Journal of Food Composition and Analysis 70 (2018) 78–88

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Journal of Food Composition and Analysis

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

Optimization of phycocyanin extraction from Spirulina platensis using T

different techniques
Işıl İltera, Saniye Akyıla, Zeliha Demirelc, Mehmet Koçb, Meltem Conk-Dalayc,
⁎
Figen Kaymak-Ertekina,
a
Faculty of Engineering, Food Engineering Department, Ege University, Bornova, 35100, İzmir, Turkey
b
Faculty of Engineering, Food Engineering Department, Adnan Menderes University, 09010, Aydin, Turkey
c
Faculty of Engineering, Bio Engineering Department, Ege University, Bornova, 35100, İzmir, Turkey

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

Keywords: Phycocyanin is an important commercially available blue food colorant. Herein we report an optimization study
Food colorants of various phycocyanin extraction methods from Spirulina platensis cyanobacterium biomass (dry, frozen and
Food composition wet). Three different solvents i.e. distilled water, Na-Phosphate pH: 7.4 suspension and 1.5% CaCl2 (w/v) water
Phycocyanin solution were applied as the extraction medium. The highest total phycocyanin content (55.33 mg/g) was ex-
ABTS scavenging activity
tracted from frozen biomass using 1.5% CaCl2 (w/v aq.) solution. Process variables of classical, ultrasound and
Ultrasound extraction
Microwave extraction
microwave extraction methods (biomass/solvent ratio, extraction time, vibration, speed, and power) were op-
Classical homogenisation timized considering the CCRD experimental design to enrich phycocyanin. The optimum conditions of extraction
methods; classical, ultrasound and microwave were determined as: 1.71% biomass/solvent ratio, 6237.66
homogenization rate and 15 min extraction time; 1% biomass/solvent ratio, 60% amplitude and 16.23 min
extraction time; 2.34% biomass/solvent ratio, 133.29 W and 165.96 s extraction time. Classical extraction
method provided vivid blue color, a higher amount of phycocyanin, and maximum antioxidant activity as
compared to other extraction methods.

1. Introduction
Spirulina platensis is a non-toxic cyanobacterium characterized by
high levels of carbonate and bicarbonate in an alkaline environment. S.
platensis has been utilized as a food supplement as it contains poly-
saccharides, vitamins, minerals, unsaturated fatty acids, carotenoids,
and phycobiliproteins (Su et al., 2014; Wu et al., 2016). Biomass of S.
platensis consists 55–70% proteins, 3–9% fats, and 15–30% carbohy-
drates in addition to fibers and pigments (Kay and Barton, 1991).
Phycobiliproteins are accessory photosynthetic pigments that are
aggregated in the cell as phycobilisomes, which are attached to the
thylakoid membrane of chloroplast (Arad and Yaron, 1992). Cyano-
bacterial phycobiliproteins can be divided into three main classes;
phycoerythrin (PE –bright pink, red), phycocyanin (PC –dark blue), and
allophycocyanin (AP –brighter blue) (Ghosh et al., 2015; Kumar et al.,
2014; Singh et al., 2015). These phycobiliproteins are widely used in
medicines, foods, cosmetics and fluorescent materials (Eriksen, 2008;
Kannaujiya and Sinha, 2016).
Phycocyanin is a water soluble natural pigment that is widely used
as color additive in foods (chewing gums, dairy products, gellies etc.),
cosmetics (lipstick and eye liners), fluorescent reagent, and as probe
and tracer in clinical diagnostic instruments as well as in immunology
(Eriksen, 2008; Kumar et al., 2014). It has significant antioxidant, anti-
inflammatory, hepatoprotective, and radical scavenging properties
(Martelli et al., 2014; Wu et al., 2016).
There are various techniques reported to extract phycocyanin (Sekar
and Chandramohan, 2008) from S. platensis biomass in various physical
forms (dry, wet and frozen). Its extraction is difficult as the cell wall of
the cyanobacteria is quite resistant (Wyman, 1992), which is made of
four layers i.e. fibril, peptidoglycan, proteins, and analogous to gram-
negative bacteria (Van Eykelenburg, 1977). There are several methods
reported to disrupt the cell wall including homogenization, sonication,
microwave, supercritical fluid extraction and lysozyme disintegration
(Deniz et al., 2016; Duangsee et al., 2009; Martinez et al., 2016).
The extraction process should be effective in terms of high extrac-
tion yield and must be environmental friendly. Ultrasonic cleaning
baths or probe systems are mainly used to breakdown the cell wall of
the bacteria (Vinatoru, 2001). Laboratory scale ultrasonic baths pro-
vides higher extraction yields as compared to the probe system
(Vinatoru et al., 1999). Microwave extraction utilizes microwaves

⁎
Corresponding author.
E-mail address: figen.ertekin@ege.edu.tr (F. Kaymak-Ertekin).

