Food Chemistry 313 (2020) 126129

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Characterization of Saccharomyces cerevisiae based microcarriers for T

encapsulation of black cumin seed oil: Stability of thymoquinone and
bioactive properties
Kevser Karaman
Erciyes University, Faculty of Agriculture, Agricultural Biotechnology Department, Kayseri, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Thymoquinone is a chief phytochemical constituent of black cumin seed oil (BCSO) and shows strong bioactivity.
Black cumin seed oil It has a weak stability against environmental conditions like heat and light. Encapsulation process by
Yeast Saccharomyces cerevisiae is a popular technique to preserve the bioactivity and increase the stability of functional
Encapsulation bioactive compounds. In the current study, BCSO was encapsulated by both plasmolysed (PYC) and non-
Thymoquinone
plasmolysed yeast cell (NPYC) and stability of thymoquinone and bioactive properties of all samples were
Stability
Storage
evaluated. And also, some physicochemical, morphological and conformational characterizations were carried
out for the encapsules. The results showed that thymoquinone concentration and its bioactivity were preserved
better in PYC during storage compared to BCSO and NPYC. The highest degradation ratio of thymoquinone
during storage for the BCSO was 96.78% while the lowest one was for the PYC sample (52.63%).

1. Introduction open air conditions, light and heat (Edris, 2011).

Black cumin (Nigella sativa) is one of the most popular aromatic seed
and has been used as a nutraceutical or medicinal food for decades. The
growing interest in cold-pressed oils also affected the use of black
cumin seed oil (Kiralan, Özkan, Bayrak, & Ramadan, 2014). Because of
positive health aspects of the black cumin (Sultan et al., 2009), it is
commonly used as an important nutraceutical in phytotherapeutic ap-
plications. Black cumin seed and also its oil contain a chief bioactive
compound called as thymoquinone which is one of the major compo-
nents of the black cumin and many activities of the seed or oil have
already been attributed to this special compound (Ali & Blunden,
2003). It is known that thymoquinone is a terpenoid based substance
and the members of terpenoids are quite sensitive against heat. The loss
of thymoquinone concentration in heated black cumin seeds (approxi-
mately higher than 150 °C) was reported to be more than 75% (Agbaria,
Gabarin, Dahan, & Ben-Shabat, 2015). Salmani, Asghar, Lv, and Zhou
(2014) investigated the effects of solvent type, pH and light on the
stability of thymoquinone during storage and reported that it was more
stable at lower pH and its stability decreased with rising alkalinity. And
also thymoquinone showed significant degradation in aqueous media
and the results clearly showed that thymoquinone was highly sensitive
to light, even after a short period of exposure. The black cumin seed oil
(BCSO) is also sensitive to the oxidative reactions due to its fatty acid
composition and it is easily deteriorated during storage especially under
There are some technological processes for the preservation of the
phytoactive compounds in the food matrix like encapsulation as alter-
native to the chemical preservation methods. Encapsulation is a process
where the entrapping of one compound into another one which is a wall
material that can be in different particle sizes (Burgain, Gaiani, Linder,
& Scher, 2011). There are many encapsulation systems used commonly
such as spray-drying, emulsion, fluidized-bed coating, coacervation and
liposome entrapment and these techniques showed efficiency to cover
the active compounds to enhance the storage stability (Pham-Hoang,
Romero-Guido, Phan-Thi, Waché, 2013). Recently, one of the most
popular encapsulation system is based on the yeast cell capsulation due
to yeasts are naturally complex, containing both a multilayered cell
wall and various internal compartments and antioxidant mechanisms
(Bishop, Nelson, & Lamb, 1998). The yeast cell envelope can be con-
sidered as a coating material. It acts like a protecting capsule control-
ling the osmotic pressure and the exchanges of the cell with its en-
vironment (Pham-Hoang, Romero-Guido, Phan-Thi, & Waché, 2013).
Cell wall of the yeast has a major role in the encapsulation process
because it provides a mechanical support and allows molecules to dif-
fuse into the cell. In fact, yeast cells are a good encapsulation medium
for fat soluble materials. The cell wall properties play an important role
because the first contact occurs between the cell and molecule. So, a
hydrophobic cell wall is very suitable for the encapsulation of hydro-
phobic molecules like oils. The main mechanism of the yeast

E-mail address: kevserkaraman@erciyes.edu.tr.

