Food Bioscience 34 (2020) 100542

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

β-Glucosidase from almonds and yoghurt cultures in the biotransformation T

of isoflavones in soy milk
Subrota Hatia, Dian W. Ningtyasb,c, Jassimran Kaur Khanujac, Sangeeta Prakashc,∗
a
Dairy Microbiology Department, SMC College of Dairy Science, Anand Agricultural University, Anand, 388110, Gujarat, India
b
Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University, Jl. Veteran Malang, 65145, Indonesia
c
School of Agriculture and Food Sciences, The University of Queensland, St. Lucia, Queensland, 4072, Australia

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

Keywords: β-Glucosidase is an enzyme that hydrolyzes the isoflavone bonds and develops the active form of aglycones
β-Glucosidase providing various health benefits. The potential of β-glucosidase from two sources: almonds and soy yoghurt
Almonds cultures (lactic cultures) to react with soy in the biotransformation of soy isoflavones was studied. This study
Prunus dulcis investigated 1) the optimum amount of culture (1, 2, 3, 4%) and incubation temperature (30, 35, 42 °C) for
Yoghurt
fermentation of soymilk (SM); 2) the production of β-glucosidase and its ability to react with isoflavones with
Soybeans
Glycine max
and without the addition of skim milk powder (SMP). The ability of β-glucosidase to convert isoflavones into
Isoflavones aglycones was measured using spectrophotometry and reverse-phase high-pressure liquid chromatography. The
results showed that a 1% lactic acid culture at 30 °C produced the highest viable count of lactic acid bacteria
(~108 CFU/ml) in fermented SM. Fermentation at 30 °C and 1% inoculation showed the maximum β-glucosidase
activity (4.28 U/ml) while 8 U/ml of β-glucosidase from almonds had the highest activity (daidzein: 1.48 g/l and
genistein: 0.78 g/l). Among the yoghurt cultures, 2% inoculation in soymilk led to the highest aglycones
(daidzein: 1.14 g/l and genistein: 0.50 g/l). Supplementing soymilk with skim milk powder led to aglycone
levels similar to β-glucosidase from almond.

1. Introduction
Soybeans (Glycine max) are an important source of essential nu-
trients, providing good quality proteins, dietary fibre, iron, manganese,
phosphorus and several B vitamins, including folate (Riaz, 2006, pp.
9–10). Originally from China, soybean is now grown worldwide. SM or
soy products have a potential to be used as functional fermented pro-
ducts (Yamanaka, Okumura, Mitsugi, & Hasagawa, 1969) due to its
various physiologically bioactive compounds like soy isoflavones, soy
proteins, and oligosaccharides (Wang & Murphy, 1994). Moreover, soy-
based products have been used as an alternative to dairy milk products
for people who are lactose intolerant or live in regions where dairy
products are not regularly available. The soy isoflavones have been
associated with the prevention of osteoporosis, cardiovascular disease,
and several hormone-dependent cancers (Gil-Izquierdo et al., 2012).
Isoflavones are the strongest flavonoid compounds found in legumes
(Fabaceae or Leguminosae), and are found particularly in soybeans or
soy products. Soy isoflavones are phytoestrogenic compounds that are
found in three different forms, i.e., daidzein, glycitein and genistein
(Donkor, Henriksson, Vasiljevic, & Shah, 2007). According to Chang
and Stone (1990), the isoflavonoid aglycones, β-, acetyl- and malonyl-
glucosides are predominant in non-fermented soy and soy products,
whereas fermented soy foods are generally higher in aglycones. Re-
searchers have reported that isoflavones, especially the aglycones,
provide various health benefits for humans, mainly antioxidative and
antihypertensive effects associated with reduced oxidative stress and
the stability of vasodilator agents; anticarcinogenic effects by control-
ling the cell growth and death through apoptosis (Dong, Xu, Sikes, &
Wu, 2013; Michlmayr & Wolfgang, 2014; Sunita & Pattanayak, 2011).
However, isoflavonoids in glucoside forms are not absorbed in the
brush borders of small intestines and must be biotransformed to agly-
cones prior to absorption in the gut (Chun, Chung, & Song, 2009;
Setchell, 2000). Hence, hydrolysis of glucosides through enzymatic
processes using β-glucosidase to increase their bioavailability in soy
products needs to be done. The metabolism of oligosaccharides and soy
proteins by lactic acid bacteria (LAB) can increase the nutritional values
and bioavailability as well as the sensory qualities of the fermented
food products in part by masking the beany flavor (Chou & Hou, 2000).
β-Glucosidase production using biocatalysis was shown to be of
industrial interest, because of its potential use in industrial applications,

∗
Corresponding author.
E-mail address: s.prakash@uq.edu.au (S. Prakash).

