Journal of Functional Foods 42 (2018) 85–94

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Journal of Functional Foods

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

Valorization of brewer’s spent grain using fungi solid-state fermentation to T

enhance nutritional value
⁎
Sachindra T. Cooraya,b, Wei Ning Chenc,
a
Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, Block S2 – B3a – 01, Singapore 639798, Singapore
b
Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, 1 CleanTech Loop, CleanTech
One #06-08, Singapore 637141, Singapore
c
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore

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

Keywords: Brewer’s spent grain (BSG) is a nitrogen and fiber rich waste generated in high volumes in beer manufacturing.
Fermentation Here we have investigated the use of fermentation for value addition to this waste material. In this study we use
Waste valorization solid-state fermentation (SSF) using food grade Rhizopus oligosporus to enhance its nutrient content by valor-
Metabolomics ization of BSG. Subsequently, untargeted GC–MS (Gas chromatography mass spectrometry) based metabolomics
GC–MS
was used to analyse the constituents. We investigated different solvents to extract extracellular metabolites and
Nutrition
best derivatization method, where methanol and silylation provided best results. We observed significant levels
Amino acids
of metabolite changes in amino acids, citric acid, vitamins and antioxidants. This data provided insight on
metabolites variations during fermentation, proving that fermentation enhanced BSG’s nutrient content. Hence,
data from metabolomics studies can be used to find novel applications for food wastes and innovative processing
methods for valorization of waste materials.

1. Introduction compounds present in BSG. BSG essentially comprise of lignocellulose

Producing high value added bio-products, including amino acids,
antibiotics, vitamins, enzymes and bulk chemicals, using microorgan-
isms has been a trending topic in the biotechnological industry. Using
biomass from agro-industrial wastes to exploit opportunities in bio-
technological applications in producing added value products is bene-
ficial, as these material do not compete with world food supply (Bilal,
Asgher, Iqbal, Hu, & Zhang, 2017). Brewer’s spent grain (BSG) is rich in
fiber and nitrogen, accounting to 70% and 20% of dry weight respec-
tively. BSG is an underutilized by-product generated in the beer man-
ufacturing process. In 2014 worldwide beer production was reported to
be 193 billion liters, and for every 100 liters of beer produced roughly
20 kg of BSG was generated (Mussatto, Dragone, & Roberto, 2006). This
waste material is known to hold substantial amounts of valuable com-
pounds that remain untouched during the brewery process. However,
majority of the spent grain is used for landfills or as animal feed
(Buffington, 2014; Nigam, 2017). Other uses for BSG are sought after
because the cost of disposal is high. A main reason hindering the pro-
cessing of BSG is due to the recalcitrance of plant cell walls. Therefore,
physical, chemical or biological treatments need to be performed.
Previous studies have been conducted to evaluate important
matter (cellulose and non-cellulose carbohydrates) protein and lignin
(Mussatto et al., 2006; Robertson et al., 2010). Major portion of total
lipids present in BSG are triacylglycerols, followed by fatty acids
(mainly linoleic, palmitic, and oleic acids). Minor amounts of dia-
cylglycelos and monoacylglycerols have also been reported (del Río,
Prinsen, & Gutiérrez, 2013; Fărcaş et al., 2015; Niemi et al., 2012). In
addition, BSG has shown to contain many biologically active com-
pounds such as polyphenols, flavonoids and antioxidants (Fărcaş et al.,
2015; Guido & Moreira, 2017). Since BSG contains many interesting
nutritional compounds as mentioned above, studies have been con-
ducted to incorporate BSG in human diet (Ktenioudaki, Chaurin, Reis, &
Gallagher, 2012; Öztürk, Özboy, Cavidoğlu, & Köksel, 2002). This
material has also been considered to be used as a substrate along with
other agro-wastes to produce microbial cell mass without the need for
extra nutrients (Aggelopoulos, Bekatorou, Pandey, Kanellaki, &
Koutinas, 2013) as well as to grow fungal strains (Wang, Sakoda, &
Suzuki, 2001).
In addition to the readily available valuable material in BSG, studies
have been conducted to use chemical processes and/or enzymatic hy-
drolysis to further obtain value-added compounds. However, the en-
zymatic treatment has added advantages of not generating toxics and

⁎
Corresponding author at: School of Chemical and Biomedical Engineering, Nanyang Technological University, Blk N1.2-B1-07, 62, Nanyang Drive, Singapore 637459, Singapore.
E-mail addresses: sach0008@e.ntu.edu.sg (S.T. Cooray), WNChen@ntu.edu.sg (W.N. Chen).

