International Journal of Food Microbiology 262 (2017) 14–22

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Biotransformation of soy whey into soy alcoholic beverage by four MARK

commercial strains of Saccharomyces cerevisiae
Jian-Yong Chuaa, Yuyun Lua, Shao-Quan Liua,b,⁎
a
Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Science Drive 3, Singapore 117543, Singapore
b
National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu 215213, China

A R T I C L E I N F O A B S T R A C T

Keywords: Soy whey is a liquid waste stream generated from tofu and soy protein manufacturing, and is commonly disposed
Soy whey of into the drainage system in food industry. Instead of disposing of soy whey as a waste, it could be used to
Alcoholic beverage produce alcoholic beverages. This study investigated the feasibility of converting soy whey into soy alcoholic
Yeasts beverage using four commercial Saccharomyces cerevisiae strains as a zero-waste approach to tackle the soy whey
Alcoholic fermentation
disposal issue. The four Saccharomyces yeasts grew by approximately 2 log CFU/mL and produced approximately
Isoflavones
7–8% (v/v) of ethanol. Isoflavone glucosides were hydrolyzed and transformed into isoflavone aglycones, in-
Zero-waste
creasing the antioxidant capacity. New aroma-active volatiles, especially esters and higher alcohols, were pro-
duced and imparted fruity and floral notes to the soy alcoholic beverage. Therefore, alcoholic fermentation
would serve as a solution toward zero-waste manufacturing by biotransforming soy whey into a world's first
novel functional alcoholic beverage naturally enriched with free isoflavones.

1. Introduction
Soybean has been known to be an economical source of protein for
human consumption (Yeo and Liong, 2010). Soybean can be consumed
as it is or be processed into other soy products. Tofu, which is a product
made from soybean, is very popular among Asian countries and it is
increasingly being accepted by consumers worldwide due to its asso-
ciated health benefits (Belén et al., 2012; Corzo-Martínez et al., 2016).
Tofu can be produced through several procedures that differ among
countries. One of the commonly used procedures of producing tofu is by
first producing soymilk from fresh soybean, followed by boiling and
then coagulation using calcium or magnesium salt (Belén et al., 2012).
Soy whey is generated when the tofu is pressed to remove excess water
(Han et al., 2001). Soy whey is also produced from soy protein con-
centrate/isolates manufacture, though tofu whey was the focus of this
study.
With increasing production of tofu, the amount of tofu whey will
increase proportionally (Corzo-Martínez et al., 2016). Tofu whey, when
discarded as an untreated waste, will create environmental pollution
due to its nutrient content (mainly protein and soluble sugars) present
in the liquid substrate (Mitra et al., 2010; Zhang et al., 2012). Instead of
disposing of tofu whey as a waste, this liquid substrate has been proven
to be a potential resource for various purposes. Tofu whey has been
used for the recovery of soy protein using batch foam fractionation (Li
et al., 2014) and isoflavone aglycones using foam fractionation and
acidic hydrolysis (Liu et al., 2013a, 2013b). Besides recovery processes,
tofu whey can be used as a fermentation or growth substrate to produce
citric acid (Khare et al., 1994), nisin (Mitra et al., 2010) and en-
dopolysaccharides (Zhang et al., 2012). However, these approaches still
generate considerable amounts of wastes at the end of the processes.
Furthermore, little research has been done to biotransform tofu whey
into directly edible food and beverage products, without generating any
waste streams (total utilization of tofu whey or zero waste).
As discussed by Mitra et al. (2010), soy whey contains sufficient
nutrients to support microbial growth. In addition, Varela (2016)
pointed out that any beverage containing sufficient fermentable sugars
can be biotransformed into alcoholic products. Therefore, tofu whey,
with an appropriate level of sugar supplementation, could serve as a
potential substrate for yeast fermentation to produce alcoholic bev-
erages. One important rationale for using yeast to biotransform tofu
whey into fermented beverages is that numerous flavor-active com-
pounds, besides ethanol, are formed by yeast and these compounds can
give the fermented beverage its characteristic aroma and flavor (Varela,
2016). Hence, alcoholic fermentation could serve as an alternative
method to transform tofu whey into directly consumable products and
at the same time, creating a zero-waste solution to the tofu whey dis-
posal issue.
In industrial wine fermentation, starters of selected Saccharomyces

⁎
Corresponding author at: Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore.
E-mail address: chmlsq@nus.edu.sg (S.-Q. Liu).