https://doi.org/10.1016/j.jfca.2018.04.007
Received 2 August 2017; Received in revised form 11 April 2018; Accepted 16 April 2018
Available online 20 April 2018
0889-1575/ © 2018 Elsevier Inc. All rights reserved.
I. İlter et al.
(300 MHz–300 GHz) to heat the extraction medium (usually solvent) to
extract most of the useful chemical contents out of bacterial biomass
(Jain et al., 2009). Classical homogenization is generally applied using
Ultra Turrax equipment to efficiently bring targeted compounds and
solvent into contact; without extracting unwanted components from
biomass. However, all of these cell disruption methods lack specificity
as cell debris and other unwanted impurities are also released. Never-
theless, phycocyanin is sensitive to light, oxygen, moisture, and tem-
perature. Therefore, it is able to readily degrade under certain physi-
cochemical conditions (Kannaujiya and Sinha, 2016).
This study was primarily aimed to determine the most effective form
of biomass (frozen, dried and fresh) and most suitable extraction
medium (distilled water, Na-phosphate buffer pH: 7.4 solution and
1.5% CaCl2 (w/v)) to obtain higher amounts of phycocyanin. The
second aim of this study was to compare classical, ultrasound and mi-
crowave extraction methods and their conditions targeting vivid blue
color, maximum phycocyanin concentration, and acceptable anti-
oxidant activity.
2. Materials and methods
2.1. Materials and chemicals
Arthrospira (Spirulina) platensis EGEMACC 38, used to extract phy-
cocyanin, was obtained from the Microalgae Culture Collection of Ege
University. In the algal biotechnology laboratory of Ege University, S.
platensis was grown in Zarrouk medium (Zarrouk, 1966) at 50 μmol
photons m−2 s−1 light intensity and aerated at an airflow rate of 2 L/
min at 22 ± 2 °C.
Folin-Ciocalteu reagent (Merck, Darmstadt, Germany) and sodium
carbonate (Fisher Science, UK) were employed to quantify total poly-
phenols, while gallic acid (Merck, Darmstadt, Germany) was used as a
calibration standard. Trolox (Hoffman-La Roche) (6-hydroxy-2,5,7,8-
tetramethychroman-2-carboxylic acid; Aldrich Chemical Co.,
Gillingham, Dorset, UK) was used as an antioxidant standard. ABTS i.e.
2,2′-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) diammonium
salt, and calcium chloride were obtained from Sigma-Aldrich (Poole,
Dorset, UK).
2.2. Extraction procedures
2.2.1. Determination of biomass form and solvent type
Phycocyanin was extracted from frozen, dried and fresh biomass of
S. platensis; in three different extraction mediums. Biomass was frozen
at −20 ± 2 °C for 24 h while it was dried in the oven at 50 ± 2 °C
with a target of about 8% moisture content. To determine the effect of
pre-treatments (freezing and drying) applied to the biomass, dry matter
content was kept constant. Classical phycocyanin extraction was per-
formed under IKA-Turrax homogenizer rotating at 7000 rpm in distilled
water with Na-phosphate buffer (pH: 7.4) and 1.5% CaCl2 (w/v) as
extraction medium at 25 °C for 10 min. The temperature was controlled
with a circulator water bath (Daihan, WCR P8, Korean). After extrac-
tion, the cell residue was centrifuged at 4000 rpm for 10 min. Crude
extracts were analyzed for phycocyanin content. The most appropriate
form of biomass was chosen considering maximum phycocyanin con-
centration and vivid blue color.
Classical (7000 rpm, 10 min), ultrasound (50% amplitude, 15 min)
and microwave (150 W, 180 s) extractions were performed at 1% bio-
mass/solvent ratio to determine the most suitable extraction solvent. A
mechanical homogenizer (Ultra Turrax, IKA, Model T25, Germany) was
used during classical extraction. Ultrasound extraction was performed
in an ultrasound bath (Daihan Wisd WUC-D06H, Korea,
290 × 150 × 150 mm) at 40 kHz. The intensity of sonication was am-
plitude controlled (independent variable). However, as reported earlier,
the ultrasonic intensity distribution inside the ultrasonic bath is not
homogeneous. Before starting extraction procedure in ultrasound bath,
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Journal of Food Composition and Analysis 70 (2018) 78–88
the most intense zone of sonication inside the bath (aluminum foil test)
was determined for the extraction experiments.
A microwave device (Milestone, Start E, Italy, programmable,
Teflon-coated built-in magnetic stirrer) was used at 2450 MHz in the
microwave extraction method. The extractions were carried out in a
100 mL-sealed vessel made of high-purity TFM, surrounded by a safety
shield (made of HTC, a new high-performance plastic) including a
“vent-and-reseal” safety valve. Temperature was monitored and con-
trolled with the aid of a shielded thermocouple, inserted directly into
the vessel. The evolution of time, temperature, and power were con-
tinuously recorded during each experiment.
Selection of solvent to be used in the classical, ultrasound and mi-
crowave extractions have been determined considering the structural
properties of the S. platensis (Jin et al., 2014; Silveira et al., 2007; Vali
Aftari et al., 2017). Extraction phase in classical and ultrasound
methods; the process temperature was kept constant at 25 ± 2 °C with
the circulator water bath. In the microwave extraction method, the
temperature didn't exceed 40 ± 2 °C in the extraction chamber. Three
different solvents (distilled water, Na-phosphate buffer pH: 7.4 solution
and 1.5% CaCl2 (w/v)) were used as extraction medium. After extrac-
tion, the cell residue was removed and phycocyanin content was ana-
lyzed.
All experiments were performed in triplicate, and the presented
results are means ± 95% confidence interval. The obtained data was
statistically analyzed by analysis of variance using SPSS version 13.0 for
Windows (SPSS Inc., Chicago, IL) and mean analysis was performed
using Duncan’s procedure. The differences were considered significant
when p ≦ 0.05.
2.2.2. Process parameters optimization of the extraction methods
Effect of specific extraction process conditions (homogenization rate
(rpm)/amplitude (%)/microwave power (W)), biomass/solvent ratio
(%) and extraction time of classical, ultrasound and microwave ex-
traction methods on total phycocyanin, total phenolic content, and
ABTS%+ scavenging activity of extracts were investigated using Central
Composite Rotatable Design (CCRD). While the range of biomass/sol-
vent ratio (% w/w, A) was the same for all extraction methods, the
extraction time (min, C) was variable for each procedure.
Homogenization rate (rpm) (B) for classical extraction, amplitude (%,
D) for ultrasound extraction and microwave power (W, E) for micro-
wave extraction methods were selected as independent extraction
method parameters. 1.5% CaCl2 (w/v) was used as extraction medium
for all the extraction methods. The boundary conditions created by the
CCRD of the independent variables for each extraction method are
given in Table 1.
Multiple regression analysis was conducted to fit the Eqs. (1)–(3) on
the experimental data; significant terms of the model were determined
by ANOVA. The CCRD and the corresponding data analysis were carried
out using the Design-Expert 7.0.0 (Stat-Ease Inc., MN, USA).
k k k−1 k
PC = β0 + ∑i =1 βi x i + ∑i =1 βii x i2 + ∑i =1 ∑j=i +1 βij x ji (k = 1,2,3)
(1)
k k k−1 k
TPC = β0 + ∑i =1 βi x i + ∑i =1 βii x i2 + ∑i =1 ∑j=i +1 βij x ji (k = 1,2,3)
(2)
k k
ABTS = β0 + ∑i =1 βi x i + ∑i =1 βii x i2
k−1 k
+ ∑i =1 ∑j=i +1 βij x ji (k = 1,2,3)
(3)
2.3. Analysis
2.3.1. Phycocyanin content measurement
Prior to spectrophotometric measurement, the extract was
I. İlter et al. Journal of Food Composition and Analysis 70 (2018) 78–88

