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2. Author's personal copy
Supercritical CO2 extraction of c-linolenic acid (GLA) from Spirulina
platensis ARM 740 using response surface methodology
M.G. Sajilata, Rekha S. Singhal *, Madhusudan Y. Kamat
Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Mumbai 400 019, India
Received 8 March 2007; received in revised form 24 April 2007; accepted 11 May 2007
Available online 26 May 2007
Abstract
Spirulina platensis, a blue-green algae, is a potential source of the nutraceutical, c-linolenic acid (GLA). The present work reports on
recovery of GLA from S. platensis ARM 740 by supercritical carbon dioxide extraction (SCE) in comparison with conventional solvent
extraction. Response Surface Methodology (RSM) was applied to optimize the operating conditions for high recovery of GLA by SCE.
The levels studied were a pressure range between 100 and 500 bars, a time period between 26 min and 94 min and an ethanol level of
9.64–16.4 ml ethanol/16 g of freeze-dried biomass. The use of ethanol as a co-solvent with CO2 considerably increased the GLA yields
compared to SCE. A recovery of 102% GLA in the supercritical extract (as compared to Bligh and Dyer extraction) was obtained in 1 h
using a minimum of 13.7 ml of ethanol as co-solvent per 16 g of biomass, a temperature of 40 °C and a pressure of 400 bars. Supercritical
CO2 with a co-solvent is therefore recommended as a better option to the conventional solvents for the complete recovery of GLA from
S. platensis.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: GLA; Supercritical CO2 extraction; Spirulina platensis; Response surface methodology (RSM)
1. Introduction
GLA, an x6-polyunsaturated fatty acid is a metabolite
of linoleic acid (LA) and the first intermediate in the con-
version of LA to arachidonic acid (AA). There is ample evi-
dence to suggest that the activity of D-6-desaturase
involved in the conversion of linoleic acid to GLA is
impaired in several diseased states, which may be amelio-
rated with an exogeneous supply of GLA. This possibility
has triggered a potential interest in GLA for commercial
production.
GLA is mainly used as a dietary supplement to increase
the production of the anti-inflammatory 1-series prosta-
glandins. Elderly patients with diminished activity of D-5-
and D-6-desaturase constitute a target group in need of
GLA-rich fat emulsions. GLA supplements have also been
indicated to be beneficial in the development of cell mem-
branes of infants. In cases where infants cannot be breast-
fed, formulae containing GLA as a precursor of AA could
be provided (Uauy & Mena, 1999). Recent studies have
shown GLA to lower the low density lipoprotein (LDL)
in hypocholesterolemic patients (Ishikawa et al., 1989),
the alleviation of the symptoms of pre-menstrual syndrome
(Horrobin, 1983), and the treatment of atopic eczema (Biagi
et al., 1988). It is also implicated in the amelioration of a
number of diseased states including schizophrenia, multiple
sclerosis, dermatitis, diabetes and rheumatoid arthritis.
The need for a rapid, efficient, and safe method for the
extraction of GLA from natural sources has been broadly
emphasized in view of it being used for human consump-
tion. The blue-green alga, Spirulina platensis, is reportedly
a potential source of GLA, which comprises about 18–21%
of the fatty acid composition of the lipids (Gunstone, 1992;
Mendes, Reis, & Palavra, 2006). Toxicity studies have
revealed Spirulina to be non-toxic and safe (Chamorro,
1980; Chamorro, Salazar, Castillo, Steele, & Salazar,
0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2007.05.028
*
Corresponding author. Fax: +91 022 24145614.
E-mail address: rekha@udct.org (R.S. Singhal).
www.elsevier.com/locate/jfoodeng
Journal of Food Engineering 84 (2008) 321–326

3. Author's personal copy
1997; Salazar, Chamorro, Salazar, & Steele, 1996; Salazar,
Martinez, Madrigal, Ruiz, & Chamorro, 1998). Spirulina
can also be cultivated commercially in open ponds in a con-
tinuous regime. All these factors have contributed to Spiru-
lina being a viable source for the production of the much
sought after GLA.
