1(40) 0.282 0.122
9 0(6,0) 0(10) 0(30) 2.496 0.200
10 0(6,0) 0(10) 0(30) 2.598 0.202
11 0(6,0) 0(10) 0(30) 2.867 0.209
12 1.68(5,2) 0(10) 0(30) 3.641 0.187
13 +1.68(6,8) 0(10) 0(30) 3.142 0.075
14 0(6,0) 1.68(2) 0(30) 0.003 0.003
15 0(6,0) +1.68(18) 0(30) 1.602 0.240
16 0(6,0) 0(10) 1.68(13) 0.005 0.000
17 0(6,0) 0(10) +1.68(47) 0.007 0.010
18 0(6,0) 0(10) 0(30) 2.321 0.281
19 0(6,0) 0(10) 0(30) 2.826 0.280
20 0(6,0) 0(10) 0(30) 2.717 0.214

X1(initial pH of DSF); X2 (mL water added to DSF); X3 (°C incubation) and YA or YM (YA = AU of b-glucosidase inDSF-fermented with Aspergillus oryzae g1 sample or YM = AU
b-glucosidase in DSF-fermented with Monascus purpureus g1 sample).
 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65 59

glycitin, glycitein and genistein (0, 1, 0.025, 0.006 and
0.002 mg mL1) and daidzein (0.1, 0.025, 0.006, 0.002 and
0.001 mg mL1). The standards were injected in triplicate to yield
the corresponding chromatograms for the chemical isoflavone
forms, each with their corresponding retention times. The peaks
for each type of isoflavone compound sample were identified by
comparing the retention times and UV spectrum in the respective
reference standard regions. The application was coupled in the
chromatograph-generated calibration curves and isoflavone con-
centrations were calculated and expressed in mg of isoflavones
per g of DSF, control or fermented DSF.
2.9. Specificity of b-glucosidase activity from M. purpureus NRRL 1992
and A. oryzae IOC 3999/1998 in relation to isoflavones extract from
DSF
The specificity of b-glucosidase activity of each fungus was ana-
lysed using as 50 mL of isoflavones extract of the DSF obtained
according to item 2.6. To eliminate the solvents, route evaporator
was used until the volume was 13 mL. To it was added 37 mL of
0.1 M phosphate-citrate buffer, pH 5.5. The specificity of b-glucosi-
dase activity was determined under optimal conditions of enzyme
reaction using 800 lL of isoflavones extract of the DSF and 200 lL
of enzyme extract containing 0.4 AU g1 sample. After 30 min of
incubation the reaction was stopped by the addition of 2 mL of
methanol. Control was used containing only the enzyme extraction
buffer. Then the contents of different forms of isoflavones were
measured according to item 2.8.
3. Results and discussion
3.1. Effects of DSF fermentation conditions with A. oryzae IOC 3999/
1998 on b-glucosidase production
From the exploratory model of the first RCCD block (Table 2),
the ANOVA and the regression analysis, the effects of variables X1
(initial pH of DSF), X2 (mL water added to 10 g of DSF) and X3 (tem-
perature in °C incubation) were observed. None of the interactions
(X1X2, X1X3, X2X3 and X1X2X3) were significant and the coefficient of
determination (R2) was 0.01. By considering the curvature and per-
forming a new regression analysis and ANOVA (Table 3), only the
curvature was found to be significant (p < 0.05), with a high R2 of
0.99. This finding indicated that the linear model was not adequate
to explain the effects of the three variables (X1, X2 and X3) on re-
sponse function YA (AU b-glucosidase from DSF fermented with
A. oryzae g1 sample). Thus, to enlarge the levels of investigated
variables and optimise the process, assays were performed on
block 2, which contained the 6 axial points and 3 central points.
A regression analysis and an ANOVA of response function YA (AU
b-glucosidase DSF fermented with A. oryzae g1 sample) indicated
that independent variables X2 (mL water added to the DSF) and X3
(°C incubation) showed significant quadratic effects and the nega-
tive sign.
In Table 2, the b-glucosidase activity in assay 12 was greater
(YA of 3.641 AU b-glucosidase from DSF fermented with A. oryzae
g1 sample), during which the fermentation conditions were as fol-
lows: an initial pH of 5.2 for DSF (X1), 10 mL of water added to the
DSF (X2) and 30 °C incubation (X3). However, assay 13 and the cen-
tre points of blocks 1 and 2 also yielded high b-glucosidase activi-
ties, although these tests differ only in variable X1 with initial DSF
pHs of 6.8 (assay 13) and 6.0 (centre points blocks 1 and 2). These
observations demonstrated that enzyme production with b-gluco-
sidase activity (YA) was independent of the pH in the investigated
range, confirming that variable X1 (initial pH of DSF) had no
significant effect on the production of enzyme activity with
b-glucosidase during the SSF of DSF.
Thus, the proposed model (Eq. (2)) can be described as follows:
^A ¼ 2:68508 þ 0:40114  0:87688x2
y 2
2  1:15850x3 ð2Þ
where ŷA (AU b-glucosidase in DSF-fermented with A.oryzae g1
sample) and x2 (variable coded X2 = mL of water added to DSF), x3
(variable coded X3 = °C incubation) and * terms were statistically
significant (p < 0.05). The model showed a lack of fit, which was
not significant (p > 0.05) and an R2 of 0.91, indicating that 91% of
the experimental results adequately matched with the proposed
model, so the model can be used for predictive purposes.
Analysing the mathematical model (Eq. (2)) response function
YA (AU b-glucosidase from DSF-fermented g1 sample) and