Table 1 2.3.2. Determination of total phenolic content (TPC)

Extraction process variables and levels for the specific CCRD experimental de- TPC of phycocyanin extracts was determined spectro-
sign. photometrically (Varian Cary 50 Bio, UV–vis Spectrophotometer) using
Extraction Independent Coded Values the Folin-Ciocalteu method with slight modifications. Sample (1 mL)
Method Variables was added to 75 mL of water and mixed with previously diluted Folin-
−1.682 −1 0 1 1.682 Ciocalteu reagent with water (1:2 ratio). Subsequently, 10 mL of satu-
rated sodium carbonate solution was added to a flask and diluted up to
Classical Biomass/ (A) 0.32 1 2 3 3.68
Extraction Solvent Ratio 100 mL with water. The samples were mixed and left in the dark at
(CE) (%) room temperature for 1 h. The absorbance was measured at 720 nm
Hom. Rate (B) 3636 5000 7000 9000 10,363 against a blank solution. Triplicate analyses were done for each extract.
(rpm)
Gallic acid was used as a standard (100–700 mg/L) for calibration
Time (min) (C) 1.59 5 10 15 18.41
curve, and TPC was expressed as milligrams of gallic acid equivalents
Ultrasound Biomass/ (A) 0.32 1 2 3 3.68 per liter of extract (mg GAE/L).
Extraction Solvent Ratio
(UE) (%)
Amplitude (%) (D) 19.77 30 45 60 70.23 2.3.3. ABTS%+ scavenging activity
Time (min) (C) 4.89 10 17.5 25 30.11 ABTS radical cation (ABTS%+) was produced by reacting ABTS stock
Microwave Biomass/ (A) 0.32 1 2 3 3.68 solution (7 mmol L−1) with 2.45 mmol L−1 potassium persulfate (final
Extraction Solvent Ratio concentration) and left in the dark at room temperature for 12–16 h. To
(ME) (%)
analyze phenolics, ABTS%+ solution was diluted with PBS, pH: 7.4, to
Power (W) (E) 65.91 100 150 200 234.09
Time (s) (C) 19.09 60 120 180 220.91 an absorbance of 0.70 ( ± 0.02) at 734 nm and equilibrated at 30 °C.
After addition of a 10 μL aliquot of each dilution into the assay, they
produced 20–80% inhibitions of the blank absorbance. After addition of
centrifuged (Nuve NF400, Turkey) at 4000 rpm for 10 min at 20 °C. 1.0 mL of diluted ABTS%+solution (A734nm = 0.700 ± 0.020) to 10 μL
Absorbance of the blue supernatant was determined by a spectro- of antioxidant compounds or Trolox standards (final concentration
photometer (Varian Cary 50 Bio, UV–vis Spectrophotometer) at 615 0–15 μM) in PBS, the absorbance was recorded at 30 °C, exactly 1 min
and 652 nm. Phycocyanin concentration (w/v) was calculated by Eq. after initial mixing and up to 6 min (Varian Cary 50 Bio, UV–vis
(4) (Bennett and Bogorad, 1973). Spectrophotometer). Appropriate solvent blanks were run in each
assay. All experiments were carried out in triplicate, on each occasion
A615 − 0.474 × A652 mg
PC = ⎛ ⎞ and at each separate concentration of the standard and samples. The
5.34 ⎝ ml ⎠ (4)
percentage inhibition of absorbance at 734 nm is calculated and plotted
After the extraction process, the biomass was centrifuged and phy- as a function of the concentration of antioxidants and of Trolox mg TE/
cocyanin content (w/w) of the extracts (dry basis) was calculated using g DW for the standard reference data (Shanab et al., 2012).
Eq. (5) (Silveira et al., 2007).
3. Results

Phycocyanin Content =
PC ( ) × SV
mg
ml
(ml)
mbiomass (g) × DMbiomass (g/g) (5) 3.1. Extraction of phycocyanin

where the phycocyanin content is calculated from the phycocyanin 3.1.1. Selection of physical form of biomass and solvent type
concentration (mg/mL) PC, solvent volume (mL) SV, mass of biomass The effects of different biomass forms and extraction solvents on the
(g) Mbiomass, and dry matter of biomass DMbiomass: phycocyanin content (w/w) during the extraction of phycocyanin from

Fig. 1. Change of the phycocyanin content with respect to solvent type (1: 1.5% CaCl2 (w/v) solution; 2: Sodium phosphate buffer solution (pH: 7.4); 3: Distilled
water) and biomass form. Values with the different letter in each column showed statistically significant difference between and within groups (p < 0.05; n = 3).