Among the extraction methods for enhanced recovery,
supercritical fluid extraction is considered one of the most
promising techniques in producing solvent-free extracts.
Mendes et al. (2006) reported a maximum recovery of
45% GLA from Spirulina maxima by supercritical carbon
dioxide extraction (SCE) with ethanol as a co-solvent at
350 bars and 60 °C. However, the SCE was carried out
only at 50 and 60 °C and pressures of 250 and 350 bars.
In the present study, attempts were made to use a wide
range of pressure and temperature conditions and ethanol
concentration to maximize the extraction of GLA by SCE.
In this context, a statistical experimental design using
response surface methodology (RSM) was attempted to
optimize the conditions most suitable for the extraction.
RSM consists of a group of empirical techniques to evalu-
ate relations existing between a cluster of controlled exper-
imental factors and the measured responses, according to
one or more selected criteria (Akhnazarova & Kefarov,
1982; Teruel, Gontier, Bienaime, Saucedo, & Barbotin,
1997). A prior knowledge and understanding of the process
and the process variables under investigation are necessary
for achieving a realistic model.
In the present study, the efficacy of SCE of GLA from S.
platensis was evaluated vis-a`-vis conventional solvent
extraction. RSM was applied to optimize the operating
conditions for the extraction of GLA. A 23
full-factorial
central composite design (CCD) was chosen to explain
the combined effects of 3 parameters, viz. pressure, time,
and ethanol concentration on GLA recovery.
2. Materials and methods
All reagents were of AR grade. GLA methyl ester stan-
dard was procured from Sigma Chemical Company (USA)
Ltd. The other fatty acid methyl ester (FAME) standards
were procured from Merck India Ltd.
2.1. Microorganisms
S. platensis ARM-740 was procured from CFTRI,
Mysore, India.
2.2. Medium
The medium used for the cultivation of S. platensis was
SOT medium containing (g/l) NaHCO3, 16.8; K2HPO4,
0.5; NaNO3, 2.5; K2SO4, 1; NaCl, 1; MgSO4 Á 7H2O, 0.2;
CaCl2 Á 2H2O, 0.04; FeSO4 Á 7H2O, 0.01, ethylene diamine
tetraacetate, 0.08 and A-5 trace metal solution, 1 ml. The
A-5 trace metal solution contained (g/l) H3BO3, 2.86;
MnCl2 Á 4H2O, 1.81; ZnSO4 Á 7H2O, 0.22; Na2MoO4 Á
2H2O, 0.039; CuSO4 Á 5H2O, 0.079 and Co(NO3)2 Á 6H2O,
0.49 (Hirano et al., 1990).
2.3. Cultivation
Stock cultures of S. platensis were maintained and inoc-
ula transferred according to Vonshak (1986). S. platensis
was cultivated in two carboys, each of 18 l capacity at
28–30 °C and harvested after 10 days for higher yield of
biomass. Cultures were illuminated with 6 Philips cool
white fluorescent tubes (40 W each) providing 1200 lux.
The cells were harvested by filtration through a nylon mesh
(33 lm). The cell paste was lyophilized and stored at
À20 °C for further use.
2.4. Extraction of lipids
2.4.1. Bligh and Dyer method (Bligh & Dyer, 1959)
One gram of dried biomass was treated with a mixture
of 100 ml methanol, 50 ml chloroform and 38 ml water,
sonicated for 10 min and shaken overnight for maximum
extraction of lipids. The mixture was vacuum-filtered using
a Buchner funnel through Whatmann No. 3 filter paper.
The supernatant after filtration was centrifuged at
8000 rpm at a low temperature of 8 °C, treated with
50 ml chloroform and 50 ml water, mixed well and the
chloroform layer retrieved after separation in a separating
funnel. The emulsion formed was removed with the addi-
tion of common salt, which helped in better recovery of
the lipids and subsequently GLA. The aqueous methanolic
upper layer in the separating funnel was further treated
with about 25 ml chloroform, mixed well and the chloro-
form layers were combined to obtain total lipids on
removal of the solvent. The lipids so obtained were stored
under nitrogen atmosphere at À20 °C, until further use.