https://doi.org/10.1016/j.jff.2017.12.027
Received 12 October 2017; Received in revised form 12 December 2017; Accepted 12 December 2017
Available online 10 January 2018
1756-4646/ © 2017 Elsevier Ltd. All rights reserved.
S.T. Cooray, W.N. Chen
being more environmentally friendly. Commercial enzymes have been
used to extract sugars (Mussatto, 2014) and to release hydroxycinnamic
acids (p-coumaric and ferulic acids) (Bartolomé & Gómez-Cordovés,
1999) from BSG. Nevertheless, the use of commercial or crude enzymes
is costly and less economical. Thus, proceeding to microbial fermen-
tation to hydrolyze biomass will be cost effective.
Microbial fermentation of BSG has reported to significantly increase
the protein (crude and soluble) content (Bekatorou, Bountas, Banat, &
Kanellakl, 2007; Canedo, de Paula, da Silva, & Vendruscolo, 2016), to
release sugars (Mussatto & Roberto, 2005) and to produce lactic acid
(Mussatto, Fernandes, Dragone, Mancilha, & Roberto, 2007). In a pre-
vious study we were able to enhance the available nitrogen content
using fungi fermentation using BSG and produce a novel media to
supply the complete nitrogen requirement for yeast growth by ex-
tracting nutrients from BSG (Cooray, Lee, & Chen, 2017). Enzymes
produced for the degradation of biomass during microbial fermentation
helps to release nutrients from BSG. Concurrently those microorganisms
produce primary and secondary metabolites, which in return can fur-
ther add more value to the original BSG.
Filamentous fungi have been used in producing fermented food and
beverages since ancient times. And today the usage of these fungi has
expanded in to biotechnological fields to produce chemicals to be used
in manufacturing food ingredients, medicine, enzymes and proteins
(Smedsgaard & Nielsen, 2005). There have been reports indicating that
secondary metabolites and extracellular enzymes produced by these
filamentous fungi comprise of bioactive compounds that are useful as
antibiotics, immunosuppressive drugs, cholesterol reducing agents and
antitumour drugs (Newman, Cragg, & Snader, 2003). Tempeh is a tra-
ditional Indonesian food produced by the fermentation of soybean re-
sidue, okara, using Rhizopus oligosporus. Tempeh is regarded as a pro-
tein rich meal that has plenty of vitamins and minerals. R. oligosporus
was reported to increase protein digestibility in the okara, decompose
anti-nutrients present and enhance its nutrient content (O'Toole, 1999;
Stodolak & Starzyńska-Janiszewska, 2008).
An untargeted global investigation of fermented products is useful
to find any potentially valuable chemicals produced as a result of fer-
mentation. Such an approach to use a metabolomics study is beneficial
to evaluate the possibility of producing interesting compounds by BSG
fermentation. Metabolomics helps to simultaneously explore hundreds
of individual compounds in a single run rather than as compound
groups. The metabolite extraction step is an integral part in a meta-
bolome investigation. Classical solvent extraction is the most popular
metabolite extraction method. The solvent most suitable for a particular
extraction depends on the application. According to previous scholars,
hexane, methanol, ethyl acetate, dichloromethane and water are some
commonly used solvents employed for metabolite extraction (Kim, Heo,
Park, Singh, & Lee, 2016).
GC–MS is a well-developed technology permitting the analysis of
many metabolites in a single run (Villas-Bôas, Mas, Åkesson,
Smedsgaard, & Nielsen, 2005). The main advantage of this method of
analysis is the availability of comprehensive commercial and in-house
MS libraries, making it easier to identify metabolites. An essential re-
quirement for a GC–MS analysis is that all analytes need to be volatile
for the separation to be possible in gas phase (Villas-Bôas et al., 2005).
Derivatization modifies the functionality of an analyte, to enable
chromatographic separation (Orata, 2012; Smart, Aggio, Van Houtte, &
Villas-Boas, 2010). In the derivatization process chemical groups are
introduced to convert analytes to increase volatility and improve
thermal stability, to decrease polarity and to enhance sensitivity of the
mass detector. Alkylation, acylation and silylation are the three general
derivatization reactions used in GC. In the alkylation reaction, the ac-
tive hydrogen is replaced by an alkyl group resulting in a less polar
product. This process is also known as esterification. Silylation involves
the replacement of active hydrogen with a trimethylsilyl (TMS) group.
Acylation introduces acyl group to the organic compounds by replacing
a hydroxyl group. Out of these three methods of derivatization,
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Journal of Functional Foods 42 (2018) 85–94
alkylation and silylation are considered to be the widely used methods
in metabolomics analysis and metabolomics profiling (Villas-Bôas,
Smart, Sivakumaran, & Lane, 2011; Zarate et al., 2016).
In this study our objective was to investigate the use of fermentation
on underutilized BSG using food grade Rhizopus oligosporus to produce
value added products and analyse the production using a metabolomics
based approach. Moreover, we explore metabolomics sample prepara-
tion strategies for metabolite extraction and derivatization for GC, to be
used in the untargeted metabolomics analysis to evaluate the changes
taking place during fermentation. The study provides important in-
formation regarding BSG fermentation and how this bioprocess is being
able to increase the nutritional quality of the waste biomaterial.
Furthermore, we aim to show how a metabolomics study is able to
evaluate the changes taking place in the fermentation process.