80
I. İlter et al.
S. platensis biomass are shown in Fig. 1. Sarada et al. (1999) reported
that various drying parameters such as temperature and drying time led
to approximately 50% loss of phycocyanin in S. platensis. The significant
loss of phycocyanin in dried samples could be due to its peripheral
position in phycobilisomes on the thylakoid membrane and attributable
to its sensitivity towards temperature (Gantt, 1981). According to the
literature, freeze-thawing method is widely used for the extraction of
phycocyanin from fresh biomass that improves the efficiency of ex-
traction (Abalde, 1998; Doke, 2005; Minkova et al., 2003; Soni et al.,
2006). The phycocyanin content of frozen biomass obtained by classical
extraction with three different solvents is higher than that of wet and
dry biomass (Fig. 1). Freezing is a way to store agricultural products to
avoid significant changes in their chemical contents and physical
properties; however, it sometimes breaks the cellular structure, which
may affect the properties of a product (Chan et al., 2013, 2009).
Nakagawa et al. (2016) reported that the cell breakage caused by
freezing improved the phycocyanin extraction yield.
Furthermore, the highest phycocyanin content of frozen biomass
was obtained using 1.5% CaCl2 (w/v) as extraction medium that can be
attributed to its ionic strength, similar to that happens inside in-
tracellular conditions and led to a better extraction yield of phyco-
cyanin (Silveira et al., 2007).
Although Silveira et al. (2007) reported that extraction of phyco-
cyanin with water did not differ much from that of 10 mmol L−1 so-
dium phosphate buffer (pH 7.4), we found two fold higher extraction
yield of phycocyanin in frozen biomass using distilled water as com-
pared to phosphate buffer. Abalde (1998) also found higher extraction
yield of phycocyanin from Synechococcus sp. in distilled water as
compared to phosphate buffer. The lowest phycocyanin content was
observed in sodium phosphate buffer (pH 7.4), while the highest was
achieved with 1.5% CaCl2 (w/v) and distilled water as shown in Fig. 2
(p < 0.05). The color of the extract was evaluated according to the
Hue angle value. Results showed that all phycocyanin extracts with
1.5% CaCl2 (w/v) solution obtained with different extraction methods
were in the blue region (300–360° hue angle) (Fig. 3). However, the
Hue angles of some extracts in PBS and distilled water were out of blue
region. Even though the phycocyanin content of the extract obtained
with distilled water was higher than that obtained with 1.5% CaCl2 (w/
v) solution, the Hue angles of these extracts were not in the blue region.
In case of using 1.5% CaCl2 (w/v) solution as a solvent, the highest
phycocyanin content was provided by microwave followed by
Fig. 2. Change of the phycocyanin content by CE, UE and ME methods with respect to solvent type (1.5% CaCl2 (w/v) solution, sodium phosphate buffer solution
(pH: 7.4), distilled water). Values with the different letter in each column showed statistically significant difference between and within groups (p < 0.05; n = 3).
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Journal of Food Composition and Analysis 70 (2018) 78–88
ultrasound and classical extractions. In microwave extraction, proteins
did not degrade as compared to other methods due to its short extrac-
tion time. However, the color values of extracts obtained with micro-
wave extraction were found to be light blue as compared to the values
of extracts obtained by other methods.
When 1.5% CaCl2 (w/v) solution was used as the extraction solvent,
cell structure of S. platensis did not damage as seen in Fig. 4. This so-
lution only dissolved the sodium-calcium channels without breaking
down the cell, leaving the phycobiliproteins present in the thylakoid
membranes centered in the cell membrane (Hatti-Kaul and Mattiasson,
2003). The blue-red color was obtained because the remaining pig-
ments (except phycocyanin) in the cell could not be released. However,
in ultrasound extraction, the highest phycocyanin content was obtained
with distilled water (p < 0.05). As the ultrasound extraction destroyed
the cell walls, all biological components in the biomass were easily
released. As a result, the blue color was suppressed, and the green color
was observed in the extracts (Fig. 5). Increase in the green color can be
considered as the extraction of more amount of chlorophyll, which is
not required.
As a solvent, distilled water causes dark green color by removing the
chlorophyll-releasing substances, since it completely breaks down the
basic growing cell. In the sodium phosphate buffer solution (pH: 7.4),
the amount of total phycocyanin obtained is considerably lower (with
light green color) than that obtained in the other two solvents.
Although the sodium phosphate buffer solution (pH: 7.4) increased the
ion density to protect the cell relative to the distilled water, the blue
color could not be obtained (Duangsee et al., 2009). Solution of 1.5%
CaCl2 (w/v) gave the darkest blue color and provided the highest
amount of phycocyanin content as compared to other extraction
methods.
3.1.2. The effect of extraction process conditions
In the second part of this study, two green techniques i.e. ultrasound
(UE) and microwave (ME) were compared with the classical extraction
(CE) in terms of total phycocyanin content (PC), total phenolic content
(TPC) and ABTS%+ scavenging activity (Table 2). They were fit to the
quadratic models (Eqs. (1)–(3)) those were the most well-matched with
the experimental data (p < 0.001). The lack of fit of these models was
insignificant that means these models were also suitable mathemati-
cally. The ANOVA results and regression coefficients of these models
are given in Table 3.
I. İlter et al. Journal of Food Composition and Analysis 70 (2018) 78–88

Fig. 3. Change of the Hue angles by CE, UE and ME methods with respect to solvent type (1.5% CaCl2 (w/v) solution, sodium phosphate buffer solution (pH: 7.4),
distilled water). Values with the different letter in each column showed statistically significant difference between and within groups (p < 0.05; n = 3).

