Mok et al.

AMB Expr (2019) 9:60

https://doi.org/10.1186/s13568-019-0786-5

ORIGINAL ARTICLE Open Access

A metabolomic approach to understand

the solid‑state fermentation of okara using
Bacillus subtilis WX‑17 for enhanced nutritional
profile
Wai Kit Mok1†, Yong Xing Tan2,3†, Jaslyn Lee1, Jaejung Kim1 and Wei Ning Chen1*

Abstract
Okara is a major agro-waste produced from the soybean industry. To hydrolyze the okara and enable nutrient release,
a strategy to valorize okara using solid-state fermentation with food grade Bacillus subtilis (B. subtilis) WX-17 was car-
ried out. The study showed that fermentation of okara with B. subtilis WX-17 improved its overall nutritional content.
The total amino acids content increased from 3.04 ± 0.14 mg/g in unfermented okara to 5.41 ± 1.21 mg/g in okara
fermented with B. subtilis WX-17. Total fatty acids content increased from 153.04 ± 5.10 to 166.78 ± 2.41 mg/g okara,
after fermentation. Antioxidant content (DPPH) also increased by 6.4 times after fermentation. To gain insight into the
mechanism, gas chromatography–mass spectrometry analysis was carried out. In total, 49 metabolites were detected,
which could be classified mainly into carbohydrates, TCA cycle metabolites, amino acids and fatty acids. The decrease
in carbohydrate metabolites, showed that glycolysis was upregulated. This would have provided the energy and met-
abolic flux towards the amino acid and fatty acid pathway. This is also in line with the increased amino acids and fatty
acid production seen in okara fermented with B. subtilis WX-17. The findings of this work demonstrated the potential
of using B. subtilis WX-17 fermentation, to enhance the nutritional profile of okara. This could serve as a potential low-
cost animal feed or incorporated into the human diet.
Keywords: Bacillus subtilis, Okara, Fermentation, Metabolomics, Food waste valorisation

Introduction physiological and therapeutic functions such as the pre-

Okara, also known as soy pulp is a major agro-waste vention of cardiovascular diseases (CVD) in humans.
produced from the soybean industry which produces However, a large amount of okara are being disposed of
soymilk and bean curd. Approximately 1.1 kg of okara in landfills and incineration plants annually due to their
are produced from 1 kg of soybean (O’Toole 1999). unpalatable and insoluble nature (Li et al. 2012b). It is
Okara is highly nutritious. It contains approximately estimated that around 14 million tonnes of okara are gen-
50% fiber, 25% protein, 10% lipid as well as a myriad of erated worldwide annually. The countries include Japan,
other high-value compounds such as isoflavones, coume- Korea, China and Singapore, which contribute 800,000
stans, saponins, phytosterols, lignins and phytates. These tonnes, 310,000 tonnes, 2.8 million tonnes and 11,000
compounds have been shown to exhibit numerous tonnes, respectively (Li et al. 2012b; Seong et al. 2015).
To utilize the highly nutritive compounds in the okara,
pre-treatment is required to release the nutrients from
*Correspondence: [email protected] the insoluble okara. Previous studies have shown that
†
Wai Kit Mok and Yong Xing Tan contributed equally to this work enzymes secreted by microorganisms during fermenta-
1
School of Chemical and Biomedical Engineering, Nanyang
Technological University, 62 Nanyang Drive, N1.2‑B1‑35, tion can hydrolyse complex macromolecules such as fatty
Singapore 637459, Singapore acids, proteins and fibres into smaller and more soluble
Full list of author information is available at the end of the article

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provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
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Mok et al. AMB Expr (2019) 9:60 Page 2 of 12

