Stress influenced increase in phenolic content and radical scavenging capacity of Rhodotorula glutinis CCY 20-2-26

Abstract

Rhodotorula glutinis CCY 20-2-26 when grown under controlled stress of either NaCl (1–5 %) or H2O2 (1–5 mM) on basal media exhibited a twofold increase in its total phenolic contents. The radical scavenging capacities (RSCs) as determined by ABTS test were found to be highest in 4 mM H2O2 (1.44 mM TEAC mg−1) and 4 % NaCl (1.13 mM TEAC mg−1) as compared to control samples (0.41 mM TEAC mg−1). Similarly, the RSCs as determined by DPPH test were also highest in 4 % NaCl (1.83 mM TEAC mg−1) and 4 mM H2O2 (1.78 mM TEAC mg−1) compared to control (0.48 TEAC mg−1). The relative RSCs from EPR spin-trapping assay for H2O2-stressed cultures were highest in 1 mM H2O2 (56.1 μM TEAC g−1) whereas in NaCl-stressed cultures it was highest in 5 % NaCl (44.6 μM TEAC g−1) as compared to control (30.9 μM TEAC g−1). Five phenolic compounds (gallic acid, benzoic acid, catechin, caffeic acid and ferulic acid) were detected for the first time in R. glutinis CCY 20-2-26.

Full text

ORIGINAL ARTICLE
Stress influenced increase in phenolic content and radical
scavenging capacity of Rhodotorula glutinis CCY 20-2-26
Raj Kumar Salar •Milan Certik •Vlasta Brezova •
Marta Brlejova •Vladimira Hanusova •
Emı
´lia Breierova
´
Received: 28 March 2012 / Accepted: 5 May 2012 / Published online: 30 May 2012
The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Rhodotorula glutinis CCY 20-2-26 when grown
under controlled stress of either NaCl (1–5 %) or H
2
O
2
(1–5 mM) on basal media exhibited a twofold increase in its
total phenolic contents. The radical scavenging capacities
(RSCs) as determined by ABTS test were found to be highest
in 4 mM H
2
O
2
(1.44 mM TEAC mg
-1
)and4%NaCl
(1.13 mM TEAC mg
-1
) as compared to control samples
(0.41 mM TEAC mg
-1
). Similarly, the RSCs as determined
by DPPH test were also highest in 4 % NaCl (1.83
mM TEAC mg
-1
)and4 mMH
2
O
2
(1.78 mM TEAC mg
-1
)
compared to control (0.48 TEAC mg
-1
). The relative RSCs
from EPR spin-trapping assay for H
2
O
2
-stressed cultures
were highest in 1 mM H
2
O
2
(56.1 lMTEACg
-1
)whereas
in NaCl-stressed cultures it was highest in 5 % NaCl
(44.6 lMTEACg
-1
) as compared to control (30.9 lM
TEAC g
-1
). Five phenolic compounds (gallic acid, benzoic
acid, catechin, caffeic acid and ferulic acid) were detected for
the first time in R. glutinis CCY 20-2-26.
Keywords Rhodotorula Phenolics Radical scavenging
capacity HPLC EPR/spin trapping
Abbreviations
ABTS 2,20-Azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt
DCW Dry cell weight
DMPO 5,5-Dimethyl-1-pyrroline N-oxide
DMSO Dimethylsulfoxide
DPPH 1,1-Diphenyl-2-picrylhydrazyl
EPR Electron paramagnetic resonance
FC Folin and Ciocalteu’s phenol reagent
GAE Gallic acid equivalent
RCorrelation coefficient
RRSC Relative radical scavenging capacity
RSC Radical scavenging capacity
SW Magnetic field sweep
TEAC Trolox equivalent antioxidant capacity
TPC Total phenolic content
UV–Vis Ultraviolet/visible
Introduction
Stress in biological tissues is known to bring about a bio-
chemical response involving an increase in the levels of
various antioxidant compounds or in the activity of
enzymes responsible for the regeneration of antioxidant
metabolites (Ramotar et al. 1998). Earlier, several studies
were carried out to show that yeast species produce
increased levels of carotenoids when they are grown under
unfavourable conditions (Certik and Breierova 2002;
Marova et al. 2004). Although, yeast is a non-photosyn-
thetic microorganism, there are yeasts that can biosynthe-
size carotenoids and phenolic compounds in the cell.