Fig. 4. Microscopic images obtained by classical homogenization method with 3 different solvents in S. platensis biomass (A: S. platensis biomass, B: S. platensis
obtained with sodium phosphate buffer solution (pH: 7.4), C: S. platensis obtained with distilled water, D: S. platensis obtained with 1.5% CaCl2 (w/v) solution).

3.1.3. Phycocyanin content (PC)
Increasing the biomass/solvent ratio may improve the diffusivity of
the solvent into the cells and provide more extract. However, an excess
of solvent has been reported to absorb cavitation energy from the ex-
traction system, resulting in a lower extraction yield (Samavati, 2013;
Silveira et al., 2007). In case of less amount of solvent, the biomass
cannot meet sufficient solvent molecules, thus results in insufficient
phycocyanin extraction from the cell; as observed in CE and ME
methods in this study. On the contrary, the changes in biomass/solvent
ratio did not affect the PC in UE method. As shown in Fig. 6, the counter
lines belong to the biomass/solvent ratio are straight.
A significant positive effect of extraction power was observed in UE
method (Table 3). Ultra-sonication provokes cavitation that further
results in cell swelling, more solvent uptake, and pore enlargement
present on the cell walls; thus increase diffusivity across the cell walls.
Enhanced extraction yields obtained from UE can also be attributed to
the fact that sonication breaks down the cell walls and facilitates the
washing out of the cell content (Vinatoru, 2001).
According to Fig. 6, PC was significantly influenced by the extrac-
tion time in all extraction methods. PC increased with the time, as the
temperature was kept constant throughout the experiment using the
circulator water bath to avoid this effect.
The maximum PC (102.97 mg/g) (Exp. No: 6) was obtained when
3% biomass/solvent ratio, 30% amplitude and 25 min of extraction
time was used in UE method, whereas the minimum PC (9.34 mg/g)
(Exp. No: 9) was obtained when biomass/solvent ratio of 0.32%, mi-
crowave power of 150 W and 120 s extraction time conditions were
used in ME method. However, using CE method, the maximum phy-
cocyanin content (74.74 mg/g) (Exp. No: 14) was obtained at biomass/
solvent ratio of 2%, homogenization rate of 7000 rpm and extraction
time of 18.41 min. The CE method was found as the most suitable
method to extract total PC as compared to the other methods

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I. İlter et al.
Fig. 5. Images of extracts obtained from 3 different methods using 3 different solvents in S. platensis biomass. (A, B and C are extraction methods; CE, UE and ME,
respectively. 1, 2 and 3 are solvent types 1.5% CaCl2 (w/v) solution, sodium phosphate buffer solution (pH: 7.4), distilled water, respectively).
considering the vivid blue color obtained together with higher PC.
3.1.4. Total phenolic content (TPC)
Phenolics are important antioxidants as they can donate a hydrogen
atom or an electron to form stable radical intermediates. The pertur-
bation of the predicted model for TPC is illustrated in Fig. 6. TPC was
significantly influenced by the biomass/solvent ratio in all extraction
methods. In all extraction methods, TPC decreased as the amount of
solvent was decreased. Less solvent caused a higher consistency in the
extraction medium, thus inhibited diffusion of phenolics to the extract.
Furthermore, the maximum TPC (368.24 mg GAE/L) (Exp. No: 8)
was provided by UE method, as it destroyed the tissues (Table 2). UE
has acoustic cavitation and thermal effects. It reduces the particle size,
damages the cell walls, modifies the microstructure, intensifies the
solvent penetration in the cells; thus instantaneously releases the
polyphenolics into the surrounding medium (Both et al., 2014; Deng
et al., 2015).
Several studies have found that TPC increases quickly with the in-
crease of biomass ratio, at room and high temperatures or in the pre-
sence of ultrasound waves (Bucic-Kojic et al., 2007; Galvan
D’Alessandro et al., 2012; Saénz et al., 2009; Su et al., 2014; Yang and
Zhang, 2008). Accordingly, the extraction of TPC is based on the energy
input to increase the chemical solubility and the rate of mass transfer,
which are important factors for efficient mass transfer in addition to the
extraction power and time variables (Prasad et al., 2009; Shouqin et al.,
2004). Experimental results indicated that extracts obtained from ME
method have lower TPC than CE due to its short extraction time.
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Journal of Food Composition and Analysis 70 (2018) 78–88
3.1.5. ABTS%+ scavenging activity
Phycocyanin is the major phycobiliprotein (Pleonsil et al., 2013; Re
et al., 1999) in S. platensis that contains high level of glutamic acid
(GLU), aspartic acid (ASP), alanine (ALA), leucine (LEU), arginine
(ARG), isoleucine (ILE), serine (SER), glycine (GLY), and threonine
(THR) (Gang et al., 1999); most of them reported as valuable anti-
oxidants (Tounkara et al., 2014). The antioxidant activity of phyco-
cyanin solution was evaluated by the ABTS methods (Ko et al., 2014;
Wang et al., 2015).
The perturbation of the predicted model for ABTS%+ is illustrated
in Fig. 6. ABTS%+ was significantly influenced by the biomass/solvent
ratio and extraction power. Wu et al. (2016) reported higher ABTS
radical-scavenging activity with the increase in phycocyanin con-
centration. It is well known that oxidative reactions and reactive
oxygen species (ROS) cause atherosclerosis, diabetes, Alzheimer’s dis-
ease etc. (Mohsenzadegan and Mirshafiey, 2012; Sharma and de Haan,
2014). ABTS scavenging activity data clearly demonstrates that food-
grade phycocyanin possesses significant antioxidant activity. It means
that food-grade phycocyanin might prevent certain diseases if con-
sumed.
The ABTS radical-scavenging activity data of S. platensis biomass
obtained using three different extraction methods are shown in Table 2.
The maximum ABTS%+ scavenging activity (43.01 mmol L−1 Trolox/
mL) (Exp. No: 8) was provided by the sample having 3% biomass/sol-
vent ratio, 9000 rpm homogenization rate and 15 min of extraction time
in CE method. Whereas the minimum ABTS%+ scavenging activity
(19.07 mmol L−1 Trolox/mL) (Exp. No: 1) was obtained from UE
 I. İlter et al.

