Optimization of supercritical carbon dioxide (CO2) extraction of sardine (Sardinella lemuru Bleeker) oil using response surface methodology (RSM)

M.A. Gedia,b, J. Bakarc,* and A.A. Mariodd,e 

aFaculty of Agriculture, Somali National University, Colubia Road, Hamarweine, Mogadishu, Somalia

bDepartment of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

cLaboratory of Halal Science, Institute of Halal Products Research, Universiti Putra Malaysia, Selangor, Malaysia

dFaculty of Sciences and Arts, University of Jeddah, PO BO 110, ALKAMIL 21931, Alkamil-Saudi Arabia

eFood Science & Technology Department, College of Agricultural Studies, Sudan University of Science & Technology, P.O. Box 71 Khartoum North, Sudan

*Corresponding author: jamilah@putra.upm.edu.my



Oil was extracted from freeze-dried sardine (Sardinella lemur) fillets using supercritical carbon dioxide (SC-CO2) and a few milliliters of ethanol were optimized with response surface methodology (RSM). The impact of extraction pressure (200–400 bars) and temperature (40–70 °C) were studied on the total extraction yields, ratios of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). The results were compared with those of Soxhlet and modified Kinsella methods (MKM). The oils obtained using the SC-CO2 and MKM methods were significantly (P<0.05) higher in oil yield (8.04% and 6.83%), EPA (5.43% and 5.45%) and DHA (18.76% and 18.54%), respectively, compared to the Soxhlet yield (5.10%), EPA (2.17%) and DHA (06.46%). Of the two independent variables, pressure had a critical effect on yield while EPA and DHA ratios were notably influenced by temperature. The combined optimal values were pressure at 328 bars and temperature at 40 °C, with corresponding responses of 7.20%, 5.68% and 20.09% for yield, EPA and DHA, respectively. The experimental values in this study were reasonably comparable to their predicted counterparts.



Optimización de la extracción mediante dióxido de carbono supercrítico (CO2) de aceites de sardinas (Sardinella lemuru Bleeker) usando la metodología de superficie de respuesta (RSM). El aceite se extrae de filetes de sardinas (Sardinella lemur) liofilizando, mediante dióxido de carbono supercrítico (SC-CO2) y unos mililitros de etanol, optimizándose mediante la metodología de superficie de respuesta (RSM). Se ha estudiado la influencia de la presión de extracción (200–400 bars) y la temperatura (40–70 °C) sobre los rendimientos de extracción total, y sobre las relaciones de ácido eicosapentaenoico (EPA) y ácido docosahexaenoico (DHA). Los resultados se compararon con los obtenidos mediante extracción con Soxhlet y el método de Kinsella modificado (MKM). Los aceites obtenidos mediante SC-CO2 y métodos MKM fueron significativamente (P<0.05) superiores en rendimientos de aceite (8,04% y 6,83%), EPA (5,43% y 5,45%) y DHA (18,76% y 18,54%), respectivamente, en comparación con rendimientos mediante Soxhlet (5,10%), EPA (2,17%) y DHA (06,46%). De las dos variables independientes, la presión tuvo un efecto crítico sobre el rendimiento, mientras que los porcentajes de EPA y DHA estuvieron notablemente influenciados por la temperatura. Los valores óptimos fueron para una presión de 328 bar y una temperatura de 40 °C, y sus correspondientes respuestas fueron 7,20%, 5,68% y 20,09% para el rendimiento, EPA y DHA, respectivamente. Los valores experimentales de este estudio fueron los previstos y son comparables razonablemente con sus homólogos.


Submitted: 09 August 2014; Accepted: 23 December 2014

Citation/Cómo citar este artículo: Gedi MA Bakar J, Mariod AA. 2015. Optimization of supercritical carbon dioxide (CO2) extraction of sardine (Sardinella lemuru Bleeker) oil using response surface methodology (RSM). Grasas Aceites 66 (2): e074. doi: http://dx.doi.org/10.3989/gya.0824142.

KEYWORDS: Docosahexaenoic acid; Eicosapentaenoic acid; Fatty acid composition; Fish oil; Response surface methodology; Supercritical carbon dioxide extraction

PALABRAS CLAVE: Aceite de pescado; Ácido docosahexaenoico; Ácido eicosapentaenoico; Composición de ácidos grasos; Extracción con dióxido de carbono supercrítico; Metodología de superficie de respuesta

Copyright: © 2015 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence.