https://doi.org/10.1016/j.foodchem.2019.126129
Received 18 September 2019; Received in revised form 17 December 2019; Accepted 25 December 2019
Available online 30 December 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
K. Karaman Food Chemistry 313 (2020) 126129

encapsulation is adhesion of the related compound by the cell wall in Table 1

terms of electrostatic properties (Ly et al., 2006) and then the per- Some physicochemical properties of loaded yeast encapsules.
meation of the substance by passive diffusion in terms of the cell por- Sample OC (%) EE (%) LC (g/kg)
osity character (Ciamponi, Duckham, & Tirelli, 2012). Different studies
a a
were performed on the potential application of yeast cell of Sacchar- PYC 29.98 ± 1.85 59.97 ± 3.70 260.34 ± 6.83a
NPYC 19.59 ± 2.98b 39.18 ± 5.97b 172.20 ± 14.5b
omyces cerevisiae as a microcarriers in the encapsulation of some
bioactive compounds or extracts like curcumin (Young & Nitin, 2019), OC: Oil content, EE: Encapsulation efficiency, LC: Loading capacity.
anthocyanins (Nguyen et al., 2018) menhaden fish oil (Czerniak, PYC: Plasmolysed loaded yeast encapsule, NPYC: Nonplasmolysed loaded yeast
Kubiak, Białas, & Jankowski, 2015) and limonene (Normand, Dardelle, encapsule. The superscript small letters in each column shows significant dif-
Bouquerand, Nicolas, & Johnston, 2005). ferences (p < 0.05).
In the present scenario, black cumin seed oil (BCSO) was en-
capsulated by using yeast cell of Saccharomyces cerevisiae which was
both plasmolysed and nonplasmolysed cell wall and thymoquinone
stability was monitored during 8 days storage at constant conditions
and decrease percentages were determined. Some morphological and
conformational analyses were also performed to characterize the dif-
ferences among the loaded and unloaded yeast encapsules.

2. Materials and methods

2.1. Materials

In this study, the black cumin seed oil (BCSO) used in the en-
capsulation process was provided from Dr. Yılmaz Medicinal Plant and
Drug Raw Materials Co. (Kayseri, Turkey). BCSO was an oil sample
produced by the process of cold pressing of the black cumin seeds and
then filtration. Some quality parameters were informed by the producer
as: specific gravity: 0.919, iodine number: 119.5, total free fatty acid:
0.9 mg KOH/g oil, and major fatty acids were: oleic acid (22.05%),
linoleic acid (59.89%) and palmitic acid (12.05%). Thymoquinone level
was determined as 39.5 mg/g oil. The yeast of Saccharomyces cerevisiae
was purchased as Baker’s yeast (Pakmaya Co. Düzce, Turkey) from a
local market in Kayseri (Turkey).

2.2. Preparation of yeasts and encapsulation of black cumin seed oil

(BCSO)

The encapsulation process was performed in two ways suggested by

Kavosi, Mohammadi, Shojaee-Aliabadi, Khaksar, and Hosseini (2017).
In the first experiment, the yeast cells were not plasmolysed and before
encapsulation, 170 g of yeast cells was washed using phosphate buffer
(pH 6.8) and then collected by centrifugation (4100 g for 10 min).
Washing procedure with distilled water was repeated five times. The
washed cells were then freeze-dried (nonplasmolysed yeast cell). The
procedure was followed for the preparation of plasmolysed cells in
which the cells were suspended in flasks containing 10% of sodium
chloride (NaCl) solution. The flasks were shaken for 48 h at 180 rpm at
55 °C in the shaking water bath. Plasmolysed cells were obtained by
centrifugation (4100 g for 10 min), then washed five times with deio-
nized water to eliminate cell/NaCl residues and finally all samples were
frozen (−18 °C) and then freeze-dried (Christ Alpha 1–2 LDplus/
1.4 mbar at −46 °C).
Oil-loaded yeast microcapsules were prepared using a modified
procedure. To prepare the oil in water emulsion, 10 g of black cumin
seed oil was homogenized with 40 g of deionized water [including 3%
(v/ v) Tween 80 (Merck, Germany)] using an Ultra-Turrax (IKA,
Germany) at 10000 rpm for 5 min. The emulsification was carried out
in an ice bath to prevent rising temperature. Yeast cells (plasmolysed,
nonplasmolysed) were added to the emulsion in a 1:1 (g/g) ratio. The
final suspension was then incubated for 12 h under controlled condi-
tions (180 rpm at 40 °C). The microcapsules were separated from the
emulsion by centrifugation at 8000 g for 15 min, followed by several
washes with distilled water to remove unencapsulated oil residues.
Finally, all microcapsules were frozen (−18 °C) and then freeze-dried
(Christ Alpha 1–2 LDplus/1.4 mbar at −46 °C). Finally, the dried
samples were ground using a coffee grinder (Bosch, Germany) and
Fig. 1. Thymoquinone levels of black cumin seed oil (BCSO), plasmolysed
loaded yeast encapsule (PYC) and nonplasmolysed loaded yeast encapsule
(NPYC) samples and decrease ratios of thymoquinone during storage.
stored at −18 °C until the analyses (Kavosi et al., 2017). All capsule
productions were performed with two repetitions.
2.3. Determination of some physicochemical properties of yeast encapsules
2.3.1. Oil content analysis, encapsulation efficiency (EE), and loading
capacity (LC)
To determine the effectiveness of the microencapsulation process,
the encapsulation efficiency and loading capacity were determined and
calculated as following equations. Oil content of the yeast micro-
capsules was determined gravimetrically and expressed as encapsula-
tion efficiency (Eq.1). Loading capacity was calculated by subtracting
the total amount of oil from the surface oil and then divided by the mass
of dry microcapsules (Eq. (2)). The total oil content of the micro-
capsules was determined by using petroleum ether (Merck, Germany)
based on the Soxhlet method. The surface oil levels of the capsules were
determined by extraction using n-hexane at room temperature. The
weighed yeast capsules were placed in hexane under stirring for 5 min.
Then, the final suspension was filtered using a filter paper and, subse-
quently, the solvent was evaporated using vacuum rotary evaporator at
40 °C. The surface oil content was calculated gravimetrically. Total oil
content was calculated by subtracting the oil content of the test samples
from the oil content of the blank yeast sample (Kavosi et al., 2017).