2.4.2. Extraction with methanol:acetyl chloride (95/5% v/v)
One gram of dried biomass was treated with 20 ml of
methanol-acetyl chloride (95:5). The mixture was sealed
in a Teflon-lined vial under a nitrogen atmosphere and
heated to 80 °C for 1 h. The vial contents were cooled
and extracted with 10 ml of hexane containing 0.01% butyl-
ated hydroxyl toluene. The extract obtained by this method
contains mixtures of FAME in the hexane layer. The hex-
ane layer was passed through a bed of Na2SO4, concen-
trated under a stream of nitrogen and injected into GC
for quantitative determination of FAME.
2.4.3. Supercritical carbon dioxide extraction (SCE)
For each experiment, 16 g of freeze-dried material with a
particle size of 250 lm was subjected to supercritical CO2
extraction. Pure carbon dioxide of >99% purity was used
for the extraction. For extractions by SCE with co-solvent,
the required level of ethanol was uniformly mixed with the
biomass and immediately filled into the extraction vessel
prior to extraction. A static time of 5 min was maintained
for all the trials undertaken.
322 M.G. Sajilata et al. / Journal of Food Engineering 84 (2008) 321–326

4. Author's personal copy
A laboratory-scale supercritical equipment (SPEED-
SFE) of Applied Separations, USA, was used. A plug of
glass wool was pushed to the closed end of a SS 316 high-
pressure extraction vessel fitted with a filter-containing
metal frit and tamped tightly in place. A known amount
of sample was placed in the vessel and a second plug of
glass wool was placed above the sample matrix. SPEED
matrix (a hydromatrix and dispersing agent) was added
to eliminate the dead volume of the extraction vessel. A
wad of glass wool was put in place just before fastening
the vessel with a filter-containing metal frit. The vessel
was packed firmly to ensure that carbon dioxide diffused
uniformly through the sample matrix. The vessel was
placed in the oven module and a thermocouple was con-
nected to the vessel body. A pressure tight collection vial
of Borosil glass with Teflon caps and two septa of 100 ml
capacity were used to collect the extract from CO2. A glass
flow meter or rotameter (LPM CO2 black glass float) with a
working range of 0.2–2.2 l/min, provided at the collection
end, was used to measure the flow rate of CO2.
2.5. Analysis
2.5.1. Lipid transmethylation (Cohen & Cohen, 1991)
Hundred milligrams of dried biomass was treated with
3 ml of methanol-acetyl chloride (95:5). The mixture was
sealed in a Teflon-lined vial under nitrogen atmosphere
and heated to 80 °C for 1 h. The vial contents were cooled
and extracted in 1 ml of hexane containing 0.01% butylated
hydroxyl toluene.
2.5.2. Fatty acid analysis
The FAMEs were analyzed by GC (Chemito 8510 HR)
using a highly polar EGSS-X column procured from Chro-
matopak, Mumbai. An injection and detection temperature
of 250 °C and a column temperature of 180 °C were used
for GC analysis. Components of FAME were identified
by comparing their retention times of the integrated peaks
with those of authentic standard FAME. Quantitative
determinations were carried out using heptadecanoic acid
methyl ester as the internal standard.
2.6. Experimental design and optimization
For SCE, the significant independent variables are tem-
perature, pressure, concentration of co-solvent, flow rate,
and time. However, in the present work, temperature was
kept a constant at 40 °C, since preliminary trials (not
reported here) showed a temperature range of 40–50 °C
to be most suitable. Also, a flow rate of 0.7 l/min for car-
bon dioxide was kept constant throughout the RSM
experiments.
For a scientific or engineering investigation concerned
with a process or system response Y that depends on the
input factors (also called input variables) X1, X2, X3 . . .XK,
the relationship between response and variables can be mod-
eled by
Y ¼ f ðX1; X2; . . . XK Þ þ e;
where e is an error term that represents the sources of var-
iability not captured by f. It is assumed that the e over dif-
ferent runs are independent, and have mean zero.