Fish and its products utilized for human consumption in different ways (fresh or frozen, whole or fish fillet) contribute to the nutrition and health of a considerable portion of the world’s population to provide vital nourishment, especially proteins, fats, vitamins and minerals.

Fats from fatty fish species consist of an important dietary source of ω-3 long-chain polyunsaturated fatty acids (PUFAs) namely eicosapentaenoic acid (EPA, 20:5ω-3), and docosahexaenoic acid (DHA, 22:6ω-3), which have shown potential benefits on the adequate growth of children and prevention of cardiovascular diseases and cancer (Shahidi and Miraliakbari, 2004). Fish fat also contributes to energy supplies and assists in the proper absorption of fat soluble vitamins namely A, D, E, and K in humans (Banda-Nyirenda et al., 2009). Health organizations have established specific guidelines for the general population to increase the intake of ω-3 PUFAs; for example, WHO advises that the total ω-3 PUFA intake should cover 1–2% of human energy, and the American Heart Association and the Scientific Advisory Committee of Nutrition (U.K.) recommend eating fish, particularly fatty fish, at least two times a week (Pazos et al., 2008).

With growing public awareness of these clinical benefits of fish lipids mainly their contents of PUFAs, particularly EPA (C20:5 n-3) and DHA (C22:6 n-3, there is a drastic need to develop efficient extraction methods to obtain these important fatty acid components. The extraction and purification of the lipids by conventional methods, such as Soxhlet extraction, vacuum distillation, urea crystallization or conventional crystallization involve some problems due to the toxicity or flammability of the solvents. These methods may have adverse health effects. They may also cause the decomposition of the PUFAs as they are used with high-temperatures during processing (Létisse et al., 2006). These drawbacks can be avoided by using supercritical carbon dioxide (SC-CO2) procedure, a more appropriate technique for the extraction and fractionation of edible oils containing thermo and light susceptible components like PUFAs. The extraction can be carried out at low temperatures away from light, besides this, carbon dioxide (with critical temperature, pressure and density of 31.18 °C, 72.0 bar 0.47 g·cm−3, respectively) is safe (generally recognized as safe, GRAS from the US Food and Drug Administration, FDA), residue free, non-flammable, inexpensive and environmentally friendly (Pyo and Oo, 2007). SC-CO2 extracted products are excellent in quality fresh-like products and are comparable to natural foods which are free of biological impurities, have a longer shelf life, and the ability to fractionate extracts in a single step besides a feasibility to extract various products by simply adjusting operating conditions (Martínez, 2008, Lang and Wai, 2001).

This study aimed to obtain oil extracts from freeze-dried sardine fillets using a supercritical fluid method with CO2 as the extraction solvent and EtOH as the co-solvent for the recovery of these extracts. The impact of extraction pressure and temperature on total extraction yields and fatty acid profiles was studied with a focus on the ratios of EPA and DHA. The results were compared with those of Soxhlet and modified Kinsella extraction methods.


2.1. Raw materialTOP

Whole fish samples (sardines) were purchased from Pasar Borong, a whole sale local market at Pugung, Selangor, Malaysia. Chemicals and solvents were either of analytical or HPLC grade were purchased from Fisher Scientific Chemical Co. (Loughborough, England) and Merck (Darmastadt, Germany).

2.2. Preparation of fishTOP

The fresh sardine samples purchased from Pasar Borong were kept in plastic bags and transported in an insulated icebox to the laboratory. The samples were immediately de-headed, gutted, and washed with copious amounts of cool water, and the flesh and bones were then separated using a de-boner (model- FD 6, Safe World Food-Tech Pvt. Ltd., Klang, Selangor Darul Ehsan, Malaysia). The flesh was stored at −25 °C and then freeze dried (Model: LABCONCO, USA) at a drying temperature of −40 °C under a 0.133 bar vacuum. The moisture content of the freeze dried samples was determined (data not given) and kept in desiccators until use.

2.3. Proximate analysis of the fishTOP

Before oil extraction, the fish was analyzed for its proximate composition such as moisture, lipid, ash and protein contents to indicate its initial nutritional qualities. Moisture, oil and ash contents were determined as described by AOAC (AOAC, 1990) with slight modifications for Soxhlet oil extraction. The oven-drying method (105 °C) was used for moisture content and furnace at 550 °C for ash content. Protein content was determined according to Pomeranz and Meloan (Pomeranz and Meloan, 2000).