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.09.007
Received 12 July 2017; Received in revised form 7 September 2017; Accepted 11 September 2017
Available online 13 September 2017
0168-1605/ © 2017 Elsevier B.V. All rights reserved.
J.-Y. Chua et al.
cerevisiae strains are used to replace traditional spontaneous fermenta-
tion (Viana et al., 2008). Currently, there is no study on the feasibility
of alcoholic fermentation of minimally supplemented tofu whey. This
research explored the usage of four commercial strains of Sacchar-
omyces cerevisiae in the fermentation of tofu whey. Different yeast
species or strains would produce different types and amounts of me-
tabolites during fermentation which would allow winemakers to select
the yeast that would best suit their desired wine style (Bisson and
Joseph, 2009; Varela, 2016).
The objective of this study was to evaluate the feasibility of using
tofu whey as a substrate for alcoholic fermentation and determine the
fermentation kinetics and flavor compounds produced by four com-
mercial strains of Saccharomyces yeasts.
2. Materials and methods
2.1. Yeast cultures
Four different strains of S. cerevisiae were used in this experiment.
Active dry yeast S. cerevisiae Merit (Chr. Hansen, Horsholm Denmark;
used mainly in wine fermentation), S. cerevisiae EC1118 (Lallemand
Inc., Brooklyn Park, Australia; used mainly in wines and ciders fer-
mentation), S. cerevisiae R2 (Lallemand Inc., Brooklyn Park, Australia;
used mainly in wine fermentation) and S. cerevisiae 71B (Lallemand
Inc., Brooklyn Park, Australia; used mainly in wine fermentation) were
propagated in sterile yeast nutrient broth containing 2.5 g of yeast ex-
tract, 2.5 g of bacteriological peptone, 2.5 g of malt extract and 20 g of
glucose in 1 L of water adjusted to pH 5.0 using 1 M HCl and incubated
statically for 72 h at 20 °C to obtain pure cultures. The pure cultures
were stored at −80 °C until use.
2.2. Tofu whey collection and treatment
Tofu whey was obtained through the making of tofu in our food
processing laboratory. Soybeans (from Canada) obtained from a local
supermarket were soaked in deionized water in 1:6 (w/v) ratio for 16 h.
After that, the soaked beans were drained and blended with deionized
water using a blender (Model MX-J210GN, Panasonic, Osaka, Japan) in
a 1:6 dry weight to volume ratio to obtain a slurry. The slurry was then
filtered through a cheese cloth to obtain soymilk. The residue (okara)
was rinsed with deionized water in a 1:2 dry weight to volume ratio.
The soymilk was boiled for 5 min and cooled to 87 °C prior to the ad-
dition of 2% (w/w with respect to the dry weight of the soybean)
commercial calcium sulfate (Home Brew Ohio, Sandusky, USA) sus-
pended in deionized water. The mixture was stirred to ensure proper
mixing of the coagulant with the soymilk before it was allowed to stand
for 15 min. The calcium ions serve as bridging ions to facilitate the
cross-linking between soy proteins to form the gel (soybean curd). The
coagulated soymilk (tofu) was transferred onto a meshed mold overlaid
with a cheese cloth. The tofu was pressed thrice for 5 min each at
maximum pressure using a manual press and the tofu whey was col-
lected below the meshed mold.
The pH of the tofu whey (initial pH of 5.78) was first adjusted to
pH 4.0 using 1 M D-/L-malic acid. The soluble solids content (°Brix; an
initial value of 2.55) was then adjusted to 15°Brix using commercial
sucrose. The pre-treated tofu whey was pasteurized at 60 °C for 20 min
and the effectiveness of the pasteurization was verified via plate
counting.
Prior to the preparation of the pre-culture, the frozen pure cultures
were reactivated by inoculating into fresh yeast nutrient broth and in-
cubated for three days at 20 °C. Four small batches (15 mL) of the
pasteurized tofu whey was inoculated with 5% (v/v) of each of the four
reactivated yeast cultures and incubated for three days at 20 °C. The
fermented tofu whey served as the pre-culture for the fermentation.
15
International Journal of Food Microbiology 262 (2017) 14–22
2.3. Fermentation design
Duplicate laboratory-scale fermentations were conducted twice in
500-mL Erlenmeyer flasks containing 300 mL of pasteurized soy whey.
Each flask was inoculated with 1% (v/v) of each pre-culture. The fer-
mentation was carried out statically at 20 °C for 10 days and samples
were withdrawn periodically for analysis. Yeast cell count was de-
termined by spread plating on potato dextrose agar (PDA; Oxoid,
Hampshire, United Kingdom). The pH and °Brix values were measured
using a pH meter (827 pH Lab, Metrohm, Herisau, Switzerland) and a
refractometer (RX-5000α, ATAGO, Tokyo, Japan), respectively.
2.4. Soluble protein determination
Soluble protein determination was carried using the Bradford
method (Bradford, 1976). A Bradford protein assay kit was obtained
from Bio-Rad (California, United States of America). The concentrated
protein reagent (Coomassie blue) was diluted with deionized water at a
ratio of 1:4 (v/v). Five milliliter of the protein reagent was added to
0.1 mL of the sample and incubated for 5 min before absorbance mea-
surement at 595 nm using a UV–Vis spectrophotometer (UVmini-1240,
Shimadzu, Kyoto, Japan). A blank was prepared using 0.1 mL of deio-
nized water with 5 mL of the protein reagent. Bovine serum albumin
(BSA) from 0.1 to 1.0 mg/mL was used as the standard for quantifica-
tion. All samples and standards readings were carried out between
5–20 min after mixing to ensure precise readings (Bradford, 1976).
2.5. Non-volatile component analysis
Sugars, glycerol, organic acids, amino acids and isoflavones were
analyzed using high performance liquid chromatography (HPLC;
Shimadzu, Kyoto, Japan). Samples used for HPLC analyses were first
centrifuged at 11,000 × g for 10 min and then filtered through an 0.20-
μm filter prior to injection. Both sugars and glycerol were detected
using evaporative light scattering detector (ELSD) connected to a
Zorbax carbohydrate column (150 × 4.6 mm; Agilent, Santa Clara, CA,
USA). Sugars were eluted at 40 °C with a mobile phase comprising of
acetonitrile/water (80:20 v/v) flowing at 1.4 mL/min. Glycerol was
eluted with the same mobile phase at 30 °C and flow rate of 0.5 mL/
min. Organic acids were separated at 40 °C using Supelcogel C-160 H
column (300 × 7.8 mm; Supelco, Bellefonte, PA, USA) with 0.1% (v/v)
sulfuric acid (H2SO4) as the mobile phase at 0.4 mL/min. For organic
acids, photodiode array (PDA) set at 210 nm was used for detection.
Amino acids and ammonia were analyzed by using reversed-phase
Waters AccQ-Tag Nova-Pak C18 column (150 × 3.9 mm; Waters,
Dublin, Ireland) and reagents from Waters (1993). Isoflavones were
separated using Zorbax Eclipse Plus C18 Column (150 × 4.6 mm;
