Hur et al.

AMB Expr (2018) 8:143

https://doi.org/10.1186/s13568-018-0675-3

ORIGINAL ARTICLE Open Access

Characterization of quinoa (Chenopodium

quinoa) fermented by Rhizopus oligosporus
and its bioactive properties
Jaewon Hur1, Thi Thanh Hanh Nguyen2, Namhyeon Park2, Jeesoo Kim1 and Doman Kim1,2,3*

Abstract 
Quinoa is a pseudocereal that contains high quality protein, minerals, vitamins, polyphenols, and phytosterols. In this
study, quinoa was fermented by Rhizopus oligosporus (R. oligosporus) up to 5 days and the functional compounds
(l-carnitine, GABA, vanillic acid and gallic acid) were analyzed by LC/MS. The amounts of l-carnitine and GABA were
0.13 mg/kg and 540 mg/kg for nonfermented quinoa (NF), 3.15 mg/kg and 1040 mg/kg for fermented quinoa at
3 days (3F), and 1.54 mg/kg and 810 mg/kg for fermented quinoa at 5 days (5F). The vanillic acid and gallic acid were
1.3 and 0.1 mg/kg for NF, 1.55 and 2.37 mg/kg for 3F, and 1.83 and 0.84 mg/kg for 5F, respectively. Total phenolic con-
tents and total flavonoids contents were 41 mg gallic acid (GAE)/kg and 13 mg quercetin equivalent (QE)/kg for NF,
74 mg GAE/kg and 16 mg QE/kg for 3F, and 80 mg GAE/kg and 19 mg QE/kg for 5F, respectively. Antioxidant activity
­(SC50) was 3.6 mg/mL for NF, 3.4 mg/mL for 3F, and 2.3 mg/mL for 5F. Nitric oxide production on RAW264.7 mac-
rophages of fermented quinoa revealed 29% and 56% inhibition of nitric oxide production for NF and 5F, respectively.
Therefore, fermented quinoa can be used as a healthy and valuable food product.
Keywords:  Anti-inflammatory activity, Antioxidant activity, Fermentation, l-Carnitine, Quinoa, Rhizopus oligosporus

Introduction 0.8  mg/kg avocado) (Steiber et  al. 2004). In addition,

Quinoa (Chenopodium quinoa) has been widely cul- l-carnitine is an antioxidant compound that can prevent
tivated in South America for 5000–7000  years (Vega- oxidative stress and regulates cellular respiration by nitric
Gálvez et  al. 2010). It has high nutritional properties oxide (Brown 1999). As an antioxidant role of l-carnitine,
and contents of antioxidant compounds; essential amino mainly three enzymes (glutathione peroxidase, catalase,
acids, vitamins, minerals, and unsaturated fatty acids and superoxide dismutase) are prevented from peroxida-
(Carciochi et  al. 2016). Lysine and methionine con- tive damage (Kalaiselvi and Panneerselvam 1998).
tents are high in quinoa, deficient in many legumes and Rhizopus oligosporus (R. oligosporus) is a dominant fun-
rice, and also can be a precursor of l-carnitine synthe- gus in special fermented soybean products like Indonesia
sis (Koeth et  al. 2013). l-carnitine is synthesized from teampeh. During the fermentation by R. oligosporus, the
lysine and methionine, serving an essential function in macromolecules were hydrolyzed by enzymes and cou-
transportation of fatty acids into the mitochondrial com- pled with the metabolism of the corresponding hydrolytic
partment for β-oxidation and subsequent energy pro- products to changes in the biochemical composition of
duction (Bremer 1983). l-carnitine is abundant in red food substrates (deReu et al. 1997; Handoyo and Morita
meat (5977  mg/kg beef ) but in lesser amounts in crops 2006). Fermented soybean with R. oligosporus showed
(0.4 mg/kg wheat seed), and plants (2.9 mg/kg tomatoes, increase amounts of γ-aminobutyric acid (GABA), one
of main fermented products from synthesizing from glu-
tamate by glutamate decarboxylase (Dhakal et  al. 2012;
*Correspondence: kimdm@snu.ac.kr Handoyo and Morita 2006). In our previous study, R. oli-
1
Graduate School of International Agricultural Technology, Seoul gosporus was used to produce l-carnitine and GABA in
National University, Pyeongchang 25354, Republic of Korea
Full list of author information is available at the end of the article buckwheat by fermentation with no additional nutrients,