2.4. Extraction of sardine oil by soxhletTOP

Soxhlet extractions were carried out (in triplicate) as described by AOAC, 1990 with minor modifications. Five grams of freeze dried fillet were extracted using 200 mL of petroleum ether for eight hours. The extracted lipids were evaporated under vacuum at 60 °C using a rotary evaporator (Rotavapor R-210, Büchi, Switzerland) and then were placed in an oven at 30 °C for 1 h before they were transferred into desiccators and reweighed. The extracted lipids were transferred into a brown bottle, flashed with nitrogen gas and stored at −25±1 °C.

2.5. Extraction of sardine oil by the modified Kinsella method (MKM)TOP

Freeze dried sardine fillets were extracted using modified Kinsella (1977) method (MKM) by Kim et al. (1991). Initially, samples were homogenized for 2 min using a warring blender with chloroform and methanol in the proportion of 1 g tissue: 1 mL chloroform: 2 mL methanol. An additional equivalent amount of chloroform and de-ionized water (1 mL·g−1 tissue) was added and the mixture was homogenized for 30 sec. The mixture was filtered through a Whatman No.1 filter paper on a No.3 Buchner funnel with a slight suction. The mixture was transferred to a decanter flask and was left to stand for a few minutes to complete the separation and clarification. The lower clear phase (chloroform and lipid) was poured into a conical flask. The extract was then concentrated using a vacuum rotary evaporator (Rotavapor R-210, Büchi, Switzerland). The extracted lipid was transferred into a brown bottle, flashed with nitrogen gas and stored at −25±1 °C until further analysis. The total lipid content was calculated gravimetrically as below:

Total lipid content (%) = W1/WS*100

Where: W1 = weight of dried lipid (g) and WS = the weight of sample (g).

2.6. Supercritical carbon dioxide (SC-CO2) extraction of sardine oilTOP

The supercritical fluid extractor (SFE) used was ABRP200, Pittsburgh, PA, USA with a 500 mL extractor vessel attached (Figure 1). Parameters were selected from a software (ICE) program, which permitted to set and control the extraction status. The liquid CO2 was pressurized to the desired pressure and heated to the targeted temperature with a pressure pump (P-50, Pittsburg, PA, USA) to reach the supercritical state prior to passing it into the extraction vessel. During this step, the pump head temperature was decreased to 4 °C. Then, the system was equilibrated until pressure, CO2 rate and temperature became constant to begin the extraction. Absolute ethanol (EtOH) was used as the co-solvent and was fixed at a flow rate of 3 mL·min−1.

Figure 1. Schematic Diagram of Supercritical Fluid Extractor.


The duration of the static and dynamic extraction times was fixed at 30 and 80 min, respectively. The thimble containing the sample (40 g) was placed in the extraction chamber equipped with a heating jacket. The components were then extracted by the pre-heated supercritical CO2 and entered the trap through a nozzle where CO2 was depressurized. The extraction was performed under various experimental conditions as generated by the RSM design. EtOH was removed from the extracts by vacuum evaporation using a rotary evaporator. The extracts were then placed in the oven at 30 °C for 30 min before being transferred into desiccators for final constant weight. The extracts were transferred into brown glass bottles, flashed with nitrogen and stored in a freezer of −25±1 °C until further analysis.

2.7. Identification of fatty acid (FA) profile by gas chromatography (GC)TOP

The fatty acid (FA) composition of the lipid extracts was analyzed based on the Christie (1993) method using Agilent gas chromatography (G1530N, USA). The column used was BPX-70 (60 m×0.32 mm i.d., 0.25 μm film thickness) with the phase composition 90% biscyanopropyl; 10% cyanopropyl phenyl polysiloxane from SGE, Melbourne, Australia. A100 μL aliquot of the test sample was thoroughly mixed by dissolving 50 μL of sample into 950 μl of n-hexane, and 50 μL of sodium methoxide was added to prepare the FA methyl esters (FAMEs). The mixture was then shaken vigorously using an auto-vortexer (Stuart, UK) for 30 s and stored for 5 min so that it formed two layers. The clear upper layer containing the FAMEs (1 μL) was pipetted off and injected into the gas chromatograph. The oven temperature was set at 115 °C, held for 2 min, raised to 180 °C at a rate of 8 °C·min−1 and held for 10 min to be finally raised to 240 °C at a rate of 8 °C·min−1 and held for 10 min and the carrier gas (helium) at a rate of 1.6 mL·min−1 was flashed through. The FA components were identified based on the standard mixture of FAMEs containing 37 mixed fatty acids.