Agilent, Santa Clara, CA, USA) with 0.1% acetic acid in DI water (sol-
vent A) and 0.1% acetic acid in methanol (solvent B). The analysis
started with 15% solvent B with gradual increments of solvent B till
50% within 46 min and held at 50% solvent B for additional 10 min
before equilibrating back to 15% solvent B. Isoflavones were detected
using PDA set at 262 nm. All analytes were identified and quantified
using pure sugars, glycerol, organic acids, amino acids and isoflavones
as standards. The coefficients of determination (r2) of the standard
curves were at least 0.98.
2.6. Volatile component analysis
Volatile components were analyzed using headspace solid-phase
micro-extraction (HS-SPME) gas chromatography (GC) coupled with
mass spectrometry (MS) and flame ionization detector (FID) (Agilent;
Santa Clara, CA, USA). A 5-mL portion of the soy alcoholic beverage
adjusted to pH 2.5 using 1 M HCl was subjected to HS-SPME extraction
at 60 °C for 50 min using a carboxen/poly(dimethylsiloxane) fiber
(Supelco, Bellefonte, PA, USA) at 250 rpm/min. Following extraction,
J.-Y. Chua et al.
the SPME fiber was desorbed at 250 °C for 3 min in the injection port.
Carrier gas (helium) flow rate was set at 1.2 mL/min and the tem-
perature program was set to increase from 50 °C (5 min) to 230 °C
(30 min) at a rate of 5 °C/min. Identification of the volatile compounds
was done through comparing their individual mass spectra with the
NIST08 Library and Wiley275 Library as well as linear retention indices
(LRI). Ethanol quantification was carried out using external standards
dissolved in 10% (v/v) unfermented soy whey. The coefficients of de-
termination (r2) of the standard curves were at least 0.98.
2.7. Antioxidant capacity assay
2,2-Diphenyl-1-picrylhydrazyl (DPPH) analysis was carried out to
determine the changes in the antioxidant activity before and after fer-
mentation. Samples used for and DPPH analyses were subjected to the
same pre-treatment as the HPLC samples. DPPH analysis, expressed as
Trolox equivalent, was carried out using a UV–Vis spectrophotometer
(UVmini-1240, Shimadzu, Kyoto, Japan) set at 515 nm. Pre-treated
samples (0.1 mL) were mixed with 3.9 mL of 25 mg/L methanolic DPPH
solution and left to stand in the dark for 2 h. A sample of 100% me-
thanol was used as a blank for the measurement and a 0.1 mL of DI
water mixed with 3.9 mL of 25 mg/L methanolic DPPH solution was
used as the control. A standard curve ranging from 0 to 0.3 mg/mL was
prepared using Trolox (dissolved in phosphate-buffered saline; PBS) as
the standard.
2.8. Statistical analysis
All analytical results were subjected to one-way analysis of variance
(ANOVA) and Tukey's test using SPSS® 17.0 software for Windows
(SPSS Inc., Chicago, IL, USA) to evaluate any statistical differences
between the treatments at P ≤ 0.05. Principal component analysis
(PCA) was carried out using Matlab R2008a (MathWorks, Natick, MA,
USA).
3. Results and discussion
3.1. Yeast growth kinetics and enological parameters
The growth kinetics of the four Saccharomyces cerevisiae strains are
shown in Fig. 1a. The four strains of S. cerevisiae showed similar growth
kinetics where the yeasts grew from day 0 to day 2, followed by stable
yeast cell counts from day 2 to day 10. The four yeast strains grew
by > 2 log CFU/mL (from 5.8–5.9 to 7.8–8.0 log CFU/mL). No decrease
(a) 8.5
Yeast Cell Count (Log CFU/mL)
8.0
7.5
7.0
6.5
6.0
5.5
0 2 4 6
Days
Fig. 1. (a) Growth of the four Saccharomyces cerevisiae strains and (b) sucrose utilization during tofu whey fermentation. (□) S. cerevisiae Merit; (♦) S. cerevisiae EC1118; (Δ) S. cerevisiae
R2; (×) S. cerevisiae 71B.
International Journal of Food Microbiology 262 (2017) 14–22
in yeast cell counts was observed during the 10-day fermentation, de-
spite the low yeast assimilable nitrogen (YAN) content (approximately
35 mg N/L) in the soy whey. This result agreed with the findings of
Mitra et al. (2010), who claimed that tofu whey was nutritionally
adequate to support the growth of microorganisms (lactic acid bacteria
was used in the mentioned study).
The initial and final pH and soluble solids content of the soy alco-
holic beverage are shown in Table 1. All four soy alcoholic beverage
samples showed a decrease in pH, with S. cerevisiae Merit samples
having the lowest pH, which coincided with the highest production of
total organic acids (Table 1). S. cerevisiae 71B samples had the highest
final pH corresponding to the lowest production of total organic acids
(Table 1). This is in line with the results reported by Chen and Liu
(2014) where strain 71B fermentation had the highest end-pH after 14-
day lychee wine fermentation.
The four strains of S. cerevisiae yeasts shared similar patterns of °Brix
changes, where there was a continuous decline in the °Brix till the end
(data not shown but trends are similar to the sucrose data presented in
Fig. 1b). The decline in the °Brix corresponded to the consumption of
the sugars by the yeasts (Table 1). Strain Merit fermented samples had
the lowest °Brix of 5.04 while strain R2 fermented samples had the
highest °Brix of 5.20.
The initial and the final concentrations of sugars, glycerol and or-
ganic acids are shown in Table 1. The four S. cerevisiae strains could
utilize the supplemented sucrose effectively with a similar consumption
rate except for strain 71B (Fig. 1b), which had the fastest sucrose uti-
lization. The results were consistent with the findings of Chen and Liu
(2014).
As discussed by Walker and Stewart (2016), S. cerevisiae yeasts are
ethanologenic in that easily ferment glucose, fructose, and sucrose. This
explains the low final concentration of glucose and fructose (Table 1).
Furthermore, the four yeast strains used in this study are wine yeasts
that have the capability to utilize sugars quickly. The slight increment
in the fructose concentration at the end of the fermentation was likely
attributed to the hydrolysis of sucrose to glucose and fructose by the
yeast invertase (Lu et al., 2016). The lower final concentration of glu-
cose than fructose indicates that S. cerevisiae yeasts preferred glucose
over fructose due to the glucophilic nature of S. cerevisiae (Viana et al.,
2008).
Glycerol was produced by the four S. cerevisiae strains with the
lowest amount being produced strain 71B (Table 1). This might suggest
that 71B was more osmotolerant than the other three strains as the
production of glycerol is related to the counteracting osmotic stress in
the yeast cells (Scanes et al., 1998).
(b) 140
120
Sucrose Concentration (g/L)
100
80
60
40
20
0
8 10 0 2 4 6 8 10
Days
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J.-Y. Chua et al. International Journal of Food Microbiology 262 (2017) 14–22