nutrients. It also reduces the amount of antinutritional mapping of metabolites unto the metabolomic pathways
factors present in okara including trypsin, phytic acid, would also provide an important insight into the mech-
lectin and tannin (Paredes-Lopez and Harry 1989). Vari- anisms behind solid-state fermentation of okara with
ous microorganisms such as Aspergillus sp., Aspergillus B. subtilis WX-17. Till date, based on our knowledge,
niger and Aspergillus ficuum had been studied for their there exist a gap in the utilization of bacteria in valoris-
ability to produce phytases which inhibited the antinu- ing okara from a metabolomic perspective. The findings
tritional factor phytate. This had been shown to reduce of this study could open up the possibility of using fer-
the bioavailability of calcium, zinc and iron (Pandey et al. mented okara as a low-cost animal feed or even supple-
2001; Schlemmer et al. 2009). ment the human diet which could go a long way towards
Solid state fermentation (SSF) is the culture of micro- alleviating the global food security issue.
organism using solid substrate in the absence of liquid to
produce desirable products. SSF has shown to be effec- Materials and methods
tive in enhancing the nutritional content of a complex Chemicals
substrate. This is because microorganism secretes abun- Glycerol, nutrient broth, methanol, ribitol, methoxamine
dant enzymes which catabolizes complex macromol- hydrochloride, N-methyl-N-(trimethylsilyl) trifluoro-
ecules into simpler forms. For example, a recent study acetamide (MSTFA), trimethylchlorosilane (TMCS),
conducted by Dessie et al. showed that fruit and vegeta- sodium chloride, acetic acid, heptadecanoic acid, etha-
ble wastes that underwent solid state fermentation using nol, chloroform, BF3-methanol, hexane, γ-aminobutyric
Aspergillus niger and Rhizopus oryzae increased in suc- acid, dimethylformamide (DMF), 1-1,-diphenyl-2-picryl-
cinic acid which is an important metabolite in the tri- hydrazil (DPPH).
carboxylic acid (TCA) cycle and have numerous health
benefits such as antioxidant properties and strengthening Microorganism
of the immune system (Dessie et al. 2018; Saif and Fumio Bacillus subtilis WX-17 was isolated from Marumiya
Hashinaga 2005). Bacteria, yeast and fungi are commonly Kyushu Ichiban Natto. This strain has been deposited in
used in SSF. Rhizopus, Lactobacillus, Streptococcus, NCIMB with the accession number NCIMB 15204. The
Aspergillus and Bacillus are some of the most common isolation was carried out by adding 20 mL of sterile water
microorganism used in solid state fermentation of food to 3 natto beans in a falcon tube and vortexed for 5 min
material (Hesseltine 1987). to extract the microorganism. The cell suspension was
In recent years, SSF have received increasing amount serial diluted and plated onto nutrient agar plates and
of attention from researchers and industrial players since incubated at 37 °C for 24 h. A single colony was inocu-
several studies performed on colourings, flavourings, lated into 5 mL of nutrient broth and incubated at 37 °C
additives and other desirable products for the food indus- for 24 h and subsequently stored in aliquots containing
try had shown that SSF can achieve higher yield com- 50% glycerol at − 80 °C.
pared to SLF (Rodriguez-Couto and Sanromán 2006). It is
considerably cheaper and more environmentally friendly Bacterium identification
as compared to the more commonly used submerged liq- A single colony was inoculated in 5 mL of nutrient broth
uid fermentation (SLF). Some of the advantages of SSF and incubated overnight. The bacterial DNA were then
over SLF include reduced probability of contamination isolated using Bio Basic EZ-10 Spin Column Fungal
due to lack of moisture and simple media composition Genomic DNA Mini-Prep Kit. Next, PCR was carried
since most nutrients are in the solid substrate. SSF also out to amplify the 16S rDNA gene of the bacteria using
allows the use of simple reactor design due to the con- the forward and reverser primer 27F (5′ AGA GTT TGA
centrated nature of solid substrate (Mienda and Idi 2011). TCM TGG CTC AG 3′) and 1492R (5′ GGT TAC CTT
Bacillus subtilis (B. subtilis) is a microorganism of GTT ACG ACT T 3′), respectively. PCR was performed
interest for fermentation of okara, due to its ability to with the following parameters: 35 cycles at 98 °C 10 s
secrete enzymes, which can break down the macromol- for denaturation, 55 °C 5 s for annealing, 72 °C 2 min
ecules in okara, as well as the ability to increase antioxi- for elongation and 68 °C 10 min for extension followed
dant activity. The objective of this study was to investigate by cooling to 4 °C. Gel electrophoresis was then carried
the effects of SSF on okara using a strain of food grade out on the PCR sample to remove other contaminants
B. subtilis WX-17, which was isolated in this study, from and the DNA purified using QIAquick Gel Extraction
Natto. Then, an untargeted metabolomic approach using Kit (250) before sequencing. 16s rRNA sequencing (Gen-
gas chromatography-mass spectrophotometry (GC–MS) bank accession number MK559744) was outsourced
was carried out to analyse the value-added products pro- to Bio Basic Asia Pacific Pte Ltd using Sanger dideoxy
duced in okara fermented with B. subtilis WX-17. The sequencing technology. The obtained 16s rRNA sequence

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Mok et al. AMB Expr (2019) 9:60 Page 3 of 12