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Hur et al. AMB Expr (2018) 8:143 Page 2 of 8

and feed for chickens to gain high l-carnitine content in Whole grain images were taken at magnification of ×100,
eggs (Park et al. 2016). The quinoa fermentation by using 5.0  kV of accelerate voltage in secondary electron (SE)
R. oligosporus have been reported (Matsuo 2005, 2006). mode. At magnification ×1.0 k, fungi hypha was removed
However, these studies only focus on antioxidant activ- and observed at 15 kV of accelerate voltage in SE mode.
ity of fermented quinoa. And there was no report for the
improvement of l-carnitine and GABA in fermented qui- Analyses of l‑carnitine and GABA
noa as well as its biological and biochemical studies. In LC/MS analysis was conducted by using the method as
this regard, we fermented quinoa with R. oligosporus DK1 our previously described (Park et al. 2016). Samples were
to improve the quality of the food regarding the enhance- dissolved in water for analysis of l-carnitine or GABA.
ment of l-carnitine, GABA, phenolic acid compounds, Samples were diluted with acetonitrile for l-carnitine
antioxidant and anti-inflammatory effects. White quinoa and GABA, and all filtered using a 0.2  µm membrane
was selected for this study because it contains more anti- (Sartorius AG, Gottingen, Germany). A 1  µL sample
oxidative compounds than those of red and black quinoa was injected into the LC/MS system (Waters, Milford,
(Masayo and Watanabe 2010). MA, USA); Waters Acquity H-Class system with Waters
QDa detector, Waters Acquity UPLC BEH HILIC 1.7 µm,
Materials and methods 2.1 mm × 100 mm column. Solvent A was 15 mM ammo-
Microbial strain and culture condition nium formate with 0.1% formic acid in distilled water
Rhizopus microspores var. oligosporus was obtained from and solvent B was 0.1% formic acid in acetonitrile. The
our previous study (Park et  al. 2016) and deposited as temperature of column was maintained at 40 °C. The fol-
KCCM 11948P (Korean Culture Center of Microorgan- lowing elution gradient was applied for l-carnitine and
isms, Seoul, Korea). It was maintained on Potato Dex- GABA analyses; 0–3 min, 10% A; 3.1–5 min, 10–30% A;
trose Agar (PDA, Difco, USA) plates. 5.1–6 min, 30–60% A; then a 4 min for equilibrium step.
Electrospray ionization (ESI) was conducted with a posi-
Preparation of fermented quinoa tive with selective ion recording (SIR) (m/z 162 for l-car-
Rhizopus oligosporus was cultured on PDA medium nitine and m/z 104 for GABA). Capillary energies were
at 30  °C for 3  days to prepare spores (Park et  al. 2016). 1.5 kV. Cone voltage was 10 V for l-carnitine and 5 V for
White quinoa was purchased from KtFood (Seoul, GABA. Acetonitrile (90%, v/v) was used as a blank. Cali-
Korea). 150  g quinoa was soaked in 150  mL water for bration curves were prepared using the external stand-
12 h and steamed for 20 min at 121 °C. Fermentation was ard method with l-carnitine concentrations ranged from
conducted by inoculating 1 × 104  spores/g steamed qui- 0.01 to 1  µg/mL, GABA ranged from 0.1 to 10  µg/mL.
noa at 30 °C for 3–5 days. Fermented quinoa was lyophi- Linearity between concentrations of standards vs area
lized at − 10 to 0  °C under 20  Pa (Tokyo Rikakikai Co., was evaluated (r2 > 0.99).
Tokyo, Japan) for further study.
Analyses of phenolic acids
Sample extraction LC/MS analysis was conducted by using the method as
300 g lyophilized quinoa was extracted with 1 L of etha- previously described (Park et  al. 2016). Samples were
nol for 1 h at 28 °C in the shaking incubator and repeated dissolved in DMSO for vanillic acid, chlorogenic acid,
seven times, then filtered using filter paper (8 micron, or gallic acid as 10  mg/mL. Samples were diluted with
11  cm) (Whatman LTD., Maidstone, England). Ethanol methanol for phenolic acids, and all filtered using a
in the sample was removed by evaporation (Heidolph 0.2  µm membrane. A 1  µL sample was injected into the
Instruments, Schwabach, Germany) with addition of LC/MS system. Solvent A was distilled with water and
400  mL distilled water. The lipid layers from extracted solvent B was acetonitrile with 1  mL formic acid/L.
sample were removed and the extracted sample were lyo- The temperature of column was maintained at 40  °C.
philized for further study. Extraction yield was calculated The following elution gradient was applied for phenolic
as follows; acids analyses; 0–0.5 min, 95% A; 0.5–3 min, 95–70% A;
3–5 min, 70–0% A; 5–6 min, 0% A; then a 4 min for equi-
extract mass (g)
Yield (g/100 g)% = × 100 (%) librium step. ESI was conducted with a negative with SIR
quinoa mass (300 g) (m/z 169 for gallic acid, m/z 353 for chlorogenic acid, and
m/z 167 for vanillic acid). Capillary energy was 0.8 kV for
Scanning electron microscopy image analyses
phenolic acids. Cone voltage was 10 V for phenolic acids
Quinoa surfaces of sample prepared before and after
and methanol was used as a blank. Calibration curves
fermentation were observed using a scanning electron
were prepared using the external standard method with
microscope (SEM, TM 3030plus, Hitachi, Tokyo, Japan).
phenolic acids concentration ranged from 0.1 to 10  µg/
Hur et al. AMB Expr (2018) 8:143 Page 3 of 8