Table 1
Enological parameters of the soy whey and the respective soy alcoholic beverage fermented with four S. cerevisiae strains.

Day 0 Day 10

Pre-treated soy whey⁎ Merit EC1118 R2 71B

pH 3.99 ± 0.01a 3.75 ± 0.01b 3.85 ± 0.00c 3.81 ± 0.02d 3.91 ± 0.01e
°Brix 14.77 ± 0.03a 5.04 ± 0.06b 5.10 ± 0.03bc 5.20 ± 0.09c 5.09 ± 0.07bc
Ethanol (% v/v) 0.00 ± 0.00 & a 7.15 ± 1.06b 8.00 ± 0.45b 7.82 ± 0.38b 7.33 ± 0.48b
Sugars (g/L)
Fructose 0.80 ± 0.26a 2.58 ± 0.67b 4.08 ± 0.73c 4.40 ± 0.69c 2.78 ± 0.49b
Glucose 1.33 ± 0.22a 0.52 ± 0.03b 0.76 ± 0.07bc 0.83 ± 0.08c 0.62 ± 0.07bc
Sucrose 125.42 ± 1.09a 0.61 ± 0.05b 0.40 ± 0.03b 0.83 ± 0.02b 0.00 ± 0.00b
Total sugars 127.54 ± 1.06a 3.71 ± 0.72bc 5.23 ± 0.65cd 6.05 ± 0.62d 3.40 ± 0.045b
Glycerol (g/L) 0.00 ± 0.00a 4.48 ± 0.35b 4.82 ± 0.35b 5.02 ± 0.74b 3.57 ± 0.16c
Organic acids (g/L)
Citric acid 1.61 ± 0.12a 2.19 ± 0.11b 2.21 ± 0.08b 2.21 ± 0.15b 2.17 ± 0.03b
Alpha-ketoglutaric acid 0.00 ± 0.00a 0.13 ± 0.01b 0.09 ± 0.01c 0.12 ± 0.01b 0.06 ± 0.00d
Malic acid 1.86 ± 0.17a 2.71 ± 0.11b 2.47 ± 0.08bc 2.52 ± 0.10b 2.24 ± 0.05c
Pyruvic acid 0.00 ± 0.00a 0.41 ± 0.02b 0.35 ± 0.01c 0.39 ± 0.04bc 0.31 ± 0.01d
Succinic acid 0.50 ± 0.15a 1.02 ± 0.10b 0.92 ± 0.11b 1.12 ± 0.13b 1.06 ± 0.06b
Acetic acid 0.00 ± 0.00a 0.34 ± 0.07b 0.22 ± 0.02c 0.26 ± 0.03bc 0.26 ± 0.02bc
Total organic acids 3.97 ± 0.28a 6.80 ± 0.36b 6.27 ± 0.25bc 6.62 ± 0.41bc 6.10 ± 0.14c

a,b,c,d
Statistical analysis using ANOVA at 95% confidence interval. Values with the same letter indicate no significant difference between the groups.
⁎
Pre-treated soy whey refers to the soy whey after sugar and malic acid adjustment.
&
0.00 ± 0.00: undetected. The value is added to facilitate statistical analysis.

Organic acids increased in concentration at the end of the fermen-
tation (Table 1). Citric, α-ketoglutaric, succinic and malic acids are
intermediate products of the TCA cycle (Veech, 2013). As briefly dis-
cussed by Whiting (1976), citric and malic acids can be produced by
Saccharomyces yeast and subsequently taken up back into the cells.
Therefore, this demonstrated that the production of the acids was
higher than the consumption for biological activity.
Pyruvic acid is a metabolite from the glycolytic pathway of the wine
yeasts (Morata et al., 2006), and the release of pyruvic acid into the
fermentation medium was expected. Acetic acid concentration present
in the four soy alcoholic beverage samples ranged between 0.22 to
0.34 g/L (Table 1) which is within the optimum range (0.2 to 0.7 g/L)
to impart favorable wine profile to the beverage (Viana et al., 2008).
The low amount of the acetic acid present could be due to diversion of
acetic acid to formation of acetate esters as reflected in Table 4.
3.2. Changes in soluble proteins and amino acids
The initial and final concentrations of the soluble proteins and
amino acids are shown in Table 2. It should be noted that pasteurized
soy whey without sucrose and malic acid additions was used as the
control due to sucrose interference with protein assay by the Bradford
method (data not shown). A substantial amount of soluble proteins was
found in the soy whey prior to fermentation. Zhang et al. (2014) re-
ported that soy whey contained soluble proteins such as lipoxygenase,
lectin, and Kunitz trypsin inhibitor. The soluble proteins decreased
significantly after fermentation in all samples, the degree of decreases
being dependent on yeast strains (Table 2). This reduction of soluble
proteins might be due to the proteolytic activity of S. cerevisiae
(Maturano et al., 2012).
Most of the amino acids decreased to undetectable levels after fer-
mentation; however, the two sulfur-containing amino acids, cysteine
and methionine, increased significantly in concentration, which varied
little with yeast strains (Table 2). The increase in the concentration of
cysteine and methionine could be attributed to protein hydrolysis by
the yeasts (Song et al., 2008). The increase in methionine and cysteine
concentration after fermentation could also imply that S. cerevisiae
yeasts have a lesser preference for sulfur-containing amino acids, being
consistent with the fact that no sulfur-containing volatile compound
was detected by GC analysis (Table 4).
As described above, the majority of the amino acids showed a
decrease in concentration after fermentation. This indicated that the
decrease in concentration of the amino acids outweighed their pro-
duction from the protein hydrolysis by the yeasts. Some of these amino
acids could be transformed into fusel alcohols through the Ehrlich
pathway (Hazelwood et al., 2008). For example, the reduction in con-
centration of leucine and phenylalanine (Table 2) corresponded to the
formation of isoamyl alcohol and phenylethanol, respectively, as shown
in Table 4.
The yeast assimilable nitrogen (YAN) content of the soy whey was
calculated to be approximately 35.3 mg N/L based on the amino acid
and ammonia content in Table 2. Vilanova et al. (2007) reported
140 mg N/L to be the minimum concentration of YAN needed for wine
fermentation. Hence, the YAN content in the soy whey was only around
~25% of the minimally required YAN by the yeasts. Nitrogen defi-
ciency in wine fermentation has been reported to lower biomass yields,
which in turn affect the fermentation rate (Vilanova et al., 2007).
However, the four Saccharomyces yeasts were able to grow by
~2 log CFU/mL within two days (Fig. 1a), being consistent with the
results of Chen and Liu (2014) who reported similar 2 log CFU/mL
increments of the yeasts after fermentation in lychee wine. Despite the
low nitrogen status of the soy whey, the residual sugar level ranged
from 3.4 to 6.1 g/L (“dry” to slightly “sweet”) (Table 1).
3.3. Changes in isoflavone content and antioxidant capacity
The changes in the isoflavones are shown in Table 3. Soy isoflavones
are naturally present in abundant amounts in soybeans (Zaheer and
Akhtar, 2015). These isoflavones, both the glucoside and the aglycone
forms, can remain soluble after coagulation of tofu (Benedetti et al.,
2016) and migrate to the soy whey (Xiao et al., 2015). All isoflavone
glucosides determined (daidzin, glycitin, genistin) decreased sub-
stantially in concentration (ranging from 79.5% to 88.8%), corre-
sponding to the significant increases (ranging from 4.4 to 7.3-fold) of
the isoflavone aglycones (daidzein, glycitein and genistein) after fer-
mentation (Table 3). This suggested that the four strains of Sacchar-
omyces yeasts possess strong β-glucosidase activities that can hydrolyze
isoflavone glucosides into their corresponding aglycones and glucose
(Hernández et al., 2003). The four Saccharomyces strains possess similar
activity in the glucoside hydrolysis, but the conversion rate differed,
with Merit having the highest conversion rate of 73.8% while the other
three strains had similar conversion rates between 43.5% to 49.0%.