https://doi.org/10.1016/j.fbio.2020.100542
Received 23 January 2019; Received in revised form 6 February 2020; Accepted 6 February 2020
Available online 14 February 2020
2212-4292/ © 2020 Published by Elsevier Ltd.
S. Hati, et al.
such as the improvement of the conversion rate, the mild reaction
conditions and easier separation of product from reaction broth as well
as being a low-cost method for commercial production of free iso-
flavones. The gut microbiome, particularly LAB, produces β-glucosidase
enzymes that convert the isoflavones from glycones to their aglycones
form (Hati, Vij, Singh, & Mandal, 2015). Strains of Streptococcus ther-
mophilus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. casei, L. plan-
tarum, L. fermentum, and several Bifidobacterium species could increase
the concentrations of genistein and daidzein in SM (Donkor & Shah,
2008; Michlmayr & Wolfgang, 2014; Rekha & Vijayalakshmi, 2011).
The microorganisms colonized in the intestine produce β-glucosidase
enzymes that hydrolyzed the glucosides and converted them into
aglycones, popularly known as phytoestrogens (Rafii, 2015). However,
the growth pattern of LAB varies in SM and cow milk due to compo-
sitional differences (Hati, Patel, & Mandal, 2018) especially the absence
or presence of lactose. SM contains prebiotics like galactooligo-
saccharides that support the growth of LAB (LeBlanc et al., 2004). A
previous study mainly focused on the activity of β-glucoside on the
reactions with isoflavones in SM (Hati et al., 2015). However, to en-
hance the reactions, SM could be enriched with skim milk powder
(SMP) (Otieno, Ashton, & Shah, 2006; Tsangalis, Wilcox, Shah, &
Stojanovska, 2005). Only two researchers have reported the increased
in the production of isoflavones aglycones in SM due to the addition of
lactose and SMP (Ding & Shah, 2010; Pham & Shah, 2007). In this
study, the effect of SMP supplementation on biotransformation of
soybean isoflavones during fermentation of SM from 30 to 45 °C using
yoghurt cultures was investigated.
This study aimed to investigate the ability of yoghurt cultures, i.e.,
L. bulgaricus and S. thermophilus, to convert the isoflavones in SM from
its glucoside form to aglycone forms during fermentation. It was hy-
pothesized that unfermented SM with pure β-glucosidase obtained from
almonds (EC Number 3.2.1.21) could increase the production of agly-
cones. Further, the use of β-glucosidase from almond allows for the use
of only plant-based materials in SM. Spectrophotometric method for
monitoring the β-glucosidase activity and reverse-phase high-pressure
liquid chromatography (RP-HPLC) for quantifying the production of
isoflavones were used in this study.
2. Materials and methods
2.1. Materials
2.1.1. Soybeans
Australian soybeans (Batch no. 09619, best before 04/2021) sold by
Golden Pulses N Foods Pty. Ltd. (Brisbane, QLD, Australia) were pur-
chased from a local supermarket in Brisbane, Australia.
2.1.2. Chemicals
Genistein, daidzein, genistin, daidzin, acetonitrile, methanol and
phosphoric acid for HPLC were of HPLC grade from Sigma-Aldrich Pty.
Ltd. (Castle Hill, NSW, Australia). β-Glucosidase from almonds was
purchased from Sigma-Aldrich with > 98% purity (EC: 3.2.1.21). p-
Nitrophenyl β-D-glucopyranoside (p-NPG) was purchased from Merck
Millipore (Bayswater, VIC, Australia).
2.1.3. Microbiological cultures
The yoghurt cultures (YCX11) – a mixed culture of L. delbrueckii ssp.
bulgaricus (LB) and S. thermophilus (ST) were obtained from Chr. Hansen
Pty. Ltd. (Bayswater, VIC, Australia) as direct vat set (DVS) cultures.
DVS cultures were stored at -40 °C for further use, a maximum of 1 yr.
2.2. Methods
2.2.1. Preparation of SM
Soybeans (200 g) were soaked in 1 l of tap water for 16 h at room
temperature (25–28 °C). The soaked beans were boiled for 15 min and
2
Food Bioscience 34 (2020) 100542
dehulled completely by kneading the beans until all the hulls came off.
The dehulled soybeans were then mixed with 500 ml of tap water and
ground in a commercial blender unit (Nutri Ninja Auto-IQ Blender
BL480, Mann & Noble, Eastern Creek, NSW, Australia) for 3 min, fil-
tered using a muslin cloth and heated to 100 °C for 5 min.
2.2.2. Fermentation of SM and analysis
One hundred ml of SM in 200 ml bottles were inoculated with 1, 2, 3
and 4% (w/v) of the pure yoghurt culture and incubated at 30, 35 and
42 °C for 18 h. According to Chandan and O'Rell (2013), the incubation
temperature for yoghurt from 31 to 45 °C even though the yoghurt
culture is thermophilic. Samples were analyzed for pH (Cube pH meter,
TPS, Brendale, QLD, Australia), titratable acidity (as a lactic acid per-
centage) by titration with 0.01 N NaOH solution (AOAC no. 947.05,
1990) and viable counts of LAB following the method of Hati et al.
(2018). The fermented SM were diluted in saline solutions. After serial
dilutions, 1 ml of samples were transferred into petri plates with molten
(42–45 °C) selective agar (LB in de Man, Rogosa and Sharpe (MRS)
medium and ST in M17 medium) from Oxoid Ltd. (Basingstoke,
Hampshire, UK). The contents were mixed thoroughly by gently swir-
ling the plates. After solidification, plates were incubated at 37 °C for
24 h and colony counts were averaged and expressed as log CFU/ml. In
addition, the fermented SM were also analyzed for β-glucosidase ac-
tivity (section 2.2.4) and isoflavones contents (section 2.2.7) after 18 h
of incubation.
2.2.3. The activity of β-glucosidase in SMP supplemented soy yoghurt
To study the effect of SMP on β-glucosidase activity of yoghurt
culture, SM was prepared by supplementing with different levels of
SMP (1.5, 2 and 2.5%). The SMP were purchased from Real Dairy
Australia Pty. Ltd. (Darra, Queensland, Australia). Subsequently, to
pasteurize the SM and inactivate lipoxygenase enzyme, SM was heated
to 85–90 °C for 15–20 min and then cooled to 30 °C for β-glucosidase
activity measurements (Ferragut, Cruz, Trujillo, Guamis, & Capellas,
2009).
A 2, 4, 6 and 8 U/ml solution of β-glucosidase was prepared sepa-
rately in 0.1 M sodium phosphate buffer (pH 7.0) for preparation of a
standard curve. p-NPG solution (5 mM) was prepared in the same buffer
then covered to avoid light. A 9 ml aliquot of p-NPG solution was in-
cubated with 1 ml of β-glucosidase separately at 37 °C for 30 min. The
amount of p-nitrophenol released was measured at 420 nm using a
spectrophotometer (SPD-10 AV, Shimadzu, Kyoto, Japan). One unit of
enzyme activity was defined as the amount of enzyme that released one
nmol of p-nitrophenol from the p-NPG/ml/min substrate with the assay
conditions as described by Tsangalis, Ashton, Mcgill, and Shah (2002).
2.2.4. Estimation of β-glucosidase activity
β-Glucosidase activity was determined by measuring the rate of
hydrolysis of p-NPG using the method of Otieno et al. (2006) and
Scalabrini, Rossi, Spettoli, and Matteuzzi (1998) with some modifica-
tions. In this method, yoghurt cultures were inoculated at 1, 2, 3 and
4% into 100 ml of SM and incubated at 30, 35 and 42 °C for 18 h. Then
500 μl of 5 mmol/l p-NPG (prepared in 100 mmol/l sodium phosphate
buffer solution, pH 7.0) was added to 5 ml aliquots of each sample and
incubated at 37 °C for 30 min. The reaction was stopped by the addition
of 250 mL cold 0.2 mol/l sodium carbonate. The resulting mixture was
centrifuged at 14,000×g for 30 min using a Sigma centrifuge (Hettich®
EBA 21Sigma-Aldrich) and filtered using a 0.45 μm Millex filter (Merck
Millipore). The amount of p-nitrophenol released was measured at
420 nm as described in section 2.2.3.
2.2.5. Hydrolysis of SM using β-glucosidase from almonds
β-Glucosidase enzymes from almonds were added at various con-
centrations (2, 4, 6 and 8 U/ml) and incubated at 30 °C for 60 min in
triplicate in heat-treated SM. Samples were withdrawn after the in-
cubation period and freeze-dried using a Christ freeze-dryer (Alpha 1–4
S. Hati, et al. Food Bioscience 34 (2020) 100542

