Food Chemistry 313 (2020) 126129

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Food Chemistry 313 (2020) 126129

Contents lists available at ScienceDirect

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).


There are some technological processes for the preservation of the
Black cumin (Nigella sativa) is one of the most popular aromatic seed phytoactive compounds in the food matrix like encapsulation as alter-
and has been used as a nutraceutical or medicinal food for decades. The native to the chemical preservation methods. Encapsulation is a process
growing interest in cold-pressed oils also affected the use of black where the entrapping of one compound into another one which is a wall
cumin seed oil (Kiralan, Özkan, Bayrak, & Ramadan, 2014). Because of material that can be in different particle sizes (Burgain, Gaiani, Linder,
positive health aspects of the black cumin (Sultan et al., 2009), it is & Scher, 2011). There are many encapsulation systems used commonly
commonly used as an important nutraceutical in phytotherapeutic ap- such as spray-drying, emulsion, fluidized-bed coating, coacervation and
plications. Black cumin seed and also its oil contain a chief bioactive liposome entrapment and these techniques showed efficiency to cover
compound called as thymoquinone which is one of the major compo- the active compounds to enhance the storage stability (Pham-Hoang,
nents of the black cumin and many activities of the seed or oil have Romero-Guido, Phan-Thi, Waché, 2013). Recently, one of the most
already been attributed to this special compound (Ali & Blunden, popular encapsulation system is based on the yeast cell capsulation due
2003). It is known that thymoquinone is a terpenoid based substance to yeasts are naturally complex, containing both a multilayered cell
and the members of terpenoids are quite sensitive against heat. The loss wall and various internal compartments and antioxidant mechanisms
of thymoquinone concentration in heated black cumin seeds (approxi- (Bishop, Nelson, & Lamb, 1998). The yeast cell envelope can be con-
mately higher than 150 °C) was reported to be more than 75% (Agbaria, sidered as a coating material. It acts like a protecting capsule control-
Gabarin, Dahan, & Ben-Shabat, 2015). Salmani, Asghar, Lv, and Zhou ling the osmotic pressure and the exchanges of the cell with its en-
(2014) investigated the effects of solvent type, pH and light on the vironment (Pham-Hoang, Romero-Guido, Phan-Thi, & Waché, 2013).
stability of thymoquinone during storage and reported that it was more Cell wall of the yeast has a major role in the encapsulation process
stable at lower pH and its stability decreased with rising alkalinity. And because it provides a mechanical support and allows molecules to dif-
also thymoquinone showed significant degradation in aqueous media fuse into the cell. In fact, yeast cells are a good encapsulation medium
and the results clearly showed that thymoquinone was highly sensitive for fat soluble materials. The cell wall properties play an important role
to light, even after a short period of exposure. The black cumin seed oil because the first contact occurs between the cell and molecule. So, a
(BCSO) is also sensitive to the oxidative reactions due to its fatty acid hydrophobic cell wall is very suitable for the encapsulation of hydro-
composition and it is easily deteriorated during storage especially under 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).
Fig. 1. Thymoquinone levels of black cumin seed oil (BCSO), plasmolysed
In the first experiment, the yeast cells were not plasmolysed and before loaded yeast encapsule (PYC) and nonplasmolysed loaded yeast encapsule
encapsulation, 170 g of yeast cells was washed using phosphate buffer (NPYC) samples and decrease ratios of thymoquinone during storage.
(pH 6.8) and then collected by centrifugation (4100 g for 10 min).
Washing procedure with distilled water was repeated five times. The
stored at −18 °C until the analyses (Kavosi et al., 2017). All capsule
washed cells were then freeze-dried (nonplasmolysed yeast cell). The
productions were performed with two repetitions.
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 2.3. Determination of some physicochemical properties of yeast encapsules
55 °C in the shaking water bath. Plasmolysed cells were obtained by
centrifugation (4100 g for 10 min), then washed five times with deio- 2.3.1. Oil content analysis, encapsulation efficiency (EE), and loading
nized water to eliminate cell/NaCl residues and finally all samples were capacity (LC)
frozen (−18 °C) and then freeze-dried (Christ Alpha 1–2 LDplus/ To determine the effectiveness of the microencapsulation process,
1.4 mbar at −46 °C). the encapsulation efficiency and loading capacity were determined and
Oil-loaded yeast microcapsules were prepared using a modified calculated as following equations. Oil content of the yeast micro-
procedure. To prepare the oil in water emulsion, 10 g of black cumin capsules was determined gravimetrically and expressed as encapsula-
seed oil was homogenized with 40 g of deionized water [including 3% tion efficiency (Eq.1). Loading capacity was calculated by subtracting
(v/ v) Tween 80 (Merck, Germany)] using an Ultra-Turrax (IKA, the total amount of oil from the surface oil and then divided by the mass
Germany) at 10000 rpm for 5 min. The emulsification was carried out of dry microcapsules (Eq. (2)). The total oil content of the micro-
in an ice bath to prevent rising temperature. Yeast cells (plasmolysed, capsules was determined by using petroleum ether (Merck, Germany)
nonplasmolysed) were added to the emulsion in a 1:1 (g/g) ratio. The based on the Soxhlet method. The surface oil levels of the capsules were
final suspension was then incubated for 12 h under controlled condi- determined by extraction using n-hexane at room temperature. The
tions (180 rpm at 40 °C). The microcapsules were separated from the weighed yeast capsules were placed in hexane under stirring for 5 min.
emulsion by centrifugation at 8000 g for 15 min, followed by several Then, the final suspension was filtered using a filter paper and, subse-
washes with distilled water to remove unencapsulated oil residues. quently, the solvent was evaporated using vacuum rotary evaporator at
Finally, all microcapsules were frozen (−18 °C) and then freeze-dried 40 °C. The surface oil content was calculated gravimetrically. Total oil
(Christ Alpha 1–2 LDplus/1.4 mbar at −46 °C). Finally, the dried content was calculated by subtracting the oil content of the test samples
samples were ground using a coffee grinder (Bosch, Germany) and from the oil content of the blank yeast sample (Kavosi et al., 2017).