Carotenoids and phenolic compounds combat various types
R. K. Salar (&)
Department of Biotechnology, Chaudhary Devi
Lal University, Sirsa 125 055, India
e-mail: rajsalar@rediffmail.com
M. Certik V. Brezova M. Brlejova V. Hanusova
Faculty of Chemical and Food Technology,
Slovak University of Technology, Radlinskeho 9,
812 37 Bratislava, Slovak Republic
E. Breierova
´
Institute of Chemistry, Slovak Academy of Sciences,
Du
´bravska
´cesta 9, 845 38 Bratislava, Slovak Republic
123
3 Biotech (2013) 3:53–60
DOI 10.1007/s13205-012-0069-1

of cancer and other diseases because of their free radical
scavenging and/or provitamin A (carotene) potential
(Bhosale and Gadre 2001). Free radicals are known to be a
product of normal metabolism. These are also involved in
organism’s vital activities including phagocytosis, regula-
tion of cell proliferation, intracellular signalling and syn-
thesis of biologically active compounds (Halliwell 1989;
Miquel and Romano-Bosca 2004).
Phenolic compounds from plant kingdom are well doc-
umented for their antioxidant properties and health pro-
moting benefits. However, little attention has been paid to
the phenolic compounds evaluation in yeasts (Rizzo et al.
2006). In the recent past, both eukaryotic and aerobic
prokaryotic organisms have been developed with an overall
antioxidant defence system for mitigating the damaging
effects of free radicals (Kullisaar 2002; Daeschel 2004;
Jaehrig et al. 2008; Chen et al. 2010). Nevertheless, all
aerobic organisms including humans have antioxidant
defences that protect against oxidative damage and repair
damaged molecules. However, the natural antioxidant
mechanisms can be inadequate, the supply of antioxidants
through dietary ingredients is of great interest for the food
industry (Scalbert and Williamson 2000; Greenwald et al.
2001; Chen et al. 2010).
Although, the yeasts have received extensive concern on
using them as starter cultures for developmentof new products
(Wouters et al. 2002) and potential probiotics (Psomas et al.
2001,2003;Kumuraetal.2004), studies related to radical
scavenging capacities (RSCs) of yeasts are scanty (Rapta et al.
2005). In order to survive in adverse environment, microor-
ganisms have developed efficient adaptation mechanisms to
tide over undesirable stress by activated synthesis of bio-
molecules (Estruch 2000; Certik and Breierova 2002). Thus,
studies related to exogenous stress and scavenging property
could explain in part the mechanism of protection against
harmful effects of the environment.The purpose of the present
investigation was to determine total phenolic compounds of
Rhodotorula glutinis CCY 20-2-26 grown under controlled
stress of NaCl or H
2
O
2
including its intact cells and cell free
extracts. Radical scavenging capacity of its extracts modu-
lated by NaCl or H
2
O
2
was evaluated by EPR spin-trapping
technique, DPPH and ABTS assays which may provide evi-
dence for exploring novel products with antioxidant activity.
Further, phenolic compounds were identified using HPLC.
Materials and methods
Microorganism and culture conditions
Rhodotorula glutinis CCY 20-2-26 was obtained from
Culture Collection of Yeasts (CCY, Institute of Chemistry,
Slovak Academy of Sciences, Bratislava, Slovak Republic)
and maintained on agar slants at 4 C. It was cultivated on
basal medium consisting of (g L
-1
): yeast extract, 5;
glucose, 20; (NH
4
)
2
SO
4
, 10; KH
2
PO
4
,1;K
2
HPO
4
3H
2
O,
0.2; NaCl, 0.1; CaCl
2
, 0.1; MgSO
4
7H
2
O, 0.5; and 0.25 mL
of microelement solution [(mg L
-1
): H
3
BO
4
, 1.25;
CuSO
4
5H
2
O, 0.1; KI, 0.25; MnSO
4
5H
2
O, 1; FeCl
3
6H
2
O,
0.5; (NH
4
)
2
Mo
7
O
24
4H
2
O, 0.5; and ZnSO
4
7H
2
O, 1]. For
the present study, R. glutinis was grown under non-lethal
and maximally tolerated concentration of either NaCl
(1–5 %) or H
2
O
2
(1–5 mM). The inoculum (10 % v/v)
consisted of 48-h-old cells of R. glutinis grown in the above
basal media. The cultures were cultivated in 500 mL
Erlenmeyer flasks containing 150 mL of cultivation med-
ium on a rotary shaker (140 rpm) at 28 C to early sta-
tionary growth phase. Cells were harvested by
centrifugation at 3,000 rpm and washed thrice with dis-
tilled water and stored at -20 C until further analysis.