Table 3
ANOVA for b-glucosidase activity produced by Aspergillus oryzae IOC 3999/1998 or Monascus purpureus NRRL 1992, Exploratory Block.

Source of variation SS DF MS F p value*

YA Curvature 14.1218 1 14.1218 384.104 0.003
x1 0.0371 1 0.0371 1.009 0.421
x2 0.0001 1 0.0001 0.003 0.959
x3 0.0874 1 0.0874 2.378 0.263
x1.x2 0.0073 1 0.0073 0.198 0.700
x1.x3 0.0316 1 0.0316 0.859 0.452
x2.x3 0.0000 1 0.0000 0.000 0.987
x1.x2.x3 0.0100 1 0.0100 0.271 0.654
Error 0.0735 2 0.0368
Total 14.3688 10
YM Curvature 0.0360 1 0.0360 1549.380 0.001
x1 0.0135 1 0.0135 579.832 0.002
x2 0.0007 1 0.0007 28.726 0.033
x3 0.0147 1 0.0147 632.628 0.002
x1.x2 0.0025 1 0.0025 106.640 0.009
x1.x3 0.0012 1 0.0012 53.323 0.018
x2.x3 0.0139 1 0.0139 597.787 0.002
x1.x2.x3 0.0010 1 0.0010 41.613 0.023
Error 0.0000 2 0.0000
Total 0.0834 10
R2A = 0.99; R2M = 0.99

x1 (initial pH of DSF); x2 (mL water added to DSF); x3 (°C incubation) and YA or YM (YA = AU of b-glucosidase in DSF-fermented with Aspergillus oryzae g1 sample or YM = AU b-
glucosidase in DSF-fermented with Monascus purpureus g1 sample); R2 = determination coefficient.
*
significant (p < 0.05).
60 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65

Fig. 1. Response surface: (a) AU of b-glucosidase in DSF-fermented with A. oryzae g1 sample, where X1 (initial pH of DSF) was fixed at 6.0. (b, c and d) AU b-glucosidase in
DSF-fermented with- M. purpureus g1 sample, where (b) X3 (°C incubation) fixed at 30 °C; (c) X2 (mL water added to DSF) fixed at 10 mL (d) X1 (initial pH of DSF) fixed 6.0.