2.8. Experimental design and statistical analysisTOP

Response surface methodology (RSM) was used to determine the optimum conditions for yield EPA and DHA of sardine oil extracted using SFE. The experimental design and statistical analysis were carried out using the Minitab V. 14 statistical package (Minitab Inc., PA,USA). Central composite design (CCD) with cube points was selected to evaluate the effects of two independent variables (extraction temperature and pressure), coded as X1 and X2, respectively, on the yield EPA and DHA of the SC-CO2 extracted sardine oil. The minimum and maximum values for the extraction temperature were set at 40 to 70 °C whereas pressure was in the range of 200 to 400 bars. It should be noted that using CCD with cube, out range values called “star points” could be found to predict the optimum point in case it lies out of the selected range. Optimization of the two independent variables was achieved by maximizing the three dependent variables i.e. yield, EPA and DHA to achieve highest values using a MINITAB numerical response optimizer. The whole design consisted of 13 combinations including five replicates of the center point as in Table 1, (Myers, 2002). The ANOVA tables were generated and the effect and regression coefficients of individual linear, quadratic and interaction terms were determined. The significances of all the terms in the polynomial were analyzed statistically by computing the F-value at a probability (p) of 0.001, 0.01 or 0.05 of the SC-CO2 extracted sardine oil. The statistically found non-significant (p>0.05) terms were removed from the initial models and only significant (p<0.05) factors were involved in the final reduced model. Experimental data were fitted to the following second order polynomial model and regression coefficients were obtained. The generalized second-order polynomial model proposed for the response surface analysis was given as follows

Where β0, βi, βii, βij were regression coefficients for intercept, linear, quadratic and interaction terms, respectively. XI and Xj were coded values of the independent variables, while k was the number of the tested factors (k=2).

For the söxhlet and modified Kinsella methods, triplicate extractions of each were considered and their means were compared with the SC-CO2 optimized sardine oil.

Table 1. Comparison of yield and n-3 PUFAs (EPA and DHA) obtained via SC-CO2 with those of Soxhlet and MKM
Techniques Run Parameters Responses
Yield (%) EPA (%) DHA (%)
X1 X2 y0 y1 y0–y1 y0 y1 y0–y1 y0 y1 y0–y1
SC-CO2 Extraction 1 400 40 6.04 6.64 −0.06 5.45 5.51 −0.06 20.23 20.69 0.05
2 300 76 5.32 6.09 −0.13 4.98 5.11 −0.13 15.70 15.69 0.01
3c 300 55 8.16 7.95 −0.09 5.39 5.48 −0.09 18.38 18.82 −0.44
4 441 55 5.76 5.42 0.02 5.54 5.51 0.02 19.12 18.86 0.25
5c 300 55 8.07 7.95 0.00 5.48 5.48 0.00 19.01 18.82 0.18
6c 300 55 8.14 7.95 0.09 5.57 5.48 0.09 18.89 18.82 0.06
7 400 70 5.43 5.18 0.04 5.61 5.56 0.04 16.50 16.94 −0.44
8c 300 55 7.62 7.95 0.01 5.49 5.48 0.01 19.06 18.82 0.23
9 300 33 6.15 5.73 0.01 5.86 5.84 0.01 20.35 20.25 0.09
10 158 55 2.39 3.08 −0.02 5.06 5.08 −0.02 17.38 17.53 −0.15
11c 300 55 7.78 7.95 0.10 5.58 5.48 0.10 18.79 18.82 −0.03
12 200 70 6.21 5.24 0.07 4.78 4.70 0.07 16.40 15.99 0.40
13 200 40 3.39 3.27 −0.03 5.74 5.77 −0.03 19.01 19.22 −0.21
Soxhlet extraction1 5.10±0.10 2.17±0.55 6.46±2.36
MKM extraction1 6.83±0.15 5.43±0.05 18.54±1.68
1Each value is the mean ±S.D; n=3; c: center point; SC-CO2: supercritical carbon dioxide; PUFAs: poly unsaturated fatty acids; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; MKM: modified Kinsella method “otherwise called Blight and Dyer (1959)”; y0: experimental value; y1: predicted value; y0–y1: residue.