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K. Karaman Food Chemistry 313 (2020) 126129

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 (1) different concentration (25–175 mg/L). All measurements were per-
formed with three replications.
LC(g/kg) = (WTO − WSO)/WDMM (2)

where EE is the encapsulation efficiency, WEO is the weight of en- 2.4.2. Yeast encapsule performance to preserve the thymoquinone stability
capsulated oil, WIO is the weight of initial amount of oil, LC is the during storage
loading capacity, WTO is the weight of total oil, WSO is the weight of Thymoquinone is a sensitive bioactive component of BCSO against
surface oil and WDMM is the weight of dry mass of microcapsules. WEO is some environmental conditions like high heat and light and it under-
the calculated by subtracting the nonencapsulated oil from total goes to formation of radicals (Ghosheh et al., 1999). Due to sensitivity
amount of oil. All measurements were performed with two repetitions. of thymoquinone, a stability determination test was carried out. For this
purpose, Schaal oven test was followed (Przybylski et al., 1999). The
2.4. Bioactive properties of yeast encapsules black cumin seed oil, plasmolysed and nonplasmolysed loaded yeast
encapsules were placed in a test tubes and put into the desiccator in-
2.4.1. Thymoquinone concentrations and degradation ratios during storage cluding the NaCl solution to provide 75% relative humidity. The oven
by HPLC temperature was set 65 °C and the samples were stored for 8 days to
Thymoquinone, the main bioactive substance of BCSO was analyzed determine the stability performance of the yeast capsules in the pre-
by HPLC according to the methodology of Ghosheh, Houdi, and Crooks servation of the thymoquinone compared to native black cumin seed
(1999). For this purpose, thymoquinone extraction from the samples oil. In all storage intervals, thymoquinone extraction was performed
(both native oil and yeast capsules) was performed. A 100 mg of native and HPLC analysis of thymoquinone was carried out to determine its
oil or yeast capsule powder was weighed into a centrifuge tube and levels and decrease ratios. All analyses were replicated with two re-
4 mL of methanol (Merck, Germany) was added. Then all samples were petitions.
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 2.4.3. DPPH radical scavenging activity
filtrates were injected into the HPLC (Shimadzu LC 2030C, Japan). Antiradical activity of the samples exposed to Schaal oven test was
Isocratic mobile phase consisting of methanol:2-propanol (Merck, determined using the DPPH radical (2,2-diphenyl-1-picrylhydrazyl-
Germany) and ultrapurified water (45:5:50%) was used. The flow rate Merck, Germany) according to the method of He et al. (2011). For this
was 1 mL/min and the column (Inertsil ODS 4 column, 5μ, 46 × 250 purpose, 100 μL of supernatant obtained for the thymoquinone analysis
mm) temperature was set at 40 °C. PDA detector was used, and the was mixed with 3900 μL of DPPH solution (0.1 mM in methanol) and
absorbance was recorded at 254 nm. Thymoquinone levels of the the samples were mixed by vortex. Then the samples were incubated for
samples were determined using a calibration curve prepared by a high 30 min at dark conditions and room temperature. Finally, the absor-
purity thymoquinone standard (Sigma Chemical Co. (St. Louis, MO) at bances of the samples were recorded at 517 nm using a