(GenBank accession number MK559744) was then com- For fatty acids analysis, 10 mg of fermented okara and
pared with other 16s rRNA sequences using the BLAST 10 mg of fresh okara (control) were weighed and placed
algorithm (https​://blast​.ncbi.nlm.nih.gov/Blast​.cgi). into eppendorf tubes. 1000 µL of 0.9% NaCl solution and
200 µL of acetic acid were then added. 10 µL of 10 mg/
mL heptadecanoic acid dissolved in ethanol was added
Source of okara
to the extraction solvent to serve as IS. The solvents were
Fresh okara samples were kindly provided by Vitasoy
then homogenized as described above. Then, 3 mL of a
International Singapore Pte Ltd, Singapore. Okara were
chloroform–methanol 2:1 mixture was added, and the
separated into aliquots and sealed in air tight polyethyl-
samples were inverted several times, vortexed vigorously
ene bags and stored at − 20 °C in the dark.
for 5 min, and centrifuged at 10,000g for 10 min at 4 °C.
The chloroform layer (bottom, 1 mL) was collected and
Fermentation dried overnight at 30 °C. The dried lipid residue was re-
Bacillus subtilis WX-17 was inoculated into 5 mL of dissolved in 500 µL BF3-methanol 10% (FLUKA, 15716)
nutrient broth and incubated at 37 °C for 24 h which and incubated in a sealed vial in a 95 °C heater for 20 min.
served as the stock culture. 10 g of okara was inoculated FAMEs were extracted with 300 µL n-hexane after the
with B. subtilis WX-17 at a concentration of ­106 CFU/g addition of 300 µL saturated NaCl in water. Samples were
of okara in a petri dish. The petri dish was then covered vortexed for 5 min and centrifuged at 14,800 rpm for
with 2 layers of cling film. The first layer was pressed onto 5 min. 150 µL of sample (top layer) was transferred into
the inoculated okara and the second layer was wrapped glass vials for GC–MS analysis.
across the surface of the petri dish. Both layers were For amino acids analysis, 4 mg of fermented okara and
punctured with numerous holes using a sterile pin to 4 mg of raw okara (control) were resuspended in 200 µL
maintain aeration and moisture content within the petri of 6 M HCl. 20 µL of γ-aminobutyric acid (10 mg/mL)
dish and subsequently fermentation was carried out at were added as IS. The tubes were sealed and baked for
37 °C for 72 h. A beaker of water was placed in the incu- 24 h in an oven at 105 °C. The cell hydrolysate was dried
bator to maintain the moisture content. The fermented at 95 °C in a heat block. After drying, 20 µL of DMF and
okara were then freeze-dried and stored at − 20 °C until 20 µL of MSTFA were added. The tubes were sealed and
further analysis. incubated at 85 °C for 1 h. Samples were then centrifuged
at 14,800 rpm for 5 min and supernatant were transferred
Sample preparation for metabolomic, fatty and amino to glass vials. 40 µL of DMF were added into the glass
acids analyses vials and the vials were inverted a few times before send-
For the metabolomic analysis, 3 mL of methanol were ing for GC–MS analysis.
added to 900 mg of fermented okara and raw okara

enized using Fastprep-24™ 5G Homogenizer. Homog-

(control), respectively. The samples were then homog- GC–MS method for metabolomic, fatty and amino acids
analyses
enizing was carried out for 30 s at 5 min interval for 5 Metabolomic analysis including carbohydrates and TCA
times. The tubes were placed in a box of ice after each cycle metabolites were carried out via GC–MS. The GC–
homogenizing cycle to cool the samples. Next, the sam- MS system (Agilent Technologies 7890A-5975C) was
ples were centrifuged at 9000g for 10 min at 4 °C. The equipped with a HP-5MS, 5% Phenyl-Methyl-Silox cap-
supernatant was extracted and filtered through a 0.22 µm illary column (30 m × 0.250 mm id.; 0.25 μm film thick-
filter. 10 µL of ribitol (2 mg/mL) were added into 1.5 mL ness; Agilent J&W Scientific, Folsom, CA, USA). 1 µL of
of filtered supernatant as the internal standard (IS). Sam- samples were injected into the system by the autosam-
ples were then vortexed for 30 s and allowed to dry in a pler in splitless mode. The injector temperature and ion
heat block at 30 °C, overnight. Then, 100 µL of methox- source temperature were set at 250 °C and 230 °C, respec-
amine hydrochloride (20 mg/mL pyridine) was added tively. The oven temperature was as follows: 75 °C for
to the lyophilized samples for methoximation to protect 4 min, ramped to 280 °C at the rate of 4 °C/min, and held
the carbonyls and incubated at 37 °C for 60 min. Next, at 280 °C for 2 min. Data were acquired in full scan mode
silylation was carried out by adding 200 µL of N-methyl- from 35 to 600 m/z with a 0.3 s of scan time. Metabolites
N-(trimethylsilyl) trifluoroacetamide (MSTFA) with 1% were identified using the NIST08 mass spectral library
trimethylchlorosilane (TMCS) and subsequently incu- based on mass spectral similarity. Samples were normal-
bated at 70 °C for 30 min. The samples were then cen- ized with respect to the IS, ribitol, before comparison.
trifuged for 30 min at 15,330g and 150 µL of supernatant For fatty acid analysis, the injector temperature and ion
were transferred to glass vials and sent for GC–MS source temperature were set at 250 °C and 230 °C, respec-
analysis. tively. The oven temperature was as follows: 80 °C for