3.1. Moisture content, protein, fat and ash of sardine filletsTOP

Moisture protein, ash and lipid contents are generally used as indicators of nutritional values of fish (Stansby, 1962). A high content of water in fish was correlated with low protein and lipid contents (Dempson et al., 2004). The moisture, protein, crude lipid and ash contents of the Malaysian sardine fillets were 77.8±0.2, 15.4±0.51, 5.1±0.10 and 1.4±0.17, respectively. These values were within the normal ranges for sardine which were reported by Payne et al. (1999), taking into account that lipid levels as well as other energy parameters fluctuate seasonally (Payne et al., 1999). Despite differences in region, which may lead to a variation in parameters (Çelik et al., 2005), our findings were in a close agreement with those reported by Serdaroğlu and Felekoğlu (2005), Payne et al. (1999), and Fernandes et al. (2014).

3.2. Response surface methodology (RSM) model fitnessTOP

The ranges of each independent variable (pressure and temperature) which affected extraction efficiency of EPA and DHA ratios were chosen. In this study, the upper and lower values for the parameters were set at +alpha (+α=1.414) and –alpha (−α=1.414), hence, all the factor levels were selected within the limits that were practical with SC-CO2 (above critical temperature of 31 °C and critical pressure of 72 bars) and desirable. In RSM, natural variables are transformed into coded variables that have been defined as dimensionless with mean zero and same standard deviation (Liyana-Pathirana and Shahidi, 2005).

The linear, quadratic and interaction effects of supercritical pressure and temperature on sardine oil extraction efficiency, and the ratio of EPA and DHA are shown in Table 1. The results suggest that the models fitted for response variables were empirically adequate due to their high coefficient of determination (R2>0.92), which means that more than 92% of the response variation could be explained as a function of the two SC-CO2 parameters (Pressure and temperature). The highly adjusted R2 (>0.89) as well as insignificance of any lack of fit in the data also indicated its reliability.

Using multiple regression analysis, the relationship between the tested parameters and the responses were explained in equations (Eq. 2, 3, and 4 for yield, EPA and DHA, respectively).

3.3. Optimization proceduresTOP

Multiple response optimizations were used to assess the optimum levels of the parameters which could achieve the desirable response areas (Mirhosseini et al., 2008). Besides the numerical optimization (Figure 2), the 3D plots (Figure 3) which were advocated for the graphical interpretation of the interaction effect of independent variables on the dependent variables (Montgomery and Wiley, 2001) were also considered using the Minitab software to locate the exact optimum point of independent variables and to obtain overall joint optimized values using the Minitab program response optimizer. The overall optimal values for maximum SC-CO2 extraction efficiency and n-3PUFAs ratios (EPA and DHA) of pressure and temperature were anticipated to be at pressure of 328 bars and temperature of 40 °C as shown in Figure 2. Under the optimized conditions, the corresponding predicted dependent variables for yield, EPA and DHA were 7.20, 5.68 and 20.09%, respectively.

Figure 2. Combined optimum conditions of yield (%), EPA (%) and DHA (%) ratios of SC-CO2 extracted sardine oil.


Figure 3. Surface plot of pressure and temperature influence on sardine fillet oil (a) extraction yield, (b) EPA ratio and (c) DHA ratio


3.4. Verification of the final reduced modelsTOP

The fitness of the response surface equation was checked from the error rate between experimental and predicted values of the reduced response regression models (Tables 2 and 3). The experimental and predicted values of yield, EPA and DHA obtained from equations 2, 3 and 4 are presented in Table 1. For each of the experimental values, Y0 was compared with the predicted values, Y1 calculated from the equation. A close agreement between the experimental and predicted values was noted and no significant (p>0.05) difference was found between those values, thus suggesting the adequate fitness of the response equations.

