LWT - Food Science and Technology 111 (2019) 211–217

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LWT - Food Science and Technology

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Optimization of lactic acid fermentation conditions for fermented tofu whey T

beverage with high-isoflavone aglycones
Yan Zhu, Zimeng Wang, Li Zhang∗
College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China

A R T I C LE I N FO A B S T R A C T

Keywords: Four lactic acid bacteria strains (Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus rhamnosus GG
Tofu whey and Lactobacillus plantarum), possessing β-glucosidase enzyme activity, were investigated for tofu whey fer-
Isoflavone aglycones mentation based on the capacity of bioconversion of isoflavones. The results suggested that the mixture of L.
Lactic acid bacteria rhamnosus GG and L. paracasei had great potential for efficient enrichment of bioactive isoflavone aglycones and
Fermentation
the percentage composition (daidzein, genistein) increased from 4.6 to 93.78% after fermentation of tofu whey.
At the same time, the viable cell counts of the mixture of L. rhamnosus GG and L. paracasei increased to 9.13
log10 CFU/mL while pH value dropped to 4.48 and titratable acidity was 0.28%. Headspace solid-phase mi-
croextraction method followed by gas chromatography-mass spectrometry analysis indicated that the odor
compounds, particularly the aldehydes and alcohols, were metabolized to trace or undetected levels after fer-
mentation. This research provided not only a new way to develop a functional probiotic beverage enriched in
isoflavone aglycones, but also provided a potentially valuable new use for soy whey protein.

1. Introduction
Soybean is known to be an economical and plentiful source of
phytoprotein and also a rich source of isoflavones. While tofu and other
soy products have been widely produced and consumed in Asian
countries for many centuries, soy and soy products are increasingly
being accepted by consumers worldwide due to its associated health
benefits (Benedetti et al., 2016). Tofu whey is a byproduct during the
production process of squeezing tofu and dried bean curd, which con-
tains high amounts of beneficial compounds, such as soluble protein,
oligosaccharide, isoflavones and so on. During the process of tofu
production, about 8–9 L of tofu whey will be produced for 1 kg of
soybean processed. Currently, tofu whey was treated by crude filtration
process before disposal in order to reduce the Chemical Oxygen De-
mand (COD) values and Biochemical Oxygen Demand (BOD) values.
However, the discharge of tofu whey would still lead to eutrophication
due to its remaining nutrient constituents. Thus, it is meaningful and
urgent to carry out the disposal of tofu whey reasonably.
Isoflavones are major and important constituents of soy and tofu
whey and have a number of associated health benefits. They have been
demonstrated with the potential to reduce the risk of age-related and
hormone-related diseases, including cancer, menopausal symptoms
(Singh & Vij, 2017), cardiovascular disease and osteoporosis
(Mahmoud, Yang, & Bosland et al., 2014). As a kind of phenolic com-
pounds, isoflavones were related to the main nonvolatile off-flavor in
soy products, because they contributed to the bitter taste and astringent
mouthfeel. In addition, aglycones have much more unpleasant flavors
than glucosides (Matsuura, Obata, & Fukushima et al., 1989; Poliseli-
Scopel, Gallardo-Chacón, Juan, Guamis, & Ferragut, 2013). The form
and content of isoflavones can be affected by certain types of soybean
processes, such as hydration, thermal treatment (Zhang et al., 2018). It
was reported that tofu whey contained 43.92% (based on dry matter) of
the isoflavones in the raw soybeans (Wang & Murphy, 1996). They exist
in the form of glucosides (daidzin, genistin and glycitin) and their
corresponding aglycones (daidzein, genistein and glycitein). In addi-
tion, as the intermediate metabolites of bioconversion, acetylglucosides
and malonylglucosides were unstable and remained relatively low in
concentration (Otieno, Ashton, & Shah, 2006). Because of the lower
molecular weight and less hydrophilicity, aglycones are absorbed faster
and better than glucosides in the human body (Nielsen & Williamson,
2007). Therefore, conversion of isoflavones to their aglycone form in-
creases their absorption and bioavailability and can be considered a
desirable processing step. The glucoside conjugates of isoflavone could
be converted to aglycones due to the presence of the enzyme β-gluco-
sidase or α-galactosidase (Toda, Sakamoto, Takayanagi, & Yokotsuka,
2001). These two enzymes are naturally present in soybeans and may

∗
Corresponding author.