LSC, Martin Christ, GmbH, Osterode, Germany) for the determination lactic cultures, followed by monitoring the β-glucosidase activity and
of isoflavones. measuring the biotransformation of isoflavones during SM fermenta-
tion.
2.2.6. Biotransformation of soy isoflavones through fermentation using
lactic cultures
Yoghurt cultures were added at 1, 2, 3 and 4% to freshly prepared 3.1. Identifying the concentration (inoculum rate) and incubation
and heat-treated SM at 30 °C for 18 h. After fermentation, 100 ml temperature during fermentation of SM
samples from each inoculation were freeze-dried for the analysis of
isoflavones. To investigate the effect of SMP, estimation of isoflavones 3.1.1. Effect of inoculation rate and incubation temperature on pH and
was also carried out in SM supplemented with different levels of SMP titratable acidity
(1.5, 2 and 2.5%). Soy yoghurts also known as sogurt or fermented soy milk are be-
coming popular due to the low level of cholesterol and saturated fat, no
2.2.7. Extraction of isoflavones lactose (Sarkar, 2006) and the presence of the functional compound
The extraction of isoflavones was done in triplicate, using the isoflavones, good quality proteins, essential minerals and vitamins. Soy
method of Griffith and Collison (2001) with some modifications. yoghurt prepared with different strains of LAB contribute specific flavor
Briefly, 1 g of freeze-dried sample was mixed thoroughly with 20 ml of and texture to the end-product (Donkor et al., 2007). Most LAB gen-
80% methanol and 100 μl of the internal standard flavone (1 mg/ml) in erally prefer a neutral pH to grow as provided by SM and during fer-
a 50 ml screw cap tube. Isoflavones were extracted at 50 °C for 120 min mentation, the pH of SM dropped with the incubation period. At all
in a water bath TWB-24D-CP (Thermoline Scientific, Wetherill Park, incubation temperatures, it was observed that after 18 h of fermenta-
NSW, Australia). After thorough shaking, the aliquots were then filtered tion the pH of SM yoghurt decreased to ~4.40 while the percentage of
using a Whatman No. 1 qualitative filter paper (Whatman®, Sigma-Al- lactic acid increased (from ~0.08 to ~0.50%) as the inoculation rate
drich). One ml of the filtered solution was passed through a 0.45 μm increased. No significant (P ≥ 0.05) reduction in pH was observed for
Millipore nylon filter into an HPLC vial then injected into the HPLC fermented SM at 30 and 42 °C with the increasing rate of inoculum
system within 4 h to avoid the degradation of isoflavones (Griffith & (from 1 to 4%). The lowest pH value (4.40) and highest total acid
Collison, 2001). (0.55%) were obtained at an inoculation rate of 4% at 30 °C (Table 1) in
agreement with the viable cell count of ST and LB observed after 18 h of
2.2.7.1. HPLC method. The HPLC method was based on Nakamura fermentation (Fig. 1). Normally, LAB grows and produces lactic acid
et al. (2001) with some modifications. A symmetry column with a C18 during fermentation using the milk sugar (lactose) present in bovine
stationary phase (4.6 mm i.d. x 250 mm, 5 μm particle size) from milk. However, in SM, devoid of lactose, the yoghurt cultures produce
Waters (Milford, MA, USA) and an Alltech® Alltima™ HP C18 HL guard α-galactosidase which uses the oligosaccharides (raffinose, stachyose,
column from Grace Discovery Science (Columbia, MD, USA) were used. sucrose) and converts them to lactic acid in various amount (Hati et al.,
A Hewlett Packard 1100 series HPLC (Agilent Technologies, Forest Hill, 2013) determined by the α-galactosidase activity of the strain.
VIC, Australia) with an autosampler, a quaternary pump, a UV detector, The inoculation rate of 3 and 4% showed maximum production of
a vacuum degasser and a thermostatic column compartment was used. acidity when incubated at 30 °C, with 0.52 and 0.55% lactic acid, re-
The column temperature was maintained at 25 °C. The injection volume spectively. Moreover, the pH also decreased from ~6.5 at the start to
was 5 μl. Mobile phase: Solvent A (water: phosphoric acid, 1000:1, v/v) 4.4 at the end of fermentation at 30 °C. In addition, the study by Wang,
and solvent B (water:acetonitrile:phosphoric acid, 200:800:1, v/v/v) Yu, Yang, and Chou (2003) measured a final pH of 4.5 in fermented SM
were used. The pH of solvent A and B was ~2.5. All the reagents used in after 18–24 h of incubation at 37 °C using ST. Similarly, Horackova
the mobile phase were filtered using a 0.45 μm nylon membrane. The et al. (2015) also observed the production of 0.43% lactic acid in soy
gradient was: Solvent A 100% at 0 → 0% (50 min) →100% (60 min). yoghurt by ST and LB after 16 h of fermentation, whereas Chang, Dong-
The flow rate was 0.8 ml/min, and the UV detector was set at 260 nm. Hyun, and Myung (2010) reported the pH of commercially available
Stock solutions for isoflavones were prepared separately by dissolving yoghurt ranged between 4.2-2.5.
1 mg of the pure crystalline compound in 10 ml of methanol. Each
solution was diluted with methanol to 5 working solutions at various Table 1
concentrations (1–40 μg/ml) to prepare standard curves. All the Effect of inoculation rates and incubation temperatures on pH and acidity at the
end of 18 h fermentation of soy milk.
working standards were injected into the HPLC system within 4 h
after preparation. Retention time and UV absorption patterns of pure Treatment pH Total acid (% lactic
isoflavonoid standards were used to identify isoflavones. Then, acid)
Incubation Rate of
isoflavones concentration were calculated back to a dry basis (μg/ml)
temperature (°C) inoculum (%)
and reported as g/l.
30 0 6.5 ± 0.1d 0.08 ± 0.01d
2.3. Statistical analysis 1 4.41 ± 0.04a 0.51 ± 0.01b
2 4.48 ± 0.01a 0.50 ± 0.00b
3 4.43 ± 0.01a 0.52 ± 0.01b
The experiments were done in triplicate. Data were expressed as 4 4.40 ± 0.01a 0.55 ± 0.00c
means with standard error (SEM) and the results were statistically 35 0 6.50 ± 0.02d 0.09 ± 0.00d
analyzed using the Statistical Package for the Social Sciences (SPSS 1 4.88 ± 0.01c 0.36 ± 0.01a
statistics version 22.0, IBM Corp., Armonk, NY, USA). To determine 2 4.85 ± 0.00c 0.40 ± 0.01 a
3 4.44 ± 0.00a 0.45 ± 0.01 a
differences among samples, one-way ANOVA and Duncan's test were 4 4.42 ± 0.01a 0.50 ± 0.01b
used at a significance level of 0.05. 42 0 6.51 ± 0.01d 0.08 ± 0.01d
1 4.71 ± 0.01b 0.48 ± 0.01b
3. Results and discussion 2 4.65 ± 0.01b 0.47 ± 0.01b
3 4.67 ± 0.01b 0.49 ± 0.01b
4 4.62 ± 0.01b 0.51 ± 0.00b
In the preparation of soy yoghurt, it is important to standardize the
inoculation rate and the incubation temperature to observe the ideal Values are expressed in mean ± standard error from three replications. Means
growth pattern of the cultures during fermentation. Hence, the first step with different superscripts between row show significant difference
is identifying the inoculation rate and incubation temperature of the (P < 0.05).