Table 2
Total phycocyanin, total phenolic content and ABTS%+ scavenging activity of three extraction methods at 20 different experimental conditions.
No A Xa C Extraction methods

CE UE ME

PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml) PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml) PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml)

1 −1 −1 −1 39.97 ± 0.70 117.76 ± 1.35 26.54 ± 0.09 86.11 ± 0.27 156.70 ± 0.86 19.07 ± 1.82 14.69 ± 0.22 78.78 ± 1.20 22.46 ± 0.34
2 1 −1 −1 39.97 ± 0.28 209.95 ± 1.64 30.20 ± 0.97 86.65 ± 0.04 329.86 ± 1.60 25.88 ± 0.39 19.66 ± 0.20 182.76 ± 1.20 28.54 ± 0.09
3 −1 1 −1 52.16 ± 1.23 97.56 ± 2.35 33.86 ± 0.13 95.04 ± 1.17 150.93 ± 0.98 26.88 ± 0.77 14.43 ± 2.67 74.71 ± 3.75 23.13 ± 0.01
4 1 1 −1 38.43 ± 0.39 226.40 ± 7.35 41.71 ± 0.38 60.44 ± 0.75 333.63 ± 0.52 32.10 ± 0.68 19.68 ± 4.76 147.76 ± 1.92 30.92 ± 0.50
5 −1 −1 1 61.30 ± 1.06 124.83 ± 1.31 31.43 ± 0.00 85.38 ± 0.97 139.52 ± 0.17 30.01 ± 0.06 18.60 ± 1.62 94.32 ± 0.53 25.08 ± 0.08
6 1 −1 1 58.83 ± 1.73 204.20 ± 2.73 40.09 ± 0.66 102.98 ± 1.14 308.92 ± 0.26 37.06 ± 1.09 25.28 ± 1.18 183.12 ± 1.75 29.07 ± 0.48
7 −1 1 1 63.49 ± 0.83 117.08 ± 0.67 30.37 ± 0.15 91.59 ± 0.03 202.49 ± 1.63 31.98 ± 0.45 18.25 ± 0.03 87.32 ± 0.18 24.78 ± 0.02

84
8 1 1 1 42.67 ± 0.33 218.52 ± 4.81 43.01 ± 0.19 83.37 ± 0.70 368.24 ± 0.49 38.34 ± 1.95 22.73 ± 0.36 183.36 ± 3.30 30.36 ± 0.01
9 −1.68 0 0 43.54 ± 1.16 47.15 ± 0.98 23.96 ± 0.25 93.77 ± 1.03 54.73 ± 1.41 20.07 ± 0.55 9.34 ± 0.08 32.81 ± 2.05 23.31 ± 1.32
10 1.68 0 0 25.83 ± 0.82 201.26 ± 1.74 42.94 ± 0.52 93.68 ± 1.60 347.23 ± 0.39 35.76 ± 0.97 19.85 ± 0.00 189.61 ± 0.84 33.43 ± 1.27
11 0 −1.68 0 48.66 ± 0.49 145.03 ± 1.61 29.97 ± 0.00 99.75 ± 1.22 268.20 ± 1.84 30.38 ± 0.75 23.25 ± 0.30 141.55 ± 0.20 23.93 ± 0.13
12 0 1.68 0 53.09 ± 0.14 197.47 ± 2.74 42.96 ± 0.13 92.86 ± 0.14 292.36 ± 1.17 36.86 ± 0.32 18.69 ± 0.04 138.74 ± 0.96 25.72 ± 0.09
13 0 0 −1.68 53.46 ± 1.14 158.55 ± 2.35 28.37 ± 0.06 70.46 ± 1.25 221.35 ± 0.13 21.73 ± 0.17 18.16 ± 0.12 116.98 ± 2.05 28.33 ± 0.09
14 0 0 1.68 74.74 ± 0.63 176.62 ± 3.94 40.28 ± 1.18 77.51 ± 0.46 278.89 ± 2.97 38.43 ± 0.24 28.54 ± 0.37 156.74 ± 0.44 29.34 ± 0.01
15 0 0 0 55.47 ± 0.86 145.84 ± 8.71 40.70 ± 0.25 91.86 ± 0.43 250.94 ± 2.86 35.43 ± 0.25 28.82 ± 1.10 135.26 ± 0.46 28.43 ± 0.09
16 0 0 0 54.81 ± 1.19 179.04 ± 3.19 39.53 ± 0.00 83.26 ± 1.60 232.85 ± 0.83 36.52 ± 1.03 27.44 ± 0.54 147.56 ± 1.20 27.61 ± 0.03
17 0 0 0 52.77 ± 0.40 154.26 ± 8.37 36.23 ± 0.45 88.12 ± 1.67 240.76 ± 2.15 33.34 ± 0.64 27.06 ± 0.71 126.50 ± 2.82 28.00 ± 1.18
18 0 0 0 65.67 ± 0.81 186.86 ± 0.95 39.88 ± 0.31 91.34 ± 1.15 244.06 ± 0.15 31.51 ± 0.30 28.22 ± 0.17 133.64 ± 1.17 29.89 ± 0.62
19 0 0 0 58.70 ± 1.18 199.68 ± 5.13 39.26 ± 1.41 84.66 ± 1.49 244.19 ± 1.83 37.20 ± 0.12 24.88 ± 3.48 126.05 ± 0.98 30.25 ± 1.29
20 0 0 0 59.29 ± 0.60 198.31 ± 1.57 39.99 ± 0.68 87.49 ± 0.16 251.59 ± 1.03 36.00 ± 8.35 26.63 ± 0.53 152.23 ± 0.28 28.75 ± 0.01