Table 2. Regression coefficients and analysis of variance of the reduced regression models for total yields
Term Regression coefficient (β)
yield EPA DHA
cons −34.7800 8.247 14.77
X1 0.1507 −0.003 0.023
X2 0.6783 −0.073 0.100
X12 −0.0002 −8.804 −3.136
X22 0.0045 * −0.001
X1X2 −0.0006 >0.001 *
Regression model (R2) 0.9250 0.947 0.968
Regression 0.0001 >0.001 >0.001
lack-of-fit 0.1100 0.343 0.306
Adj R-sq 0.8710 0.92 0.952
Cons: constant; X1: pressure (bars); X2: temperature (°C); *: its values were not significant and thus reduced from the model; EPA: eicosapentaenoic acid and DHA: docosahexaenoic acid.
Table 3. Significant probability (p-values and t-ratio) of the independent variable effects in the final reduced models of sardine oil from SFE
Variable Main effects Quadratic effects Interaction effects
X1 X2 X12 X22 X1X2
yield p-value >0.001a 0.002b >0.001a 0.004a 0.034c
t-ratio −6.335 7.854 4.909 −7.481 −2.631
EPA P-value 0.001b >0.001a 0.028c * >0.001a
t-ratio 4.940 −8.300 −2.680 * 6.43
DHA p-value 0.003b >0.001a 0.031c 0.007b *
t-ratio 4.235 −14.466 −2.620 0.119 *
EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; X1 and X2: the main effects; X12 X22: the quadratic effect and x1x2: the interaction effect of pressure (bars) and temperature (°C), respectively; *: its values were not significant and thus reduced from the model. Values with small superscript letters are statistically significant at ap<0.001, bp<0.01, cp<0.05.

3.5. Extraction of the oilTOP

The sardine oils obtained from the various SC-CO2 (run orders 1–13) and solvent (Soxhlet or MKM) extracts are given in Table (1). Although some SC-CO2 runs (center points) gave higher extraction efficiency than MKM, overall results of both were statistically similar (p>0.05) and the two techniques gave a higher yield (>6%) than that of the söxhlet extraction (5.1%). SC-CO2 extraction results were in agreement with those reported by Létisse et al. (2006). The MKM method initially established by Bligh and Dyer (1959) could be extracted with all lipids, including polar lipids, phospholipids and possibly lipids bound with other components from cellular membranes and with high yield. Yet application of this method for food supplementation has raised questions on its safety owing to toxicity of the solvents used (Létisse et al., 2006). Hence, the SC-CO2 method using the environmentally-friendly and non-toxic CO2 with small milliliters of non-hazardous solvents like ethanol or even nil could be a more attractive technique, provided that the conditions whereby maximum yield of lipid and/or fatty acids can be achieved, are optimized.

3.6. Impact of supercritical pressure and temperature on the dependent variablesTOP

The individual influence and efficient interaction between pressure and temperature were investigated. The impact of pressure and temperature on the SC-CO2 sardine oil extraction was determined at pressures of 200 and 400 bars, temperatures of 40 and 70 °C respectively, and at a constant CO2 flow rate (18 g·min−1). Similar trends of pressure and temperature influence on the extracts with SC-CO2 were observed in previous studies (Mariod et al., 2010, Jamilah et al., 2011). The high extraction yield (>8%) was obtained at 300 bars and 55 °C followed by 200 bars and 70 °C, while the lower yield was obtained at 158 bars and 55 °C. Based on these results a significant (p<0.05) effect of pressure and temperature on the sardine extraction efficiency was obvious. At low and extremely high pressures (200>p>350 bars), the oil extraction efficiency was decreased. The consequence of the extraction pressure and temperature on the yield of sardine oil at a steady CO2 flow rate (18 g·min−1) is demonstrated in Figure 3a. In view of the above results, it is clear that there was a significant joint (p<0.05) effect between the two parameters; i.e., when low pressure (200 bars) but high temperature (70 °C) and vice versa (400 bars and 40 °C) was applied, a good yield of 6.21% and 6.04, respectively (Table 1) was achieved. A reciprocal impact of SFE pressure-temperature inter-relationship, similar to the present findings, was reported by Wie et al. (2009). By increasing the temperature or pressure of the solvent, the rate of extraction with SC-CO2 can be improved (Zaidul et al., 2007). The center points (300 bars and 55 °C), however, showed the highest yields of sardine oil extraction with SC-CO2. This was in line with what Pan et al. (2012) reported.