Extraction of phenolic compounds
The extraction of phenolic compounds was performed
directly on the microbial biomass. Dry yeast biomass
(DCW—dry cell weight) was prepared gravimetrically. For
preparing methanol extracts, 200 mg of DCW was sus-
pended in 20 mL of methanol in 100-mL conical flasks.
The samples were shaken for 10 min and then centrifuged
at 5,000 rpm for 10 min. The supernatants were collected.
The DMSO extracts were prepared using yeast biomass
with 1 mL DMSO (Merck, Germany) at 60 C for 60 min
in 2-mL Eppendorf tubes and then centrifuged at
5,000 rpm for 10 min. The extracts obtained were filtered
through membrane filters (0.22 lm). All extracts were
stored at -20 C until further analysis of total phenolic
content and RSC.
Total polyphenolic content determination
Total polyphenol contents were determined on the biomass
of harvested cells (herein after called ‘‘cells’’) and DMSO
and methanol extracts (herein after called ‘‘extracts’’) of
R. glutinis using Folin–Ciocalteu (FC) reagent following
Yu et al. (2004) and Commission Regulation (EEC) No.
2676/90 (1990) with slight modifications. Briefly, 10 mg of
yeast biomass was taken and suspended in 10 mL volu-
metric flask with 0.5 mL of distilled water. Then 0.5 mL of
FC reagent (Merck) and 1.5 mL of aqueous sodium car-
bonate anhydrous solution 20 % (w/v) was added. The
flask was filled with distilled water to volume and the
suspension was poured off into a centrifuge tube. After
120 min, the suspension was centrifuged at 4,000 rpm for
5 min. The absorbance was read at 765 nm, subtracting the
value of a control solution consisting of distilled water
instead of biomass. For determination of total polyphenol
54 3 Biotech (2013) 3:53–60
123

content in DMSO and methanol extracts, 100 lLof
extracts were taken instead of yeast biomass. The amount
of total polyphenol was calculated as gallic acid equiva-
lents (GAE) from the standard calibration curve of gallic
acid (Sigma-Aldrich) and expressed as milligram gallic
acid equivalents per gram of yeast. It should be noted that
no significant polyphenolic activity was observed in
methanol extracts and thus, only DMSO extracts were used
for analysis of RSC.
DPPH radical scavenging assay
The free RSC of different fractions was measured by the
DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging method
according to Yen and Chen (1995) with some modifica-
tions. Briefly, 200 lL of DMSO extract was taken in
spectrophotometric cell and then 3 mL of 100 lM DPPH
(Sigma-Aldrich) (4 mg DPPH in 100 mL methanol) was
added. In the reference sample, 200 ll of DMSO was used
instead of extracts. The changes in absorbance at 519 nm in
minute 10 relative to the reference sample were recorded
using a Implen NanoPhotometer 1890 (version, 7122
V2.0.0). The DPPH RSCs were expressed as trolox
equivalent antioxidant capacity (TEAC) in lmol g
-1
of
yeast biomass.
ABTS
•?
radical cation depolarization assay
Antioxidant activity was measured using a modified method
of Re et al. (1999) and Arts et al. (2004). 2,20-Azino-bis
(3-ethylbenzothiazoline-6-sulfonate) (ABTS, Sigma) was
used for production of the corresponding radical cation
(ABTS
•?
) by dissolving 17.2 mg ABTS and 3.3 mg
K
2
S
2
O
8
(Aldrich) in 5-mL distilled water, and the resulting
solution was left to stand for 16 h in dark at room temper-
ature. A stock solution of ABTS
•?
was prepared by mixing
1 mL of this reaction mixture with 60-mL water. The con-
centration of ABTS
•?
was determined by UV–Vis spec-
troscopy using the characteristic value of molar absorption
coefficient at 732 nm, 1.5910
4
mol
-1
Lcm
-1
. After
rigorously mixing 2.3-mL ABTS
•?
solution with 200 lLof
DMSO extracts, the UV–Vis spectra were taken in 0.5 s
intervals for 10 min using UV-3600 UV–Vis spectropho-
tometer (Shimadzu, Japan, 1-cm square quartz cell). UV-
Vis spectrum of initial ABTS
•?
solution measured against
distilled water was taken as a reference spectrum. The dif-
ference in the absorbance in 10th min at 732 nm relative to
reference spectrum was used to calculate the antioxidant
activity. The ability of samples to eliminate ABTS
•?
is
expressed using 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (trolox, Aldrich) as reference antioxidant
and the results were expressed as TEAC in lmol g
-1
of
yeast biomass (DCW).