2
K. Karaman
Fig. 2. Antiradical activities of black cumin seed oil (BCSO), plasmolysed loaded yeast encapsule (PYC) and nonplasmolysed loaded yeast encapsule (NPYC) (on the
left) and decrease levels of bioactivity performance during storage (on the right).
EE(%) = (WEO/WIO) × 100
LC(g/kg) = (WTO − WSO)/WDMM
where EE is the encapsulation efficiency, WEO is the weight of en-
capsulated oil, WIO is the weight of initial amount of oil, LC is the
loading capacity, WTO is the weight of total oil, WSO is the weight of
surface oil and WDMM is the weight of dry mass of microcapsules. WEO is
the calculated by subtracting the nonencapsulated oil from total
amount of oil. All measurements were performed with two repetitions.
2.4. Bioactive properties of yeast encapsules
2.4.1. Thymoquinone concentrations and degradation ratios during storage
by HPLC
Thymoquinone, the main bioactive substance of BCSO was analyzed
by HPLC according to the methodology of Ghosheh, Houdi, and Crooks
(1999). For this purpose, thymoquinone extraction from the samples
(both native oil and yeast capsules) was performed. A 100 mg of native
oil or yeast capsule powder was weighed into a centrifuge tube and
4 mL of methanol (Merck, Germany) was added. Then all samples were
mixed by vortex for 2 min and centrifuged at 7500 g and 10 °C for
5 min. Finally, the supernatant was filtered using 0.45 μm filter and the
filtrates were injected into the HPLC (Shimadzu LC 2030C, Japan).
Isocratic mobile phase consisting of methanol:2-propanol (Merck,
Germany) and ultrapurified water (45:5:50%) was used. The flow rate
was 1 mL/min and the column (Inertsil ODS 4 column, 5μ, 46 × 250
mm) temperature was set at 40 °C. PDA detector was used, and the
absorbance was recorded at 254 nm. Thymoquinone levels of the
samples were determined using a calibration curve prepared by a high
purity thymoquinone standard (Sigma Chemical Co. (St. Louis, MO) at
Food Chemistry 313 (2020) 126129
(1) different concentration (25–175 mg/L). All measurements were per-
formed with three replications.
(2)
2.4.2. Yeast encapsule performance to preserve the thymoquinone stability
during storage
Thymoquinone is a sensitive bioactive component of BCSO against
some environmental conditions like high heat and light and it under-
goes to formation of radicals (Ghosheh et al., 1999). Due to sensitivity
of thymoquinone, a stability determination test was carried out. For this
purpose, Schaal oven test was followed (Przybylski et al., 1999). The
black cumin seed oil, plasmolysed and nonplasmolysed loaded yeast
encapsules were placed in a test tubes and put into the desiccator in-
cluding the NaCl solution to provide 75% relative humidity. The oven
temperature was set 65 °C and the samples were stored for 8 days to
determine the stability performance of the yeast capsules in the pre-
servation of the thymoquinone compared to native black cumin seed
oil. In all storage intervals, thymoquinone extraction was performed
and HPLC analysis of thymoquinone was carried out to determine its
levels and decrease ratios. All analyses were replicated with two re-
petitions.
2.4.3. DPPH radical scavenging activity
Antiradical activity of the samples exposed to Schaal oven test was
determined using the DPPH radical (2,2-diphenyl-1-picrylhydrazyl-
Merck, Germany) according to the method of He et al. (2011). For this
purpose, 100 μL of supernatant obtained for the thymoquinone analysis
was mixed with 3900 μL of DPPH solution (0.1 mM in methanol) and
the samples were mixed by vortex. Then the samples were incubated for
30 min at dark conditions and room temperature. Finally, the absor-
bances of the samples were recorded at 517 nm using a
3
K. Karaman
Fig. 3. Comparison of FTIR spectrum for a) Plasmolysed loaded yeast, b) Nonplasmolysed loaded yeast, c) Plasmolysed unloaded yeast and d) Nonplasmolysed
unloaded yeast and e) Black cumin seed oil.
spectrophotometer (UV-vis 1800 Shimadzu spectrophotometer, Shi-
madzu Corp., Japan). The results were expressed as % inhibition which
was calculated using the following formula (Eq.3).
% Inhibition = ((A control − A sample)/A control) × 100 (3)
where Asample is the absorbance of sample; Acontrol is the absorbance of
DPPH solution. All measurements were performed with four replica-
tions.
2.4.4. ABTS.+ radical scavenging activity
In addition to the DPPH radical scavenging activity, ABTS.+ radical
scavenging activity test was also performed according to the method of
Gong et al. (2012). For this aim, an ABTS.+ stock solution was prepared
by dissolving the ABTS.+ radical (Sigma, St. Louis, MO, USA) in
2.45 mmol/L potassium persulfate to create the radical substance
during the storage for 16 h at dark conditions and room temperature. At
the end, the ABTS.+ radical solution was diluted by using a phosphate
buffer solution (pH 7.4) until the absorbance value of 0.7 ± 0.05 at
734 nm was recorded. Then 30 μL of undiluted supernatant obtained for
the thymoquinone analysis was mixed by ABTS.+ solution and the
4
Food Chemistry 313 (2020) 126129
mixture was exposed to incubation for 6 min at room temperature.
Finally, the absorbance values of the samples were measured at 734 nm
by a spectrophotometer (UV-vis 1800 Shimadzu spectrophotometer,
Shimadzu Corp., Japan). The ABTS.+ radical scavenging activity of the
samples as % inhibition was calculated using the following equation
(Eq.4).
% Inhibition = ((A control − A sample)/A control) × 100 (4)
.+
where Asample is the absorbance of ABTS with sample; Acontrol refers to
the absorbance of ABTS.+ without sample. The % inhibition values
were converted into the Trolox values and all results were expressed as
Trolox equivalent antiradical capacity (microgram Trolox/g sample).
2.5. Morphological and conformational characterization of microcapsules
2.5.1. Scanning electron microscopy imaging (SEM)
To monitor the surface and microstructure of both freeze dried
microcapsules and unloaded yeast cells, Scanning Electron Microscopy
system (LEO 440 Computer-Controlled Digital Scanning Electron
Microscope, Zeiss, Oberkochen, Germany) was used. The samples were
K. Karaman Food Chemistry 313 (2020) 126129