3
S. Hati, et al.
Fig. 1. Effect of inoculation rates (1, 2, 3, 4.%) and incubation temperatures (30, 35 and 42 °C) on viable counts of: L. bulgaricus (A) and S. thermophilus (B) after
fermentation of soy milk (mean ± standard error, n = 3).
It can be inferred from Table 1 that total acidity increases while pH
decreases with the increase of incubation temperature (30 and 35 °C)
and the rate of inoculation (from 1 to 4%). However, at 42 °C, the
highest total acid was obtained at an inoculation rate of 4%, although
the increase of acidity is not significantly different (P ≥ 0.05) for all
inoculation rates. In this study, yoghurt culture inoculated at 1% and
incubated at 35 °C showed the lowest production of acidity during
fermentation of SM. Although numerous studies have reported the op-
timum growth temperature for yoghurt culture at 37–45 °C (Donkor
et al. (2007); Radke-Mitchell and Sandine (1986)), the use of low
temperature (31–45 °C) in fermentation of yogurt has been reported by
Chandan and O'Rell (2013) while no references are available on fer-
mented SM incubated at low temperature. However, according to
Angeles and Marth (1971), acid production in SM was not always di-
rectly related to the growth rates of microorganism. Hence, the acidity
production from fermented SM incubated at low temperature needs to
be investigated further.
3.1.2. Effect of inoculation concentration and incubation temperature on
the viable count of LB and ST
The viability counts of ST and LB in fermented SM at different in-
oculation rates (1, 2, 3 and 4%) and incubation temperature (30, 35 and
42 °C) for 18 h as shown in Fig. 1. Although not significantly different
(P ≥ 0.05), at an incubation temperature of 30 °C and 1% cultures, the
highest viable cell counts for both cultures were 8.49 log CFU/ml for LB
(Fig. 1A) and 8.62 log CFU/ml for ST (Fig. 1B). There was no significant
difference (P ≥ 0.05) at 2 and 3% inoculum rate of LB counts when the
SM was incubated at all three temperatures.
It was found that the cell count of ST was slightly higher (8.50 log
CFU/ml) than the LB (8.20 log CFU/ml) when inoculated at 1% yoghurt
cultures during the fermentation of SM. This is possibly due to the in-
ability of LB to use sucrose, the major sugar in SM, compared to SM, in
agreement with Pinthong, Macrae, and Rothwell (1980). Bordignon,
Nakahara, Yoshihashi, and Nikkuni (2004) also reported that LB
showed the lowest α-galactosidase activity in the medium containing
raffinose. This study was consistent with that reported by Donkor et al.
(2007), which found that ST strains had higher viable counts than LB in
soy yoghurt. Other studies have shown that fermentation of SM using
lactic cultures at 37–42 °C for a particular incubation period resulted in
a decrease of pH value (3.9–4.3) (Wang, Lin, Wang, & Chen, 2013) and
4
Food Bioscience 34 (2020) 100542
an increase in total titratable acidity (0.64–0.97%). From this study,
30 °C and a 1% rate of inoculum gave the maximum viable counts for
ST and LB and considerably lowered the pH and produced acid after
fermentation of SM.
3.2. β-Glucosidase activity
β-Glucosidase activity of yoghurt culture as affected by different
inoculation rates at various incubation temperatures and different le-
vels of SMP at 1% of culture are shown in Fig. 2A and B. Yuksekdag,
Cinar Acar, Aslim, and Tukenmez (2017) stated that the enzymatic
activity depends on the strain, growth medium and culture conditions,
such as temperature and pH.
3.2.1. β-Glucosidase activity of yoghurt cultures in SM
The β-glucosidase activity in fermented SM with optimal growth
conditions of yoghurt cultures were significantly (P < 0.05) different
(Fig. 2A). The highest β-glucosidase activity was obtained from culture
inoculated at 1% (4.23 U/ml), at 30 °C due to high numbers of total
viable counts for both cultures (Fig. 1A). At inoculation rates of 2 and
3%, the culture produced a similar amount of β-glucosidase enzymes.
The lowest activity (P < 0.05) was found when the SM was fermented
at 35 °C irrespective of the inoculation rate (Fig. 2). These could be due
to different inoculation rates and incubation temperatures used for
determination of β-glucosidase activity that is dependent on each other.
However, the production of β-glucosidase and their activity appeared to
be species-specific and highly influenced by the interaction between
fermentation parameters (strain, growth medium and culture condi-
tions). Usually the enzymatic activity of β-glucosidase corresponds with
the optimal temperature for microorganism growth (Ahmed, Nasim,
Batool, & Bibi, 2017; Choi, Kim, & Rhee, 2002; Yuksekdag et al., 2017).
Although different cultures were used, the results showed similar trends
of β-glucosidase activity after 18 h of fermentation using LB and ST.
Similar findings were also observed by Tsangalis et al. (2005) who re-
ported that fermented SM showed various degrees of β-glucosidase
activity depending on the starter organism and growth behavior.
3.2.2. Effect of SMP supplementation on β-glucosidase activity of yoghurt
cultures in SM
SM was supplemented with different percentages of SMP to support
S. Hati, et al.
Fig. 2. β-Glucosidase activity of yoghurt culture in soy milk as influenced by: (A) inoculation rates (1, 2, 3, 4%, and control: unfermented soy milk) at different
incubation temperatures (30, 35 and 42 °C) without SMP; and (B) at 1% inoculation rate of culture in soy milk with SMP (1.5; 2; 2.5%, and control: fermented soy