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Mok et al. AMB Expr (2019) 9:60 Page 4 of 12

1 min, ramped to 250 °C at the rate of 7 °C/min, and held Results

at 250 °C for 8 min. Data were acquired in full scan mode Metabolic profiles of fermented and unfermented okara
from 50 to 600 m/z at 2.66 scans per second. Metabolites A metabolomics analysis using GC–MS provided an
were identified using the NIST08 mass spectral library overview on the metabolic profiles between the fer-
based on mass spectral similarity. Samples were normal- mented and unfermented okara. Statistical analysis (PLS-
ized with respect to the IS, heptadecanoic acid before DA and heatmap) was carried out to understand the
comparison. changes between the samples. These changes observed
For amino acid analysis, solvent delay was set at 2 min in the level of each metabolite, helped to shed light on
30 s. The injector temperature and ion source tempera- the effects of B. subtilis WX-17 fermentation on okara.
ture were set at 250 °C and 230 °C, respectively. The oven In total, 49 metabolites were detected. Figure 1 showed
temperature was as follows: 160 °C for 1 min, ramped the PLS-DA score plot of the fermented and unfer-
to 290 °C at the rate of 20 °C/min, ramped again to 310 mented okara. The green and red highlights denoted
at 20 °C/min and held at 310 °C for 1 min. Data were the 95% confidence region. The first principal compo-
acquired in full scan mode from 180 to 550 m/z at 3.85 nent accounted for 76.5% of the total variance while
scans per second. Metabolites were identified using the the second principal component accounted for 5.3% of
NIST08 mass spectral library based on mass spectral the total variance which combined to explain a total of
similarity. Samples were normalized with respect to the 81.8% of the variance. This showed that the first compo-
IS, γ-aminobutyric acid before comparison. nent largely explained most of the variance between the
samples. From the PLS-DA score plot (­R2 = 99.8% and
­Q2 = 98.3%), clear and distinct separations between the
Antioxidants analysis unfermented okara and the fermented okara along the
300 µL of ethanol was added to 100 mg of sample and first principal component were observed. This showed
homogenized as described above. The samples were then that during fermentation, the metabolic profile of okara
centrifuged at 10,000 rpm for 5 min. 150 µL of the super- had changed significantly.
natant were transferred to new tubes and added with Although the PLS-DA score plot provided a visual
100 µL of 1,1,-diphenyl-2-picryl-hydrazil (DPPH) solu- representation of the difference in the metabolic profile
tion and 250 µL of ethanol. The samples were then incu- between fermented and unfermented okara, it did not
bated in a dark place for 30 min at room temperature. provide details about the specific metabolites. Hence a
The absorbance of the mixture was measured at 515 nm clustering heatmap was constructed to provide a more
with ethanol as blank using Nanodrop 2000c Spectro-
photometer. The activities of the samples were evaluated
with respect to trolox equivalent- % signal inhibition cali-
bration curve whereby % signal inhibition is defined as:
As
% Signal Inhibition = 1 − Ao × 100.
As is defined as the absorbance of the samples and A
­ o is
defined as the absorbance of pure DPPH.

Statistical analysis
All experiments were conducted in triplicates. Statistical
analysis was carried out using MetaboAnalyst 4.0 (Xia
et al. 2011, 2012; Xia and Wishart 2016). Data scaling
was carried out using mean-centering and divided by the
standard deviation of each variable prior to partial least
squares discriminant analysis (PLS-DA) and heatmap
analysis. The heatmap was also constructed using Euclid-
ean distance measurement and ward clustering algorithm.