Fig. 4. Comparison of DSC spectrum for plasmolysed and nonplasmolysed loaded and unloaded yeast microcapsules. a) Plasmolysed loaded yeast, b)
Nonplasmolysed loaded yeast, c) Plasmolysed unloaded yeast and d) Nonplasmolysed unloaded yeast.

analyzed while operating at an accelerating voltage of 5 kV with dif- 2.5.2. Fourier transform infrared spectroscopy (FT-IR)
ferent magnifications of 5KX, 10KX, 20KX, and 30KX. IR spectrum and the state of the bonds in the structure, binding sites
of both unloaded and loaded yeast cell structures and black cumin seed
oil were monitored by using Perkin Elmer 400 FT-IR Spectrometer

5
K. Karaman
Fig. 5. Comparison of XRD properties for plasmolysed and nonplasmolysed
loaded and unloaded yeast capsules.
Spotlight 400 Imaging System (France) and spectrums were compared.
2.5.3. Differential scanning calorimetry (DSC)
All samples were weighed approximately 2.5 mg and placed in
aluminum pan and heated from 25 to 350 °C at a scanning rate of
10 °C min−1. Nitrogen was used as a purge gas at a flow rate of 30 mL/
min. Analysis was performed by using Differential Scanning
Calorimetry (TA Instruments Thermo DSC Q20, England) equipment.
2.5.4. X-ray diffraction spectrum (XRD)
XRD patterns of both loaded and unloaded yeast cells and black
cumin seed oil were observed with using Bruker Axs D8 diffractometer
equipment (Bruker AXS Inc., Madison, WI, USA) and measurements
were performed at angle 2ɵ, ranging between 10◦ and 100◦.
2.6. Statistical analysis
Statistical analysis of the samples was carried out using SAS statis-
tical software (SAS Institute, 2000). The comparative analyses were
conducted using Duncan’s multiple range tests. A significance level of
0.05 was used for all comparisons.
3. Results and discussion
3.1. Oil content, encapsulation efficiency and loading capacity of the yeast
encapsules
In the present study, as a microcarrier of BCSO, both plasmolysed
and nonplasmolysed yeast cells were used and their performance and
properties were compared. Table 1 shows the oil content (OC), en-
capsulation efficiency (EE) and loading capacity (LC) values of pro-
duced microcapsules with plasmolysed (PYC) and nonplasmolysed
yeast cells (NPYC). According to the Table 1, encapsulation process was
more successful where the samples were covered with plasmolysed
yeast compared to nonplasmolysed ones. It could be seen that en-
capsulation efficiency and loading capacity values of PYC were higher
than those of NPYC. Oil content of PYC (29.98%) was higher than that
of NPYC (19.59%). Plasmolysis is a modified autolysis process in the
presence of a reactive substance, such as an inorganic salt (sodium
chloride) or organic solvent (toluene, ethanol, ethyl acetate) (Milić,
Rakin, & Šiler-Marinković, 2007). And in the encapsulation processes
performed with yeast cells, to obtain higher loading capacity removing
the cytoplasmic substances, a plasmolysis process, generally has been
carried out (Kavosi et al., 2017). Our results are in accordance to those
obtained by Shi et al. (2007) and Kavosi et al. (2017) while Paramera,
Konteles, and Karathanos (2011) did not monitored any significant
increase in curcumin loaded by plasmolysed yeast capsules.
6
Food Chemistry 313 (2020) 126129
3.2. Thymoquinone stability and degradation ratios of the yeast encapsules
Thymoquinone concentration levels of both BCSO and yeast en-
capsule samples were analyzed by HPLC and the results were illustrated
in Fig. 1. As is seen from the Fig. 1, the highest thymoquinone level
(39.5 mg/g oil) was determined for the native black cumin seed oil
which is untreated control one while the lowest values were determined
for the microcapsule prepared by nonplasmolysed yeast (1.24 mg/g
oil). As is expected, thymoquinone level was determined to be highest
in native BCSO because it was the control sample and as it was seen, the