response surface in Fig. 1a, it was observed that there is a region in
which b-glucosidase activity is greater than 2.0 AU b-glucosidase in
DSF-fermented with A. oryzae g1 sample, i.e., x2 was between 0.8
and +0.8, or X2 was between 6.0 and 14.0 mL, and the water added
to the DSF and x3 was between 0.8 and +0.8 or X3 was between 22
and 38 °C during incubation. The desirability parameters (Fig. 2)
indicate that the maximum production of b-glucosidase activity
would be YA = 3.0862 AU of b-glucosidase from A. oryzae DSF-fer-
mented g1 sample, which occurred when x2 = 0 or X2 = 10.0 mL
water added to the DSF and x3 = 0 or X3 = 30 °C incubation. The
model was validated by conducting an assay in triplicate with X2
(10 ml of water added to the DSF), which corresponded to 50%
moisture and X3 (30 °C incubation) and the X1 variable (initial pH
of DSF of 6.0) was kept constant. The experimental b-glucosidase
activity average was equal to YA exp. = 2.6347 ± 0.2688 AU of
b-glucosidase in DSF-fermented by the A. oryzae g1 sample. This
result showed no significant difference (p > 0.05) of the estimated
value by t-test, i.e., ŷA = 3.0862 AU of b-glucosidase in
DSF-fermented with A. oryzae g1 sample.
Therefore, to achieve maximum b-glucosidase production by
the SSF of DSF with A. oryzae IOC 3999/1998, the recommendations
are to use 10 mL of water added to 10 g of DSF (X2), with an incu-
bation temperature of 30 °C (X3) and a pH (X1) between 5.2 and 6.8.
Qian et al. (2012) found that the maximum b-glucosidase pro-
duction in an SSF of wheat bran with A niger occurred at a pH
6.0, with 70% moisture for 72 h incubation at 28 °C, whereas lower
b-glucosidase activity was observed between 32 and 36 °C. How-
ever, when using SSF on sorghum bagasse with the same fungus,
conditions including a pH of 5.0, 70% moisture and 96 h incubation
at 30 °C produced maximum b-glucosidase activity (Lavudi,
Harinder, & Laximi, 2003). When using a mixed culture of
Trichoderma reesei and A. oryzae in the SSF of soybean hulls
 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65
Fig. 2. Parameter desirability estimated in the condition of maximum b-glucosidase production (YA = AU of b-glucosidase in DSF-fermented with Aspergillus oryzae g1 sample
or YM = AU of b-glucosidase in DSF-fermented with M. purpureus g1 sample).
supplemented with wheat bran, the maximum b-glucosidase
activity occurred when the pH was 5.0, with 70% moisture and
96 h of incubation at 30 °C (Brijwani, Oberoi, & Vadlani, 2010).
The YA exp. condition of maximum b-glucosidase production was
2.6347 ± 0.26884 AU of b-glucosidase from DSF fermented with A.
oryzae g1 sample. However, considering the initial volume of the
extract and fermented DSF wet basis, it was possible to convert
the value of YA exp. to 0.0878 AU of b-glucosidase from DSF fer-
mented with A. oryzae mL1 crude extract. This result represented
approximately half of the activity described by Aguiar, Suzuki,
Paredes-Guzmán, Alencar, and Park (2003), who obtained
0.18 AU with b-glucosidase from A. oryzae fermented DSF mL1
and 0.15 AU with b-glucosidase from Aspergillus awamori fer-
mented DSF mL1 after DSF fermentation (1:1, w/v; DSF/deionised
water), but with a 96 h incubation at 30 °C and 107 spores. How-
ever, Georgetti et al. (2009) used the same conditions as Aguiar
et al. (2003), but after 48 h of DSF incubation, they observed that