2. Materials and methods
2.1. Chemicals
Type-1 ultrapure water was prepared using Elga PURELAB® Pulse.
Hexane, methanol, ethyl acetate and dichloromethane were bought
from Sigma-Aldrich (St. Louis MO, USA). All derivatizing agents were
bought from Sigma-Aldrich (St. Louis MO, USA).
2.2. Strains
Rhizopus microsporus var. oligosporus (DSM 1964, German Collection
of Microorganisms and Cell Culture, DSMZ) were maintained on potato
dextrose agar (PDA) plates at 37 °C.
2.3. Brewer’s spent grain and fermentation
BSG was kindly provided by Asia Pacific Breweries (Singapore) Pte,
Ltd. and was stored in airtight plastic containers at −80 °C until used.
Fermentation of BSG was performed according to a method proposed
with minor modifications (Ikasari & Mitchell, 1994). For the analysis
10 g of BSG was inoculated with 1 ml spore suspension (107 CFU/ml) of
R. oligosporus and incubated at 37 °C for three days. The experiment was
conducted with multiple replicate samples. Samples were collected at
different time periods: the beginning (0 h), 24 h, 48 h and 72 h. The BSG
samples were freeze-dried to remove all moisture present within and
stored at −20 °C until the metabolites were extracted. The freeze-
drying helps to remove the excess water and to avoid any problems due
to dilution effects.
2.4. Composition analysis of BSG before and after fermentation
Analysis of the composition of BSG before and after fermentation
was outsourced to ALS (Australian Laboratory Services), Singapore. Fat
content was measured using solvent extraction; Total dietary fiber using
enzymatic-Gravimetric method; Total sugars (as invert sugar) using
Lane-Eynon method; Starch content using enzymatic methods and
HPLC; and ash content by incinerating at 550 °C.
Amino acid analysis was performed according to a previous study
(Zamboni, Fendt, Rühl, & Sauer, 2009). Freeze-dried BSG samples were
used for the analysis. 200 μl of 6 M HCl (Sigma-Aldrich, St. Louis MO,
USA) was added to approximately 0.4 mg of samples. These samples
were baked in a 105 °C oven for 18 h in sealed tubes. Then the hydro-
lysate was dried at 95 °C in a heat block until completely dried. 20 μl of
dimethylformamide (DMF) (Sigma-Aldrich, St. Louis MO, USA) was
added and the dried hydrolysate was resuspended in DMF. This DMF
was transferred into a new 2 ml tube. 20 μl of N-tertbutyldimethylsilyl-
N-methyltrifluoroacetamide with 1% (wt/wt) tertbutyldimethyl-chlor-
osilane (TBDMSTFA) (Sigma-Aldrich, St. Louis MO, USA) was added to
the DMF solution and sealed well. The sample was incubated in a heat
block at 85 °C for 1 h. The derivatized samples were injected to the
S.T. Cooray, W.N. Chen
GC–MS. Amino acid standard AAS18 (Sigma-Aldrich, St. Louis MO,
USA) was used for quantitation.
Chromatography was performed using Agilent Technologies 7890A
GC-5975C inert MS system. 1 μl samples were injected into the HP-5MS
capillary column by splitless mode by the auto-injector. Helium was
used as a carrier gas at a flow rate of 1.5 ml/min. The inlet source
temperature was maintained at 230 °C and MS source temperature at
250 °C. The oven temperature was maintained at 160 °C for 1 min and
ramped to 290 °C at a rate of 20 °C/min and held for 1 min. Then once
again it was ramped to 310 °C at a rate of 20 °C/min and held for 1 min.
Data were acquired in full scan from 180 to 550 m/z with a solvent
delay of 2.7 min.
2.5. Selection of extraction solvent and extracellular metabolites extraction
Suitability of an appropriate solvent for metabolite extraction was
investigated using ultrapure water, hexane (Sigma-Aldrich, St. Louis
MO, USA), methanol (Sigma-Aldrich, St. Louis MO, USA), ethyl acetate
(Sigma-Aldrich, St. Louis MO, USA) and dichloromethane (Sigma-
Aldrich, St. Louis MO, USA). Metabolites were extracted using the
method suggested by Rauf et al. with slight modifications (Rauf, Irfan,
Nadeem, Ahmed, & Iqbal, 2010). 100 ml of the solvent was added to
30 g of the fermented substrate. The mixture was placed in a rotary
shaker for 30 min at 180 rpm in order to homogenize the flasks.
Afterwards the broth was centrifuged (9000g, 10 min, 4 °C). The su-
pernatant containing the metabolites was filtered through a 0.22 μm
filter.
2.6. GC–MS sample preparation and sample analysis
1.5 ml supernatant of the sample media prepared as mentioned in
the previous step was spiked with 10 μL internal standard (ribitol
(Sigma-Aldrich, St. Louis MO, USA), 2 mg/mL dissolved in water). The
sample that used ultrapure water as the extraction solvent was freeze-
dried, but the other samples were left in a heat block at 30 °C to dry
overnight.
2.6.1. Derivatization and metabolite analysis
For Silylation, the lyophilized/dried samples were derivatized ac-
cording to Wang et al. before the GC–MS analysis (Wang, Bai, Chen, &
Ching, 2010). Methoximation was performed by dissolving the samples
in 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine)
(Sigma-Aldrich, St. Louis MO, USA) to protect the carbonyls and in-
cubating at 37 °C for 60 min. Afterwards, silylation was carried out by
adding 100 μL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide
(MSTFA) with 1% trimethylchlorosilane (TMCS) (Sigma-Aldrich, St.
Louis MO, USA) to each sample and incubating at 70 °C for 30 min.
Subsequently the samples were shaken for 60 min at room temperature
and then analysed in GC–MS within 24 h in a random order.
For Alkylation, the lyophilized samples were derivatized for analysis
according to Horak (Horak et al., 2009). The dried residue was redis-
solved in 500 μL of 10% BF3-methanol (Sigma-Aldrich, St. Louis MO,
USA) and incubated in a sealed screw cap tube in a heat block at 95 °C