In developing the regression equation, the test variables
were coded according to the equation
xi ¼
Xi À Xcp
DXi
where xi is the independent variable coded value. Xi is the
independent variable real value. Xcp is the independent var-
iable real value at the centre point and DXi is the step
change of the real value of the variable ‘i’ corresponding
to a variation of a unit for the dimensionless value of the
variable ‘i’.
The response variable (recovery of GLA) was fitted by a
second order model in order to correlate the response vari-
ables to the independent variables. The general equation of
the second degree polynomial equation is
Y i ¼ bo þ
X
biXi þ
X
bijXiXj þ
X
biiX2
i þ e;
where Yi is the predicted response; Xi, Xj are input vari-
ables which influence the response variable Y; bo is the
ith linear coefficient; bii is the quadratic coefficient and bij
is the linear-by-linear interaction between Xi and Xj, where
‘i’ tends from 1 to 3.
A 23
full factorial central composite design (CCD) for
three independent variables each at five levels with six star
points and six replicates at the centre points was employed
to fit a second order polynomial model which indicated 20
experiments to be required for this procedure. The ‘Design
Expert’ software (version 6.0.10, Stat-Ease, Inc., Minneap-
olis, USA) was used for regression and graphical analysis
of the data obtained. The statistical analysis of the model
was performed in the form of analysis of variance
(ANOVA). This analysis includes the Fisher’s F-test (over-
all model significance), its associated probability P(F), cor-
relation coefficient R, determination coefficient R2
which
measures the goodness of fit of regression model. It also
includes the t-value for the estimated coefficients and asso-
ciated probabilities, P(t). For each variable, the quadratic
models are represented as response surface plots.
3. Results and discussion
The total lipid content of S. platensis ARM 740 was
found to account for 8.63% of the freeze-dried biomass.
The GLA content of the lyophilized biomass was 0.64%
on the basis of dry weight of biomass. The composition
of the fatty acid methyl esters (FAME) assessed by direct
transesterification of the freeze-dried material was found
to be: myristic acid – 4.38%, palmitic acid – 53.09%, oleic
acid – 18.37%, GLA – 15.8%, linoleic acid – 6.57%, and
stearic acid – 1.8%.
In the extraction of GLA from biomass, the Bligh and
Dyer extraction was compared with extraction using a
M.G. Sajilata et al. / Journal of Food Engineering 84 (2008) 321–326 323

5. Author's personal copy
mixture of methanol:acetyl chloride in the ratio of 95:5.
The Bligh and Dyer method involves the use of many sol-
vents and is a tedious and time-consuming process.
Although, extraction using methanol and acetyl chloride
(95:5) is rapid, it involves the use of toxic solvents such
as acetyl chloride and hexane. Extraction using supercriti-
cal CO2 is worthwhile compared to methanol:acetyl chlo-
ride extraction, since transmethylation of SCE extracts
requires lower quantity of hexane (1 ml hexane per g sam-
ple) compared to direct extraction of FAME from biomass
by transmethylation (8–10 ml of hexane per g sample).
Transmethylation becomes essential when further purifica-
tion of GLA methyl ester from the extracted FAMEs is
required.
The GLA methyl ester obtained by transmethylation of
the freeze-dried biomass was 0.64%. The recovery of GLA
by Bligh & Dyer method was 78% of that obtained by
transmethylation. However, since Bligh & Dyer is the con-
ventional method for lipid extraction and hence GLA, it
was considered the benchmark for the recovery purpose.
The amount of GLA extracted with the solvent mixture
(chloroform, methanol and water) according to Bligh and
Dyer (1959) was considered to be 100% of the total GLA
present in S. platensis. With GLA being present in the
polar lipid fraction, namely, the galactolipids, and reports
indicating poor solubility of polar lipids in CO2, several
SCE trials were undertaken using ethanol as a co-solvent.
Ethanol being GRAS was selected as a co-solvent com-
pared to methanol. One of the main objectives of the work
was to minimize the use of co-solvent. Too much of co-sol-
vent would not be a relevant aspect in the extraction tech-
nique employed.