plasmolysed yeast capsule and nonplasmolysed yeast capsules had
lowest oil as encapsulated form (Table 1). Due to lower oil content, they
showed lower thymoquinone level compared to control oil sample.
These results could be seen also from Fig. S1 showing the peak areas of
the thymoquinone for all samples. Thymoquinone was reported as the
main bioactive substance of the BCSO (Nigella sativa) and it represented
18.4–24% of the oil (Herlina, Kurniawati & Faridah, 2003). Herlina,
Aziz, Kurniawati, and Faridah (2003) investigated the thymoquinone
content of black cumin seeds cultivated at different altitudes and re-
ported that the level of thymoquinone ranged between 1039 and
2940 mg/kg seed. In another study, Kiralan et al. (2014) compared the
thymoquinone content of BCSO samples obtained by different extrac-
tion methods such as cold pressing, microwave and Soxhlet extraction
systems and reported that the highest thymoquinone level (0.014 mg/g
oil) was determined for the sample produced by cold pressing approach.
Solati, Baharin, and Bagheri (2014) reported that the thymoquinone
level was 4.07 mg/g oil for the sample extracted by supercritical CO2
technique while Lutterodt, Slavin, Whent, Turner, and Yu (2010) re-
ported the thymoquinone level of BCSO was 8.73 mg/g oil. To see the
effect of encapsulation using plasmolysed or nonplasmolysed yeast cell
on the quality of the BCSO, the samples were stored for 8 days at 65 °C
and 75% relative humidity in a desiccator where placed in an oven.
Fig. 1 also shows the thymoquinone levels of the samples during storage
periods. It is clear that a sharp and significant (p < 0.05) decrease in
the thymoquinone concentrations of all samples was observed. Fig. S2
also shows the decrease in the HPLC peak areas of thymoquinone
during storage clearly. Thymoquinone level decreased from 39.5 to
1.27 mg/g oil for control BCSO sample after 8 days storage while it was
0.72 and 0.51 mg/g oil for the PYC and NPYC sample at the end of the
storage while the levels were 1.52 and 1.24 mg/g oil at the beginning of
the storage, respectively. It was determined that the thymoquinone
level decreased during storage because it has weak stability at un-
suitable conditions like heat, humidity or light presence. The highest
decrement level was monitored for the BCSO sample while the lowest
was for PYC sample which means the encapsulation process especially
by using plasmolysed yeast showed a stronger preserving effect on
thymoquinone compared to control BCSO and NPYC (p < 0.05). De-
gradation percentage of BCSO after 2 days storage was 21.8% while it
was calculated as to be 8.55 and 29.03% for PYC and NPYC, respec-
tively. After 5 days storage, degradation percentage significantly in-
creased and it was determined as 86.09, 41.4 and 51.61% for BCSO,
PYC and NPYC, respectively. At the end of the storage for 8 days, the
highest degradation ratio was calculated for the BCSO as 96.78% while
the lowest one was for the sample of PYC (52.63%). It could be said that
the encapsulation showed better thymoquinone stability compared to
control sample and also, the plasmolysis process increased the micro-
capsule stability compared to nonplasmolysed samples. In a study
conducted by Czerniak et al. (2015), yeast cell of Saccharomyces cere-
visiae was used as microencapsulating agent for the menhaden fish oil
and they determined the oxidative stability of the samples during sto-
rage. They reported that the peroxide value of the samples which was
encapsulated by yeast cell was significantly lower than that of the na-
tive oil after 30 days storage. Nguyen et al. (2018) investigated the
anthocyanin stability of Hibiscus sabdariffa L. by using yeast cell as
microcapsule agent and they reported that the yeast cells were good
materials for the encapsulation of pigments to preserve the coloring
K. Karaman Food Chemistry 313 (2020) 126129