Nucleotide sequence accession number

The 16s rRNA sequence of WX-17 was deposited in
Fig. 1 PLS-DA score plot of all metabolites found for fermented and
the GenBank database with the accession number unfermented okara. The green and red highlights denoted the 95%
MK559744. confidence region. Explained variance are shown in brackets

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Mok et al. AMB Expr (2019) 9:60 Page 5 of 12

visual breakdown of the metabolites that changed after growth and might have produced proteases and lipases
fermentation (Fig. 2). From the heatmap, unfermented to break down proteins and lipids in okara, into simpler
okara had high amount of carbohydrates such as fruc- amino acids and fatty acids (Lesuisse et al. 1993; Yang
tose, ribose, glucose, galactose, mannose and maltose. et al. 2000).
In comparison, the levels of carbohydrates were lower
in fermented okara. In addition, fermented okara had Carbohydrates and TCA cycle metabolites
higher amounts of amino and fatty acids as compared To better illustrate the effects of fermentation on fer-
to unfermented okara. This suggested that B. subti- mented and unfermented okara, the results for carbohy-
lis WX-17 consumed the carbohydrates in okara for its drate metabolites and metabolites involved in the TCA

Fig. 2 Heatmap analysis correlating the metabolites of fermented and unfermented okara

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Mok et al. AMB Expr (2019) 9:60 Page 6 of 12

cycle were presented in Figs. 3 and 4 respectively. In total, cycle that can unblock succinate dehydrogenate which
7 types of carbohydrates were detected. They were ribose, would promote cell respiration even under stressful
fructose, mannose, galactose, glucose, sucrose, and malt- environment (Kamzolova et al. 2018).
ose. This result agreed with previous findings by Li et al.
and Hou et al. which showed that the polysaccharides in
soybeans and okara were ribose, galactose, glucose, fruc- Amino acids and fatty acids metabolites
tose, sucrose, mannose and other minor components From the heat map, it was shown that amino acids and
such as verbascose, pinitol and myo-inositol (Hou et al. fatty acids levels increased after fermentation. To reaf-
2009; Li et al. 2012a). The abundance of all the different firm these findings, the amount of amino acids and
types of carbohydrates were reduced after fermentation fatty acids before and after fermentation were deter-
(Fig. 3). mined using GCMS protocols which were specific for
From the metabolomics analysis, it was shown that amino and fatty acids. These specific protocols are
isocitric acid and malic acid increased while succinic more sensitive towards their respective target metabo-
acid and fumaric acid decreased (Fig. 4). Most nota- lites. Table 1 showed the absolute value of amino acids
bly, isocitric acid increased by approximately 3 times. (mg/g okara) in fermented and unfermented okara. The
Recent studies have postulated on the potential benefits results in Table 1 showed that all amino acids increased
of isocitric acid. For example, Omar et al. suggested after fermentation with the total amount increasing by
that isocitric acid contains antioxidant properties that almost two-fold after fermentation. Notably, the essen-
can help to combat oxidative stress of the brain and tial amino acids leucine, phenylalanine and glutamic
liver in mice by decreasing the brain lipid peroxidation acid increased the most at 2.26, 2.42 and 2.12 times,
and inflammation, liver damage as well as DNA frag- respectively.
mentation (Abdel-Salam et al. 2014). Another study Fatty acids specific analysis suggested that there was
suggested that isocitric acid can help in combating negligible change in stearic and palmitic acids levels.
hypoxia or hypoxic conditions such as fatigue, dizzi- However, linoleic and oleic acids levels were shown to
ness to more serious conditions such as hypercapnia increase by 2.93 and 2.37 times, respectively (Table 2).
and organ failure as it is the only metabolite in the TCA

Fig. 3 Abundance of all the carbohydrates detected during analysis

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Mok et al. AMB Expr (2019) 9:60 Page 7 of 12