mL. Linearity between concentrations of standards vs were conducted in duplicate. Results were expressed as
area was evaluated (r2 > 0.99). mean ± standard error (SEM).

Cell cytotoxicity tests

Total phenolic contents analysis (TPC) RAW264.7 mouse macrophage cell line was purchased
The total phenolic contents were determined by Folin from Korean Cell Line Bank (Seoul, Korea) and cul-
ciocalteu’s method (Masayo and Watanabe 2010) with tured in Dulbecco’s modified Eagle’s medium (DMEM,
gallic acid as the standard (Sigma). 10 mg quinoa or fer- Gendepot, USA) supplemented with 10% (v/v) fetal
mented quinoa extracted powder was dissolved in 1 mL bovine serum (FBS, Gendepot, USA), 100  U/mL peni-
water. Each 120 µL of sample or gallic acid (0–50 µg/mL) cillin and 100  µg/mL streptomycin (Invitrogen, USA) at
was added into 96 wells plate and 15 µL of Folin ciocal- 37 °C in 5% ­CO2 (Choi et al. 2018b; Maxwell et al. 2017).
teu’s reagent (Sigma) was mixed together for 3  min in RAW264.7 macrophage cell was seeded on 96 wells
dark condition. Then, 15  µL of 10% (w/v) N ­ a2CO3 was plate at 2 × 104  cells/well and cultured for 48  h. Cells
added and reacted for 30 min in dark condition. The TPC were rinsed with phosphate-buffered saline (PBS) and
were determined by spectrophotometry at 750 nm (Spec- then treated with quinoa or fermented quinoa extract in
traMax M3, Molecular Devices, USA) and presented as DMEM medium without Fetal bovine serum (FBS) rang-
gallic acid equivalent (GAE). ing from 1.56 to 1600 µg/mL obtained by diluting quinoa
or fermented quinoa extract with the culture medium.
RAW 246.7 cells cultured in a medium without adding
Total flavonoid contents (TFC) analysis
samples were used as controls. After 24 h at 37 °C, 90 µL
TFC was determined by aluminum chloride colorimetric
of medium was mixed with 10  µL of Ez-CyTox solution
method (Chang et al. 2002) with quercetin as the stand-
(Daeil Lab Service, Seoul, Korea) and then incubated at
ard (Sigma). 20 mg quinoa or fermented quinoa extracted
37 °C for 1 h. Absorbance was measured at 450 nm using
powder was dissolved in 1  mL DMSO, and then five-
SpectraMax M3. Percent viability was calculated as cell
folds diluted in methanol to give final concentration as
viability relative to the control.
1 mg/mL and volume of 2 mL. Each sample or quercetin
(0–15 µg/mL) was mixed with 100 μL of 10% (w/v) A ­ lCl3
and 100 μL of 0.1 mM C ­ H3CO2K. TFC was determined
Measurement of nitric oxide production
by spectrophotometry at 415  nm using SpectraMax M3
Nitric oxide production was determined as previously
and presented as quercetin equivalent (QE).
described method (Kim et  al. 1999). RAW 264.7 cells
were seeded to 96 wells plate at 2 × 104 cells/well and cul-
Determination of DPPH radical scavenging activity tured at 37 °C for 48 h. The sample treated with 1 µg LPS/
Antioxidant activities of quinoa or fermented quinoa mL and 100 µM indomethacin was used as positive con-
were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) trol. Cells were then treated with quinoa or fermented
radical scavenging method (Nguyen et al. 2015). Quinoa quinoa extract in DMSO ranging from 12.5 to 50 µg/mL
extracted powder were diluted in 70% ethanol and centri- without effects on cytotoxicity under testing, and cul-
fuged at 13,572×g for 10 min. Supernatants were reacted tured at 37 °C for 24 h. Then, 80 μL of culture superna-
with 100  μM DPPH (Sigma) in ethanol solution to give tant was mixed with 80  µL of Griess reagent containing
a final concentration of 0.2–7  mg quinoa extract/mL, 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid, and
then, kept at room temperature for 30  min in darkness. 0.1% (w/v) naphthylethylenediamine, for 20  min, and
Absorbance of each sample was measured at 517 nm on absorbance was measured at 540  nm using SpectraMax
a microplate reader, SpectraMax M3. DPPH radical-scav- M3. The amount of nitrite in the sample was evaluated
enging activity was converted into percentage of anti- from a standard curve generated with a sodium nitrite
oxidant activity using the following equation (Choi et al. standard curve (0–500 µM in cell culture medium).
2018a):
DPPH radcal − scavenging activity (%)
Statistical analysis
=
(Absorbance of control − Absorbance of test sample) Experimental results were statistically analyzed by t-test
Absorbance of control (IBM SPSS Statistics 22, IBM, USA). Values are presented
× 100 as mean ± standard error of the mean (SEM). Signifi-
A linear regression curve was established to deter- cant differences between the groups were evaluated and
mine the amount of sample necessary to decrease 50% indicated by different lower-case letters (p < 0.05 and
of the absorbance of DPPH (­SC50 value). All analyses p < 0.01).
Hur et al. AMB Expr (2018) 8:143 Page 4 of 8