EPR spin-trapping technique
The thermal decomposition of potassium persulfate
(K
2
S
2
O
8
) in DMSO at 333 K was used as a source of
reactive radicals. To measure the RSC of yeast extracts, the
EPR spin-trapping technique (Rapta et al. 2005) was used,
employing 5,5-dimethyl-1-pyrroline N-oxide (DMPO,
Sigma-Aldrich) as a spin trap. Sulfate radical anions
(SO
4
•-
) generated upon thermal decomposition of K
2
S
2
O
8
represent reactive species with high reduction potential,
capable to react with a variety of organic compounds
(Wardman 1989). In DMSO solvent these paramagnetic
species are added to the double bond of DMPO spin-
trapping agent producing the corresponding spin adducts
(Zalibera et al. 2009). All EPR measurements were carried
out in a 4-mm flat quartz cell in a Bruker TE
102
(ER 4102
ST) cavity using the EMX EPR spectrometer (Bruker,
Rheinstetten, Germany) working in the X-band. The ER
4111 VT temperature unit (Bruker, Germany) was used for
temperature regulation. The reaction mixture consisted of
100 lL of DMSO extracts (pure DMSO in reference),
100 lL DMSO, 25 lL of 0.2 M DMPO dissolved in
DMSO and 25 lL of 0.01 M K
2
S
2
O
8
(DMSO). A time
course of EPR spectra of the DMPO spin adducts was
recorded in 66-s intervals for 22 min at 333 K (each
spectrum represents an accumulation of three scans). The
integral EPR intensity (double integral) found after 22 min
of thermal treatment for the sample solution was compared
with the reference measurement. The difference between
the integral EPR intensities of the reference and the sam-
ples in 22nd min characterises the amount of radicals
scavenged by the various components present in the sample
acting as radical scavengers. The RSC values were calcu-
lated as a percentage of scavenged radicals relative to the
reference sample (DMSO). These values were recalculated
to TEAC using calibration curve measured analogously for
trolox solutions in K
2
S
2
O
8
/DMPO/DMSO systems, and so
obtained radical scavenging characteristics of investigated
samples were evaluated in lmol of trolox/1 g of extract.
HPLC analysis of phenolic compounds
Phenolic compounds were analysed by HPLC on an Agi-
lent 1100 series HPLC unit with computer-controlled
software and system controller. Mobile phase consisted of
acetonitrile (A) and water/acetic acid (pH 2.8) (B). Linear
gradient from 5 to 100 % Ain 35 min and flow rate
1 mL min
-1
was applied. An autoinjector was used to
inject 10 lL of extracts or standards into the HPLC system.
Absorbance data were recorded at 272 nm over a period of
35 min. Phenolic compounds’ identification was achieved
by the absorbance recorded in the chromatograms relative
to external standards. Standards used included gallic acid,
3 Biotech (2013) 3:53–60 55
123

benzoic acid, catechin, caffeic acid and ferulic acid. The
data were further quantified by ChemStation software B 01
03 (Agilent Technologies).
Results and discussion
Production of total phenolic compounds under different
culture conditions
In an earlier study conducted by Rapta et al. (2005)
increased free radical scavenging and antioxidant activities
of metabolites (carotenoids) produced by yeasts under
heavy metal stress was reported. However, it is important
to know whether only carotenoids are responsible for this
increase or some other metabolites, particularly phenolic
compounds are also involved. As phenolic compounds are
well known for their antioxidant activities, in the present
investigation total phenolic contents (TPC) of R. glutinis
were determined directly on the ‘‘cells’’ as well as from the
‘‘extracts’’. To the best of our knowledge, this is the first
report of occurrence of phenolic compounds in yeasts,
particularly in the investigated species.
The extracts of R. glutinis cells were prepared in DMSO
to observe the effect of stress factors on phenolic contents
and antioxidant activities. Under control culture conditions
with no medium supplementation with stress factors, TPC
produced by R. glutinis were 30.8 and 37.69 mg GAE g
-1
in ‘‘cells’’ and ‘‘extracts’’, respectively. A substantial
increase in TPC was observed when R. glutinis was grown
under non-lethal and maximally tolerated concentration of
either NaCl (1–5 %) or H
2
O
2
(1–5 mM) on basal media.