the SSF with A. awamori ATCC 22342, A. niger and Aspergillus niveus
produced b-glucosidase activity with different specific activities.
3.2. Effects of DSF fermentation conditions with M. purpureus NRRL
1992 on the production of b-glucosidase
From the exploratory model of the first RCCD block (Table 2),
the ANOVA and the regression analysis, the effects of variables X1
(initial pH of DSF), X2 (mL water added to 10 g of DSF) and X3 (°C
incubation) and all their interactions (X1X2, X1X3, X2X3 and
X1X2X3) were not significant, and the R2 was 0.56. Considering
the curvature was performed along with a new regression analysis
and ANOVA (Table 3) in which the bending and effects of variables
X1 (initial pH of DSF), X2 (mL water added to the DSF) and X3
(°C incubation) and all interactions X1X2, X1X3, X2X3 and X1X2X3
were found to be significant, with an R2 of 0.99 (Table 3), the linear
model was shown to not be adequate for explaining the effects of
the three variables (X1, X2 and X3) on response function YM (AU
b-glucosidase in DSF-fermented with M. purpureus g1 sample).
Thus, to increase the investigated variable levels and process
61
optimisation, assays were performed in block 2, which contained
6 axial points and 3 central points. By using a regression analysis
of the response function YM, independent variables X1 and X2
showed significant linear and quadratic effects, and variable X3
showed a significant quadratic effect.
It was observed (Table 2) that b-glucosidase activity was higher
at the centre point of the second block (YM = 0.2585 AU of b-gluco-
sidase from DSF fermented with M. purpureus g1 sample), and in
assays 18, 19 and 20 with X1 (initial pH of DSF of 6.0), X2 (10 mL
of water added to the DSF) and X3 (30 °C incubation). However,
the three fermentation conditions (assays 3, 7 and 16) in which
there was no b-glucosidase enzyme activity occurred when vari-
able x3 was at its lowest levels (1 or 1.68), i.e., X3 = 20 or 13 °C.
Considering the high contribution of the X2.X3 interaction to the
proposed model, this interaction was maintained in the equation
and the other non-significant terms were removed. Therefore, the
proposed model (Eq. (3)) can be described as follows:
^M ¼ 0:23064 þ 0:00894  0:03776x1 þ 0:034508x2
y
 0:035110x2 2 2
1  0:038388x2  0:079504x3
þ 0:041666x2 x3 ð3Þ
where ŷM (AU b-glucosidase in DSF-fermented with M. purpureus
g1 sample), x1 (coded variable X1 = initial pH of DSF), x2 (variable
coded X2 = mL water added to the DSF), x3 (variable X3 = coded °C
incubation) and *significance (p < 0.05). The model showed no sig-
nificant lack of fit with an R2 of 0.81, indicating that the experimen-
tal results showed good agreement with the proposed model, so the
model can be used for predictive purposes.
Analysing the proposed model (Eq. (3)) for YM and response sur-
faces in Fig. 1b, c and d showed the regions with maximum b-glu-
cosidase production (YM) when (Fig. 1b) x1 is between 1.3 and
+0.2, or the X1 initial pH of DSF was between 5.4 and 6.1 and x2
was between 0.3 and +1.2, or X2 was 8.5 and 16 mL of water
was added to the DSF with a 30 °C incubation, (Fig. 1c) x1 is be-
tween 1.2 and +0.2 or the X1 pH was between 5.4 and 6.1 and
x3 was between 0.4 and +0.3, or X3 was between 26 and 33 °C
62 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65