for 20 min. Then the tube was cooled down to near room temperature.
Afterwards, 300 μL of saturated NaCl in water and 300 μL of n-hexane
were added. Then the samples were centrifuged (14,000 rpm, 10 min,
room temperature). The upper hexane layer was transferred to glass
vials for GCMS analysis.
GC–MS analysis for metabolites was performed according to a pre-
vious study (Cooray et al., 2017) using Agilent 7890A GC – 5975C inert
MSD (with Triple Axis Detector) system (Agilent Technologies, CA,
USA).
2.7. Data visualization and statistical analysis
Data extraction was performed by deconvolution algorithm using
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Journal of Functional Foods 42 (2018) 85–94
Agilent MassHunter Qualitative Analysis software (B.07.00). The ex-
tracted compounds were aligned, normalized (according to the IS, ri-
bitol) and filtered using Agilent Mass Profiler Professional software
(B.02.01). Multivariate data analysis and statistical analysis was per-
formed using MetaboAnalyst 3.0 (Xia, Psychogios, Young, & Wishart,
2009; Xia & Wishart, 2016). Data scaling was performed using pareto
scaling prior to principal component analysis (PCA) and orthogonal
partial least squares discriminant analysis (OPLS-DA). PCA and OPLS-
DA were used to identify significantly changed features in the samples.
Metabolites displaying significant changes between unfermented BSG
and fermented BSG samples were determined and their accurate masses
were compared with spectra in the National Institute of Standards and
Technology (NIST) mass spectral library with similarity above 75% for
feature identification. Statistical analysis of the data was performed
using one-way ANOVA by the post hoc Tukey's Honest Significant
Difference (HSD) to determined significant metabolic changes; wherein
p < 0.001 was considered significant. Clustering heatmaps were con-
structed to obtain an overview of the fermentation process, which was
created using Ward clustering algorithm and Euclidean distance cal-
culation. Finally, a list of metabolites with tentative assignments was
created.
3. Results and discussion
3.1. Constituent variation in BSG with the fermentation
After 3 days of fermentation the content of BSG was changed as
reported in Table 1. A noticeable decrease was seen in the fat content
(5.57%), dietary fibers (7.82%), sugar content (> 1.04%) and starch
content (1.74%), but ash content was not altered significantly.
With reference to Table 1, the reduction in fat content could be due
to the hydrolysis of fat by lipases. According to previous studies fungi
are reported to produce lipases during fermentation (Mahapatra,
Kumari, Garlapati, Banerjee, & Nag, 2010). The significant decrease in
dietary fibers could be as a result of degradation by pectinase and
cellulase enzymes produced during fermentation. Similarly the in-
vertase and amylase produced as the fermentation progressed could
have broken down the sugars and starch molecules. Proteases produced
during fermentation can breakdown complex proteins into simpler
amino acids (Hsiao et al., 2014). Even if there was a decrease in the
level of dietary fiber, the final amount of fiber is significant. Thus, the
fermented BSG is still favourable as a source of dietary fiber for food
and other related applications. Filamentous fungi demonstrate myce-
lium growth in which it branches out to form a network of hypae. This
growth pattern enables the fungi a greater surface area for nutrient
uptake. Fungi are known to efficiently degrade plant biomass, which is
comprised mainly of polysaccharides and lignin. However, fungi cannot
take up complex polymeric compounds. Therefore, extracellular en-
zymes are produced to degrade the polymers to mono- or short oligo-
mers (Khosravi, Benocci, Battaglia, Benoit, & de Vries, 2015, chap. 1;
Mäkelä, Donofrio, & de Vries, 2014).
Amino acid content of the BSG was increased due to the fermenta-
tion by the fungi (Table 2). The analysis method was able to detect
essential amino acids, valine, leucine, threonine, phenylalanine and
lysine, together with other amino acids, alanine, glycine, proline,
Table 1
Composition of brewer’s spent grain (BSG) before fermentation and after fermentation of
3 days reported based on dry matter content in g/100 g.
Unfermented BSG Fermented BSG
Fat content (g/100 g dry) 10.09 4.52
Total dietary fiber (g/100 g dry) 45.65 37.83
Total sugar content (g/100 g dry) 3.04 <2
Starch content (g/100 g dry) 2.17 0.43
Ash content (g/100 g dry) 3.35 3.39
S.T. Cooray, W.N. Chen
Table 2
Amino acid (mg/g BSG) changes observed during fermentation in absolute values for the
unfermented BSG and fermented BSG after 3 day.
Unfermented BSG Fermented BSG
Alanine 0.100 0.223
Glycine 0.100 0.131
Valine 0.233 0.396
Leucine 0.311 0.496
Proline 1.907 4.253
Serine 0.024 0.087
Threonine 0.056 0.337
Phenylalanine 0.158 0.362
Aspartic acid 0.316 0.481
Glutamic acid 0.402 0.743
Lysine 0.100 0.144
Tyrosine 0.100 0.119
Total amino acids 3.807 7.772
serine, aspartic acid, glutamic acid and tyrosine. All detected amino
acids showed increased levels after the fermentation period. Threonine
(6.018 fold) and serine (3.597 fold) showed the maximum change.
Total amino acid levels changed from 3.807 mg/g in unfermented BSG
to 7.772 mg/g in fermented BSG after three days of fermentation.
3.2. Selection of extraction solvent and derivatization method
One of the aims of this study was to establish a sample preparation
methodology to analyse the fermentation products of BSG by GC–MS.
The goal in the metabolomics study was to analyse as many as possible
metabolites in a single detection run. Therefore, selection of both the
extraction solvent and derivatization method was based on the number