a
Factor X represented the extraction power depend on the method, homogenisation rate (rpm) for CE, amplitude (%) for UE, power (W) for ME.
Journal of Food Composition and Analysis 70 (2018) 78–88
 I. İlter et al.

Table 3
ANOVA results for each response variables of the optimization process in terms of total phycocyanin, total phenolic content, and ABTS%+ scavening activity with three extraction methods.
Source df CE UE ME

PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml) PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml) PC (mg/g) TPC (mg GAE/L) ABTS (mM trolox/ml)

SS p-value SS p-value SS p-value SS p-value SS p-value SS p-value SS p-value SS p-value SS p-value

Model 9 2244 < 0.000 37,289 0.000 671.2 < 0.000 1612.7 < 0.000 114,000 0.000 673.0 < 0.000 544.5 < 0.000 31,560 < 0.000 163.3 < 0.000
A 1 326.6 < 0.000 31,995 < 0.000 307.1 < 0.000 32.9 0.120 103,000 < 0.000 196.7 < 0.000 111.7 < 0.000 28,656 < 0.000 119.8 < 0.000
a
X 1 1.20 0.660 606.8 0.240 132.5 < 0.000 110.9 0.010 1896 0.000 58.2 0.000 8.55 0.050 187.3 0.180 3.64 0.040
C 1 613.6 < 0.000 137.6 0.560 77.9 0.000 138.9 0.010 1536 0.000 277.2 < 0.000 84.0 < 0.000 1256.5 0.000 2.59 0.070
AX 1 128.6 0.000 430.8 0.316 8.34 0.170 479.4 < 0.000 4.34 0.830 0.650 0.670 0.46 0.620 70.1 0.400 1.36 0.170
AC 1 11.4 0.200 202.2 0.488 12.0 0.110 246.6 0.000 53.7 0.470 0.240 0.790 0.11 0.800 7.63 0.780 2.30 0.090

85
XC 1 75.7 0.010 13.3 0.857 36.0 0.010 2.96 0.620 1931 0.000 14.5 0.060 0.88 0.490 130.4 0.260 0.530 0.380
2
A 1 884.8 < 0.000 3846 0.010 58.7 0.000 44.9 0.080 2895 0.000 92.1 0.000 285.6 < 0.000 1183 0.000 0.950 0.250
2
X 1 64.3 0.010 1.2 0.956 13.1 0.100 101 0.010 2769 0.000 3.75 0.310 69.5 < 0.000 19.7 0.650 32.8 < 0.000
2
C 1 94.7 0.000 14.5 0.851 42.0 0.010 396.4 0.000 147.5 0.240 44.7 0.000 26.5 0.000 0.001 1.000 0.130 0.670
Residual 10 61.26 3893 38.4 115.5 956.5 32.9 17.5 903.4 6.35
Lack of Fit 5 15.4 0.870 2557 0.750 26.2 0.210 55.7 0.530 715.9 0.130 9.65 0.820 8.09 0.560 315.7 0.740 0.870 0.970
Pure Error 5 45.86 2557 12.2 55.76 240.6 23.2 9.42 587.7 5.48
Cor Total 19 2305 41,182 710 1728 115,000 706 562 32,464 170
2
R 0.973 0.906 0.946 0.933 0.992 0.953 0.969 0.972 0.963
2
Radj 0.950 0.820 0.897 0.873 0.984 0.912 0.941 0.947 0.929
C.V. % 4.77 11.94 5.43 3.89 3.98 5.72 6.09 7.23 2.89
PRESS 187 13,848 217 516 5815 107 76 3530 14
Adeq Precision 27.9 12.4 14.8 16.1 45.0 16.2 19.0 22.9 18.5

a
Factor X represented the extraction power depend on the method, homogenisation rate (rpm) for CE, amplitude (%) for UE, power (W) for ME.
Journal of Food Composition and Analysis 70 (2018) 78–88
I. İlter et al. Journal of Food Composition and Analysis 70 (2018) 78–88

Fig. 6. Perturbation plots of process variables; biomass/solvent ratio (%), homogenization rate (rpm), amplitude (%), power (W), extraction time on PC, TPC and
ABTS (Factors K, L, M represented the extraction methods (CE, UE and ME) and subunit numbers for the responses (PC, TPC and ABTS)).