E-mail addresses: 17863903422@163.com (Y. Zhu), namwoomeng@163.com (Z. Wang), qdzhangli@ouc.edu.cn (L. Zhang).

https://doi.org/10.1016/j.lwt.2019.05.021
Received 27 December 2018; Received in revised form 9 April 2019; Accepted 6 May 2019
Available online 07 May 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
Y. Zhu, et al.
also be produced by various microorganisms (Kuda, Kataoka, & Nemoto
et al., 2016; Singh & Vij, 2018). LAB is used widely due to their pro-
biotic impartment of beneficial effects towards human metabolism and
immunological health. Previous research has demonstrated that the
antioxidant capacity of fermented soy whey by LAB and kefir was en-
hanced prominently (Moraes Filho, Busanello, & Garcia, 2016; Yang
et al., 2017). Hence, there are reasons to believe that LAB fermentation
could be used as an alternative method to transform tofu whey into
directly consumable products.
Different LAB strains could produce different types and amounts of
metabolites during fermentation which allows people to select the best
strains suitable for their desired beverage type. There are numerous
flavor-active compounds formed by LAB fermentation, which con-
tribute to the characteristic aroma and flavor of fermented beverage
(Peralta et al., 2016; Ricci et al., 2018). Thus, the objective of this work
was to produce a new fermented probiotic beverage with a pleasant
flavor, which is rich in isoflavone aglycones. This study will serve as an
initial investigation to understand the fermentation characteristics of
LAB in tofu whey fermentation and provide the theoretical basis for the
further development of non-dairy probiotic tofu whey beverage.
2. Materials and methods
2.1. Microorganism and materials
Four lactic acid bacteria (LAB) strains were used for the fermenta-
tion of tofu whey. Lactobacillus paracasei 22709 (LP) and Leuconostoc
mesenteroides 22264 (LM) were purchased from China Center of
Industrial Culture Collection (CCIC, Beijing, China). Lactobacillus
rhamnosus GG (LGG) and Lactobacillus plantarum (PL) were provided by
Laboratory of Functional Dairy Food & Probiotic Engineering, Ocean
University of China.
Tofu whey was supplied by a local tofu factory (Sanjucheng Bean
Products Factory of Qingdao). The pH of fresh tofu whey varied with
batch processing, thus its pH was adjusted to 6 after centrifugation, and
it was sterilized at 115 °C for 10 min.
p-nitrophenol-β-D-glucopyranoside, p-nitrophenol-α-D-galactopyr-
anoside, four isoflavone standards (daidzin, genistin, daidzein and
genistein), alkane standard solution (C7–C30) and cyclohexanone (in-
ternal standard) were purchased from Sigma-Aldrich Chemical CO. (St.
Louis, MO, USA).
2.2. Determination of α-galactosidase and β-glucosidase activity
The tofu whey was inoculated with 2% (v/v) subcultured four
strains. The enzyme extract was collected at set intervals during the
cultivation for total 36 h. The α-galactosidase and β-glucosidase ac-
tivity were determined by calculating the rate of hydrolysis for p-ni-
trophenol (pNP)-β-D-glucopyranoside and p-nitrophenol (pNP)-α-D-ga-
lactopyranoside with some modifications (Maitan-Alfenas et al., 2014).