Fig. 4 Abundance of all the TCA cycle key metabolites detected

Table 1 Changes in amino acids in absolute value (mg/g the DPPH radical scavenging activity of unfermented
dried okara) for fermented and unfermented okara okara at 6.79 µg Trolox equivalent/g dried okara, fer-
mg/g okara Control (raw okara) Fermented okara mented okara displayed an increased in DPPH radical
scavenging activity by approximately 6.4 times.
Glycine 0.183 ± 0.0441 0.329 ± 0.104
Valine 0.0228 ± 0.00291 0.0458 ± 0.00457
Discussion
Proline 1.28 ± 0.442 2.15 ± 0.591
Metabolomics analysis showed that the overall metabo-
Leucine 0.303 ± 0.0684 0.685 ± 0.175
lite profile of okara changed after B. subtilis WX-17
Serine 0.130 ± 0.0309 0.141 ± 0.0188
fermentation (Figs. 1 and 2). Most of the carbohydrate
Threonine 0.138 ± 0.0391 0.151 ± 0.00157
metabolites decreased after fermentation. This indi-
Phenylalanine 0.0799 ± 0.0306 0.194 ± 0.0179
cated that it was being consumed by B. subtilis WX-17
Aspartic acid 0.200 ± 0.0703 0.292 ± 0.0278
which could utilize the carbohydrates through glycolysis,
Glutamic acid 0.611 ± 0.0211 1.30 ± 0.182
to produce increased amounts of the energy molecule
Lysine 0.0694 ± 0.00989 0.0856 ± 0.0156
acetyl-CoA. This precursor could then enter the amino
Tyrosine 0.0235 ± 0.00215 0.0439 ± 0.00352
acids and fatty acid pathway. This suggested that the
Total amino acids 3.04 ± 0.136 5.41 ± 1.21
microorganism was able to utilise the carbon source pre-
Results are as mean ± standard deviation (3 replicates) sent within okara and use it for metabolism to produce
other components. It had been suggested that glucose is
the preferred carbon source for B. subtilis (Singh et al.
Antioxidant activity
2008; Tian et al. 2015). In this study, analysis of the vari-
The DPPH radical scavenging activity of fermented
ous sugar pathways suggested that most other forms of
okara was shown to have increased during fermentation
carbohydrate were converted into glucose before being
(Fig. 5). Fermented okara showed the highest DPPH radi-
used for other processes.
cal scavenging activity at 43.68 µg Trolox equivalent/g
The increase in amino acids were confirmed as shown
dried okara after 72 h of fermentation. As compared to
in Table 1. This could be due to B. subtilis producing

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Mok et al. AMB Expr (2019) 9:60 Page 8 of 12

Table 2 Changes in fatty acids in absolute value (mg/g heart disease due to its ability to reduce LDL-cholesterol,
dried okara) for fermented and unfermented okara thrombogenicity, LDL-oxidative susceptibility as well
mg/g okara Control (raw okara) Fermented okara as insulin sensitivity factors (Lopez-Huertas 2010). The
increase in linoleic acid and oleic acid were expected as
Stearic acid 60.3 ± 2.00 55.0 ± 5.24 B. subtilis are known to produce lipases that catalyses the
Oleic acid 3.39 ± 1.02 8.04 ± 2.87 hydrolysis of fatty acids (Ma et al. 2006; Sánchez et al.
Linoleic acid 9.61 ± 3.31 28.2 ± 9.55 2002).
Palmitic acid 79.8 ± 1.93 75.6 ± 5.37 Endogenous metabolic processes or exogenous chemi-
Total fatty acids 153.04 ± 5.09 166.78 ± 2.41 cals in food system may generate free radicals which
Results are as mean ± standard deviation (3 replicates) may cause oxidative damages by oxidizing biomolecules
resulting in tissue damage or even cell death. Numerous
traditional fermented food such as miso, tempeh, sufu
extracellular proteases which would breakdown the pro-
and douche have free radical scavenging ability (Zhu
teins in okara into amino acids thereby contributing to
et al. 2008). Therefore, it is of interest to analyse if B. sub-
their increase after fermentation. The increase in amino
tilis WX-17 fermented okara displayed the same ability.
acids is important as they can provide as a rich source of
In this regard, analysis showed that antioxidant activity
nitrogen that are essential to living organisms. For exam-
increased by 6.4 times which strengthened the case of
ple, yeast such as Rhodosporidium toruloides and Saccha-
fermented okara as a functional food for animals.
romyces cerevisiae requires nitrogen for the synthesis of
To better understand the metabolic flux during fermen-
amino acids, proteins, DNA and RNA (Cruz et al. 2002;
tation, a metabolic pathway analysis was performed that
Evans et al. 1984).
would allow a hypothetical insight into the various meta-
Fatty acid level also increased after okara fermenta-
bolic pathways which were up regulated or down regu-
tion (Table 2). Particularly, the increase in both linoleic
lated during fermentation (Fig. 6). The pathway analyses
acid and oleic acid levels, were desirable as studies have
were performed with reference to the Kyoto Encyclopae-
reported various health benefits when these fatty acids
dia of Genes and Genomes (KEGG) database (Kanehisa
were consumed. Linoleic acid is a polyunsaturated fatty
et al. 2017; Kanehisa and Goto 2000; Kanehisa et al. 2016;
acid that contains numerous purported health benefits
Ogata et al. 1999).
such as anti-obesity, anti-carcinogenesis, anti-atheroscle-
The precursor for glycolysis is glucose. It is converted
rosis, anti-diabetic, osteosynthetic and immunomodu-
into pyruvate through the intermediate glucose 6-phos-
lation effects (Benjamin and Spener 2009; Nagao and
phate (G6P) and phosphoenolpyruvate (PEP). The anal-
Yanagita 2005). Likewise, oleic acid is a monounsaturated
ysis showed that apart from ribose, all other forms of
fatty acid that had been linked to a reduction in coronary