of compounds detected after analyzing the chromatographs of the
samples, once the background noise and solvent ions were removed.
The number of extracted metabolites by Agilent MassHunter
Qualitative Analysis software varied according to the solvent used and
the derivatization method employed (Fig. 2). According to the results,
methanol was able to extract more extracellular metabolites, compared
to water, hexane, dichloromethane and ethyl acetate. Furthermore an
organic solvent, such as methanol, is easier to use in down processing
compared to water. Methanol being a mild nonpolar solvent can help in
maximizing the metabolome coverage (Kirkwood, Maier, & Stevens,
2013). Moreover, methanol is known to effectively precipitate proteins
in solutions (Gowda & Raftery, 2014), which is advantageous in the
GC–MS analysis. Therefore methanol was selected for the extracellular
metabolite extraction in this analysis. Metabolites have diverse prop-
erties. Thus, to conduct a comprehensive untargeted analysis it is best
to use multiple separation methods, such as to separate polar and
nonpolar compounds. Even though separate methods provide better
coverage, it is time consuming and high in terms of manpower usage
and cost. Therefore, our approach in this study was to use a single
method for metabolite extraction.
Derivatization is essential in gas chromatography (GC) to improve
the chromatographic characteristics of analytes. Derivatization is able
to decrease analytes’ polarity and/or increase detector sensitivity of the
targets. According to our results, when alkylation and silylation deri-
vatization methods were compared, the latter provided a greater
number of hits over alkylation. As this study was aiming towards con-
ducting a global analysis on metabolomics, silylation was selected,
since it was the derivatization method that showed more peaks in the
chromatograph (Fig. 1). Silylation is one of the classical and broadly
used derivatization method in metabolomics analysis by GC–MS. Sugars
and their derivatives (sugar alcohols, amino sugars etc.), phenols,
simple carboxylic acids (mono- and di-) and fatty acids are efficiently
derivatized in silylation (Villas-Bôas et al., 2011). In the reaction the
active hydrogen is replaced with a silyl group [–Si(CH3)3 –]. N-methyl-N-
(trimethylsilyl) trifluoroacetamide (MSTFA) is considered the most
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Journal of Functional Foods 42 (2018) 85–94
Fig. 1. Number of metabolites extracted by Agilent MassHunter Qualitative Analysis
software when different extraction solvents (water, hexane, methanol, ethyl acetate (EA)
and dichloromethane (DCM)) were used and when the two derivatization methods, si-
lylation and alkylation, were used. Key: Alkylation of unfermented BSG (light blue), al-
kylation of fermented BSG (dark blue), silylation of unfermented BSG (yellow) and sily-
lation of fermented BSG (orange). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
volatile from the trimethylsilyl acetamides (Orata, 2012). In the reac-
tion of derivatives from MSTFA, the N-methyltrifluoroacetamide group,
is replaced by the analyte. It should be noted that the silylation reaction
mixture is not separated from the derivatives before injecting to the GC.
Thus, injected samples could contain derivatives, residual agents as
well as non-derivatized non-volatile compounds. However, in silylation
samples need to be completely free of water, as anhydrous reaction
conditions are required. Derivatization by alkylation is primarily used
in analyzing polyfunctional amines and organic acids. Boron trifluoride
(BF3) in methanol or n-Butanol (formula F3 B: HOCn H2n + 1) is a con-
venient and inexpensive reagent that can be used for alkylation (Orata,
2012).
3.3. Metabolite variation during the fermentation process
Fermentation metabolome was examined to gain insight into the
changes that has taken place during the bioprocess. Untargeted meta-
bolomics analysis was performed for the four time points using GC–MS
together with a multivariate data analysis. Fig. 2 depicts the overlay
spectra obtained by GC–MS from samples collected every 24 h during
fermentation, starting day zero. As the fermentation progressed, the
chromatograph pattern changed and the number of peaks detected in-
creased. This indicates that more and different metabolites were pro-
duced with time as the fungi grew.
Even if the spectra differences depicts a change in the metabolites, a
more reliable method such as multivariate analysis is essential to find
further details about the exact changes that have taken place. Principal
components analysis (PCA) and orthogonal partial least square-dis-
criminant analysis (OPLS-DA) are two examples of such multivariate
analysis that can be used for reducing dimensionality. PCA is able to
create two and three-dimensional ordination plots after dimensionality
reduction using linearly uncorrelated principle components. PCA score
plot generated in our study, clearly displayed metabolic discrimination
between the BSG fermentation samples (Fig. 3A), in which separation
took place between principal components (PCs) one and two accounting
for 80.1% of the variance. It can be clearly seen that distinguishable
differences exist among the samples when the fermentation progressed,
except for the variation between days two and three that are not as
significant.
Citric acid and alanine were the dominant differently expressed
compounds according to the PCA loading plot (Fig. 3B), showing a
differentiation in the fermentation samples along the first principal
S.T. Cooray, W.N. Chen Journal of Functional Foods 42 (2018) 85–94