17
J.-Y. Chua et al. International Journal of Food Microbiology 262 (2017) 14–22

Table 2
Total soluble proteins and amino acids contents in soy whey before and after fermentation.

Day 0 Day 10

Untreated soy whey@ Merit EC1118 R2 71B

Soluble Protein (mg/mL) 1.041 ± 0.039a 0.803 ± 0.061b 0.490 ± 0.083c 0.597 ± 0.043cd 0.681 ± 0.020d
Amino acid (mg/L)
&
Aspartic acid 0.00 ± 0.00 a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a
Serine + asparagine 8.83 ± 0.38a 2.67 ± 0.42b 1.16 ± 0.43c 2.88 ± 0.78b 1.32 ± 0.30c
Glutamic acid 19.65 ± 4.47a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Glycine 2.22 ± 0.62a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Histidine + glutamine 2.56 ± 0.78a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Ammonia 0.48 ± 0.12a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Arginine 36.69 ± 4.98a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Threonine 16.12 ± 2.27a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Alanine 7.96 ± 1.36a 3.30 ± 0.45b 4.32 ± 0.35b 4.45 ± 0.11b 4.73 ± 0.55b
Proline 39.05 ± 4.85a 2.50 ± 0.25b 1.80 ± 0.17b 2.04 ± 0.06b 2.27 ± 0.81b
Cysteine 5.50 ± 0.89a 8.08 ± 0.74b 7.12 ± 0.66ab 7.18 ± 0.74ab 6.61 ± 1.02ab
Tyrosine 7.95 ± 1.67a 2.22 ± 0.17b 2.77 ± 0.44b 2.17 ± 0.21b 2.02 ± 0.29b
Valine 4.95 ± 0.74a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Methionine 3.30 ± 0.63a 8.55 ± 1.90b 5.97 ± 1.55ab 7.21 ± 1.02b 5.90 ± 0.81ab
Lysine 4.57 ± 0.52a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Isoleucine 2.92 ± 0.37a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Leucine 3.65 ± 0.30a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Phenylalanine 6.45 ± 0.61a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
Tryptophan 14.52 ± 1.22a 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b

a,b,c,d
Statistical analysis using ANOVA at 95% confidence interval. Values with the same letter indicate no significant difference between the groups.
@
Untreated soy whey refers to the soy whey prior to sugar and malic acid adjustment.
&
0.00 ± 0.00: undetected; the value is added to facilitate statistical analysis.

The increase in isoflavone aglycone concentration may have con-
tributed to the remarkable increases (about 2-fold) in the antioxidant
capacity of the soy alcoholic beverage after fermentation (Table 3). This
result is consistent with that of Marazza et al. (2012), where the in-
crease in the isoflavone aglycone concentration in soymilk after the
fermentation by Lactobacillus rhamnosus corresponded to an increase in
the antioxidant capacity measured by DPPH scavenging assay. Marazza
et al. (2012) also suggested that isoflavone aglycones are more active
antioxidant compounds than their corresponding β-glucoside pre-
cursors. Furthermore, the hydrolysis of the isoflavone β-glucosides to
the aglycone form may help to increase the bioavailability of the iso-
flavones (Hati et al., 2015). Therefore, the hydrolysis of the isoflavone
glucosides to their corresponding aglycones may improve the health
function of the soy alcoholic beverage.
3.4. Changes in volatile compounds
Table 4 shows the volatile composition of the soy whey before and
after fermentation. Six groups of volatiles (acids, alcohols, esters,
aldehydes, ketones, and furan) were identified. In general, almost all
indigenous aroma compounds (mainly aldehydes) were metabolized to
low or undetectable levels while new aroma compounds, especially
esters and alcohols, were formed. As such, the beany off-flavor of the
soy whey was replaced with the fruity and alcoholic notes typical of a
wine profile.
Hexanoic acid, which was originally present in the soy whey, was
completely metabolized to trace levels at the end of the fermentation
and this observation is similar to that reported by Lee et al. (2010) in
papaya wine where indigenous hexanoic acid was diminished after
fermentation. Hexanoic acid is formed from hexanal present in the
soymilk when exposed to air (Wilkens and Lin, 1970). The drop in
hexanoic acid may be partially attributed to the formation of its cor-
responding esters, ethyl hexanoate and isoamyl hexanoate (Table 4).
Octanoic, decanoic and 9-decenoic acids were not present in the soy
whey but present in the soy alcoholic beverage (Table 4). It was re-
ported that S. cerevisiae yeasts, in general, are producers of octanoic and
decanoic acids (Garde-Cerdan and Ancin-Azpilicueta, 2007).
Indigenous alcohols, such as 1-penten-3-ol, 1-pentanol, 1-hexanol

Table 3
Changes in the isoflavone content and the antioxidant capacity of the soy whey before and after the fermentation.