In the initial studies before undertaking RSM trials,
appropriate levels for pressure, ethanol and time period
of extraction that would be suitable for the second phase
of the study were determined by carrying out several trials
by varying a single factor at a time while maintaining the
other factors at a constant, using a wide range of levels
for each factor. The levels studied were a temperature
range between 40 and 80 °C, a pressure range between
100 and 500 bars, an ethanol level of 0–16 ml ethanol/
16 g dried biomass, and a time period up to 120 min. Tem-
peratures above 50 °C reduced the recovery of GLA in the
SCE extract, and hence the temperature was kept constant
at 40 °C for the RSM trials. Also, an extraction beyond
90 min did not enhance the recovery of GLA. In an
attempt to use as minimum a co-solvent as possible, an eth-
anol level of 0–16 ml/16 g of freeze-dried biomass was cho-
sen for the trials.
Accordingly, the levels of the variables for maximum
recovery of GLA were selected as the central points in
the more elaborate second-order experiment. In the RSM
study, the three variables studied were pressure, level of
ethanol, and time of extraction. The main goal of the sec-
ond phase of the response surface was to obtain an accurate
approximation to the response surface in a small region
around the optimum and to identify optimum process
conditions. The range and the levels of the variables inves-
tigated in the study are listed in Table 1. The central values
(zero level) chosen for experimental design were a pressure
of 400 bars, an ethanol level of 13 ml/16 g of biomass and a
time period of 60 min. In the quest for the optimum combi-
nation of the variables, experiments were performed
according to the CCD experimental plan (Table 2). The
experiment used a CCD which consists of three parts.
The eight runs involving the ‘1’ and ‘À1’ coded values
(Table 2) form a 23
design. Because they are on the corners
of the 23
cube, they are called cube points or corner points.
The six runs involving the ‘À1.68’ and ‘0’ coded values
form three pairs of points along the three coordinate axes
and are therefore called the axial points or star points.
The six runs involving the ‘0’ coded values are at the centre
of the design region and are called the centre points. This
design is a second-order design, and it allows all the linear
and quadratic components of the main effects and the lin-
ear-by-linear interactions to be estimated.
The results of the response surface model fitting in the
form of ANOVA are shown in Table 3. The ANOVA of
the quadratic regression model demonstrated the model
Table 1
Experimental range and levels of the independent variables
Variables Range and levels
À1.68 À1 0 1 1.68
Pressure (bars) 316 350 400 450 484
Ethanol (ml) 9.64 11 13 15 16.36
Time (min) 26.4 40 60 80 94
Table 2
CCD plan in coded values and observed response (% recovery of GLA)
Experiment
run no.
A B C % Recovery
1 0 0 0 101.28
2 0 0 À1.68 42.00
3 0 0 0 97.00
4 0 1.68 0 69.23
5 1.68 0 0 55.64
6 1 1 À1 11.54
7 À1 1 1 85.90
8 1 À1 1 43.60
9 0 0 0 98.72
10 0 0 1.68 100.00
11 À1 À1 1 44.23
12 0 0 0 101.28
13 À1.68 0 0 98.72
14 1 1 1 45.13
15 À1 À1 À1 36.92
16 0 0 0 98.07
17 0 À1.68 0 40.77
18 0 0 0 98.00
19 À1 1 À1 76.92
20 1 À1 À1 41.00
The results are expressed as average of three readings: A – pressure (bar),
B – ethanol (ml), C – time (min).
324 M.G. Sajilata et al. / Journal of Food Engineering 84 (2008) 321–326

6. Author's personal copy
to be significant with a low probability value (Pmodel >
F = 0.0004). The goodness of the fit of the model was
checked by determination coefficient (R2
). In this case,
the value of the determination coefficient, R2
was 0.907.
The value of the adjusted determination coefficient viz.
adj R2
= 0.823. Fig. 1 compares the RSM predicted and
experimental recovery of GLA from S. platensis.
The application of RSM yielded the following regression
equation which is the empirical relationship between GLA
recovery (Yi) and the test variables in coded units.