About 200 μL of enzyme extract was added to 5 mmol/L pNP-β-D-glu-
copyranoside or pNP-α-D-galactopyranoside, which was dissolved in
100 mmol/L sodium acetate buffer (pH 5.5), then incubated at 37 °C for
30 min. Enzyme extract with inactivation treatment was set up as
control group. The reaction was terminated by adding 1 mL of 1 mol/L
cold sodium carbonate. The amount of pNP released was measured with
a spectrophotometer (UV–2802PC, Unico (Shanghai) instrument CO.,
LTD) at 420 nm. One unit of enzyme activity (mU) was defined as the
amount of enzyme required to liberate 1.0 mmol of substrate per mL per
min under the assay conditions.
2.3. Screening bacteria strains for tofu whey fermentation
The tofu whey was inoculated with 2% (v/v) subcultured single LAB
or mixed strains, and fermented for 24 h at 37 °C. The single LAB was
LGG, LM, LP, PL, respectively. Mixed strains were the combination of
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LWT - Food Science and Technology 111 (2019) 211–217
any two strains (1:1, v/v), included LGG + LP, LGG + LM, LGG + PL,
LM + LP, LM + PL and LP + PL. The control group was unfermented
tofu whey without strains. Samples were taken for the determination of
viable cell counts, pH, the titratable acidity, and isoflavones after 24 h.
2.4. Extraction and quantification of isoflavones from tofu whey
The extraction of isoflavones, including glucosides and aglycones
from fermented and nonfermented tofu whey was carried out according
to Lee, Renita, & Fioritto et al. (2004) with some modifications. Briefly,
each freeze-dried sample was extracted with 60% acetonitrile-bidis-
tilled water shaking for 2 h. The insoluble residue was discarded by
centrifugation, and the supernatant was vaporized until all the solvent
was removed. The dried powder was then dissolved in 80% methanol
and filtered through a syringe filter for HPLC analysis.
The mobile phase consisted of solvent (A) (0.05% acetic acid water
solution) and solvent (B) (acetonitrile). The solvents flow rate was
0.6 mL/min. After 20 μL of sample or isoflavone standard was injected
into the column (Agilent ZORBAX Eclipse Plus C18)
(4.6 mm × 100 mm, 3.5 μm), the following gradient for solvent A was
applied: 85% at 0 min, steady for 5 min, 85-65% from 6 to 28 min, 65-
55% over the next 14 min and 55-10% over 5 min, increasing to 85% A
for 3 min and steady at 85% until completing the gradient program
within 50 min. The column temperature was 30 °C, and the diode array
UV–visible detector was set at 260 nm.
The behaviour of bacteria strains in tofu whey during fermentation
was compared with respect to hydrolysis of isoflavone glucosides into
aglycones. Changes of isoflavone aglycone contents were expressed as a
relative percentage to total isoflavone (daidzin, genistin, daidzein and
genistein) according to the formula: X (%) = (C1+C2) × 100/Ct, X (%):
the percentage composition of aglycones, C1: the concentration of
daidzein (μg/mL), C2: the concentration of genistein (μg/mL), Ct: the
concentration of total isoflavone (μg/mL).
2.5. Effects of pH and temperature on transition
The best performing strains based on bioconversion was employed
for the optimization studies. They were investigated with different pH
values (4.0–8.0) of tofu whey for 24 h at 37 °C. Besides, fermentation
inoculated with screened strains was carried out in tofu whey (pH 6.0)
for 24 h at different temperatures (27 °C-47 °C). The transformation of
isoflavones and viable cell counts were determined at the end of fer-
mentation process.
2.6. Determination of the optimum indices for fermented tofu whey
About 500 mL of tofu whey was inoculated with screened strains,
then was fermented at the optimal temperature and pH for 36 h ac-
cording to the 2.5. Samples were examined for cell viability, pH, ti-
tratable acidity, content of isoflavone aglycones and enzyme activity
every 4 h. Noninoculated tofu whey incubated under the same condi-
tion was used as control.