Day 0 Day 10

Treated Soy whey Merit EC1118 R2 71B

Isoflavone (mg/L)
Daidzin 18.79 ± 0.85a 3.53 ± 0.22b 2.05 ± 0.08c 3.37 ± 0.23b 3.88 ± 0.32b
Glycitin 3.28 ± 0.31a 0.72 ± 0.21b 0.00 ± 0.00 & c 0.00 ± 0.00c 0.00 ± 0.00c
Genistin 23.45 ± 0.59a 5.09 ± 0.18b 3.04 ± 0.13c 4.56 ± 0.43b 4.82 ± 0.24b
Total glucosides 45.52 ± 1.56a 9.34 ± 0.44b 5.09 ± 0.19c 7.93 ± 0.63b 8.68 ± 0.44b
Daidzein 2.16 ± 0.17a 14.79 ± 1.04b 12.90 ± 0.81c 12.06 ± 0.37cd 11.37 ± 0.67d
Glycitein 0.69 ± 0.10a 4.99 ± 0.43b 3.68 ± 0.26c 4.80 ± 0.23b 3.36 ± 0.33c
Genistein 0.83 ± 0.04a 10.58 ± 0.98b 5.87 ± 0.39c 5.24 ± 0.09c 4.97 ± 0.40c
Total aglycones 3.67 ± 0.24a 30.36 ± 1.66b 22.46 ± 1.41c 22.10 ± 0.59cd 19.70 ± 1.23d
Total isoflavones 49.19 ± 1.78a 39.70 ± 1.70b 27.55 ± 1.59c 30.04 ± 1.09c 28.39 ± 1.59c
Trolox equivalent@ (mg/mL) 0.014 ± 0.002a 0.023 ± 0.002b 0.030 ± 0.002c 0.026 ± 0.001bd 0.028 ± 0.002cd

a,b,c,d
Statistical analysis using ANOVA at 95% confidence interval. Values with the same letter indicate no significant difference between the groups.
@
Antioxidant capacity measured using DPPH scavenging assay. Antioxidant capacity is expressed as Trolox equivalent.
&
0.00 ± 0.00: undetected; the value is added to facilitate statistical analysis.

18
 Table 4
Volatile compounds (mean GC-FID peak area × 106) and their relative peak area (RPA, %) in before and after fermentation by the different Saccharomyces yeasts.

Compound identified Identification methods LRI# Soy whey Soy alcoholic beverage
J.-Y. Chua et al.

Day 0 Merit EC1118 R2 71B

Peak area RPA⁎ Peak area RPA⁎ Peak area RPA⁎ Peak area RPA⁎ Peak area RPA⁎

Acids
Acetic acid MS, LRI 1450 4.88 ± 1.06a 3.75% 4.03 ± 0.40a 0.11% 2.54 ± 0.28b 0.06% 7.57 ± 0.75c 0.17% 3.58 ± 0.14ab 0.08%
Hexanoic acid MS, LRI 1881 11.65 ± 1.08a 8.95% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
Octanoic acid MS, LRI 2046 0.00 ± 0.00 & a 0.00% 24.63 ± 1.05b 0.66% 48.99 ± 6.18c 1.07% 39.08 ± 7.27c 0.86% 48.44 ± 6.24c 1.05%
Decanoic acid MS, LRI 2262 0.00 ± 0.00a 0.00% 12.92 ± 2.93b 0.34% 24.25 ± 2.47cd 0.53% 20.40 ± 1.50c 0.45% 30.54 ± 5.28d 0.66%
9-Decenoic acid MS, LRI 2322 0.00 ± 0.00a 0.00% 4.52 ± 0.37bc 0.12% 3.84 ± 0.46b 0.08% 4.98 ± 0.11c 0.11% 5.14 ± 0.48c 0.11%
Subtotal 16.53 12.70% 46.10 1.23% 79.61 1.74% 72.02 1.59% 87.69 1.90%

Alcohols
Ethanol MS 0.00 ± 0.00a 0.00% 2762.31 ± 193.17b 73.60% 3415.85 ± 506.42b 74.70% 3214.20 ± 901.05b 71.11% 3181.55 ± 329.71b 69.03%
Isobutanol MS, LRI 1100 0.00 ± 0.00a 0.00% 6.23 ± 0.47b 0.17% 2.69 ± 0.35c 0.06% 5.34 ± 0.58b 0.12% 4.18 ± 0.46d 0.09%
1-Penten-3-ol MS, LRI 1164 3.32 ± 0.26a 2.55% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
Isoamyl alcohol MS, LRI 1218 0.00 ± 0.00a 0.00% 33.55 ± 4.68b 0.89% 25.20 ± 2.96c 0.55% 35.47 ± 5.38bd 0.78% 41.97 ± 2.87d 0.91%
1-Pentanol MS, LRI 1259 7.45 ± 0.57a 5.72% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
1-Hexanol MS, LRI 1392 24.49 ± 0.93a 18.81% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
1-Octen-3-ol MS, LRI 1463 7.81 ± 1.47a 6.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
Phenylethanol MS, LRI 2067 0.00 ± 0.00a 0.00% 54.40 ± 10.38b 1.45% 52.67 ± 6.36b 1.15% 81.84 ± 14.56c 1.81% 42.63 ± 10.45b 0.92%
Subtotal 43.06 33.08% 2856.49 76.11% 3496.40 76.46% 3336.85 73.82% 3270.33 70.96%

Esters
Ethyl Acetate MS 0.00 ± 0.00a 0.00% 8.07 ± 0.40b 0.22% 9.33 ± 0.81b 0.20% 8.34 ± 0.54b 0.18% 9.30 ± 0.86b 0.20%
Isoamyl acetate MS, LRI 1112 0.00 ± 0.00a 0.00% 2.90 ± 0.70b 0.08% 1.65 ± 0.17c 0.04% 2.26 ± 0.41bc 0.05% 2.29 ± 0.28bc 0.05%