Results Table 1  l-carnitine, GABA and  phenolic acids in  regular

Scanning electron microscopic observation and fermented quinoa
The general quinoa and degradation of quinoa after fer- Group NF 3F 5F
mentation were observed by the SEM. Figure  1 shows
l-carnitine (mg/kg of extracts) 0.13 3.15 ± 0.06** 1.54 ± 0.06**
the surface of general and fermented quinoa. General
quinoa (Fig. 1a) was observed with smooth surface. Fer- GABA (mg/kg of extracts) 540 ± 3 1040 ± 10** 810 ± 3**
mented quinoa (Fig. 1b, c) was covered by R. oligosporus Phenolic acids
and mycelium was observed on fermented quinoa. The  Vanillic acid (mg/kg of 1.3 ± 0.04 1.55 ± 0.06** 1.83 ± 0.06**
extracts)
washed off fermented quinoa (Fig. 1e, f ) revealed surface
 Gallic acid (mg/kg of extracts) 0.01 2.37 ± 0.08** 0.84 ± 0.02**
degradation comparable to the general quinoa (Fig.  1d),
 Chlorogenic acid (mg/kg of 0.002 0.03 0.002
and exposed starch granule. extracts)
(NF non-fermented, 3F 3 days, 5F 5 days of fermented quinoa extracts); (NF vs 3F
Ethanol extraction of fermented quinoa and 5F, **p < 0.01)
The extraction yields of NF, 3F, and 5F were 23.4%, 45.9%,
and 39.1%, respectively. Among them, 3  days fermented
810 mg/kg of quinoa extracts at 3F and 5F, respectively
quinoa showed highest extraction yield.
(Table 1). Concentration of vanillic acids was increased
during fermentation as 1.3, 1.55, and 1.83 mg/kg in NF,
Analyses of l‑carnitine, GABA, and phenolic acids
3F, and 5F extracts, respectively (Table  1). Concentra-
The l-carnitine content was enhanced from 0.13 mg/kg
tion of gallic acid was 0.01, 2.37, and 0.84 mg/kg in NF,
to 3.15 and 1.54 mg/kg of quinoa extracts at 3F and 5F,
3F, and 5F extracts, respectively (Table 1). Chlorogenic
respectively (Table  1). GABA was produced by the R.
acid was found 0.002 mg/kg for NF and 5F, but 0.03 mg/
oligosporus fermentation, from 540 mg/kg to 1040 and
kg was detected at 3F extract (Table 1).