Fig. 5 DPPH scavenging activity of fermented and unfermented okara across 72 h expressed in terms of Trolox equivalent (µg/g okara)

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Mok et al. AMB Expr (2019) 9:60 Page 9 of 12

Fig. 6 Metabolic pathway analysis of all detected carbohydrates, amino acids and fatty acids after fermentation of okara

carbohydrates can be converted into glucose. Mannose One of the main intermediates of glycolysis, G6P
can be converted into fructose through the enzyme can be converted into myo-inositol, a carboxylic sugar
mannose isomerase in a reversible reaction. Fructose through myo-inositol 1-phosphatase (inositol phosphate
is in turn converted into glucose via xylose isomer- metabolism pathway). During fermentation, B. subtilis
ase. Both mannose and fructose were also produced WX-17 releases phytase which hydrolyses phytic acid,
through glycolysis through the intermediates mannan an antinutrient present in okara to produce myo-inositol
and d-fructose-2-phosphate respectively. The over- which might explain the increased amount of myo-inosi-
all decrease in both mannose and fructose suggested tol detected after fermentation (Chen et al. 2015; Kero-
that the rate of consumption is greater than the rate of vuo et al. 1998). Myo-inositol can then be converted into
production. acetyl-coA through malonate-semialdehyde dehydroge-
Glycolysis also produced sucrose through the inter- nase which can then enter the TCA cycle (inositol phos-
mediate UDP-glucose and sucrose synthase. Sucrose phate metabolism pathway).
can then be converted into fructose and glucose through Another intermediate in glycolysis, phosphoenolpyru-
sucrose alpha-glucosidase. UDP-glucose can also be vate (PEP) was involved in the shikimate pathway that
converted to maltose via maltodextrin which were then produce tryptophan through a reversible reaction with
converted to glucose through maltose phosphorylase. (3-indole)-glycerol phosphate. The increased level of
Galactose can be converted into UDP-glucose via UDP tryptophan detected suggested that the forward reaction
glucose pyrophosphorylase. (3-indole-glycerol phosphate to tryptophan) occurred at
The KEGG database suggested no direct pathway for a higher rate than the backward reaction. Both phenyla-
conversion of ribose to glucose. This suggested that the lanine and tyrosine are involved in the shikimate pathway
reduction in ribose after fermentation is due to direct as well and can be interconverted through prephenate
consumption of ribose by B. subtilis WX-17. This was which is an intermediate in the shikimate pathway. Their
supported by studies which showed that B. subtilis exhib- increased levels after fermentation suggested that reac-
ited increased sporulation when provided with a mixture tions along the pathway are skewed towards phenylala-
of glucose and ribose as carbon source compared to a nine and tyrosine metabolism rather than quinate.
medium with glucose as the sole carbon source (War- Both valine and leucine which are essential amino
riner and Waites 1999). acids were produced from the glycolysis intermediate,

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Mok et al. AMB Expr (2019) 9:60 Page 10 of 12