and 10 mL of water were added to the DSF; (Fig. 1d) x2 was be-
tween -0.1 and + 1.1, or X2 had between 9.5 and 15.5 mL water
added to the DSF and x3 was between 0.3 and +0.5, or X3 was be-
tween 27 and 35 °C, with an initial DSF pH of 6.0. It is noteworthy
that the maximum YM included assays of the centre point.
The desirability parameters (Fig. 2) for the optimum condition
of response function YM occurred when x1 = 1.682, 0.8409, 0 or
+0.84 (X1 = pH 5.2, 5.6, 6.0 or 6.4); x2 = 0.84, 0, + 0.84 or 1.68
(x2 = 5.8, 10.0, 14.2 or 18.0 mL of water added to the DSF); and
x3 = 0 or + 0.84 (X3 = 30 or 38 °C). Thus, an assay in triplicate,
which coincided with the centre point (X1 = pH 6.0, X2 = 10 mL
and X3 = 30 °C), was performed experimentally, and the resulting
YM exp. = 0,245468 ± 0,002213 AU of b-glucosidase in DSF-
fermented with M. purpureus g1 sample after 48 h of incubation,
the results of which did not differ by t-test (p > 0.05) with
Fig. 3. Effect of pH on b-glucosidase activity from enzymes extracts from DSF
ŷM = 0,24838 AU of b-glucosidase in DSF-fermented with the M. fermented with M. purpureus NRRL 1992 or A. oryzae IOC 3999/1998 using the
purpureus g1 sample, thereby confirming the validity of the pro- substrate p-NPG.
posed model.
Thus, the results demonstrated that, to obtain maximum b-glu-
cosidase production by SSF of DSF with M. purpureus NRRL 1992,
the following fermentation conditions should be used: X1 (initial
DSF pH of 6.0), X2 (10 mL of water added to the DSF) and X3
(30 °C incubation). Furthermore, these fermentation conditions
were similar to those used with A. oryzae IOC 3999/1998 to obtain
maximum b-glucosidase production, except variable X1 had no sig-
nificant effect on the investigated levels.
A b-glucosidase with greater activity (2.78 AU mL1) after
9 days of incubation at 27 °C was described by Daroit et al.
(2007), who used submerged fermentation with the same
M. purpureus NRRL 1992 strain, but with grape residue (50 g/L)
and peptone (20 g/L) as nitrogen sources.
The variables associated with the greatest b-glucosidase pro-
duction effect were (X2), the amount of water added to the DSF
and (X3), the incubation temperature. These results were expected,
because during fermentation, the initial substrate moisture con-
tent generally ranged from 35% to75% and may change in response
to evaporation and metabolic activity (Nishio, Tai, & Nagai, 1979).
In studies conducted by Yadav (1988), the moisture content and
temperature were considered relevant to the SSF process. During
fermentation, the high moisture content decreases the porosity
and diffusion of oxygen, while the low content may negatively
influence microorganism growth because of lower nutrient solubil-
ity and rapid water loss by evaporation. Moreover, the incubation
temperature influences spore germination, microorganism growth,
product formation and sporulation (Lonsane, Ghildyal, Budiatman,
& Ramakrishnat, 1985).
In comparing the maximum b-glucosidase production between
the two fungi that were experimentally obtained during the valida-
tion of the proposed models, A. oryzae IOC 3999/1998 showed a
b-glucosidase activity 10.7 times higher than that of M. purpureus
NRRL 1992. Aspergillus species are among the most promising fungi
for b-glucosidase production; however, different fermentation
Fig. 4. Polyacrylamide gel electrophoresis and zymogram of the different enzyme
conditions and different strains may influence its production extract. Lane 1: extract of the DSF fermented with M. purpureus NRRL 1992. Lane 2:
(Solovyeva et al., 1997). extract of the DSF fermented with A. oryzae IOC 3999/1998.