The phenolic contents as determined directly on the ‘‘cells’’
ranged from 38.22 to 62.72 mg GAE g
-1
in cultures stressed
with different molar concentrations of H
2
O
2
compared to
control (30.8 mg GAE g
-1
). However, it ranged from 18.92
to 40.45 mg GAE g
-1
in cultures stressed with different
concentrations of NaCl (Table 1). On the contrary, the phe-
nolic contents from ‘‘extracts’’ ranged from 34.94 to
47.49 mg GAE g
-1
in NaCl-stressed cultures whereas it
ranged from 29.04 to 50.15 mg GAE g
-1
in H
2
O
2
-stressed
cultures (Table 1). The highest levels of TPC were observed
in 4 mM H
2
O
2
(50.15 mg GAE g
-1
)and1%NaCl
(47.49 mg GAE g
-1
) -stressed cultures compared to control
(37.69 mg GAE g
-1
). The increase in phenolic contents in
R. glutinis reflects operation of some defence mechanism
under stress.
Radical scavenging capacities
The results of RSC estimations are strongly dependent on
the testing system. No single method can assure the com-
plete examination of antioxidant capacity in the samples
under investigation. Antioxidant activity may be more reliably
assessed by a combination of several tests and assays. In the
present study, RSC of extracts was determined using ABTS,
DPPH and EPR spin-trapping techniques and correlated with
TPC. While ABTS and DPPH tests were used to determine
total antioxidant activity, EPR spin-trapping technique was
applied to investigate the ability of various DMSO extracts to
scavenge the reactive radicals.
Total antioxidant activity measured by ABTS and DPPH
assays were evaluated for extracts obtained from stressed
R. glutinis and compared with the control. The antioxidant
activity established by ABTS test was found to be the
highest in culture extracts stressed with 4 mM H
2
O
2
(1.4 lM TEAC g
-1
) and 4 % NaCl (1.1 lM TEAC g
-1
)
as compared to control samples (0.4 lM TEAC g
-1
)
(Fig. 1a). Similarly, the total antioxidant capacity as
determined by DPPH test was also the highest in cultures
stressed with 4 % NaCl (1.8 lM TEAC g
-1
) and 4 mM
H
2
O
2
(1.8 lM TEAC g
-1
) compared to control
(0.5 lM TEAC g
-1
) (Fig. 1b). A significant correlation
was obtained between total phenolic content vs. ABTS
(R
2
=0.9237) and DPPH (R
2
=0.9586) considering the
values of 2–5 mM H
2
O
2
. Similarly, a good correlation (the
correlation coefficient R
2
=0.8784) was observed between
ABTS and DPPH tests. It provides strong evidence that the
predominant source of antioxidant activity derives proba-
bly from phenolic compounds in yeasts. However, no
correlation was observed between phenolic contents from
NaCl-stressed cultures and ABTS/DPPH tests. This
inconsistency might be due to a change in phenolic profile
during cultivation under osmotic stress, for example
carotenoids as reported previously (Edge et al. 1997;
Marova et al. 2004) might contribute considerably to the
antioxidant activity. As stated earlier, stress in biological
Table 1 Total phenolic content of cells and cell free DMSO extracts
of Rhodotorula glutinis grown under stress of NaCl or H
2
O
2
Stress factor Total phenolic contents (GAE mg g
-1
) DCW
Cells Cell free DMSO
extracts
Control 30.80 37.69
1 % NaCl 30.06 47.49
2 % NaCl 18.92 38.26
3 % NaCl 40.45 34.94
4 % NaCl 24.86 46.15
5 % NaCl 40.45 40.37
1mMH
2
O
2
62.72 44.29
2mMH
2
O
2
50.84 34.60
3mMH
2
O
2
38.22 33.65
4mMH
2
O
2
54.55 50.15
5mMH
2
O
2
43.42 29.04
56 3 Biotech (2013) 3:53–60
123

systems might induce the formation of new metabolites or
change the behaviour of organisms under stress. Our results
are in conformity with Rapta et al. (2005) who also reported an
increased level of total antioxidant capacity of R. glutinis
stressed with heavy metal ions Ni (II) and Zn (II).