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K. Karaman Food Chemistry 313 (2020) 126129

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- mixture was exposed to incubation for 6 min at room temperature.
madzu Corp., Japan). The results were expressed as % inhibition which Finally, the absorbance values of the samples were measured at 734 nm
was calculated using the following formula (Eq.3). by a spectrophotometer (UV-vis 1800 Shimadzu spectrophotometer,
Shimadzu Corp., Japan). The ABTS.+ radical scavenging activity of the
% Inhibition = ((A control − A sample)/A control) × 100 (3)
samples as % inhibition was calculated using the following equation
where Asample is the absorbance of sample; Acontrol is the absorbance of (Eq.4).
DPPH solution. All measurements were performed with four replica- % Inhibition = ((A control − A sample)/A control) × 100 (4)
tions.
.+
where Asample is the absorbance of ABTS with sample; Acontrol refers to
2.4.4. ABTS.+ radical scavenging activity the absorbance of ABTS.+ without sample. The % inhibition values
In addition to the DPPH radical scavenging activity, ABTS.+ radical were converted into the Trolox values and all results were expressed as
scavenging activity test was also performed according to the method of Trolox equivalent antiradical capacity (microgram Trolox/g sample).
Gong et al. (2012). For this aim, an ABTS.+ stock solution was prepared
by dissolving the ABTS.+ radical (Sigma, St. Louis, MO, USA) in 2.5. Morphological and conformational characterization of microcapsules
2.45 mmol/L potassium persulfate to create the radical substance
during the storage for 16 h at dark conditions and room temperature. At 2.5.1. Scanning electron microscopy imaging (SEM)
the end, the ABTS.+ radical solution was diluted by using a phosphate To monitor the surface and microstructure of both freeze dried
buffer solution (pH 7.4) until the absorbance value of 0.7 ± 0.05 at microcapsules and unloaded yeast cells, Scanning Electron Microscopy
734 nm was recorded. Then 30 μL of undiluted supernatant obtained for system (LEO 440 Computer-Controlled Digital Scanning Electron
the thymoquinone analysis was mixed by ABTS.+ solution and the Microscope, Zeiss, Oberkochen, Germany) was used. The samples were

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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

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

6
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 3.3. Evaluation of bioactive performance of the yeast encapsules by
process for the preserving of chlorogenic acid against to the unsuitable antiradical scavenging tests
conditions and they observed no significant chemical changes during
the encapsulation, and a good stability for the yeast-encapsulated After the determination of thymoquinone level and stability of the
chlorogenic acid was also monitored. In a similar study, Young and active substance in microcapsules during storage, change in radical
Nitin (2019) compared to plasmolysed and nonplasmolysed yeast cells scavenging activity of the yeast microcapsule samples was also in-
on the thermal stability of curcumin encapsulated by S.cerevisiae cell vestigated. The ABTS.+ and DPPH radical scavenging activities of BCSO
and they reported that the results presented in this study showed that at the beginning of the storage were calculated as 9.75 μg Trolox/g
plasmolysed yeast microcarriers performed better barrier against sample and 20.2%, respectively. For the PYC and NPYC samples, the
thermal degradation compared to native yeast microcarriers. radical scavenging activity was also lower compared to native oil be-
cause of the lower bioactive constituents like thymoquinone. After