Fig. 6. Comparison of SEM images of plasmolysed loaded (a and b), nonplasmolysed loaded (c and d), plasmolysed unloaded (e and f) and nonplasmolysed unloaded
(g and h) yeast encapsules. Magnification for a, c, e and g was 5KX and for b, d, f and h was 10 KX.

properties. Shi et al. (2007) used yeast cells-based microencapsulation
process for the preserving of chlorogenic acid against to the unsuitable
conditions and they observed no significant chemical changes during
the encapsulation, and a good stability for the yeast-encapsulated
chlorogenic acid was also monitored. In a similar study, Young and
Nitin (2019) compared to plasmolysed and nonplasmolysed yeast cells
on the thermal stability of curcumin encapsulated by S.cerevisiae cell
and they reported that the results presented in this study showed that
plasmolysed yeast microcarriers performed better barrier against
thermal degradation compared to native yeast microcarriers.
3.3. Evaluation of bioactive performance of the yeast encapsules by
antiradical scavenging tests
After the determination of thymoquinone level and stability of the
active substance in microcapsules during storage, change in radical
scavenging activity of the yeast microcapsule samples was also in-
vestigated. The ABTS.+ and DPPH radical scavenging activities of BCSO
at the beginning of the storage were calculated as 9.75 μg Trolox/g
sample and 20.2%, respectively. For the PYC and NPYC samples, the
radical scavenging activity was also lower compared to native oil be-
cause of the lower bioactive constituents like thymoquinone. After