milk without SMP) incubated at 30 °C (mean ± standard error, n = 3).
the growth of yoghurt cultures at 1% inoculation rate during the fer-
mentation of SM. It can be seen from Fig. 2 that yoghurt cultures in SM
supplemented with SMP significantly (P < 0.05) increased the β-glu-
cosidase activity, with 2 and 2.5% of SMP leading to the highest activity
(~4.97 U/ml) compared to control without SMP and SM with 1% SMP.
Moreover, the addition of SMP to SM provided extra nutrient to the
yoghurt cultures that increased bacterial growth during the fermenta-
tion process. Hati, Patel, Patel, and Prajapati (2017) studied the effect
of whey protein concentrate (WPC70) supplementation on the pro-
duction of β-glucosidase enzymes in fermented SM, where the supple-
mentation enhanced the enzyme probably due to more nutrient to the
lactic cultures.
3.3. Biotransformation of soy isoflavones
3.3.1. Effect of fermentation on biotransformation of soy isoflavones by
yoghurt cultures
Foods rich in soy protein contains isoflavones that are found as a
complex mixture of genistin and daidzin (Donkor & Shah, 2008). The
metabolic fate of soy isoflavones after consumption, as well as their
biological activities, depends on their chemical structure. To exert a
biological effect, isoflavone glucosides should be hydrolyzed by both
intestinal mucosal and bacterial β-glucosidase releasing the aglycones
(Donkor & Shah, 2008). Several studies have shown that LAB with β-
glucosidase activity can increase the aglycone content during SM fer-
mentation (Donkor et al., 2007). In this study, yoghurt cultures were
inoculated at different rates in SM and incubated for 18 h at 30 °C. The
biotransformation of isoflavones in fermented SM with various in-
oculation rate of culture (without SMP) and different concentration of
SMP at 1% inoculation rate are shown in Fig. 3A and B, respectively.
Fig. 3A shows that yoghurt culture (ST and LB) produced maximum
aglycones (daidzein and genistein) at 2% followed by the 1% inoculum
rate. However, the highest conversion occurred at 1%, as the amount of
the glucoside form of daidzin (0.02 g/l) was bio-converted to daidzein
(0.74 g/l). In the case of 3 and 4%, the conversion of isoflavones sig-
nificantly decreased compared to 1 and 2%. The maximum bio-
transformation occurred at 2% is not only due to the maximum total
viable counts that observed in 1 and 2% for both the cultures but also
because of the competitive growth behavior of 3 and 4% (more cell
numbers). At concentrations of 3 and 4%, the cell numbers were higher
than the substrate sources, resulting in a lack of nutrients needed for the
5
Food Bioscience 34 (2020) 100542
cell to grow and produce β-glucosidase. According to Ahmed et al.
(2017), β-glucosidases are produced by microorganisms in low amounts
and inhibited by their end-product (glucose). The amount of enzymes
that are secreted from the cell depend on fermentation conditions such
as carbon source and concentration, nitrogen source, fermentation
period and inoculum size. The lower biotransformation with the in-
creased cell numbers is due to the decline in enzyme production and
activity. It was also observed that unfermented SM (control) also con-
tained a small amount of aglycones of isoflavones due to the overnight
soaking of soybeans in water (Fig. 3A). Matsurra, Obata, and
Fukushima (1989) reported that soybean itself contains β-glucosidase
enzymes and can produce aglycones.
The maximum biotransformation of soy isoflavones occurred at a
2.5% level of SMP supplementation in SM (Fig. 3B). Highest production
of daidzein (1.30 g/l) and genistein (0.65 g/l) occurred at a 2.5% of
SMP supplementation compared to 1.5 and 2%. Supplementation of
SMP with at least 2.5% increased the biotransformation rate as well as
the production of β-glucosidase enzymes (Fig. 3B) more than without
supplemented fermented SM with 1% inoculum (Fig. 3A). The enhan-
cing effect of the SMP supplementation on the biotransformation is
possibly due to the presence of lactose in SMP. Supplementation with
lactose will promote the growth of L. bulgaricus and thus enhance the
production of β-galactosidase. According to Pham and Shah (2009), β-
galactosidase has been found to hydrolyze isoflavone glycosides to
isoflavone aglycones. Ding and Shah (2010) also reported that at the
early stages of fermentation (48 h), the lactose-supplemented SM gen-
erated higher production of biologically active isoflavone aglycones
(glycitein, daidzein, and genistein) compared to SM without lactose.
3.3.2. Effect of β-glucosidase from almonds on the conversion of soy
isoflavones in SM
Conversion of isoflavones in SM was done by the addition of β-
glucosidase enzymes from almonds at different concentration (2, 4, 6, 8
U) followed by incubation for 60 min at 30 °C (Fig. 4). It is seen that 8 U
of β-glucosidase showed the highest biotransformation of isoflavones in
SM compared to the other concentrations (2, 4, 6 U).
With the addition of 8 U of β-glucosidase, the production of daid-
zein was greater (1.49 g/l) than genistein (0.80 g/l). Similarly, when
added at the rate of 6 and 8 U, β-glucosidase enzymes show a maximum
production of genistein (0.74 and 0.80 g/l, respectively). Matsurra et al.
(1989) showed that β-glucosidase enzymes are stable at pH 4.0–5.5,
S. Hati, et al. Food Bioscience 34 (2020) 100542