Table 4
Results of statistical analysis for verification of optimization.
Extraction Method Responses Predicted Value Experimental Valuea SEb Difference % Errorc p-Value

CE PC (mg/g) 67.61 68.12 0.583 0.551 0.808 0.432

TPC (mg GAE/L) 162.93 168.75 0.491 5.789 3.43 0.000
ABTS (mM trolox/ml) 37.64 37.09 0.495 0.551 1.48 0.327

UE PC (mg/g) 98.84 98.17 0.602 0.672 0.684 0.327

TPC (mg GAE/L) 163.24 245.39 0.619 81.68 33.35 0.000
ABTS (mM trolox/ml) 29.96 33.17 0.437 3.21 9.67 0.002

ME PC (mg/g) 28.90 29.14 0.474 0.241 0.826 0.637

TPC (mg GAE/L) 159.57 156.77 0.667 2.897 1.784 0.014
ABTS (mM trolox/ml) 29.65 29.75 0.381 0.958 3.339 0.066

a
Experimental values were expressed as mean ± standard deviation.
b
Mean standard error.
c
The % error = (|yexp − ypre|/yexp) × 100, yexp: experimental value, ypre: predicted value.

method with the sample having 1% biomass/solvent ratio, 30% am- process conditions were determined as follows: 1.71% of biomass/sol-
plitude and 10 min of extraction time. vent ratio, 6237.66 rpm of homogenization rate and 15 min of extrac-
tion time. PC, TPC, and ABTS%+ scavenging activity at these optimum
conditions were predicted as 67.61 mg/g, 162.93 mg GAE/L and
3.1.6. Optimization
37.64 mmol L−1 Trolox/mL, respectively. On the other hand, the op-
The optimum extraction conditions i.e., biomass/solvent ratio (%),
timum extraction process conditions for UE were determined as follows:
ultrasound (amplitude) (%) and microwave power (W), homogeniza-
1.00% of biomass/solvent ratio, 60% of amplitude, and 16.23 min of
tion rate (rpm), extraction time for all three extraction methods were
extraction time. PC, TPC and ABTS%+ scavenging activity at the op-
determined targeting the maximum PC, acceptable TPC and ABTS%+
timum conditions were predicted as follows: 98.84 mg/g, 163.24 mg
scavenging activity. A second order polynomial model fit well to the
GAE/L and 29.96 mmol L−1 Trolox/mL, respectively. ME method for
experimental data with lower standard error and higher regression
phycocyanin extraction gave the optimum process conditions as fol-
coefficient (R2) values. Regression analysis was performed on the ex-
lows: 2.34% of biomass/solvent ratio, 133.29 W and 165.96 s of ex-
perimental data and the coefficients of model were evaluated for sta-
traction time. PC, TPC and ABTS%+ scavenging activity at the optimum
tistical significance using ANOVA analysis. Regression coefficients of
conditions were predicted as: 28.90 mg/g, 159.57 mg GAE/L and
predicted second order polynomial models are outlined in Table 3.
29.65 mmol L−1 Trolox/mL, respectively.
When desirability function approach was applied, the optimum CE

86
I. İlter et al.
After performing five validation experiments at optimum extraction
process conditions, PC and ABTS%+ scavenging activity of the obtained
samples were found insignificant (p > 0.05). Conversely, TPC of the
obtained samples were found to be significantly (p < 0.05) different
from the predicted values determined by Design Expert- version 7.0
software (Table 4).
Considering that, equipments and methods have their specificities
and that it was impossible to perform the experiments under the same
conditions, a comparison was made to observe the results of each
method. As shown in Table 2, the highest PC of extract was obtained
using UE method where TPC was higher than those obtained from other
methods. However, a comparison of the three methods examined in the
present study indicated that the CE method resulted in targeting vivid
blue color, maximum PC, and maximum antioxidant activity simulta-
neously.
4. Conclusion
Herein this study, we have extracted phycocyanin – an antioxidant
and commercial blue colored food dye – from Spirulina platensis using
various extraction methods (classical homogenization, microwave
treatment and ultra-sonication), physical form of biomass (wet, dried
and frozen) and solvents. Effects of process variables of extraction
methods such as biomass/solvent ratio, extraction time, amplitude,
speed or/and power and Hue angles of extracts (color values) were
investigated. As a conclusion, phycocyanin extraction from frozen S.
platensis biomass was found as more suitable than dried and wet bio-
masses, while the most appropriate extraction solvent was found as
aqueous 1.5% CaCl2 (w/v). The optimum conditions of classical, ul-
trasound and microwave extractions were found as 1.71% biomass/
solvent ratio, 6237.66 homogenization rate and 15 min extraction time;
1% biomass/solvent ratio, 60% amplitude and 16.23 min extraction
time; 2.34% biomass/solvent ratio, 133.29 W and 165.96 s extraction
time, respectively; keeping in mind the maximum phycocyanin content
(PC), total phenolic content and antioxidant activity. The optimum
conditions for all three extraction methods showed that the maximum
PC of the extracts was obtained with ultrasound extraction method.
However, phycocyanin extraction with classical method resulted in a
more vivid blue color as well as in a higher phycocyanin content and
antioxidant activity. An extraction process should not only be effective
in terms of high extraction yield but also be highly selective, as pur-
ification of phycocyanin would need time and cost. This study showed
that the vivid blue color of the extracts was not related to a high PC in
extracts. Hence, the physical form of the biomass, the type of solvent
extraction methods and the level of their process variables should be
determined considering the targets in the extraction process.
Acknowledgments
This study was a part of Cost action ES1408 and the authors would
like to thank The Scientific and Technological Research Council of
Turkey (TUBITAK-115O578) for financial support and Council of
Scientific Research Projects (BAP-15MUH025) of Ege University,
Turkey.
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