Fig. 1  Scanning electron microscope of fermented quinoa. a NF, ×100; b 3F, ×100; c 5F, ×100; d NF, ×1000; e hypha removed 3F, ×1000; f hypha
removed 5F, ×1000. (NF Non-fermented, 3F; 3 days, 5F; 5 days of fermented quinoa extracts)
Hur et al. AMB Expr (2018) 8:143 Page 5 of 8

Total phenol content, total flavonoid content,

and antioxidant activity of quinoa
Antioxidant activity was mainly investigated based on
analysis of TPC, TFC, or DPPH radical-scavenging
activity of each sample. After fermentation, TPC was
increased from 41  mg GAE/kg to 74 and 80  mg GAE/
kg of quinoa extracts at 3F and 5F, respectively (Table 2).
TFC was increased from 13 mg QE/kg to 16 and 19 mg
QE/kg of quinoa extract at 3F and 5F, respectively
(Table  2). Antioxidant activity ­(SC50) of quinoa extracts
prepared with NF, 3F, and 5F were 3.6, 3.4, and 2.3 mg/
mL, respectively (Table 2, Additional file 1: Figure S1).

Cell viability of RAW264.7 cells

Cell viabilities of RAW 264.7, macrophages cells, are
shown in Fig.  2a. Cell viabilities were reached at 100%
at the concentration of 100  µg/mL so that the nitric
oxide assay was conducted from 50, 25, and 12.5 µg/mL
(Fig. 2a).

Nitric oxide production

Production of nitric oxide was investigated by lipopoly-
saccharides (LPS) stimulation. At the concentration of
50  μg/mL, the 5F extracts had significantly high anti-
inflammatory activities. Production of nitric oxide
was decreased 22.8, 19,7 and 14.0 µM in NF, 3F, and 5F
extracts, respectively (Fig.  2b). These patterns were also
shown at 25  μg/mL as 27.4, 24.5 and 23.6  µM of nitric Fig. 2  Cell viability (a) and nitric oxide production (b) on RAW264.7
oxide production in NF, 3F, and 5F extracts, respectively. of quinoa and fermented quinoa extract. (NF;Non-fermented, 3F;
As for 12.5  μg/mL, the nitric oxide was produced 30.8, 3 days, 5F; 5 days of fermented quinoa extracts) (NF vs 3F and 5F,
28.0 and 29.8 µM at NF, 3F, and 5F extracts, respectively. **p < 0.01)
Since nitric oxide production is generated from inflam-
mation, fermented quinoa had therapeutic abilities by
reducing nitric oxide production. functionalities, nutrition values and taste of quinoa,
we fermented quinoa grains using R. oligosporus. Mor-
Discussion phological characteristics by using the SEM reveals
Traditionally, quinoa has been cooked in salads, soups, degradation of quinoa by R. oligosporus enzymes after
porridges, and stews. Quinoa are found in forms of fermentation (Fig. 1a–c). Raw quinoa seeds had polyg-
flakes, grains, and flours and have increasingly become onal granules (0.6–2.0  µm diameter) present singly
incorporated into products such as noodles and energy and as spherical aggregates (Ruales and Nair 1993).
bars. Recent developments of quinoa flour in small- After fermentation, the polygonal granule was hardly
scale products include bread, muffins, pasta, snacks, observed and degraded into low-molecule-substances
drinks, flakes, baby foods, beer and extrudates (Diaz that brought different biological values to fermented
et  al. 2013; Matsuo 2006). In order to improve the

Table 2  Total phenolic and flavonoids contents and DPPH-radical scavenging activity of fermented quinoa extract
NF 3F 5F

Total phenolic contents (mg GAE/kg of quinoa extract) 41 ± 1 74** 80 ± 1**
Total flavonoid contents (mg QE/kg of quinoa extract) 13 16** 19**
DPPH radical-scavenging activity ­(SC50 (mg/mL) 3.6 3.4 ± 0.5 2.3 ± 0.1**
(NF non-fermented, 3F 3 days, 5F 5 days of fermented quinoa extracts); (NF vs 3F and 5F, **p < 0.01)
Hur et al. AMB Expr (2018) 8:143 Page 6 of 8