7
K. Karaman
8 days storage, the ABTS.+ and DPPH radical scavenging activities of
the samples decreased dramatically for BCSO and recorded as 1.81 μg
Trolox/g sample and 2.77%, respectively (p < 0.05, Fig. 2). Similarly,
both radicals were also tested to determine the antiradical performance
of the samples and it was observed that the ABTS.+ and DPPH radical
scavenging activities of PYC and NPYC were determined as 5.61 μg
Trolox/g sample and 5.45% and 16.5 μg Trolox/g sample and 55.9%,
respectively. As is seen, the highest radical scavenging activity was
measured for the sample encapsulated by nonplasmolysed yeast cell
(NPYC) compared to BCSO and PYC although it had the lowest thy-
moquinone concentration. Similar to the change in BCSO, increase of
the storage period caused a significant decrement in the radical
scavenging activity of both PYC and NPYC samples but the decrease
percentage was of course significantly lower than that of the BCSO
during storage (p < 0.05, Fig. 2). The main reason behind the higher
antiradical performance of NPYC compared to others is the yeast cell
content. The antiradical activities of plasmolysed and nonplasmolysed
unloaded yeast cells by ABTS.+ and DPPH test were also determined. It
was determined that ABTS.+ and DPPH radical scavenging activities of
plasmolysed and nonplasmolysed unloaded yeast capsules were
0.613 μg Trolox/g sample and 2.56% and 2.13 μg Trolox/g sample and
5.39%, respectively. It is clear from the results that the unloaded
(empty) yeast capsule bioactivity was affected by the plasmolysis pro-
cess and it was resulted that the plasmolysis caused a decrease in the
bioactivity of the yeast cells. Plasmolysis which means the loss of cy-
toplasmic material because of water loss due to osmosis caused re-
moving of the yeast cell components. It is usually performed by im-
mersing cells in strong saline or sugar solutions (Pham-Hoang et al.,
2013). It was reported that the yeasts (Baker’s or dry yeast) have strong
antioxidant and antiradical activity and could be evaluated as novel and
natural antioxidant ingredient (Hassan, 2011). It was also reported that
the antioxidant and antiradical activity of yeast were related to glu-
tathione, Maillard reaction products and sulfur containing amino acids
present in the yeast cell (Soomer & Jamieson, 1996). In a study about
the antioxidant performance of the yeasts, the beef patties were added
with yeast extract and it was observed that the yeast extracts prevented
the thiobarbituric acid reactive substances formation in the sample
(Wang & Xiong, 2005). In the light of this knowledge, it could be in-
ferred from that the plasmolysis process increased the oil absorption
capacity and encapsulation efficiency and also increased the stability of
active ingredient but caused a decrease in the bioactivity of the material
due to the removal of the some antioxidant components in the cell of
the yeasts. In another similar study, oxidative and thermal stability of
curcumin encapsulated with yeast cell were investigated and plasmo-
lysed yeast capsule showed weaker antioxidant performance compared
to nonplasmolysed yeast capsule and they expected lower antioxidant
activity for the nonplasmolysed yeast capsule because they stated that
caustic conditions and high heat would oxidize and degrade any innate
antioxidant systems within the cytoplasm. But the high level of glu-
tathione up to 10 mM (Penninckx, 2002) which is an essential in-
tracellular oxidative stress regulator had a strong antioxidant activity.
Based on the plasmolysis of the yeast cell, a desiccation process results a
decrement in the ratio of the reduced form of glutathione to the oxi-
dized form (GSH:GSSG) and this result causes a decrease in the Trolox
equivalent antioxidant performance (Espindola Ade, Gomes, Panek, &
Eleutherio, 2003).
3.4. Morphological and conformational characterization of the yeast
encapsules
3.4.1. FT-IR spectrum of the samples
Fig. 3 shows FT-IR spectrum of plasmolysed loaded yeast encapsules
(a), nonplasmolysed loaded yeast encapsules (b), plasmolysed unloaded
yeast cells (c), nonplasmolysed unloaded yeast cells (d) and black
cumin seed oil (e) samples. According to FT–IR spectra (Fig. 3) the
absorption bands were observed at approximately 1741 cm−1 and only
8
Food Chemistry 313 (2020) 126129
monitored in Fig. 3a, b and e indicated that C]O ester functional
groups entered into the main ingredient structure. That situation can be
considered as proof of encapsule formation given that black cumin seed
oil is the source of ester bonds. Dadkhodazade, Mohammadi, and
Shojaee-Aliabadi (2018) stated that they observed bands at 2925 cm−1
for CeH groups of lipids, stretching vibrations of amide Ι, amide ΙΙ and
mannans absorption band at 1647, 1547 and 1057 cm−1, respectively.
These results were generally in accordance to the current research re-
sults while bands obtained around 1547 cm−1 did not observed at
processed yeasts for encapsulation (Fig. 3a and b). Galichet,
Sockalingum, Belarbi, and Manfait (2001) and Burattini et al. (2008)
informed that component bands at around 1026 cm−1 were related to
mainly β-glucans (β(1 → 4)) and in the current study, those bands could
be observed for plasmolysed and nonplasmolysed loaded yeast cells
while the band obtained from nonplasmolysed one (Fig. 3b) was more
intense. Regarding to Fig. 3c and d, it could be said that plasmolysis
process shifted to left the bands.
3.4.2. DSC characteristics of the samples
DSC spectrum of loaded and unloaded microcapsule cells were il-