2.7. Measurement of volatile components
Changes in volatile components of tofu whey before or after fer-
mentation were analyzed using headspace solid-phase microextraction
(HS-SPME) method followed by gas chromatography-mass spectro-
metry (GC–MS) (7890N/5975C, Agilent, Santa Clara, USA) as described
by Dragone, Mussatto, Oliveira, and Teixeira (2009) with some mod-
ifications. The capillary column, HP-INNOWax (30 m × 0.25 mm,
0.25 μm; Agilent Technologies) was used. About 5 mL portion of the
tofu whey samples added with 0.5 g NaCl was incubated for 20 min and
subjected to HS-SPME extraction at 40 °C for 40 min using a Divi-
nylbenzene/Carboxen/Polydimethylsiloxane fiber (50/30 μm, 24 ga,
1 cm, Supelco, Bellefonte, PA, USA) at 200 rpm/min. Following
Y. Zhu, et al. LWT - Food Science and Technology 111 (2019) 211–217

extraction, the adsorbed volatiles were desorbed in the GC injector port Table 1
in splitless mode at 250 °C for 5 min. The oven temperature was held at Changes of cell growth, pH, and % TA in tofu whey fermented with lactic acid
40 °C, for 3min, then raised at 6 °C/min until 100 °C, and then pro- bacteria for 24 h at 37 °C.
gramed at 10 °C/min to 230 °C for 10 min, finally increased to 250 °C at Strains log10 CFU/mL pH Titratable acidity
10 °C/min and held for 3 min. Compounds were identified based on (% TA)
comparing their individual mass spectra with the NIST14 library and Control – 6.00 ± 0.10d 0.05 ± 0.02d
LGG 8.87 ± 0.04a 4.82 ± 0.04c 0.18 ± 0.00c
retention times. The Kovats retention indices (KI) of detected com-
LM 7.80 ± 0.04c 3.87 ± 0.02a 0.36 ± 0.05a
pounds were determined by injecting 1 μL of alkane standard solutions LP 7.96 ± 0.09c 4.985 ± 0.02c 0.12 ± 0.03c
(Alkane standard solution C7–C30 from Sigma-Aldrich). The relative PL 8.05 ± 0.01b 3.90 ± 0.01a 0.32 ± 0.10a
concentrations of volatile compounds were calculated from the peak LGG + LM 8.72 ± 0.04a 4.47 ± 0.02c 0.24 ± 0.02b
area relative to the internal standard (cyclohexanone 1.894 μg/mL). LGG + LP 8.81 ± 0.03a 4.38 ± 0.00b 0.28 ± 0.02a
LM + LP 8.07 ± 0.05b 4.30 ± 0.02b 0.28 ± 0.01a
LGG + PL 8.716 ± 0.03a 3.95 ± 0.04a 0.30 ± 0.03a
2.8. Statistical analysis LP + PL 8.36 ± 0.05ab 4.04 ± 0.02a 0.29 ± 0.02a
LM + PL 7.84 ± 0.04c 4.02 ± 0.02a 0.29 ± 0.03a

All experiments were conducted in triplicate, and results were ex-

pressed as means ± standard deviations (SD). All significant differ-
ences among samples were subjected to one-way analysis of variance
(ANOVA), followed by Duncan's multiple range test (P < 0 0.05) using
SPSS® 22 software for Windows (SPSS Inc., Chicago, IL, USA). Values of
P < 0.05 were considered statistically significant. The data were pre-
viously tested for homogeneity of variance (Hartley's test).
3. Results and discussion
3.1. α-galactosidase and β-glucosidase activity in fermented tofu whey
The α-galactosidase and β-glucosidase enzyme activities of four
strains were assayed from 8 h to 36 h. Unexpectedly, the α-galactosi-
dase activity was not detected or very low during overall fermentation
process. It can be inferred that the conditions for producing α-ga-
lactosidase were not appropriate in tofu whey (Cao, Xiong, True, &
Xiong, 2016; Li, Loman, Callow, Islam, & Ju, 2018). Notably, another
mechanism of converting isoflavone glucosides was found. Fig. 1
showed a wide variation of β-glucosidase activity during fermentation.