quinoa (Fig.  1b, c). Degradation of cell structures of xylanase when degrade the cell wall matrix (Huynh
fermented quinoa was due to lipases, amylases, pro- et al. 2014). Thus, it was probably metabolized with the
tease, and glucoamylase that produced from R. oli- bioconversion of phenolic compounds by the fermen-
gosporus (Handoyo and Morita 2006; Jin et  al. 1999). tation leads the cell-wall degrading enzymes to hydrol-
The 3F and 5F quinoa extracts had higher content of ysis of glycosidic bonds and produces bound phenolics
l-carnitine (24.1 and 11.8 times) than non-fermented and aglycone forms (Huynh et  al. 2014). Also, TFC
quinoa. Handoyo and Morita (2006) reported that R. (Table 2) were enhanced by 1.2 and 1.5 times increase
oligosporus hydrolyzed protein into amino acids and at 3F and 5F. The fermentation processes releasing
small peptides. In other study, Matsuo (2006) reported phenolic compounds from plant matrixes followed by
that the amount of lysine was increased from non- the metabolic pathways of flavonoids: glycosylation,
detection to 0.31  g/kg dry quinoa and lysine from deglycosylation, ring cleavage, methylation, glucuroni-
0.11 to 0.51 g/kg dry quinoa during R. oligosporus fer- dation, and sulfate conjunction which are way of pro-
mentation. l-carnitine is synthesized from lysine and ducing new bioactive compounds (Huynh et al. 2014).
methionine thus the synthesis of l-carnitine depends These increases in phenolic compounds effected to
on the amount of lysine and methionine in quinoa. The the enhancement of antioxidative activity (Kaur and
GABA contents in fermented quinoa showed 1.9 and Kapoor 2002). Increase of flavonoids contents has also
1.5 times higher than that of NF. GABA is synthesized influenced on DPPH-radical scavenging activity result-
from glutamate catalyzing by glutamate decarboxy- ing in higher antioxidant activity of fermented quinoa
lase while quinoa contained high glutamate (0.71 g/kg extracts than non-fermented ones. Since antioxidant
dry quinoa) (Matsuo 2006). Fermented quinoa extract properties of fermented quinoa extracts were found,
revealed 36.1% enhanced anti-oxidative activity in anti-inflammatory effect on mammalian cells was
DPPH-radical scavenging activity level. This result further investigated. Anti-inflammatory function of
was agreed with previous reported by Mastuo (2006). fermented quinoa extract was studied on RAW 264.7,
The enhancement of antioxidant activities probably macrophages, with LPS-stimulation. Nitric oxide pro-
resulted in the increased amounts of phenolic com- duction was inhibited 29.3% for NF, 38.9 for 3F and
pounds by the fermentation. Contents of phenolic 56.4% for 5F, resulting in improvement of 192.6% in
acids such as vanillic acids, gallic acids, chlorogenic anti-inflammatory activity (Fig.  2b). Kim et  al. (1999)
acids, and TPC, TFC were also changed and possibly reported that the flavonoids inhibit NO production
effected to improvement of DPPH-radical scavenging in lipopolysaccharide-activated RAW264.7 cells and
activity. Vanillic acid was known for main phenolic reduce of iNOS enzyme expression. Thus, the increase
acids in quinoa, and other phenolic acids were also anti-inflammatory effect of fermented quinoa com-
analyzed such as gallic acids and chlorogenic acids. pared to the regular quinoa due to increasing of total
Vanillic acids contents were increased by fermentation phenol and total flavonoids contents in fermented
as 1.3–1.83  mg/kg by NF and 5F, respectively. In case quinoa by R. oligosporus. Biscuits made by substitut-
of gallic acids were increased at 3F by 2.37 mg/kg from ing 20% of the flour with  R. oligosporus  fermented
0.01  mg/kg of NF, but decreased at 5F by 0.84  mg/ quinoa powder increased its iron and α-tocopherol
kg compared to 3F. As for chlorogenic acid, the pat- contents  by a factor  more than 2.5 and scored high
tern was similarly revealed as gallic acids for increased in sensory analysis of flavor hardness and taste in
amount at 3F by 0.03  mg/kg from 0.02  mg/kg of NF, soft biscuits  (Matsuo 2006). The absorption of iron
and decreased at 5F as 0.02 mg/kg from 3F. It is prob- from R. oligosporus fermented quinoa was higher than
able that phenolic acids are derivatives for other phe- that of quinoa powder in rats for partial digestion of
nolic acids (Rice-Evans et al. 1996). In this study, TPC phosphoric compounds (Matsuo 2006). Therefore, the
(Table  2) were enhanced (1.8 and 2.0 times increase increased concentration of l-carnitine and GABA in
at 3F and 5F) during the R. oligosporus fermentation fermented quinoa by R. oligosporus could be improved
and were probably obtained from formation of higher by the beneficial functionalities and nutritional values
contents of phenolic compounds such as vanillic acids, in fermented quinoa supplemented food materials.
gallic acids and chlorogenic acids. McCue and Shetty In conclusion, fermented quinoa extract has effec-
(2003) reported that α-amylase and endogenous tive antioxidant and anti-inflammatory activities. These
carbohydrate-cleaving enzymes activities had a role activities may be due to presence of phenolic com-
to generate polyphenols from carbohydrates-conju- pounds, flavonoids and the other products produced
gated phenolic compounds. R. oligosporus is a known during fermentation by R. oligosporus. Although the
strain to produce β-glucosidase, β-glucuronidase and 3rd  day fermentation revealed optimal conditions to
produce l-carnitine and GABA, the 5-day fermented
Hur et al. AMB Expr (2018) 8:143 Page 7 of 8