3.3. Effect of pH on the b-glucosidase activity 3.4. Electrophoresis and zymogram

The optimum pH of b-glucosidase activity was between 5.0 and
5.5 (p < 0.05) and between 5.5 and 6.0 (p < 0.05) for M. purpureus
NRRL 1992 and A. aryzae IOC 3999/1998, respectively (Fig. 3).
The optimum pH between 4.0 and 5.0 was described for b-glucosi-
dase activity produced by recombinant A. oryzae (Horii et al., 2009)
and pH 4.5 for the activity of b-glucosidase isolated from okara
(Chiou, Lin, Su, & Lee, 2010). Thus, pH 5.5 was used for the deter-
mination of b-glucosidase activity of both fungi.
The b-glucosidase zymogram of M. purpureus NRRL 1992 or A.
oryzae IOC 3999/1998 indicates the presence of a single isoform
(Fig. 4). One isoform was also detected in zymograms with differ-
ent intensities of b-glucosidase activity of extracts of fermented
DSF by A. awamori, A. niger or A. niveus (Georgetti et al., 2009). It
is observed that the bands showed activities with different
intensities when using 4-MUG as a substrate. It is noted that the
band corresponding to b-glucosidase extract DSF fermented by
 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65 63

M. purpureus NRRL 1992 was less intense, however, this enzyme
showed the highest specificity for glycosides isoflavones (Table 5).
On the other hand, the b-glucosidase zymogram produced by A.
oryzae IOC 3999/1998 was most active as the result found in the
test of the using as the substrate p-NPG item 3.1. Thus, although
the enzyme produced by A. oryzae IOC 3999/1998 have demon-
strated greater activity on the substrate p-NPG and 4-MUG, it
showed lower specificity in the conversion of glucoside isoflavones
to their aglycone forms than the enzyme produced by fermentation
of DSF with M. purpureus NRRL 1992.
3.5. Bioconversion of isoflavone glucosides to aglycones of DSF
fermented with A. oryzae IOC 3999/1998 or M. purpureus NRRL 1992
The different forms of DSF isoflavones were quantified by UPLC,
and their contents were 185.04 mg g1 of daidzin, 206.72 mg g1 of
genistin, 88.26 mg g1 of glycitin, 655.39 mg g1 of malonyl daid-
zin, 1535.14 mg g1 of malonyl genistin, 575.79 mg g1 of malonyl
glycitin, 107,71 mg g1 acetyl daidzin, 54.22 mg g1 of acetyl gen-
istin, 1.48 mg g1 of daidzein and 135.67 mg g1 of genistein. Gen-
istein and its conjugates are the dominant fractions of DSF at 54%,
with malonyl forms predominating. The acetyl glycitin and glyci-
tein forms were not detected.
After sterilisation by autoclave of DSF, a reduction of 73.1% and
73.8% malonyl daidzin and malonyl genistin, was seen, respec-
tively, and malonyl glyticin was not detected. The malonyl isoflav-
ones are present in larger quantities in soybeans and are
thermolabile, converting to their glycosidic forms under high tem-
peratures (Coward, Smith, Kirk, & Barnes, 1998). Park, Alencar,
Nery, Aguiar, and Pacheco (2001) found that isoflavone extraction
with DSF after heat treatment at 121 °C decreased the malonyl
isoflavone content. The DSF is a solid matrix with a high protein
content (49%), and according to Murphy, Barua, and Hauck
(2002) and Malaypally and Ismail (2010), these factors may have
contributed to the protection of malonyl isoflavones daidzin and
genistin, during sterilisation by autoclaving. In addition, the time
or temperature or a combination of both may also influence the
reduction of malonyl isoflavones (Lee & Lee, 2009).
The isoflavone malonyl daidzin and malonyl genistin from DSF
fermented with fungi showed no significant differences relative to
their respective controls. The glucoside isoflavones and acetyl iso-
flavones decreased during fermentation with two fungi (p < 0.05);
however, this reduction was more pronounced in DSF fermented
with M. purpureus NRRL 1992. However, the aglycone isoflavone
contents increased after fermentation with the two fungi
(p < 0.05). DSF fermented with A. oryzae IOC 3999/1998 decreased
by 47.25%, 30.20% and 25.58% for daidzin, genistin and glycitin and
increased by 193.90% and 72.51% of the aglycones daidzein and
genistein. DSF fermented with M. purpureus NRRL 1992 decreased
by 99.90%, 69.74% and 75.10% of the daidzin, genistin and glycitin
content and increased by 979.98% and 371.75% of the daidzein and
genistein aglycones content (Table 4).
The fermentation conditions for higher b-glucosidase produc-
tion from A. oryzae with DSF IOC 3999/1998 or M. purpureus NRRL
1992 decreased the total isoflavone content in relation to their
respective controls (Table 4). According to Aguiar et al. (2003), a
reduction in total isoflavone content may be caused by other reac-
tions that occurred during the fermentation process.
The isoflavones malonyl daidzin and malonyl genistin were not
bioconverted with the DSF fermentation process (Table 4). These
results confirmed the observations of Park et al. (2001), verifying
that the b-glucosidase from A. oryzae was not efficient for hydroly-
sing malonyl glucosides, but was effective for hydrolysing
glucoside isoflavones. The b-glucosidase extracted from okara
(a residue of soybean aqueous extracts) also showed no specificity
activity with malonyl forms; however, it was effective for isoflav-
ones glycosidic (Chiou et al., 2010).
DSF fermentation with M. purpureus NRRL 1992 reduced the
conjugated acetyl genistin and acetyl daidzin forms at a greater
proportion than A. oryzae IOC 3999/1998 (Table 4). The enzyme

Table 4
Isoflavone contents from DSF fermented with Aspergillus oryzae IOC 3999/1998 or Monascus purpureus NRRL 1992 and controls.