The enzyme activity from all four strains increased during the ex-
ponential growth phase (between 4 and 12 h). LGG displayed the
highest and the longest sustained growth in the enzyme activity, and it
reached a maximum level of 6.72 ± 0.16 mU/mL at 20 h. Subse-
quently, the β-glucosidase activity exhibited the declining tendency
after 20 h. It was deduced that the carbohydrates and other essential
Fig. 1. Changes of β-glucosidase activity from four lactic acid bacteriain tofu
whey fermented for 36 h at 37 °C.
Lactobacillu. rhamnosusGG (LGG).
Lactobacillus paracasei 22709 (LP).
Leuconostoc mesenteroides22264 (LM).
Lactobacillu. plantarum (PL).
LGG: Lactobacillus rhamnosus GG; LM: Leuconostoc mesenteroides 22264
LP: Lactobacillus paracasei 22709; PL: Lactobacillus plantarum.
Values were expressed as mean ± standard deviations (n = 3) for all analyses.
a-d
Different superscript lowercase letters in the same column denote significant
differences (P < 0.05).
substances for producing β-glucosidase were consumed due to the
growth of strains. For LM, it showed a maximum activity of
2.95 ± 0.09 mU/mL after 14 h of fermentation, then it kept decreasing
continually. The fact that the β-glucosidase activity of LGG was almost
more than 10 times higher than LP and PL suggested that LGG could
have more potential for isoflavones transformation.
3.2. Screening bacteria strains with high bioconversion of isoflavone in
fermented tofu whey
After fermentation at 37 °C for 24 h, strains included LGG exhibited
higher population (8.07–8.87 log10 CFU/mL) than those (7.80–8.36
log10 CFU/mL) without LGG (Table 1). The cell number of LGG + LP
(1:1) and LGG + LM (1:1), which exhibited significant increases of 0.85
and 0.92 log10 CFU/mL (P < 0.05), was higher than that of LP and LM
alone. There was no obvious difference in the cell number between
LGG + LP (1:1), LGG + LM (1:1), LGG + PL (1:1), and LGG group. The
pH value reduced prominently in various degrees. The acid develop-
ment of tofu whey fermented with LM and PL increased to 0.32% and
0.36% (P < 0.05), respectively, which was much higher than LGG
group or LP group. Moreover, the pH detected in mixed LGG and LP
group (pH 4.38) was lower than that fermented with LGG or LP (pH
4.82, pH 4.98). Meanwhile, the titratable acidity of mixed strains group
(0.28%) was higher than that fermented by LGG or LP alone
(0.12%–0.18%). Thus, the mixture of bacteria strains remedied the
defect of the insufficient acidity by LGG or LP alone. Significantly, this
reduction in pH was enough to cause protein flocculation, hence the
appearance of tofu whey became turbid together with the growth of
bacteria strains after fermentation.
As shown in Fig. 2, daidzin and genistin, were the predominant
isoflavones in the unfermented tofu whey extract, and the peaks for
their aglycones (daidzein and genistein) were relatively low. The peaks
of 5 and 6 (Fig. 2b and c) were considered as glycitin and its aglycone
glycitein (Chuankhayan, Rimlumduan, Svasti, & Cairns, 2007). Among
all forms of isoflavones, daidzein was eluted first, followed by genistin,
in agreement with Rodríguez-Roque, Rojas-Graü, Elez-Martínez, and
Martín-Belloso (2013).
It was found that the bioconversion of isoflavone glucosides into
corresponding aglycones occurred in four bacteria strains and their
mixed strains, compared with the unfermented control group, which
contained mainly the isoflavone glucosides. Furthermore, the bio-
conversion by the mixture of bacteria strains was better than that by
single bacteria strain during fermentation. For single bacteria strain
fermentation, LGG group showed the highest contents of aglycones
(80.94 ± 3.16%), which was notably higher than other strains

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Y. Zhu, et al.