7
K. Karaman Food Chemistry 313 (2020) 126129

8 days storage, the ABTS.+ and DPPH radical scavenging activities of monitored in Fig. 3a, b and e indicated that C]O ester functional
the samples decreased dramatically for BCSO and recorded as 1.81 μg groups entered into the main ingredient structure. That situation can be
Trolox/g sample and 2.77%, respectively (p < 0.05, Fig. 2). Similarly, considered as proof of encapsule formation given that black cumin seed
both radicals were also tested to determine the antiradical performance oil is the source of ester bonds. Dadkhodazade, Mohammadi, and
of the samples and it was observed that the ABTS.+ and DPPH radical Shojaee-Aliabadi (2018) stated that they observed bands at 2925 cm−1
scavenging activities of PYC and NPYC were determined as 5.61 μg for CeH groups of lipids, stretching vibrations of amide Ι, amide ΙΙ and
Trolox/g sample and 5.45% and 16.5 μg Trolox/g sample and 55.9%, mannans absorption band at 1647, 1547 and 1057 cm−1, respectively.
respectively. As is seen, the highest radical scavenging activity was These results were generally in accordance to the current research re-
measured for the sample encapsulated by nonplasmolysed yeast cell sults while bands obtained around 1547 cm−1 did not observed at
(NPYC) compared to BCSO and PYC although it had the lowest thy- processed yeasts for encapsulation (Fig. 3a and b). Galichet,
moquinone concentration. Similar to the change in BCSO, increase of Sockalingum, Belarbi, and Manfait (2001) and Burattini et al. (2008)
the storage period caused a significant decrement in the radical informed that component bands at around 1026 cm−1 were related to
scavenging activity of both PYC and NPYC samples but the decrease mainly β-glucans (β(1 → 4)) and in the current study, those bands could
percentage was of course significantly lower than that of the BCSO be observed for plasmolysed and nonplasmolysed loaded yeast cells
during storage (p < 0.05, Fig. 2). The main reason behind the higher while the band obtained from nonplasmolysed one (Fig. 3b) was more
antiradical performance of NPYC compared to others is the yeast cell intense. Regarding to Fig. 3c and d, it could be said that plasmolysis
content. The antiradical activities of plasmolysed and nonplasmolysed process shifted to left the bands.
unloaded yeast cells by ABTS.+ and DPPH test were also determined. It
was determined that ABTS.+ and DPPH radical scavenging activities of 3.4.2. DSC characteristics of the samples
plasmolysed and nonplasmolysed unloaded yeast capsules were DSC spectrum of loaded and unloaded microcapsule cells were il-
0.613 μg Trolox/g sample and 2.56% and 2.13 μg Trolox/g sample and lustrated in Fig. 4. When plasmolysed loaded encapsules (a) and plas-
5.39%, respectively. It is clear from the results that the unloaded molysed unloaded yeast cells (c) were compared with each other and it
(empty) yeast capsule bioactivity was affected by the plasmolysis pro- is clear that the loading caused a decrement in Tg and melting peak.
cess and it was resulted that the plasmolysis caused a decrease in the And opposite behavior was observed in crystallization peak and as a
bioactivity of the yeast cells. Plasmolysis which means the loss of cy- result of oil loading, crystallization temperature decreased. When
toplasmic material because of water loss due to osmosis caused re- compared with Fig. 4c and d, due to plasmolysis treatment, Tg, Tm and
moving of the yeast cell components. It is usually performed by im- Tc showed a decrement in yeast cells. While Tg of nonplasmolysed yeast
mersing cells in strong saline or sugar solutions (Pham-Hoang et al., was 77.19 °C and after plasmolysis that value decreased to 56.29 °C. Tm
2013). It was reported that the yeasts (Baker’s or dry yeast) have strong and Tc also decreased from 120.12 °C and 309.07 °C to 108.48 °C and
antioxidant and antiradical activity and could be evaluated as novel and 278.92 °C, respectively. Salari, Bazzaz, Rajabi, and Khashyarmanesh
natural antioxidant ingredient (Hassan, 2011). It was also reported that (2013) informed that exothermic peak was observed at 352 °C by
the antioxidant and antiradical activity of yeast were related to glu- maxium occurrence for freeze-dried nonplasmolysed yeast and also