Isoflavones (lg/g) Monascus purpureus Aspergillus oryzae

Control DSF ferm. Control DSF ferm.
a b a
Daidzin 284.603 ± 3.407 0.294 ± 0.449 270.608 ± 18.673 142.112 ± 15.491b
Genistin 697.800 ± 18.600a 211.176 ± 8.460b 699.806 ± 56.468a 488.444 ± 34.337b
Glycitin 110.308 ± 0.442a 27.474 ± 0.047b 109.444 ± 4.936a 87.142 ± 3.316b
Malonyl daidzin 151.834 ± 1.612 162.955 ± 8.438 200.859 ± 7.799 194.183 ± 9.971
Malonyl genistin 324.884 ± 12.398 374.005 ± 28.376 483.384 ± 23.624 475.640 ± 31.267
Acetyl daidzin 117.288 ± 4.435a NDb 112.953 ± 4.357a 90.096 ± 6.535b
Acetyl genistin 132.156 ± 2.230a 54.069 ± 2.887b 108.900 ± 7.174a 90.597 ± 5.551b
Daidzein 8.209 ± 0.614b 88.656 ± 1.553a 14.432 ± 1.827b 42.416 ± 4.884a
Genistein 147.064 ± 1.484b 693.517 ± 11.260a 185.574 ± 10.014b 320.132 ± 25.938a
Total aglycones 155.273 ± 2.098b 782.173 ± 12.813Aa 200.006 ± 11.841b 362.548 ± 30.82Ba
TOTAL 1974.147 ± 45.222a 1617.072 ± 61.978b 2185.958 ± 134.872a 1930.762 ± 137.289b

Average of three replicates and standard deviation ± SD.

Different lowercase letters show a significant difference between fermented DSF and its respective control (p < 0.05). Different uppercase letters for total aglycones show a
significant difference between fermented DSF (p < 0.05); ND = not detected.

Table 5
Hydrolysis of glycosidic isoflavones to aglycones by enzyme extracts of DSF fermented with Monascus purpureus NRRL 1992 or Aspergillus oryzae IOC 3999/1998.

Samples Content (lg mL1)

Daidzin Glycitin Genistin Daidzin m. Glycitin m. Genistin m. Daidzein Genistein
Control 35.78 ± 0.12a 9.12 ± 0.12a 23.23 ± 0.51a 38.61 ± 0.78a 22.22 ± 0.87a 78.89 ± 3.71a 4.22 ± 0.08c 7.98 ± 0.03c
M. purpureus 12.95 ± 0.63c NDb 3.05 ± 0.38c 38.99 ± 0.70a 22.92 ± 0.40a 76.52 ± 2.04a 15.13 ± 0.51a 27.73 ± 0.85a
A. oryzae 22.50 ± 0.99b 9.03 ± 0.08a 9.15 ± 0.28b 38.47 ± 0.12a 23.56 ± 0.46a 78.79 ± 4.82a 10.71 ± 0.18b 21.13 ± 0.27b

Average of three replicates and standard deviation ± SD.