Fig. 2. HPLC chromatogram of the isoflavones profile of four standard iso-
flavones (a) unfermented tofu whey (b) and fermented tofu whey (c). 1. Daidzin
(6.400 min) 2. Genistin (13.952 min) 3. daidzein (23.501 min) 4. genistein
(30.522 min).
(P < 0.05). The content of aglycones fermented by LM was the lowest
(28.90 ± 0.54%), although the β-glucosidase activity of this group was
higher than LP and PL (Fig. 1). This suggested that it was not reliable to
use the β-glucosidase activity as the only screening indicator when
considering the bioconversion. For the mixture of bacteria strains fer-
mentation, the percentage of isoflavone aglycones reached
92.03 ± 0.92% and 90.25 ± 0.73% by LGG + LP and LGG + LM,
respectively(see Fig. 3).
It was important for a probiotic beverage to acquire the abundant
viable cell counts and pleasant flavor. Although LM produced more
acidity than LGG, it was not suitable for a probiotic beverage because of
the unpleasant flavor and lower biotransformation capacity of iso-
flavone. Thus, LM was not considered a promising strain for subsequent
fermentation experiments. Recently, it was found that the mixed strains
had good acid production rate and good sensory preference for con-
sumers, which were more suitable for industrial production, compared
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LWT - Food Science and Technology 111 (2019) 211–217
Fig. 3. The percentage of isoflavone aglycones in tofu whey fermented with
lactic acid bacteria after 24 h of cultivation at 37 °C.
LGG: Lactobacillus rhamnosus GG; LM: Leuconostoc mesenteroides22264.
LP: Lactobacillus paracasei 22709; PL: Lactobacillus plantarum.
a-g different superscript lowercase letters denote significant differences
(P < 0.05).
with a single LAB strain (Bujna, Farkas, Tran, Mai, & Nguyen, 2018; Sun
et al., 2013). Our result demonstrated that there were no observable
differences in viability, acidity and bioconversion during tofu whey
fermentation by LGG and LP mixtures with different ratios (data not
shown). Therefore, the optimum fermentation strain was a combination
of LGG and LP with a ratio of 1:1 (v/v) to obtain fermented tofu whey
with abundant isoflavone aglycones.
3.3. The optimum pH and temperature
As shown in Fig. 4a, when the initial pH was 6.0, the cell number
was 8.92 ± 0.01 log10 CFU/mL, which was significantly higher than
other groups (P < 0.05). As regards the bioconversion, there was no
significant difference in pH 5.0 and pH 6.0 group (daidzein
46.19 ± 0.05%, 46.90 ± 0.5%; genistein 44.86 ± 0.4%,
48.39 ± 0.07%) (P > 0.05), which agreed with the report by
Matsuura, Obata, & Fukushima et al. (1989), i.e.,they found that the
optimum pH for the production of daidzein and genistein was around
6.0 in soybean milk. The bioconversion of isoflavone glucosides into
aglycones was weak because acid or alkali (pH 4, 7 and 8) environment
was not suitable for microbial survival. Moreover, changes of pH would
influence the adjustment of conformational structure of the protein,
resulting in a modulation of the hydrolytic reactions properties of the
enzyme (Jankowiak, Kantzas & Boom et al., 2014; Li et al., 2018).