Y i ¼ 99:63 À 12:83A þ 7:44B þ 10:99C À 11:46A2
À 19:31B2
À 13:61C2
À 13:70ðA Â BÞ þ 2:49ðA Â CÞ
þ 4:08ðB Â CÞ;
where Yi is the predicted response. A, B and C are the
coded values of the test variables viz. pressure, mL of eth-
anol, and time, respectively.
The significance of each coefficient determined by t-test
and P-values are listed in Table 4. The larger the magni-
tude of the t-value and smaller the P-value, the more signif-
icant the corresponding coefficient. Values of P < 0.0500
indicate model terms to be significant. This implies that
A, B (to some extent), C, and AB are the significant model
terms i.e, the effect of pressure, ml of ethanol used as a co-
solvent, and time and the interactive effect of pressure and
ethanol are more significant compared to other factors.
Table 3
ANOVA for the quadratic model
Source SS DF MS F-value P > F
Model 14766.5 9 1640.73 10.87 0.0004
Residual (error) 1509.37 10 150.94
Pure error 16.32 5 3.26
Total 16275.93 19
SS – sum of squares; DF – degrees of freedom; MS – mean square.
22
Actual
Predicted
11.54
34.09
56.65
79.20
101.75
11.54 34.09 56.65 79.20 101.75
Fig. 1. RSM predicted vs. experimental recovery of GLA from Spirulina
platensis ARM 740.
Table 4
The least-squares fit and coefficient estimates (significance of regression
coefficients)
Model term Coefficient estimate Standard error t ratio P-value
Intercept 99.63 5.01 19.88 –
A-Pressure À12.83 3.32 À3.86 0.0032
B-Ethanol 7.44 3.32 2.24 0.0492
C-Time 10.99 3.32 3.31 0.0080
A2
À11.46 3.24 À3.54 0.0053
B2
À19.31 3.24 À5.96 0.0001
C2
À13.65 3.24 À4.21 0.0018
AB À13.70 4.34 À3.56 0.0103
AC 2.49 4.34 0.57 0.5795
BC 4.08 4.34 0.94 0.3695
49.77
64.07
78.36
92.65
106.94
%recovery
-
1.00
-0.50
0.00
0.50
1.00
-1.00
-0.50
0.00
0.50
1.00
A: PressureB: Ethanol
Fig. 2. Response surface plot for GLA recovery (%): the effect of level of
ethanol added and pressure on GLA recovery (time at zero level: 60 min).
48.22
62.38
76.55
90.72
104.89
%recovery
-1.00
-0.50
0.00
0.50
1.00
-1.00
-0.50
0.00
0.50
1.00
A: Pressure
C: Time
Fig. 3. Response surface plot for GLA recovery (%): the effect of pressure
and the time period of extraction on GLA recovery (ethanol at zero level:
13 ml).
M.G. Sajilata et al. / Journal of Food Engineering 84 (2008) 321–326 325

7. Author's personal copy
The 3D response surface plots are generally the graphi-
cal representations of the regression equation and are pre-
sented in Figs. 2–4 from which the values of GLA recovery
for different levels of the variables can be predicted. Each
response plot represents an infinite number of combina-
tions of two test variables with the other maintained at
its respective zero level.
From the solutions predicted by the model, the experi-
mental conditions set at a pressure of 400 bars, an ethanol
level of 13.7 ml, and a time period of 60 min could give a
recovery of 102.5% GLA (considering the recovery of
GLA by Bligh and Dyer method as 100%). This emphasizes
the efficacy of SCE along with a co-solvent in the efficient
extraction of GLA from S. platensis.
4. Conclusion
RSM proved to be fairly accurate in predictive modeling
and optimization of conditions for recovery of GLA, and
that the recovery of GLA to be reasonably approximated
by quadratic non-linearity. Extraction of GLA from S.
platensis with SCE alone was lower as compared to extrac-
tion using a mixture of chloroform, methanol, and water as
per Bligh and Dyer method. However, the use of ethanol as
a co-solvent in SCE considerably increased GLA yields,
comparing well with the conventional extraction method,
and is therefore proposed for the extraction of GLA from
S. platensis.
References
Akhnazarova, S., & Kefarov, V. (1982). Experiment optimization in
chemistry and chemical engineering. Moscow: Mir Publishers.