pyruvate (valine, leucine, isoleucine biosynthesis path- pathway)(Ruth and Field 2013). Lastly, aspartate can
way). Valine is produced from the intermediate, 2-oxois- be converted to threonine through threonine synthase
ovalerate which can also be irreversibly converted into (glycine, threonine and serine metabolism pathway).
leucine. In addition, pyruvate can also be converted into Threonine is also produced from the reversible reaction
leucine through pyruvate metabolism. Increased levels of of glycine through catalysis by threonine aldolase (gly-
valine and leucine suggested that their rate of synthesis cine, serine, threonine metabolism pathway). Glycine,
is greater than their rate of degradation as leucine can be another essential amino acid is in turn produced from
broken down into acetyl-coA. This implied that the bulk the reversible reaction of serine through catalysis by gly-
of the acetyl-coA were converted from pyruvate rather cine hydroxymethyltransferase (glycine, serine, threonine
than leucine. metabolism pathway). In addition, serine can also be con-
TCA cycle is a chain of reactions that are used by aer- verted to cysteine through cysteine synthase (cysteine
obic organisms to release stored energy in acetyl-coA and methionine metabolism pathway).
through oxidation into adenosine triphosphate (ATP) It was also observed that all the amino acids detected
and carbon dioxide. 4 of the key components in the TCA were glucogenic amino acids which means that they can
cycle were detected, of these both isocitrate and malate be converted into glucose through gluconeogenesis. This
increased while fumarate and succinate decreased. could explain why although all the amino acids were up
Results also showed that isocitrate increased the most (3 regulated after fermentation, the overall amount detected
times) after fermentation. This is in line with studies that after fermentation were much lesser compared to fatty
had shown that B. subtilis produced the enzyme aconitate acids.
hydratase which catalysed the stereo-specific isomerisa- The metabolic pathways for the biosynthesis of fatty
tion of citrate to isocitrate (Cox and Hanson 1968; Ding- acids involved the conversion of acetyl-coA to malonyl-
man et al. 1987). The large amount of isocitrate produced coA through acetyl-coA carboxylase which were then
likely drove reactions forward to produce succinate and converted to the saturated fatty acids, palmitate and
fumarate which are consumed to produce malate. stearate through the fatty acid biosynthesis pathway.
The intermediates of the TCA cycle are involved in Palmitate and stearate then underwent elongation and
numerous reactions that produced important com- unsaturation process to yield oleate and linoleate (bio-
pounds. Isocitrate are broken down by the action of synthesis of unsaturated fatty acids pathway). Both pal-
isocitrate dehydrogenase into 2-oxoglutarate which can mitate and stearate were down regulated while oleate and
then be converted into the non-essential amino acid, glu- linoleate were up regulated. This suggested that a larger
tamate. Glutamate are in turn converted into ornithine proportion of the saturated fatty acids were converted
via acetylornithine deacetylase which is part of the urea into unsaturated fatty acids. The reduction in saturated
cycle. Critically, ornithine cyclodeaminase catalyses the fatty acids are desirable as it is well known that consump-
conversion of ornithine to proline which is an essential tion of high amount of saturated fatty acids are associ-
amino acid (arginine and proline metabolism pathway). ated with increased risk of coronary heart diseases (Zong
Glutamate, ornithine and proline were all upregulated et al. 2016).
after fermentation which strengthens the hypothesis that Overall, from the metabolic pathway analysis (Fig. 6), it
isocitrate were produced in excess. 2-oxoglutarate can can be summarized that the amount of various carbohy-
also be converted into lysine through the lysine biosyn- drates in okara decreased. This suggested that carbohy-
thesis pathway. drates were consumed and utilised by B. subtilis WX-17
From the pathway analysis, aspartate played a vital role through glycolysis, to produce energy for its metabo-
in the synthesis of numerous important compounds. lism and cellular process. Subsequently, this led to the
Firstly, in the lysine biosynthesis pathway, aspartate is increased antioxidant amount, amino acids and fatty
converted into lysine through catalysis by diaminopime- acids in fermented okara.
late decarboxylase. Aspartate can also be directly con- In conclusion, with the world’s population predicted to
verted into the TCA cycle intermediate, fumarate by reach 9 billion by 2050, food security is becoming a rising
aspartase (alanine, aspartate and glutamate metabolism global issue (Godfray et al. 2010). One strategy to combat
pathway). Next, aspartate is also directly converted into these issues is the biovalorisation of food waste. With the
asparagine, a non-essential amino acid by asparagine growing popularity of soy-based products, the amount of
synthetase (alanine, aspartate and glutamate metabo- okara produced are increasing rapidly, approximately 14
lism pathway). Aspartate is also involved in the synthe- million tonnes globally every year. This work showed the
sis of the essential amino acid, methionine which had possibility of reintroducing okara, a food waste back into
been linked with optimising the immune function of the the food chain through biovalorisation by fermentation.
human intestines (cysteine and methionine metabolism As the microorganism used is food grade, this study also

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Mok et al. AMB Expr (2019) 9:60 Page 11 of 12

presents the potential application of fermented okara Funding

The funding of this research was supported by the School of Chemical and
into human diets in various forms. The study revealed Biomedical Engineering (SCBE) at Nanyang Technological University (NTU)
that fermentation of okara using food grade B. subtilis (Tier1).
WX-17 enhanced its nutrient profile. GC–MS and meta-
bolic pathway analysis showed that both amino acids and Publisher’s Note
fatty acids production increased after fermentation due Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
to the release of hydrolases by B. subtilis WX-17 to break
down complex macromolecules into simpler molecules Received: 27 March 2019 Accepted: 25 April 2019
which are easier to digest. Furthermore, antioxidant anal-
ysis also showed that post fermentation, fermented okara
displayed higher antioxidant activities which indicated a
higher phenolic content. Future work will include char- References
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