19
Hexyl acetate MS, LRI 1258 0.00 ± 0.00a 0.00% 1.53 ± 0.36b 0.04% 0.75 ± 0.10c 0.02% 1.08 ± 0.07c 0.02% 0.91 ± 0.15c 0.02%
2-Phenylethyl acetate MS, LRI 1814 0.00 ± 0.00a 0.00% 6.61 ± 0.38b 0.18% 5.82 ± 0.76b 0.13% 5.82 ± 0.42b 0.13% 9.98 ± 0.50c 0.22%
Ethyl hexanoate MS, LRI 1215 0.00 ± 0.00a 0.00% 64.28 ± 14.55b 1.71% 43.23 ± 1.64c 0.95% 60.22 ± 13.89bc 1.33% 47.64 ± 6.80bc 1.03%
Ethyl heptanoate MS, LRI 1318 0.00 ± 0.00a 0.00% 1.33 ± 0.10b 0.04% 0.80 ± 0.09c 0.02% 1.19 ± 0.13b 0.03% 0.97 ± 0.04c 0.02%
Ethyl Octanoate MS, LRI 1427 0.00 ± 0.00a 0.00% 202.41 ± 14.28bc 5.39% 211.39 ± 25.00b 4.62% 217.06 ± 13.32b 4.80% 165.92 ± 26.18c 3.60%
Ethyl nonanoate MS, LRI 1523 0.00 ± 0.00a 0.00% 5.33 ± 1.49b 0.14% 2.01 ± 0.57c 0.04% 2.30 ± 0.26c 0.05% 2.12 ± 0.24c 0.05%
Ethyl decanoate MS, LRI 1630 0.00 ± 0.00a 0.00% 352.61 ± 54.66b 9.40% 475.76 ± 101.81bc 10.40% 513.39 ± 60.02cd 11.36% 659.07 ± 78.35d 14.30%
Ethyl benzoate MS, LRI 1667 0.00 ± 0.00a 0.00% 7.24 ± 1.21b 0.19% 7.94 ± 0.87b 0.17% 6.88 ± 0.46b 0.15% 14.89 ± 2.15c 0.32%
Ethyl 9-decenoate MS, LRI 1682 0.00 ± 0.00a 0.00% 124.96 ± 13.35b 3.33% 136.74 ± 32.98b 2.99% 193.22 ± 16.80c 4.27% 150.80 ± 26.42bc 3.27%
Ethyl undecanoate MS, LRI 1728 0.00 ± 0.00a 0.00% 0.69 ± 0.10b 0.02% 0.71 ± 0.09b 0.02% 0.63 ± 0.07b 0.01% 0.71 ± 0.07b 0.02%
Ethyl dodecanoate MS, LRI 1833 0.00 ± 0.00a 0.00% 46.95 ± 5.20b 1.25% 68.37 ± 5.89b 1.50% 70.26 ± 13.33b 1.55% 141.00 ± 20.58c 3.06%
Isobutyl decanoate MS, LRI 1744 0.00 ± 0.00a 0.00% 1.33 ± 0.37b 0.04% 1.11 ± 0.27b 0.02% 1.37 ± 0.13b 0.03% 1.99 ± 0.26c 0.04%
Isoamyl hexanoate MS, LRI 1446 0.00 ± 0.00a 0.00% 0.64 ± 0.10b 0.02% 0.43 ± 0.08c 0.01% 0.84 ± 0.11d 0.02% 0.61 ± 0.07b 0.01%
Isoamyl octanoate MS, LRI 1647 0.00 ± 0.00a 0.00% 7.91 ± 1.65b 0.21% 10.74 ± 2.07bc 0.23% 8.75 ± 0.84bc 0.19% 11.16 ± 1.37c 0.24%
Isoamyl pentadecanoate MS, LRI 1852 0.00 ± 0.00a 0.00% 10.11 ± 1.22b 0.27% 14.59 ± 1.60c 0.32% 13.15 ± 2.01bc 0.29% 24.73 ± 2.23d 0.54%
Subtotal 0.00 0.00% 844.91 22.51% 991.36 21.68% 1106.75 24.48% 1244.09 26.99%

Aldehydes
3-Methyl butanal MS 5.22 ± 0.43a 4.01% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
Hexanal MS, LRI 1077 50.77 ± 9.40a 39.01% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
2-Hexenal MS, LRI 1217 2.91 ± 0.51a 2.23% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
2-Heptenal MS, LRI 1321 1.75 ± 0.38a 1.35% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
2-Octenal MS, LRI 1426 1.90 ± 0.25a 1.46% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
(E,E)-2,4-Heptadienal MS, LRI 1498 0.53 ± 0.10a 0.40% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
Benzaldehyde MS, LRI 1531 0.65 ± 0.04a 0.50% 0.83 ± 0.13ab 0.02% 0.86 ± 0.13ab 0.02% 1.09 ± 0.14bc 0.02% 1.17 ± 0.18c 0.03%
3,4-Dimethyl-benzaldehyde MS, LRI 1830 0.65 ± 0.16a 0.50% 4.25 ± 0.83bc 0.11% 3.83 ± 0.47bc 0.08% 3.07 ± 0.29b 0.07% 5.05 ± 1.01c 0.11%
Subtotal 64.38 49.46% 5.08 0.14% 4.69 0.10% 4.16 0.09% 6.22 0.13%

Ketones
2-Heptanone MS, LRI 1174 2.68 ± 0.22a 2.06% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00% 0.00 ± 0.00b 0.00%
(continued on next page)
International Journal of Food Microbiology 262 (2017) 14–22
J.-Y. Chua et al. International Journal of Food Microbiology 262 (2017) 14–22

and 1-octen-3-nol, detected in the soy whey mainly arise from the ac-

0.00%
0.00%
0.01%
0.01%

0.00%
0.00%
RPA⁎
tion of the soybean lipoxygenase (Wilkens and Lin, 1970). The loss of
indigenous alcohols can be deemed as beneficial to the soy alcoholic
beverage flavor. For example, 1-hexanol has a grassy and beany odor
(Wolf, 1975). Hence, the metabolism of 1-hexanol to undetectable le-
0.00 ± 0.00b
0.00 ± 0.00b
0.51 ± 0.08b

0.00 ± 0.00b
vels helps to reduce the undesirable beany odor of the soy whey.
As seen from Tables 1 and 4, the four S. cerevisiae yeasts produced
Peak area

4608.84
0.51 similar amounts of ethanol, ranging between 7 to 8% (v/v). Besides

0.00
71B

being a good producer of ethanol, S. cerevisiae yeasts are good produ-

cers of fusel alcohols (Regodón Mateos et al., 2006), which are pro-
duced mainly by the Ehrlich pathway where amino acids serve as the
0.00%
0.00%
0.01%
0.01%

0.00%
0.00%
RPA⁎

respective precursors (Hazelwood et al., 2008). The different levels of

fusel alcohols (Table 4) are largely the result of the differences in the
production rate of the alcohols by the yeasts as well as the differences in
the amount of the respective esters formed.
0.00 ± 0.00b
0.00 ± 0.00b
0.44 ± 0.03b