Biagi, P. L., Bordoni, A., Masi, M., Ricci, G., Fanelli, C., Patrizi, A., et al.
(1988). Evening primrose oil (Efamol) in the treatment of children with
atopic eczema. Drugs and Experimental Clinical Research, 14, 291–297.
Bligh, E. G., & Dyer, W. J. (1959). A rapid method for total lipid
extraction and purification. Canadian Journal of Biochemical Physiol-
ogy, 67, 911–917.
Chamorro, G., (1980). Toxicological research on the alga Spirulina.
Report: United Nations International Development Organization
(UNIDO) UF/MEX/78/048.
Chamorro, G., Salazar, S., Castillo, L., Steele, C., & Salazar, M. (1997).
Reproductive and peri- and postnatal evaluation of Spirulina maxima
in mice. Journal of Applied Phycology, 9, 107–112.
Cohen, Z., & Cohen, S. (1991). Preparation of eicosapentaenoic acid
concentrate from Porphyridium cruentum. Journal of the American Oil
Chemists’ Society, 68, 16–19.
Gunstone, F. D. (1992). c-Linolenic acid. Occurrence and physical and
chemical properties. Progress in Lipid Research, 31, 145–161.
Hirano, M., Mori, H., Miura, Y., Matsunaga, N., Nakamura, N., &
Matsunaga, T. (1990). c-Linolenic acid production by microalgae.
Applied Biochemistry and Biotechnology, 24, 183–191.
Horrobin, D. F. (1983). The role of essential fatty acids and prostaglandin
in the premenstrual syndrome. Journal of Reproductive Medicine, 28,
465–468.
Ishikawa, T., Fujiyama, Y., Igarashi, C., Morino, M., Fada, N., Kagami,
A., et al. (1989). Clinical features of familial hypercholesterolemia.
Atherosclerosis, 75, 95.
Mendes, R. L., Reis, A. D., & Palavra, A. F. (2006). Supercritical CO2
extraction of c-linolenic acid and other lipids from Arthrospira
(Spirulina) maxima: Comparison with organic solvent extraction.
Food Chemistry, 99(1), 57–63.
Salazar, M., Chamorro, G., Salazar, S., & Steele, C. (1996). Effect of
Spirulina maxima consumption on reproductive and peri- and postna-
tal development in rats. Food and Chemical Toxicology, 353–359.
Salazar, M., Martinez, E., Madrigal, E., Ruiz, L. E., & Chamorro, G.
(1998). Subchronic toxicity study in mice fed Spirulina. Journal of
Ethnopharmacology, 62, 235–241.
Teruel, M. L. A., Gontier, E., Bienaime, C., Saucedo, J. E. N., &
Barbotin, J. N. (1997). Response surface analysis of chlortetracycline
and tetracycline production with K-carrageenan immobilized Strepto-
myces aureofaciens. Enzyme and Microbial Technology, 21, 314–320.
Uauy, R., & Mena, P. (1999). Requirements for long-chain polyunsatu-
rated fatty acids in the preterm infant. Current Opinion Pediatrics,
11(2), 115–120.
Vonshak, A. (1986). Laboratory techniques for the cultivation of
microalgae. In A. Richmond (Ed.), Handbook of microalgal mass
culture (pp. 117–145). Boca Raton: CRC Press.
52.33
64.97
77.61
90.24
102.88
%recovery
-1.00
-0.50
0.00
0.50
1.00
-1.00
-0.50
0.00
0.50
1.00
B:Ethanol
C: Time
Fig. 4. Response surface plot for GLA recovery (%): the effect of level of
ethanol added and the time period of extraction on GLA recovery
(pressure a zero level: 400 bar).
326 M.G. Sajilata et al. / Journal of Food Engineering 84 (2008) 321–326