Different lowercase letters in the same column represent significant difference (p < 0.05).
ND = not detected.
64 C.L. Handa et al. / Food Chemistry 152 (2014) 56–65
produced by M. purpureus NRRL 1992 showed higher specificity to
the acetyl glucoside form than that produced by A. oryzae IOC
3999/1998. The b-glucosidase from A. oryzae produced by recombi-
nant Saccharomyces cerevisiae showed low specificity to acetyl
glucosides (Kaya et al., 2008).
The SSF of DSF with both fungi produced b-glucosidase that bio-
converted glucosides (daidzin, genistin and glycitin) into their cor-
responding aglycone forms (daidzein, genistein and the absence of
glycitein), thereby yielding a fermented DSF rich in aglycones
(Table 4). Thus, using the SSF process with A. oryzae IOC 3999/
1998 or M. purpureus NRRL 1992 may be an option for bioconvert-
ing isoflavones glucoside to aglycone, which, according to Izumi
et al. (2000), has the greatest bioactive and absorptive potential
in the intestine relative to their corresponding glycosidic forms.
The bioconversion of isoflavones caused an increase in aglycone
forms by 5 and 1.8 times the DSF fermented with M. purpureus
NRRL 1992 or A. oryzae IOC 3999/1998, respectively (Table 4). It
is noteworthy that in the DSF fermented with M. purpureus NRRL
1992, the content of aglycones isoflavones (daidzein and genistein)
increased 3.86 times in comparison to the DSF fermented with A.
oryzae IOC 3999/1998, although the A. oryzae IOC 3999/1998 has
presented b-glucosidase activity at 10.7 times that of M. purpureus
NRRL 1992. These results indicate that the b-glucosidase produced
by M. purpureus NRRL 1992 was more selective for the bioconver-
sion of isoflavones glucoside to aglycones than that produced by A.
oryzae IOC 3999/1998. This result is consistent with Lee and Chou
(2006), who showed that the fermentation of kojis from black soy
with different fungi (A. awamori, A. oryzae, A. sojae, Rhizopus azy-
gosporus and Rhizopus sp.) at 30 °C for a 72 h incubation decreased
the glucoside isoflavones (daidzin, genistin and glycitin) and in-
creased the content of aglycones isoflavones (daidzein, genistein
and glycitein) when compared with non-fermented products. In
addition, the koji fermented with Rhizopus sp. showed lower
b-glucosidase activity and higher bioconversion of aglycones than
A. oryzae and Aspergillus sojae. Georgetti et al. (2009) compared
the bioconversion of genistin in genistein DSF fermented with
A. niger, A. niveus and A. awamori and observed that A. niveus
showed higher bioconversion capacity and lower b-glucosidase
activity. This finding was possibly explained by some species of
Aspergillus b-glucosidase that were produced with different speci-
ficities or other enzymes that also hydrolysed glycosidic bonds.
However, Aguiar et al. (2003) reported that various microorgan-
isms can produce b-glucosidase by acting on the hydrolysis of
glycosidic bonds of cellulose materials or of p-NPG; however, they
do not show much specificity for binding glycosidic isoflavones.
3.6. Specificity of b-glucosidase activity from M. purpureus NRRL 1992
and A. oryzae IOC 3999/1998 in relation to isoflavones extract from
DSF
The extracellular b-glucosidase produced by fungi, hydrolysed
glucoside isoflavones daidzin and genistin and produced aglycone
isoflavones daidzein and genistein (Table 5). The specificity of b-
glucosidase activity produced by M. purpureus NRRL 1992 was
1.44 times higher in the production of daidzein and 1.35 times
higher in the production of genistein than the specificity of b-glu-
cosidase activity produced by A. oryzae IOC 3999/1998. Regarding
glycitin, there is a highly specificity of b-glucosidase activity from
M. purpureus NRRL 1992. While the specificity of b-glucosidase
activity from A. oryzae IOC 3999/1998 was not detected, being that
the glycitin did not differ from control (p > 0.05). The activity of
b-glucosidase from A. oryzae recombinant, showed specificity for
the three glucoside isoflavones converting them into aglycones
(Horii et al., 2009). Low specificity of b-glucosidase activity from
E. coli was detected for glycitin (Ismail & Hayes, 2005). With regard
to malonyl isoflavones the b-glucosidase from M. purpureus NRRL
1992 and A. oryzae IOC 3999/1998 they did not present any
specificity and did not differ from the control (p > 0.05) (Table 5).
These results demonstrated that malonyl isoflavones were less
susceptible to the action of these enzymes which were also
observed on DSF fermented by both fungi (item 3.5).
4. Conclusions
The highest production of b-glucosidase by SSF in the DSF with
A. oryzae IOC 3999/1998 or M. purpureus NRRL 1992 occurred when
adding 10 mL of water to the DSF, incubating at 30 °C and using 6.0
as the initial DSF pH. For the A. oryzae IOC 3999/1998, the pH effect
was independent of the 5.2–6.8 range.
Under the same solid state fermentation conditions with DSF A.
oryzae IOC 3999/1998, b-glucosidase was produced at 10.7 times
greater than the M. purpureus NRRL 1992. However, DSF fermenta-
tion with M. purpureus NRRL 1992 produced a more efficient bio-
conversion of isoflavones glucoside to aglycone.
Acknowledgments
This work was partially fundend by Fundação Araucária/CNPq,
PRONEX Program. CLH would like to thank Foundation CAPES/
MEC for a graduate scholarship, and EII is a CNPq Research Fellow.
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