0.00 ± 0.00b

There were no esters detected in the soy whey before fermentation

Peak area

(Table 4). This result differs from the findings of Achouri et al. (2006),
4520.22

who reported that esters such as hexanoic acid esters and benzoic acid
0.44

0.00
R2

esters were present in soy milk. Furthermore, it has been reported that
esters are not the key aroma compounds in soymilk (Kaneko et al.,
0.00%
0.00%
0.02%
0.02%

0.00%
0.00%

2011). The esters originally present in the soy milk could be lost during
RPA⁎

the boiling process, resulting in no esters being detected. Esters de-

tected in the soy alcoholic beverage, therefore, were the outcome of the
yeast fermentation. Ethyl esters and acetate esters formed the two main
groups of esters detected in the soy alcoholic beverage, probably due to
0.00 ± 0.00b
0.00 ± 0.00b

0.00 ± 0.00b
0.73 ± 0.18c

the abundance of ethanol and acetic acid available (Walker and

Peak area

4572.80

Stewart, 2016).
EC1118

0.73

0.00

Most of the indigenous aldehydes detected were metabolized to

Statistical analysis using ANOVA (n = 3) at 95% confidence interval. Same letter indicates no significant difference between sample.

undetected levels at the end of the fermentation, except for benzalde-

hyde and 3,4-dimethyl-benzaldehyde (Table 4). Indigenous aldehydes,
0.00%
0.00%
0.01%
0.01%

0.00%
0.00%
RPA⁎

particularly hexanal, 2-heptenal and 2-octenal, are products from the

catalytic breakdown of linoleic acid by lipoxygenase in the soybean
Soy alcoholic beverage

(Wilkens and Lin, 1970). Other indigenous aldehydes such as 2,4-hep-

tadienal arise from the catalytic breakdown of linolenic acid by lipox-
0.00 ± 0.00b
0.00 ± 0.00b
0.42 ± 0.09b

0.00 ± 0.00b

ygenase as well (Wilkens and Lin, 1970). Aldehydes can be oxidized to

their respective acids or be reduced to their respective alcohols during
Peak area

3753.00

the fermentation. For example, hexanal can be reduced to hexanol or

Merit

0.42

0.00

oxidized to hexanoic acid which can then be transformed into hexyl

acetate or ethyl hexanoate, respectively.
LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons.

Indigenous ketones present in the soy whey were also metabolized

0.59%
1.07%
0.00%
3.72%

1.04%
1.04%
RPA⁎

to undetectable levels at the end of the fermentation (Table 4). Hep-

tanone and octanone can arise from the breakdown of linoleic acid by
the lipoxygenase activity (Wilkens and Lin, 1970). Ketones were likely
0.77 ± 0.11a
1.39 ± 0.05a
0.00 ± 0.00a

1.35 ± 0.36a

reduced to alcohols by yeasts, too. 2-Nonanone was the only ketone

0.00 ± 0.00: undetected; the value is added to facilitate statistical analysis.
Peak area
Soy whey

detected after the fermentation (Table 4).

130.15
Day 0

The only furan detected was 2-pentylfuran, which has a fruity,

4.84

1.35

green, beany flavor on its own and is formed from the breakdown of
linoleic acid by the soy lipoxygenase (Sessa, 1979). Together with
Subtotal

Subtotal

RPA (%) = (peak area of compound/total peak area) × 100%.

hexanol, 2-hexenal, ethyl vinyl ketone, 2-pentylfuran can contribute to

Total
1197
1314
1379

1211
LRI#

the grassy and beany odor of soy products (Sessa, 1979; Wolf, 1975).
Little research has been done to determine how wine yeasts metabolize
Identification methods

2-pentylfuran and hence further research is needed to understand the

mechanism of 2-pentylfuran transformation by the wine yeasts.

3.5. Principal component analysis

MS, LRI
MS, LRI
MS, LRI

MS, LRI

Principal component analysis (PCA; Fig. 2) was conducted using

data from Table 4 and acetic acid and ethanol from Table 1 to under-
stand the profiles of the different fermentation set-ups. The first two
principal components (PCs) accounted for 93.60% of the total variance
4-Methyl-2-heptanone
Compound identified

2-Methyl-3-octanone

observed, with PC1 and PC2 accounting for 83.17% and 10.43% re-
Table 4 (continued)

spectively. As seen from the bi-plot, the unfermented soy whey is po-
2-Pentyl furan
2-Nonanone

sitioned on the positive semi-axis of PC1 with indigenous aldehydes,

particularly hexanal, as the main characteristic compound. The four soy
Furan

alcoholic beverage samples are situated on the negative semi-axis of

a,b,c,d

&
#

PC1 with strains Merit and R2 (on the positive semi-axis of PC2)

20
J.-Y. Chua et al.
samples having higher amounts of phenylethanol, isobutanol, ethanol,
ethyl hexanoate, ethyl octanoate, isoamyl acetate and acetic acid, while
strains EC1118 and 71B (on the negative semi-axis of PC2) samples
have higher amounts of ethyl acetate, ethyl decanoate, ethyl benzoate,
2-phenylethyl acetate, benzaldehyde, octanoic acid and decanoic acid.
Hence, fermentation using strains Merit and R2 generated similar al-
coholic flavor profiles, while strains EC1118 and 71B generated similar
profiles.
4. Conclusion
Soy whey generated from tofu production was fermented using four
commercial Saccharomyces yeasts. With only sucrose supplementation
and pH adjustment, the four Saccharomyces yeasts grew well in the soy
whey. The soy whey was low in yeast assimilable nitrogen with only
about 35 mg N/L. Isoflavone glucosides were hydrolyzed by the four
yeasts to form isoflavone aglycones, which boosted antioxidant capa-
city. Indigenous beany odor compounds, particularly the aldehydes and
alcohols, were metabolized to trace or undetected levels. New com-
pounds, especially the esters and higher alcohols, were formed to pro-
vide the alcoholic beverage with a fruity and floral note. Therefore,
alcoholic fermentation using wine yeasts could serve as a zero-waste
solution to biotransforming soy whey into value-added soy alcoholic
beverage with naturally enriched free isoflavones.
Acknowledgement
The research work was supported, in part, by a research grant from
Ministry of Education of Singapore (WBS No. R143-000-664-114).
Conflict of interest
The authors declare that there is no conflict of interest.
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