tathione, Maillard reaction products and sulfur containing amino acids stated that Tc decreased after plasmolysis with 20% (w/v) NaCl solu-
present in the yeast cell (Soomer & Jamieson, 1996). In a study about tion, similar to the current results. Paramera et al. (2011) stated that
the antioxidant performance of the yeasts, the beef patties were added plasmolysis altered the lipid form of the membrane and caused to form
with yeast extract and it was observed that the yeast extracts prevented mobility and, thus, fluidity that’s why plasmolysed yeast cells had lower
the thiobarbituric acid reactive substances formation in the sample Tm compared to nonplasmolysed ones. Regarding to Fig. 4a and b,
(Wang & Xiong, 2005). In the light of this knowledge, it could be in- there were significant differences between the DSC thermograms due to
ferred from that the plasmolysis process increased the oil absorption plasmolysis treatment for encapsulation. In Fig. 4b, it was observed two
capacity and encapsulation efficiency and also increased the stability of endothermic peaks that the first one at 98.10 °C and the second one at
active ingredient but caused a decrease in the bioactivity of the material 208.51 °C while that situation was not detected completely in plas-
due to the removal of the some antioxidant components in the cell of molysed loaded yeast cell (Fig. 4a). It was concluded that the first en-
the yeasts. In another similar study, oxidative and thermal stability of dothermic peak was due to yeast phospholipid bilayer and the second
curcumin encapsulated with yeast cell were investigated and plasmo- one was due to black cumin seed oil (Fig. 4b) and besides not performed
lysed yeast capsule showed weaker antioxidant performance compared of plasmolysis treatment on the yeast cell limited the interaction of
to nonplasmolysed yeast capsule and they expected lower antioxidant yeast cell and oil. These results were in accordance with the report of
activity for the nonplasmolysed yeast capsule because they stated that Paramera et al. (2011).
caustic conditions and high heat would oxidize and degrade any innate
antioxidant systems within the cytoplasm. But the high level of glu- 3.4.3. XRD spectrum of the samples
tathione up to 10 mM (Penninckx, 2002) which is an essential in- XRD results of plasmolysed loaded, nonplasmolysed loaded yeast
tracellular oxidative stress regulator had a strong antioxidant activity. encapsules and also plasmolysed unloaded, nonplasmolysed unloaded
Based on the plasmolysis of the yeast cell, a desiccation process results a yeast cells and the black cumin seed oil were shown in Fig. 5. de Barros
decrement in the ratio of the reduced form of glutathione to the oxi- Fernandes et al. (2016) informed that a crystalline material exhibits
dized form (GSH:GSSG) and this result causes a decrease in the Trolox sharp peaks while amorphous materials provide a broader peak pattern.
equivalent antioxidant performance (Espindola Ade, Gomes, Panek, & In the present study, samples behaved as an amorphous material with
Eleutherio, 2003). poor crystallinity generally. Guler and Sarioglu (2014) also informed
that raw S.cerevisiae cells had amorphous phase similar to the results of
3.4. Morphological and conformational characterization of the yeast the present research. It was observed that black cumin seed oil showed
encapsules 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-
3.4.1. FT-IR spectrum of the samples spectively. When plasmolysis process occurred, two peaks were mon-
Fig. 3 shows FT-IR spectrum of plasmolysed loaded yeast encapsules itored at 2θ = 6.71°, 19.00° for loaded cells and at 2θ = 5.79°, 18.90°
(a), nonplasmolysed loaded yeast encapsules (b), plasmolysed unloaded for unloaded cells. XRD pattern of black cumin seed oil had the highest
yeast cells (c), nonplasmolysed unloaded yeast cells (d) and black intensity and loaded yeast cells followed it. That situation could be
cumin seed oil (e) samples. According to FT–IR spectra (Fig. 3) the explained by black cumin seed oil incorporated the yeast cell and in-
absorption bands were observed at approximately 1741 cm−1 and only tensity values were observed as to be higher than those of the unloaded