Fig. 3. Biotransformation of soy isoflavones by yoghurt culture as influenced by: (A) inoculation rates (1, 2, 3, 4%, and control) without SMP; and (B) at 1%
inoculation rate of culture in soy milk with SMP (1.5; 2; 2.5%) incubated at 30 °C (mean ± standard error, n = 3).

Fig. 4. Effect of different level β-glucosidase from almond on the bioconversion

of soy isoflavones in soy milk (mean ± standard error, n = 3).
Fig. 5. Mean of isoflavone content (daidzein and genistein) after incubation at
30 °C with 2% of yoghurt culture and 4 U of almonds β-glucosidase (mean ±
and its pH range of action was 3.5–7.0. It was also found that although standard error, n = 3).
the range of the reaction temperature was 10–50 °C, there was a sig-
nificant reaction even at 5 °C. Another study by Pandjaitan et al. (2000)
biotransformation of isoflavones by the yoghurt cultures was consistent
also reported the ability of β-glucosidase activity from almond to con-
with the growth of cultures in SM. The results from this study confirm
vert most of the isoflavone glucosides in soy protein isolate to their
that β-glucosidase, regardless of sources (microbial or almond), can
aglycones.
hydrolyze the isoflavones bonds and produce aglycones that depend on
To understand the difference between soy aglycones bio-
the β-glucosidase activity and its concentration. At the same level of β-
transformation by β-glucosidase from different sources (yoghurt cul-
glucosidase activity (~4 U/ml), biotransformation of daidzein and
tures and almond), a comparison was made based on the same level of
genistein from the 2% of yoghurt culture was higher compared with β-
β-glucosidase activity. Among all treatments, β-glucosidase activity
glucosidase from almond. The β-glucosidase enzymes from almonds
from yoghurt culture at an inoculation rate of 2% was ~3.6 U/ml hence
showed the highest biotransformation of isoflavones in SM after 30 min
comparable with β-glucosidase from almond at 4 U/ml. It can be in-
of incubation whereas soy yoghurt culture showed the biotransforma-
ferred from Fig. 5 that the amount of daidzein (~0.3 g/l) and genistein
tion after 18 h of incubation at 30 °C. It was observed that soy aglycones
(~0.16 g/l) produced from SM treated with almond β-glucosidase was
enriched SM could be produced simply by adding β-glucosidase en-
lower than those from yoghurt culture (daidzein 1.18 and genistein
zymes. However, further study is required to investigate and optimize
0.55 g/l). These results are similar to the previous study reported by
the biotransformation of aglycones using β-glucosidase enzymes from a
Ismail and Hayes (2005). A lower level of β-glucosidase activity was
plant source (almond).
possibly due to the difference in incubation time when SM was treated
with almond β-glucosidase (30 min) and yoghurt culture β-glucosidase
(18 h).
CRediT authorship contribution statement