The viable cell counts at 32 °C and 37 °C (8.80 ± 0.07 log10 CFU/
mL, 8.86 ± 0.09 log10 CFU/mL) were superior to other groups
(P < 0.05), and the contents of isoflavone aglycones were also higher
(Fig. 4b). There was no significant difference of viable cell counts be-
tween 47 °C group and the control group (P > 0.05), which meant the
growth of LGG + LP mixtures was difficult at high temperatures. The
main reason was that temperature of fermentation would influence the
growth and metabolism of strains. Sestelo, Poza, and Villa (2004) re-
ported that β-glucosidase activity in L. plantarum strain was reduced by
50% when it was heated at 50 °C during 5 min. It is worthwhile to note
that there was no remarkable difference in the percentage composition
of isoflavone aglycones at 32 °C and 37 °C (94.03 ± 0.5%,
95.29 ± 1.2%) (P > 0.05). These results suggested that the combi-
nation of LGG and LP was the best strain for bioconversion at moderate
temperatures. In fact, during the actual manufacture, the cost should be
Y. Zhu, et al.
Fig. 4. Effects of the initial pH (a) and fermented temperature (b) on viable cell
counts and the percentage composition of isoflavone aglyconesin tofu whey
fermented by the mixture of Lactobacillu. rhamnosus GGandLactobacillus para-
casei22709.
Viable cell counts Daidzein Genistein.
a-candA-DDifferent superscript lowercase letters denote significant differences.
taken into account, as well as energy conservation and emission re-
duction. Thus, in a follow-up experiment, the optimum fermentation
temperature (32 °C) and the appropriate pH (6.0) were selected as the
fermentation conditions for LGG + LP.
3.4. Changes in indices during the production of fermented tofu whey
3.4.1. The growth and acidification performance of LGG + LP
Fig. 5a shows the viable cell counts, pH values and titratable acidity
during tofu whey fermentation by LGG + LP. The multiplication of LAB
showed that the length of the induction phase was about 6 h since these
strains needed an adaptive phase in tofu whey. The viable cell counts of
strains enhanced apparently from 6 h to 20 h and the maximum at-
tained to 9.13 ± 0.08 log10 CFU/mL. But after 20 h, the viable cell
counts decreased gradually and dropped to 8.83 ± 0.05 log10 CFU/mL
at 36 h. The pH decline rate was more pronounced during the first 12 h
and then slowed down at later period of fermentation, which was
mainly due to the accumulation of lactic acid. At the same time, the
increase of titratable acidity confirmed this hypothesis. According to
Menezes et al. (2018), the pH decline was strain dependent based on
the metabolic activity and the growth requirement of strains. During
the fermentation, this decline in pH was acted as a preservative factor
against spoilage and pathogenic microorganisms which were not able to
proliferate under these conditions (Torino et al., 2013).
3.4.2. β-Glucosidase activity and bioconversion of isoflavones
The LGG + LP fermentation caused a significant increase in the
percentage of isoflavone aglycones via the β-glucosidase catalyzed hy-
drolysis of isoflavone glucosides from 6 h to 16 h (Fig. 5b). Simulta-
neously, the percentage of isoflavone glucosides significantly reduced
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LWT - Food Science and Technology 111 (2019) 211–217
Fig. 5. Growth curves, pH values, titratable acidity (a) and the concentration of
total isoflavones, the percentage of isoflavone aglycones and β-glucosida-
seactivity(b) infermented tofu whey by the mixture of Lactobacillu. rhamnosus
GGand Lactobacillus paracasei22709.
Viable cell counts pH Titratable acidity.
β-glucosidaseactivity Isoflavone aglycones.
Total isoflavones.
(P < 0.05), and the contents of aglycones reached the highest value of
93.78 ± 2.1% at 16 h. This result was expected because the enzyme
activity from LGG + LP increased gradually with fermentation time,
and it displayed the highest β-glucosidase activity (6.91 ± 0.13 mU/
mL) after 14 h. Comparatively, the β-glucosidase activity of other single
strain, such as LGG, was 6.71 ± 0.32 mU/mL, which was the highest
level until fermentation of 20 h (Fig. 1). A similar trend in the changes
of β-glucosidase activity, isoflavone glucosides, and aglycones was also
observed in soymilk fermented by Lactobacillus rhamnosus CRL981
(Marazza, Garro, & Giori, 2009). What's more, it was verified that the
total isoflavones content was reduced during the whole fermenting
process. It could be due to the interaction between the isoflavones as a
polyphenolic compound and some components in the matrix, such as
the proteins and inorganic salts (Kašparovská et al., 2017). According to
Fig. 5b, there was a clear decline of β-glucosidase activity from 16 h to
36 h. However, the viable cell counts, acid values and the contents of
isoflavone aglycones were the highest and there was no significant
changes from 20 h to 36 h. Therefore, it was rational to limit the fer-
mentation period to 20 h for the enrichment of aglycone forms.