The characteristic experimental EPR spectrum of
•
DMPO-
SO
4-
spin adduct recorded during the thermally initiated
decomposition of K
2
S
2
O
8
in DMSO at 333 K, along with its
simulation (a
N
=1.296 mT, a
H
b
=0.938 mT, a
H
c
=0.139
mT; g=2.0059) are shown in Fig. 2a. Figure 2brepresents
the original sets of 20 individual EPR spectra monitored in the
presence of DMPO during heating of K
2
S
2
O
8
at 333 K for the
reference sample DMSO (200 lL DMSO, 25 lL0.2M
DMPO in DMSO, 25 lL0.01MK
2
S
2
O
8
in DMSO) and for
DMSO extracts of R. glutinis grown under stress of either
NaCl or H
2
O
2
(200 ll extract in DMSO, instead of DMSO in
reference sample). It should be noted here that the decrease in
the directly monitored EPR signal intensity of spin adducts is
influenced by the concentration of individual extracts
(Fig. 2b). Figure 3shows a time dependence of EPR integral
intensities (evaluated by double integration of sets of 20
individual EPR spectra for each measurement, e.g. Fig. 2b)
representatively for the extracts of R. glutinis.TheEPRinte-
gral intensity after 22 min detected for the extracts was
compared to that of the reference. The difference between
these EPR intensities isproportional to the amount ofradicals
terminated by the scavengers present in the investigated
extract sample (Fig. 3), and is called relative radical scav-
enging capacity (RRSC, expressed in %). Finally, RRSC
values were recalculated to the TEAC values (molar amount
of trolox/1 g of dry extract inducing the identical changes in
RRSC) using calibration curve obtained under strictly iden-
tical conditions for trolox solutions in K
2
S
2
O
8
/DMPO/DMSO
systems.
The radical scavenging ability from EPR spin-trapping
assay of H
2
O
2
-stressed cultures ranged from 38.6 to
56.1 lmol trolox g
-1
with the highest in 1 mM H
2
O
2
(56.1 lmol TEAC g
-1
). Whereas in NaCl-stressed cul-
tures, it ranged from 35.8 to 44.6 lM TEAC g
-1
with the
highest in 5 % NaCl (44.6 lM TEAC g
-1
) as compared to
control samples (30.9 lM TEAC g
-1
) (Fig. 4). The EPR
spin-trapping and ABTS/DPPH spectrophotometric results
are not correlated. This may be explained due to operation
of different mechanism of action in the tests used, as most
probably distinct reaction pathways are involved in the
termination of a stable DPPH and ABTS
•?
radical species
and the reactive SO
4
•-
radical anion (Zalibera et al. 2009)
by active compounds present in the investigated samples.
In EPR experiments, we generate the SO
4
•-
with high redox
potential, which are capable to react with a variety of
organic compounds present in the extracts (e.g. glucan,
polysaccharides and carotenoids) other than phenolics.
Whereas in DPPH and ABTS assays, the dominating
compounds scavenging radical species are H-donor anti-
oxidants. However, in general it was observed that extracts
of cultures grown under stress showed higher radical
scavenging ability than unstressed cultures. Overall, there
was a significant increase in phenolic content and RSC of
R. glutinis when stressed with NaCl or H
2
O
2
. This is a first
report of occurrence of phenolic compounds in R. glutinis.
HPLC determination of phenolic compounds
The DMSO extracts of R. glutinis were evaluated using
HPLC. Preliminary studies with mobile phase of acetoni-
trile and acidified water with acetic acid (pH 2.8) were
conducted. The phenolic compounds were detected at
272 nm. All the samples reported positive for various
Fig. 1 Trolox equivalent antioxidant capacity of DMSO extracts of R. glutinis as determined by aABTS assay and bDPPH assay
3 Biotech (2013) 3:53–60 57
123

phenolic compounds viz., gallic acid, benzoic acid, cate-
chin, caffeic acid and ferulic acid in the experiential peaks
(Fig. 5). It was observed that there is a metabolic shift in
cultures when stressed with either NaCl or H
2
O
2
. Using
HPLC, Rizzo et al. (2006) determined phenolics adsorbed
on yeasts grown on different media. Phenolic compounds
produced by sclerotia of the fungus Inonotus obliquus have
a
Control
0 5 10 15 20
4 % NaCl
IEPR
IEPR
IEPR
Time [min]
2 mM H2O2
1 mM H2O2
0 5 10 15 20
Time [min]
4 mM H2O2
5 mM H2O2
DMSO
b
Fig. 2 a Experimental (full
line) and simulated (dotted line)
EPR spectrum (SW =6 mT) of
•
DMPO-SO
4-
spin adduct
recorded during the thermally
initiated decomposition of
K
2
S
2
O
8
in DMSO at 333 K.