4. Conclusions Subrota Hati: Conceptualization, Investigation, Writing - original

draft. Dian W. Ningtyas: Data curation, Writing - review & editing.
The viability of the yoghurt cultures (LB and ST) was significantly Jassimran Kaur Khanuja: Writing - review & editing. Sangeeta
increased in fermented SM supplemented with SMP during the fer- Prakash: Supervision, Writing - review & editing.
mentation period. The production of β-glucosidase, enzyme activity and

6
S. Hati, et al.
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported under the Indo-Australia Career Boosting
Gold Fellowship: 2016–2017 sponsored by the Department of
Biotechnology, Govt. of India.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fbio.2020.100542.
References
Ahmed, A., Nasim, F., Batool, K., & Bibi, A. (2017). Microbial β-glucosidase: Sources,
production and applications. Journal of Applied & Environmental Microbiology, 5(1),
31–46.
Angeles, A. G., & Marth, E. H. (1971). I. Growth and acid production. Journal of Milk and
Food Technology, 34(1), 30–36.
AOAC (1990). Official methods of analysis (15th ed.). Washington, D.C, USA: The
Association of Official Analytical Chemists.
Bordignon, J. R., Nakahara, K., Yoshihashi, T., & Nikkuni, S. (2004). Hydrolysis of iso-
flavones and consumption of oligosaccharides during lactic acid fermentation of
soybean milk. Japan Agricultural Research Quarterly, 38(4), 259–265.
Chandan, R. C., & O'Rell, K. (2013). Principles of yogurt processing. In R. C. Chandan, &
A. Kilara (Eds.). Manufacturing yogurt and fermented milks (pp. 239–261). Chichester,
West Sussex, UK: John Wiley & Sons, Ltd.
Chang, S. Y., Dong-Hyun, K., & Myung, J. H. (2010). Physicochemical and sensory
characteristics of soy yogurt fermented with Bifidobacterium breve K-110,
Streptococcus thermophilus 3781, or Lactobacillus acidophilus Q509011. Food
Science and Biotechnology, 19(1), 107–113.
Chang, C. Y., & Stone, M. B. (1990). Effect of total soymilk solids on acid production by
selected Lactobacilli. Journal of Food Science, 55, 1643–1647.
Choi, Y. B., Kim, K. S., & Rhee, J. S. (2002). Hydrolysis of soybean isoflavone glucosides
by lactic acid bacteria. Biotechnology Letters, 24(24), 2113–2116.
Chou, C. C., & Hou, J. W. (2000). Growth of Bifidobacteria in soymilk and their survival in
the fermented soymilk drink during storage. International Journal of Food
Microbiology, 56, 113–121.
Chun, O. K., Chung, S. J., & Song, W. O. (2009). Urinary isoflavones and their metabolites
validate the dietary isoflavone intakes in US adults. Journal of the American Dietetic
Association, 109, 245–254.
Ding, W. K., & Shah, N. P. (2010). Enhancing the biotransformation of isoflavones in
soymilk supplemented with lactose using probiotic bacteria during extended fer-
mentation. Journal of Food Science, 75(3), M140–M149.
Dong, X., Xu, W., Sikes, R. A., & Wu, C. (2013). Combination of low dose of genistein and
daidzein has synergistic preventive effects on isogenic human prostate cancer cells
when compared with individual soy isoflavones. Food Chemistry, 141(3), 1923–1933.
Donkor, O. N., Henriksson, A., Vasiljevic, T., & Shah, N. P. (2007). Rheological properties
and sensory characteristics of set-type soy yogurt. Journal of Agriculture and Food
chemistry, 55, 9868–9876.
Donkor, O. N., & Shah, N. P. (2008). Production of β-glucosidase and hydrolysis of iso-
flavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and
Lactobacillus casei in soymilk. Food Microbiology Safety, 73, 15–20.
Ferragut, V., Cruz, N. S., Trujillo, A., Guamis, B., & Capellas, M. (2009). Physical char-
acteristics during storage of soy yogurt made from ultra-high pressure homogenized
soymilk. Journal of Food Engineering, 92(1), 63–69.
Gil-Izquierdo, A., Penalvo, J. L., Gil, J. I., Medina, S., Horcajada, M. N., Lafay, S., et al.
(2012). Soy isoflavones and cardiovascular disease epidemiological, clinical and
-omics perspectives. Current Pharmaceutical Biotechnology, 13(5), 624–631.
Griffith, A. P., & Collison, M. W. (2001). Improved methods for the extraction and ana-
lysis of isoflavones from soy-containing foods and nutritional supplements by re-
versed-phase high-performance liquid chromatography and liquid chromatography-
mass spectrometry. Journal of Chromatographic Science, 913, 397–413.
Hati, S., Patel, N., & Mandal, S. (2018). Comparative growth behaviour and bio-
functionality of lactic acid bacteria during fermentation of soy milk and bovine milk.
Probiotics Antimicrobial Proteins, 10(2), 277–283.
Hati, S., Patel, N., Patel, K., & Prajapati, J. B. (2017). Impact of whey protein concentrate
on proteolytic lactic cultures for the production of isoflavones during fermentation of
Food Bioscience 34 (2020) 100542
soy milk. Journal of Food Processing and Preservation, 41, 1–9.
Hati, S., Vij, S., Mandal, S., Malik, R. K., Kumari, V., & Khetra, Y. (2013). α‐galactosidase
activity and oligosaccharides utilization by Lactobacilli during fermentation of soy
milk. Journal of Food Processing and Preservation, 38(3), 1065–1071.