Fig. 2. Chromatogram of the spectra observed by GC–MS when the fermentation progressed on the BSG from day 0 (red), day 1 (green), day 2 (blue) to day 3 (purple). The changes in the
peak patterns indicate the changes happening in metabolite content. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

component. Therefore, it can be inferred that production levels in-
creased with fermentation time when the unfermented and fermented
BSG samples were compared. Moreover according to this loading plot,
Glucose, D-fructose, D-gluconic acid, galactose, D-mannitol and D-
(+)-Talose displayed higher correlation to the unfermented BSG over
the other samples. Thus, we can deduce that microorganisms must have
consumed these compounds for their growth, and have accumulated
citric acid and alanine.
PCA, being an unsupervised model, in some instances may tend to
divert the focus due to systematic variations, such as instrument drift
due to temperature fluctuations, artifacts or contaminants present and
other experimental variations as a result of sample collection and me-
tabolite extraction (Wiklund et al., 2008). In contrast OPLS-DA, a su-
pervised multivariate analysis, has the ability to eliminate discrimina-
tion patterns due to external factors. The OPLS-DA model demonstrated
better separation for the analysed samples, with the total explained

Fig. 3. (A) PCA score plot and (B) PCA loading plot, for extracted metabolites as the fermentation progressed from day 0 to day 3. Component 1 (65%) vs. component 2 (15.1%) of Day 0
(red), Day 1 (green), Day 2 (dark blue) and Day 3 (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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S.T. Cooray, W.N. Chen Journal of Functional Foods 42 (2018) 85–94