8
K. Karaman Food Chemistry 313 (2020) 126129

(empty) ones. Burattini, E., Cavagna, M., Dell’Anna, R., Malvezzi Campeggi, F., Monti, F., & Rossi, F. A.
(2008). FTIR microspectroscopy study of autolysis in cells of the wine yeast
Saccharomyces cerevisiae. Vibrational Spectroscopy, 47, 139–147.
3.4.4. SEM images of the samples Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic living
Fig. 6 illustrates the scanning electron microscope (SEM) images of cells: From laboratory scale to industrial applications. Journal of Food Engineering,
all samples. SEM images of nonprocessed S. cerevisiae (Fig. 6 (g–h)) 104, 467–483.
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with yeast cells. Regarding to the Fig. 6 a–b and c–d, it could be said
de Barros Fernandes, R. V., Borges, S. V., Silva, E. K., da Silva, Y. F., de Souza, H. J. B., do
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bioactive components of black cumin seed oil is thymoquinone and it hibiting a pink-colored cell phenotype. FEMS Microbiological Letters, 197(2), 179–186.
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This work has been supported by Erciyes University Scientific Kavosi, M., Mohammadi, A., Shojaee-Aliabadi, S., Khaksar, R., & Hosseini, S. M. (2017).
Research Projects Coordination Unit under grant number of FHD-2019- Characterization and oxidative stability of purslane seed oil microencapsulated in
yeast cells biocapsules. Journal of the Science of Food and Agriculture, 98, 2490–2497.
8862. Kiralan, M., Özkan, G., Bayrak, A., & Ramadan, M. F. (2014). Physicochemical properties
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Declaration of Competing Interest methods. Industrial Crops and Products, 57, 52–58.
Lutterodt, H., Slavin, M., Whent, M., Turner, E., & Yu, L. L. (2011). Fatty acid composi-
tion, oxidative stability, antioxidant and antiproliferative properties of selected cold-
The author declares that she has no known competing financial pressed grape seed oils and flours. Food Chemistry, 128(2), 391–399.
interests or personal relationships that could have appeared to influ- Ly, M. H., Naitali-Bouchez, M., Meylheuc, T., Bellon-Fontaine, M. N., Le, T. M., Belin, J.
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Acknowledgments (Saccharomyces cerevisiae) for the production of yeast extract: Effects of different
enzymatic treatments on solid, protein and carbohydrate recovery. Journal of. Serbian
Chemical Society, 72, 5.
The author would like to thank to Dr. Mahmut KAPLAN and Dr. Morris, G. J., Winters, L., Coulson, G. E., & Clarke, K. J. (1986). Effect of osmotic stress on
Perihan GÜRBÜZ for providing the opportunity to work in their la- the ultrastructure and viability of the yeast Saccharomyces cerevisiae. Journal of
boratories. The author also would like to thank to Erciyes University General Microbiology, 132, 2023–2034.
Nguyen, T. T., Phan-Thi, H., Pham-Hoang, B. N., Ho, P. T., Tran, T. T. T., & Waché, Y.
Scientific Research Projects Coordination Unit for their financial sup- (2018). Encapsulation of Hibiscus sabdariffa L. anthocyanins as natural colours in
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Supplementary data to this article can be found online at https:// cumin in cells of Saccharomyces cerevisiae. Food Chemistry, 125, 892–902.
Penninckx, M. J. (2002). An overview on glutathione in Saccharomyces versus non-
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