3.4.3. Changes of volatile compounds
As shown in Table 2, the most abundant compounds in unfermented
tofu whey were alcohols and aldehydes, which was in agreement with
the study of Achouri, Boye, & Zamani et al. (2006). Most of alcohols and
aldehydes in tofu whey were formed from oxidation of linoleic acid and
linolenic acid by the catalytic action of lipoxygenase in soybean
(Mizutani & Hashimoto, 2004). Those compounds, such as 1-hexanol, 3-
octanol, 2-pentylfuran, 1-pentanol, hexanal, benzaldehyde, and (E)-2-
hexenal, which all were described as causing unpleasant flavor, aroma
Y. Zhu, et al. LWT - Food Science and Technology 111 (2019) 211–217

Table 2 Hioe, Frank, & Arcot, 2018b).

Relative concentration (μg/mL) of volatile compounds in tofu whey.
Compounds Retention Retention Concentration (μg/mL) 4. Conclusions
time (min) index
Unfermented Fermented Among different LAB groups, the mixture of LGG and LP (1:1)
strains presented the most promising industrial applications since it
Acids
acetic acid 15.29 1457 0.391 1.533 showed great ability to hydrolyze isoflavone glucosides into aglycones.
hexanoic acid 20.62 1843 0.119 1.337 The bioconversion of isoflavones, the viable cell counts and acidifica-
octanoic acid 22.93 2060 0.000 0.169 tion reached the maximum in fermented tofu whey at 32 °C for 20 h.
Alchols Interestingly, the objectionable odor of tofu whey, such as, beany,
ethanol 3.63 936 0.000 0.056
grassy and green flavors, was improved obviously after fermentation,
1-penten-3-ol 8.64 1164 0.000 0.023
1-pentanol 10.87 1252 0.475 0.301 and the mixture of all compounds provided characteristic odor of the
(E)-2-pentenol 12.19 1307 0.047 0.038 fermented tofu whey. Hence, it is noteworthy that fermented tofu whey
(Z)-2-pentenol 12.38 1316 0.356 0.210 by mixed lactic acid bacteria is a good source of isoflavone aglycones.
1-hexanol 13.12 1349 0.719 0.473
(Z)-3-hexenol 13.80 1379 0.090 0.078
3-octanol 13.99 1388 0.209 0.110 Conflicts of interest
3-octenol 15.07 1445 0.807 0.426
2-ethyl-1-hexanol 15.75 1482 0.185 0.168 The authors declare that they have no conflict of interest.
1-octanol 16.81 1549 0.071 0.044
(E)-2-Octen-1-ol 17.65 1605 0.000 0.303
Acknowledgements
2-furanmethanol 18.27 1652 0.052 0.000
1-nonanol 18.35 1658 0.000 0.040
benzyl alcohol 20.90 1868 0.085 0.204 This study was supported by The Key Research and Development
phenylethyl alcohol 21.29 1903 0.185 0.063 Program of Shandong Province, China (2017YYSP013) and The Natural
Aldehydes
Science Foundation of Shandong Province, China (ZR2015CM010).
hexanal 6.55 1062 0.469 0.000
(E)-2-hexenal 9.88 1210 0.863 0.000
furfural 15.36 1461 0.219 0.000 References
benzaldehyde 16.42 1523 1.050 0.574
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