Fig. 4. (A) OPLS-DA score plot and (B) S-plot loadings for extracted metabolites when the fermentation progressed from day 0 to day 3. Key: metabolites captured in Day zero (red), Day 1
(green), Day 2 (dark blue) and Day 3 (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

variance R2 (cumulative) of 95.5% and the cross validation predictive fermentation day 2.
ability Q2 (cumulative) of 91.5% (Fig. 4A). The OPLS-DA loading plot and heat map correlation was able to
A better separation of the sample classes was seen in the OPLS-DA show the significantly changing metabolites in BSG during the fer-
score plot compared to the PCA model. S-plot represents the covariance mentation process (Figs. 4 and 5B). In summary, carbohydrates such as
and correlation loadings generated using the OPLD-DA model (Fig. 4B). glucose, fructose, galactose and mannitol were reduced in the con-
Compounds contributing most for the separation between the un- centrations observed after the fermentation and other nutrient com-
fermented BSG samples and the Day-3 fermented BSG samples ac- ponents such as, citric acid, alanine, L-tyrosine, L-isoleucine, pan-
cording to the S plot are summarized in Table 3. tothenic acid, 3-hydroxyanthranilic acid, myo-inositol and L-norvaline
According to the results, the most significant change in terms of were increased in the fermented biomass.
reported abundances were seen in citric acid, L-Norvaline and the car-
bohydrates. The intensity variation of the above-mentioned metabolites
3.4. Metabolic response for BSG fermentation
during the fermentation can be visualization as illustrated in Fig. 5A.
The generated heat map depicts metabolites accountable for dis-
Decreased levels of D-glucose, D-galactose and D-fructose in the fer-
crimination patterns observed (Fig. 5B). Details about the time point of
mented BSG indicates the possible utilization of these carbohydrates in
the changes taken place during the fermentation can also be understood
glycolysis. In carbohydrate metabolism D-glucose is initially converted
using the heat map. According to the heat maps, the available carbo-
to D-glucose-6-phosphate (D-glucose-6P). D-glucose-6P can either be
hydrate levels have started dropping from fermentation day 1 and the
converted to D-fructose-6P or enter pentose phosphate pathway (PPP) to
other interesting metabolites have commenced accumulating from
proceed with glycolysis. D-fructose is converted to D-fructose-6P and
enters glycolysis. D-galactose is converted to D-glucose-6P (Leloir
Table 3
pathway) and follows the above mentioned route (Khosravi et al., 2015,
List of tentatively identified metabolites with significant changes when the fermentation chap. 1). Therefore, according to the observed results, glycolysis can be
progressed through the three days. The intensity changes were calculated based on the considered a dominant catabolism occurring during fungi growth.
unfermented BSG samples against the fermented samples. One-way ANOVA followed by Mannitol is reported to function as a reserve for carbohydrate and a
Turkey’s post hoc test was performed to find the significantly changing metabolites (*
protective agent against osmotic and oxidative stress for fungi in
p < .05, ** p < .01, *** p < .001). Intensity change represented as fold change; abun-
dance (Day3): abundance (Day0). coenzyme regulation, formation of spores and storing reducing power
(Patel & Williamson, 2016). According to our study reducing levels in
Annotated compound Class Fold change in Regulation mannitol were seen. Thus, it could be that the fungi grown in our study
name intensity has utilized mannitol as a carbon source as well.
Citric acid Tricarboxylic acids 36.51 ↑*** Pyruvate, a key intermediate in glycolysis, is useful in the bio-
Alanine Amino acid 2.233 ↑*** synthesis of amino acids such as tyrosine, tryptophan and dihydrox-
L-Isoleucine 1.590 ↑** yphenylalanine. Complex B vitamins, such as thiamine, pyridoxine
L-Tyrosine 1.190 ↑** hydrochloride, calcium D-pantothenate and nicotinic acid, constituents
3-Hydroxyanthranilic acid Aminophenol 5.0192 ↑***
of pyruvate dehydrogenase coenzyme help to transform pyruvate to
L-Norvaline Amino fatty acids 56.439 ↑***
Myo-inositol Carbohydrates 6.8123 ↑*** acetyl-CoA. Acetyl-CoA may then enter the citric acid (TCA) cycle to
Pantothenic acid Vitamin 1.8389 ↑*** perform cell growth and metabolism (Wang, He, Lu, Shen, & Jiang,
Glucose Carbohydrates 0.0068 ↓*** 2002). As indicated in our study, the reporting increased levels of
D-Fructose 0.0104 ↓*** pantothenic acid, could be acting as a precursor for CoA production.
D-Galactose 0.0036 ↓*
Our results indicate a clear increase in the levels of citric acid after
D-(+)-Talose 0.1051 ↓***
D-Mannitol 0.0242 ↓* 3-days of fermentation. Filamentous fungi are known producers of or-
ganic acids such as citric acid (Currie, 1917; Max et al., 2010). Glucose

90
S.T. Cooray, W.N. Chen Journal of Functional Foods 42 (2018) 85–94

Fig. 5. (A) Fold change of the shortlisted

metabolites of interest (B) Heatmap analysis
correlating from Day 0 (left) to Day 3 (right)
as the fermentation progress in BSG.
Metabolites shaded in red are up regulated
while those in blue are down regulated. Key:
metabolites captured in Day zero (red), Day
1 (green), Day 2 (dark blue) and Day 3 (light
blue). (For interpretation of the references
to colour in this figure legend, the reader is
referred to the web version of this article.)

is catabolized by glycolysis to produce pyruvate in the cytosol. A Kubicek, 2003; Wakai, Arazoe, Ogino, & Kondo, 2017). Out of the
fraction of this pyruvate is converted to citric acid by citrate synthesis worldwide citric acid production of 1.6 million tones in 2007 (Berovic
in mitochondria. The other fraction is converted to oxaloacetate, which & Legisa, 2007), 70% is used in food and beverage industry (as an
is then converted to malate and finally to citrate (TCA cycle) (Karaffa & acidifier or antioxidant to preserve and enhance the flavour and