Simulation spin Hamiltonian
parameters: a
N
=1.296 mT,
a
H
b
=0.938 mT,
a
H
c
=0.139 mT; g=2.0059.
bTime course of 20 individual
EPR spectra obtained for
samples of DMSO extracts of
NaCl and H
2
O
2
-stressed
Rhodotorula glutinis CCY 20-2-
26. All sets of 20 EPR spectra of
DMPO adducts monitored
during the thermal (333 K)
decomposition of K
2
S
2
O
8
in the
presence of DMSO extracts
were taken for 22 min under the
same experimental conditions as
for reference sample (DMSO,
instead of DMSO extracts).
Extracts concentrations
(lgmL
-1
): control (257), 4 %
NaCl (115), 1 mM H
2
O
2
(207),
2mMH
2
O
2
(207), 4 mM H
2
O
2
(131) and 5 mM H
2
O
2
(216)
05101520
0
2x106
4x106
6x106
8x106
RSC [%]
DMSO
Control
4 % NaCl
1 mM H2O2
2 mM H2O2
4 mM H2O2
5 mM H2O2
Integral EPR intensity
Time [min]
100
80
60
40
20
0
Fig. 3 Time course of EPR integral intensities of DMPO adducts
(spectra shown in Fig. 2b) recorded during first 22 min of the thermal
decomposition of K
2
S
2
O
8
in the presence of DMSO extracts of
R. glutinis grown under stress of NaCl or H
2
O
2
1 %2 %3 %4 %5 %1 mM2 mM3 mM4 mM5 mM
0
10
20
30
40
50
60 control
NaCl
H2O2
TEAC [µmol Trolox g–1]
DMSO extracts
Fig. 4 Radical scavenging capacities (equated to actual dry weight
of the samples) expressed as TEAC and evaluated by EPR spin
trapping of DMSO extracts of R. glutinis grown under various
concentrations of NaCl or H
2
O
2
58 3 Biotech (2013) 3:53–60
123

been reported to be the active constituents responsible for
antioxidant activities (Zheng et al. 2009). However, there is
no report of occurrence of phenolics in yeasts. In the
present investigation, extracts prepared from yeasts grown
under stress of H
2
O
2
or NaCl reported five peaks indicating
a significant amount of phenolic compounds in the inves-
tigated species.
Conclusions
The results of the present study reported that R. glutinis when
grown under stress of either NaCl or H
2
O
2
resulted in an
increased amount of total phenolic compounds. The results
were further supported by the enhanced RSC of the extracts
obtained from stressed cultures. A total of five phenolic
compounds viz., gallic acid, benzoic acid, catechin, caffeic
acid and ferulic acid were detected for the first time in
R. glutinis. This investigation will further form a basis for the
use of yeasts as a source of antioxidants and in formulation of
functional foods and pharmaceutical preparation.
Acknowledgments The work was supported by grants VEGA No.
1/0747/08, No. 1/0018/09, No. 2/0005/10 and No. 1/0975/12 from the Grant
Agency of Ministry of Education, Slovak Republic. RKS is also thankful to
Slovak Academic Information Agency (SAIA) for awarding scholarship
under National Scholarship Programme of the Slovak Republic.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
References
Arts MJTJ, Haenen GRMM, Voss HP, Bast A (2004) Antioxidant
capacity of reaction products limits the applicability of the trolox
equivalent antioxidant capacity (TEAC) assay. Food Chem
Toxicol 42:45–49
Bhosale P, Gadre RV (2001) Carotene production in sugarcane
molasses by a Rhodotorula glutinis mutant. J Ind Microbiol
Biotechnol 26:327–332
Certik M, Breierova E (2002) Adaptation responses of yeasts to
environmental stress. Chem Listy 96:147
Chen LS, Ma Y, Chen LJ, Zhao CH, Maubois JL, Jiang TM, Li HM,
He SH (2010) Antioxidant activity of two yeasts and their
attenuation effect on 4-nitroquinoline 1-oxide induced in vitro
lipid peroxidation. Int J Food Sci Technol 45:555–561
Daeschel M (2004) Live yeast in beer increases its antioxidant
activity. Emerg Food Res Dev Rep 9:1
Edge R, McGarvey DJ, Truscott TG (1997) The carotenoids as
antioxidants. J Photochem Photobiol B 41:189–200
Estruch F (2000) Stress controlled transcription factors, stress induced
genes and stress tolerance in budding yeasts. FEMS Microbiol
Rev 24:469–486
Greenwald P, Clifford CK, Miner JA (2001) Diet and cancer
prevention. Eur J Cancer 37:948–965
Halliwell B, Gutteridge JMC (1989) Free radicals in biology
and medicine, 2nd edn. Japan Scientific Societies Press,
Tokyo
Jaehrig SC, Rohn S, Kroh L, Wildenauer FX, Lisdat F, Fleischer LG,
Kurz T (2008) Antioxidative activity of (1–3), (1–6)-b-d-glucan
from Saccharomyces cerevisiae grown on different media. LWT
Food Sci Technol 41:868–877
Kullisaar T, Zilmer M, Mikelsaar M, Vihalemm T, Annuk H, Kairane
C, Kilk A (2002) Two antioxidative lactobacilli strains as
promising probiotics. Int J Food Microbiol 72:215–224
Kumura H, Tanoue Y, Tsukahara M, Tanaka T, Shimazaki K (2004)
Screening of dairy yeast strains for probiotic applications.