lustrated in Fig. 4. When plasmolysed loaded encapsules (a) and plas-
molysed unloaded yeast cells (c) were compared with each other and it
is clear that the loading caused a decrement in Tg and melting peak.
And opposite behavior was observed in crystallization peak and as a
result of oil loading, crystallization temperature decreased. When
compared with Fig. 4c and d, due to plasmolysis treatment, Tg, Tm and
Tc showed a decrement in yeast cells. While Tg of nonplasmolysed yeast
was 77.19 °C and after plasmolysis that value decreased to 56.29 °C. Tm
and Tc also decreased from 120.12 °C and 309.07 °C to 108.48 °C and
278.92 °C, respectively. Salari, Bazzaz, Rajabi, and Khashyarmanesh
(2013) informed that exothermic peak was observed at 352 °C by
maxium occurrence for freeze-dried nonplasmolysed yeast and also
stated that Tc decreased after plasmolysis with 20% (w/v) NaCl solu-
tion, similar to the current results. Paramera et al. (2011) stated that
plasmolysis altered the lipid form of the membrane and caused to form
mobility and, thus, fluidity that’s why plasmolysed yeast cells had lower
Tm compared to nonplasmolysed ones. Regarding to Fig. 4a and b,
there were significant differences between the DSC thermograms due to
plasmolysis treatment for encapsulation. In Fig. 4b, it was observed two
endothermic peaks that the first one at 98.10 °C and the second one at
208.51 °C while that situation was not detected completely in plas-
molysed loaded yeast cell (Fig. 4a). It was concluded that the first en-
dothermic peak was due to yeast phospholipid bilayer and the second
one was due to black cumin seed oil (Fig. 4b) and besides not performed
of plasmolysis treatment on the yeast cell limited the interaction of
yeast cell and oil. These results were in accordance with the report of
Paramera et al. (2011).
3.4.3. XRD spectrum of the samples
XRD results of plasmolysed loaded, nonplasmolysed loaded yeast
encapsules and also plasmolysed unloaded, nonplasmolysed unloaded
yeast cells and the black cumin seed oil were shown in Fig. 5. de Barros
Fernandes et al. (2016) informed that a crystalline material exhibits
sharp peaks while amorphous materials provide a broader peak pattern.
In the present study, samples behaved as an amorphous material with
poor crystallinity generally. Guler and Sarioglu (2014) also informed
that raw S.cerevisiae cells had amorphous phase similar to the results of
the present research. It was observed that black cumin seed oil showed
peaks at 2θ = 19.74°. Nonplasmolysed loaded and unloaded yeast cells
showed two peaks at 2θ = 8.21°, 19.41° and 2θ = 9.18°, 19.16° re-
spectively. When plasmolysis process occurred, two peaks were mon-
itored at 2θ = 6.71°, 19.00° for loaded cells and at 2θ = 5.79°, 18.90°
for unloaded cells. XRD pattern of black cumin seed oil had the highest
intensity and loaded yeast cells followed it. That situation could be
explained by black cumin seed oil incorporated the yeast cell and in-
tensity values were observed as to be higher than those of the unloaded
K. Karaman
(empty) ones.
3.4.4. SEM images of the samples
Fig. 6 illustrates the scanning electron microscope (SEM) images of
all samples. SEM images of nonprocessed S. cerevisiae (Fig. 6 (g–h))
showed spheroidal or slightly elongated spheroidal shapes, regular and
smooth surfaces. Besides plasmolysis treatment caused minor de-
formation on cells. Morris, Winters, Coulson, and Clarke (1986) in-
dicated that exposure of the yeast S. cerevisiae to hypertonic solutions
resulted in cell shrinkage while Paramera et al. (2011) also showed that
yeast cells wall remained thick, while curcumin molecules interacted
with yeast cells. Regarding to the Fig. 6 a–b and c–d, it could be said
that encapsulation and black cumin seed oil entrapment caused to
thickening to cell wall.
4. Conclusions
This research demonstrated that black cumin seed oil could be en-
capsulated successfully by using the yeast cell of S. cerevisiae and in-
crease the stability against unsuitable conditions. One of the main
bioactive components of black cumin seed oil is thymoquinone and it
could be preserved within the yeast capsules as microcarrier against to
heat and humidity. Encapsulation by using a yeast cell of S. cerevisiae is
an effective approach preventing to decrease the biofunctional activity
of thymoquinone. The results of the current work showed that the
plasmolysis is an important pre-treatment for the yeast cell to enhance
the oil absorption capacity and increase the stability of the capsulated
substance against unsuitable conditions. By using the yeast cell mi-
crocapsules, thymoquinone degradation because of the environmental
parameters like heat and light could be limited. And also further re-
searches are needed to determine the conditions to decrease the de-
gradation ratios for the capsulated samples during storage.
Funding sources
This work has been supported by Erciyes University Scientific
Research Projects Coordination Unit under grant number of FHD-2019-
8862.
Declaration of Competing Interest
The author declares that she has no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The author would like to thank to Dr. Mahmut KAPLAN and Dr.
Perihan GÜRBÜZ for providing the opportunity to work in their la-
boratories. The author also would like to thank to Erciyes University
Scientific Research Projects Coordination Unit for their financial sup-
port of the research.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.126129.
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