91
S.T. Cooray, W.N. Chen
aromas) and 20% in pharmaceuticals (as antioxidant) (Max et al.,
2010). Its global production is still reported to be increasing (Berovic &
Legisa, 2007).
The metabolic analysis shows a significant increase in alanine and
the amino acid analysis performed a clear increase in many other es-
sential and non-essential amino acids (Table 2). Amino acids act as
building blocks of proteins and they are important in regulating key
metabolic pathways (D'Este, Alvarado-Morales, & Angelidaki, 2017).
The increase in amino acid levels indicates the potential use of fer-
mented BSG as a food resource with enhanced nutrition. From the
global amino acid market of US$7 billion, 56% comprise of animal feed
supplement segments (Sanchez & Demain, 2014). Thus, the fermented
BSG shows a potential supplication in such sector.
3-Hydroxyanthranilic acid and myo-inositol are two more inter-
esting compounds reported in our fermentation. 3-Hydroxyanthranilic
acid is a product of tryptophan catabolism in the nicotinamide
pathway. This compound is an antioxidant, which acts as anti-in-
flammatory and neuro-protective (Perez-Gonzalez, Alvarez-Idaboy, &
Galano, 2017). A study reports that 3-hydroxyanthranilic acid is the key
compound responsible for the antioxidant activity in tempeh (Esaki,
Onozaki, Kawakishi, & Osawa, 1996). This antioxidant has been ef-
fective for preventing autoxidation in soybean oil and soybean powder.
Myo-inositol, which has shown a noteworthy level of production in the
fermented BSG is biosynthesised from glucose-6P, derivative of glucose.
Myo-inositol is regarded as an important compound in terms of pro-
moting female fertility and treating polycystic ovary syndrome (PCOS)
(Sortino, Salomone, Carruba, & Drago, 2017). Therefore, fermented
BSG can be regarded as a potential food ingredient with high levels of
functional activities.
Similar to many metabolomics studies only some of the peaks ex-
tracted were putatively annotated to a metabolite with a good score,
while many could not be identified. Annotations were done at level 2 in
the requirements by the Metabolomics Standards Initiative (MSI)
(Sumner et al., 2007). Metabolomics has been found useful in com-
prehensive understanding and quantitation of fermentation effect in
terms of fermentative capacity of microorganisms and benefits of pre-
biotics and functional food (Mozzi, Ortiz, Bleckwedel, De Vuyst, &
Pescuma, 2013). Our study is an extension of metabolomics analysis to
explore interesting compounds generated by fungi fermentation on
BSG.
Rhizopus oligosporus is considered as a generally regarded as safe
(GRAS) microorganism, and used in the production of the Indonesian
traditional food, tempeh. Rhizopus species have been known to produce
amylase, galactosidase and glucosidase during fermentation, which
helps to digest oligosaccharides and starch (Egounlety & Aworh, 2003).
Many studies have looked into how R. oligosporus can be used in pro-
ducing lipases to be used in food and biotechnology related applications
(Iftikhar, Niaz, Afzal, Ikram-ul-Haq, & Rajoka, 2008; Iftikhar et al.,
2010; Mahapatra, Kumari, Kumar Garlapati, Banerjee, & Nag, 2009).
Fermentation of these fungi has the potent to increase the phenolic
content, isoflavone aglycone content and antioxidant activity (Chang
et al., 2009; Cheng, Wu, Lin, & Liu, 2013; Starzyńska-Janiszewska,
Stodolak, & Jamróz, 2008). Moreover, R. oligosporus has been in-
vestigated in producing protein rich fungal biomass production for
aquaculture feed applications (Nitayavardhana & Khanal, 2010). Due to
the ability of R. oligosporus to convert the original substrate to a protein
rich material and to produce other interesting nutritional ingredients
we selected this filamentous fungi for the BSG fermentation. And as
expected conferring previous studies R. oligosporus was able to enhance
functional properties of our substrate according to the obtained meta-
bolomics results.
4. Conclusions
This study provides evidence of valorization in BSG, an under-
utilized waste material, using fermentation techniques with R.
92
Journal of Functional Foods 42 (2018) 85–94
oligosporus. We observed increased levels of amino acids, citric acid,
vitamin and antioxidant level in fermented BSG when compared with
unfermented BSG, while reducing levels of carbohydrates, fats and
dietary fibers. According to the results, fermentation seems to enhance
the nutritional value of BGS and its potential to be used in food and
nutrition related applications as a value added source. The data pro-
vided insight on the changes in metabolites observed during fermen-
tation, proving that fermentation enhanced BSG’s nutrient content. In
conclusion, our study reveals the importance of a metabolomics study
to provide information to shed light on valorization in fermentation.
Furthermore, data from metabolomics studies can be used to find novel
applications for food wastes and innovative processing methods for
valorization of waste materials to be used for other food applications.
Acknowledgements
The authors would like to thank the Nanyang Environment and
Water Research Institute (NEWRI), Singapore and the Interdisciplinary
Graduate School (IGS), Nanyang Technological University, Singapore
for the award of research scholarship to Sachindra T. Cooray and the
support for this research.
Author contribution
Sachindra T. Cooray conceived, designed and performed the ex-
periments; Sachindra T. Cooray analysed the data; Sachindra T. Cooray
and Wei Ning Chen reviewed and wrote the paper. All authors have
read and approved the final version of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
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