J Dairy Sci 87:4050–4056
Fig. 5 HPLC profile of the DMSO extracts of R. glutinis showing presence of various phenolic compounds at 272 nm
3 Biotech (2013) 3:53–60 59
123

Marova I, Breierova E, Koci R, Friedl Z, Slovak B, Pokorna J (2004)
Influence of exogenous stress factors on production of carotenoids
by some strains of cartenogenic yeasts. Ann Microbiol 54:73–85
Miquel J, Romano-Bosca A (2004) Oxidative stress and antioxidant
diet supplementation in ageing, arterosclerotic and immune
dysfunction processes. ARS Pharmacy 45(2):91–109
Psomas EI, Andrighetto C, Litopoulou-Tzanetaki E, Lombardi A,
Tzanetakis N (2001) Some probiotic properties of yeast isolates
from infantfaeces and feta cheese.Int J Food Microbiol 69:125–133
Psomas EI, Fletouris DJ, Litopoulou-Tzanetaki E, Tzanetakis N
(2003) Assimilation of cholesterol by yeast strains isolated from
infant feces and feta cheese. J Dairy Sci 86:3416–3422
Ramotar D, Belanger E, Brodeur I, Masson JY, Drobetsky EA (1998)
A yeast homologue of the human phosphotyrosyl phosphatase
activator PTPA is implicated in protection against oxidative
DNA damage induced by the model carcinogen 4-nitroquinoline-
1-oxide. J Biol Chem 273:21489–21496
Rapta P, Polovka M, Zalibera M, Breierova E, Zitnanova I, Marova I,
Certik M (2005) Scavenging and antioxidant properties of
compounds synthesized by cartenogenic yeasts stressed by heavy
metals-EPR spin trapping study. Biophy Chem 116:1–9
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C
(1999) Antioxidant activity applying an improved ABTS radical
cation decolorization assay. Free Rad Biol Med 26:1231–1237
Rizzo M, Ventrice D, Varone MA, Sidari R, Caridi A (2006) HPLC
determination of phenolics adsorbed on yeasts. J Pharm Biomed
Sci 42:46–55
Scalbert A, Williamson G (2000) Dietary intake and bioavailability of
polyphenols. J Nutr 130:2073–2085
Wardman P (1989) Reduction potentials of one-electron couples
involving free-radicals in aqueous-solution. J Phys Chem Ref
Data 18:1637–1755
Wouters JTM, Ayad EHE, Hugenholtz J, Smit G (2002) Microbes
from raw milk for fermented dairy products. Int Dairy J
12:91–109
Yen GC, Chen HY (1995) Antioxidant activity of various tea extracts
in relation to their mutagenicity. J Agric Food Chem 43:27–32
Yu L, Haley S, Perret J, Harris M (2004) Comparison of wheat flours
grown at different locations for their antioxidant properties. Food
Chem 86:11–16
Zalibera M, Rapta P, Stas
ˇko A, Brindzova
´L, Brezova
´V (2009)
Thermal generation of stable SO
4
•-
spin trap adducts with super-
hyperfine structure in their EPR spectra: an alternative EPR spin
trapping assay for radical scavenging capacity determination in
dimethylsulphoxide. Free Rad Res 43:457–469
Zheng W, Zhang M, Zhao Y, Wang Y, Miao K, Wei Z (2009)
Accumulation of antioxidant phenolic constituents in submerged
cultures of Inonotus obliquus. Biores Technol 100:1327–1335
60 3 Biotech (2013) 3:53–60
123