Hati, S., Vij, S., Singh, B. P., & Mandal, S. (2015). β-glucosidase activity and bioconver-
sion of isoflavones during fermentation of soymilk. Journal of the Science of Food and
Agriculture, 95(1), 216–220.
Horáčková, S., Andrea, M., Marcela, S., Věra, S., & Milada, P. (2015). Fermentation of
soymilk by yoghurt and Bifidobacteria strains. Czech Journal of Food Sciences, 33(4),
313–319.
Ismail, B., & Hayes, K. (2005). β-glycosidase activity toward different glycosidic forms of
isoflavones. Journal of Agricultural and Food Chemistry, 53(12), 4918–4924.
LeBlanc, J. G., Silvestroni, A., Connes, C., Juillard, V., deGiori, G. S., Piard, J. C., et al.
(2004). Reduction of non-digestible oligosaccharides in soymilk: Application of en-
gineered lactic acid bacteria that produce alpha-galactosidase. Genetics and Molecular
Research, 3(3), 432–440.
Matsurra, M., Obata, A., & Fukushima, D. (1989). Objectionable flavor of soymilk de-
veloped during the soaking of soybeans and its control. Journal of Food Science, 54(3),
602–605.
Michlmayr, H., & Wolfgang, K. (2014). β-glucosidase activities of lactic acid bacteria:
Mechanisms, impact on fermented food and human health. FEMS Microbiology Letters,
352, 1–10.
Nakamura, Y., Kaihara, A., Yoshii, K., Tsumura, Y., Ishimitsu, S., & Tonogai, Y. (2001).
Content and composition of isoflavonoids in mature or immature beans and bean
sprouts consumed in Japan. Journal of Health Science, 47(4), 394–406.
Otieno, D. O., Ashton, J. F., & Shah, N. P. (2006). Evaluation of enzymic potential for
biotransformation of isoflavone phytoestrogen in soymilk by Bifidobacterium animalis,
Lactobacillus acidophilus and Lactobacillus casei. Food Research International, 39,
394–407.
Pandjaitan, N., Hettiarachchy, N., JU, Z. Y., Crandall, P., Sneller, C., & Dombek, D.
(2000). Evaluation of genistin and genistein contents in soybean varieties and soy
protein concentrate prepared with 3 basic methods. Journal of Food Science, 65(3),
399–402.
Pham, T. T., & Shah, N. P. (2007). Biotransformation of isoflavone glycosides by
Bifidobacterium animalis in soymilk supplemented with skim milk powder. Journal of
Food Science, 72(8), M316–M324.
Pham, T. T., & Shah, N. P. (2009). Hydrolysis of isoflavone glycosides in soymilk by β-
galactosidase and β-glucosidase. Journal of Food Biochemistry, 33(1), 38–60.
Pinthong, R., Macrae, R., & Rothwell, R. (1980). The development of a soya-based yo-
ghurt: I. Acid production by lactic acid bacteria. Journal of Food Technology, 15,
647–652.
Radke-Mitchell, L. C., & Sandine, W. E. (1986). Influence of temperature on associative
growth of Streptococcus thermophilus and Lactobacillus bulgaricus. Journal of Dairy
Science, 69(10), 2558–2568.
Rafii, F. (2015). The role of colonic bacteria in the metabolism of the natural isoflavone
daidzin to equol. Metabolites, 5(1), 56–73.
Rekha, C. R., & Vijayalakshmi, G. (2011). Accelerated fermentation of 'Idli' batter using
soy residue okara. Journal of Food Science & Technology, 48(3), 329–334.
Riaz, M. N. (2006). Soy applications in food. Boca Raton, FL, USA: CRC Press.
Sarkar, S. (2006). Potential of soyoghurt as a dietetic food. Nutrition & Food Science, 36,
43–49.
Scalabrini, P., Rossi, M., Spettoli, P., & Matteuzzi, D. (1998). Characterization of
Bifidobacterium strains for use in soymilk fermentation. International Journal of Food
Microbiology, 39, 213–219.
Setchell, K. D. (2000). Absorption and metabolism of soy isoflavones-from food to dietary
supplements and adults to infants. Journal of Nutrition, 130(3) 654S-5S.
Sunita, P., & Pattanayak, S. P. (2011). Phytoestrogens in postmenopausal indications: A
theoretical perspective. Pharmacognosy Reviews, 5(9), 41–47.
Tsangalis, D., Ashton, J. F., Mcgill, A. E. J., & Shah, N. P. (2002). Enzymic transformation
of isoflavone phytoestrogens in soymilk by β-glucosidase-producing Bifidobacteria.
Journal of Food Science, 67(8), 3104–3113.
Tsangalis, D., Wilcox, G., Shah, N. P., & Stojanovska, L. (2005). Bioavailability of iso-
flavone phytoestrogens in postmenopausal women consuming soya milk fermented
with probiotic Bifidobacteria. British Journal of Nutrition, 93, 867–877.
Wang, J., Lin, Q., Wang, Y., & Chen, X. (2013). Research on soybean curd coagulated by
lactic acid bacteria. SpringerPlus, 2(250), 354–361.
Wang, H., & Murphy, P. (1994). Isoflavone content in commercial soybean foods. Journal
of Agricultural and Food Chemistry, 42, 1666–1673.
Wang, Y. C., Yu, R. C., Yang, H. Y., & Chou, C. C. (2003). Sugars and acid contents in
soymilk fermented with lactic acid bacteria alone or simultaneously with
Bifidobacteria. Food Microbiology, 20, 333–338.
Yamanaka, Y., Okumura, S., Mitsugi, K., & Hasagawa, Y. (1969). Process for preparing a
sour milk beverage or yoghurt. Food Science and Technology, 1, 1308–1310.
Yuksekdag, Z., Cinar Acar, B., Aslim, B., & Tukenmez, U. (2017). β-glucosidase activity
and bioconversion of isoflavone glycosides to aglycones by potential probiotic bac-
teria. International Journal of Food Properties, 20(sup3), S2878–S2886.