quinoa extract had higher TPC, TFC, antioxidant activ- Received: 19 June 2018 Accepted: 3 September 2018
ity and improved reduction in inflammation than regu-
lar quinoa extract. In this regard, fermented quinoa can
be used as a healthy and valuable food product.
References
Bremer J (1983) Carnitine–metabolism and functions. Physiol Rev
63:1420–1480
Brown GC (1999) Nitric oxide and mitochondrial respiration. Biochim Biophys
Acta 1411:351–369
Additional file Carciochi RA, Galván-D’Alessandro L, Vandendriessche P, Chollet S (2016) Effect
of germination and fermentation process on the antioxidant compounds
Additional file 1: Fig. S1. DPPH radical scavenging of nonfermented of quinoa seeds. Plant Foods Hum Nutr 71:361–367
quinoa (NF), 3-day fermented quinoa (3F), 5-day fermented quinoa (5F). Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total flavonoid
content in propolis by two complementary colorimetric methods. J Food
Drug Anal 10:178–182
Abbreviations Choi ES, Kang YY, Mok H (2018a) Evaluation of the enhanced antioxidant activ-
R. oligosporus: Rhizopus oligosporus; GABA: γ-aminobutyric acid; NF: nonfer- ity of curcumin within exosomes by Fluorescence monitoring. Biotechnol
mented quinoa; 3F: fermented quinoa at 3 days; 5F: fermented quinoa at Bioproc Eng 23:150–157
5 days; GAE: gallic acid equivalent; QE: quercetin equivalent; PDA: potato Choi MH, Jo HG, Kim MJ, Kang MJ, Shin HJ (2018b) Fruit juice supplementation
dextrose agar; SEM: scanning electron microscope; DMSO: dimethyl sulfoxide; alters human skin antioxidant levels in vivo: case study of Korean adults
LC/MS: liquid chromatography–mass spectrometry; TPC: total phenolic con- by resonance raman spectroscopy. Biotechnol Bioproc Eng 23:116–121
tent; TFC: total flavonoid content; DPPH: 2,2-diphenyl-1-picryhydrazyl; DMEM: deReu JC, Linssen VAJM, Rombouts FM, Nout MJR (1997) Consistency, polysac-
Dulbecco’s modified Eagle’s medium. charidase activities and non-starch polysaccharides content of soya
beans during tempe fermentation. J Sci Food Agric 73:357–363
Authors’ contributions Dhakal R, Bajpai VK, Baek K-H (2012) Production of GABA (γ-aminobutyric acid)
DK designed and coordinated the study. JH performed fermentation and mor- by microorganisms: a review. Braz J Microbiol 43:1230–1241
phological characterization. NP and JK performed preparation of extracts and Diaz JMR, Kirjoranta S, Tenitz S, Penttila PA, Serimaa R, Lampi AM, Jouppila
component analyses. TN carried out antioxidant assay and cell test. JH and TN K (2013) Use of amaranth, quinoa and kaniwa in extruded corn-based
drafted manuscript. All authors read and approved the final manuscript. snacks. J Cereal Sci 58:59–67
Handoyo T, Morita N (2006) Structural and functional properties of fermented
Author details soybean (Tempeh) by using Rhizopus oligosporus. Int J Food Prop
1 9:347–355
 Graduate School of International Agricultural Technology, Seoul National
University, Pyeongchang 25354, Republic of Korea. 2 Institutes of Food Indus- Huynh NT, Van Camp J, Smagghe G, Raes K (2014) Improved release and
trialization, Institutes of Green Bio Science & Biotechnology, Seoul National metabolism of flavonoids by steered fermentation processes: a review. Int
University, Pyeongchang 25354, Republic of Korea. 3 Center for Food and Bio- J Mol Sci 15:19369–19388
convergence, Seoul National University, Seoul 08826, Republic of Korea. Jin B, van Leeuwen HJ, Patel B, Doelle HW, Yu Q (1999) Production of fungal
protein and glucoamylase by Rhizopus oligosporus from starch processing
Acknowledgements wastewater. Process Biochem 34:59–65
Not applicable. Kalaiselvi T, Panneerselvam C (1998) Effect of l-carnitine on the status of lipid
peroxidation and antioxidants in aging rats. J Nutr Biochem 9:575–581
Competing interests Kaur C, Kapoor HC (2002) Anti-oxidant activity and total phenolic content of
The authors declare that they have no competing interests. some Asian vegetables. Int J Food Sci Tech 37:153–161
Kim HK, Cheon BS, Kim YH, Kim SY, Kim HP (1999) Effects of naturally occurring
Availability of data and materials flavonoids on nitric oxide production in the macrophage cell line RAW
All data generated or analyzed during this study are included in this manu- 264.7 and their structure–activity relationships. Biochem Pharmacol
script and Additional material. 58:759–765
Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li
Consent for publication L (2013) Intestinal microbiota metabolism of l-carnitine, a nutrient in red
Not applicable. meat, promotes atherosclerosis. Nat Med 19:576–585
Masayo A, Watanabe K (2010) Functional and bioactive properties of quinoa
Ethics approval and consent to participate and amaranth. Food Sci Technol Res 16:163–168
Not applicable. This paper does not contain any studies with human partici- Matsuo M (2005) In vivo antioxidant activity of methanol extract from quinoa
pants or animal performed by any of the authors. fermented with Rhizopus oligosporus. J Nutr Sci Vitaminol 51:449–452
Matsuo M (2006) Suitability of quinoa fermented with Rhizopus oligosporus as
Funding an ingredient of biscuit. J Jpn Soc Food Sci 53:62–69
This work was partially supported by the Basic Science Research Pro- Maxwell T, Lee KS, Chun SY, Nam KS (2017) Mineral-balanced deep sea
gram through the National Research Foundation of Korea (NRF) funded water enhances the inhibitory effects of chitosan oligosaccharide on
by the Ministry of Education (NRF-2015R1D1A1A01056929; D. Kim, and atopic dermatitis-like inflammatory response. Biotechnol Bioproc Eng
2018R1D1A1B07049569; T.T. Hanh Nguyen), by Korea Institute of Planning 22:120–128
and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through McCue P, Shetty K (2003) Role of carbohydrate-cleaving enzymes in phenolic
Agriculture, Food and Rural Affairs Research Center Support Program, funded antioxidant mobilization from whole soybean fermented with Rhizopus
by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (710012031HD220), oligosporus. Food Biotechnol 17:27–37
and under the framework of International Cooperation Program managed by Nguyen TTH, Yu S-H, Kim J, An E, Hwang K, Park J-S, Kim D (2015) Enhance-
the NRF (2016K1A3A1A19945059), and OTTOGI Corporation through Research ment of quercetin water solubility with steviol glucosides and the studies
and Publication Project. of biological properties. Funct Foods Health Dis 5:437–449
Park N, Lee TK, Nguyen TTH, An EB, Kim NM, You YH, Park TS, Kim D (2016) The
effect of fermented buckwheat on producing L-carnitine and Gamma-
Publisher’s Note aminobutyric acid (GABA) enriched designer eggs. J Sci Food Agric
Springer Nature remains neutral with regard to jurisdictional claims in pub- 97:2891–2897
lished maps and institutional affiliations.
Hur et al. AMB Expr (2018) 8:143 Page 8 of 8

Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity Steiber A, Kerner J, Hoppel CL (2004) Carnitine: a nutritional, biosynthetic, and
relationships of flavonoids and phenolic acids. Free Radic Biol Med functional perspective. Mol Aspects Med 25:455–473
20:933–956 Vega-Gálvez A, Miranda M, Vergara J, Uribe E, Puente L, Martínez EA (2010)
Ruales J, Nair BM (1993) Content of fat, vitamins and minerals in quinoa (Che- Nutrition facts and functional potential of quinoa (Chenopodium quinoa
nopodium quinoa, Willd) seeds. Food Chem 48:131–136 willd.), an ancient Andean grain: a review. J Sci Food Agric 90:2541–2547