Despite the fact that the comparison of fatty acid composition in fish oils is quite difficult due to several probable affecting factors like season (Celik, 2008, Rasoarahona et al., 2005, Shirai et al., 2002) the EPA and DHA overall values extracted with MKM or SC-CO2 in this study were in good agreement with those of South east Asian waters sardines (Chaijan et al., 2006). A significant (p<0.05) effect of pressure and temperature, with the latter being more influential on EPA and DHA, was noted. There was an inverse relationship of temperature increment with EPA (Figure 3b) and DHA (Figure 3c). For instance, when an extraction temperature as low as 33 °C with an intermediate pressure of 300 bars (Table 1) was used, the highest values of EPA (5.84) and DHA (20.35) were achieved. In contrast, the lowest portions were reached with high temperatures of up to 70 °C and a low pressure at 200 bars (Table 1). The severe susceptibility of poly unsaturated fatty acids to higher temperatures or light is a well addressed concern.

3.7. Fatty acid profileTOP

Lipids from freeze-dried sardine extracted through various extraction methods (Soxhlet, MKM and SC-CO2) were analyzed in order to determine the relationship between changes in the lipid fatty acid (FA) profile and method of extraction. The extraction efficiency (%) of the sardine oil obtained from SC-CO2 was varied based on the extraction conditions (Table 1), therefore, triplicate analyses of SC-CO2 optimum extraction efficiency (pressure: 321 bars and temperature 54 °C with a corresponding yield of 8.04%) were compared with the conventional extraction methods. The percentage values of methyl ester FA analyses in the söxhlet, MKM and SC-CO2 extracted oils are presented in Table (4), while their typical chromatographic peaks are shown in Figure 4. No significant difference (p>0.05) in FA composition between MKM and SC-CO2 was apparent. However, both formers were significantly (p<0.05) different from the soxhlet extracted oil FA composition. The prolonged extraction time (8 h) at a relatively high temperature (60 °C) with Soxhlet had perhaps decreased the heat sensitive unsaturated fatty acids, especially the more susceptible PUFAs such as, EPA and DHA in the oil. From the analysis, the main components of sardine oil are shown in Table 4.

Figure 4. Chromatograph peaks of the fatty acid composition of S. lemuru oil obtained via (A) SOXHLET, (B) MKM and (C) SC-CO2 [pressure =321 bars; temperature =54 °C; time =80 min; CO2 flow rate =18 g·min−1; modifier (EtOH) =3 mL/min]. For identification refer to the retention time in Table (4)

Table 4. Fatty acid profile of sardine (Sardinella lemuru) fillet lipids as affected by different extraction methods
Fatty acids Average RT Extracted by Soxhlet (%) Extracted by MKM (%) Extracted by BSC-CO2 (%)
C14:0 4.97±0.01 6.79±1.56a 5.24±0.36a 5.37±0.35a
C15:0 6.35±0.01 1.77±0.28a 1.38±0.08a 1.33±0.07a
C16:0 8.04±0.04 30.93±3.46a 29.89±1.17a 29.57±1.02a
C16:1n-7 8.41±0.03 6.12±0.64a 5.17±0.31a 5.12±0.18a
C17:0 9.47±0.01 3.01±0.02a 2.39±0.15a 2.30±0.05a
C17:1 9.87±0.01 0.68±0.20a 0.55±0.05a 0.63±0.02a
C18:0 11.18±0.05 15.14±1.33a 11.19±0.35a 10.97±0.02a
C18:1 n-9c 11.53±0.03 14.37±0.86a 12.05±0.40a 12.46±0.03a
C18:2 n- 6c 11.63±0.03 2.89±0.18a 2.48±0.09a 2.34±0.07a
C20:4 n- 6 16.48±0.01 0.94±0.20a 2.40±0.11b 2.19±0.03b
C20:5 n- 3 (EPA) 18.01±0.03 2.17±0.55a 5.43±0.05b 5.45±0.14b
C22:5-n-6 21.72±0.01 0.68±0.24a 1.63±0.14a 1.65±0.09a
C22:5 n- 3 22.70±0.01 0.57±0.24a 1.57±0.14a 1.81±0.30a
C22:6 n- 3 (DHA) 23.04±0.05 6.46±2.36a 18.54±1.68b 18.76±1.28b
Others 7.46 0.09 0.05
Σ SFA 65.07 50.09 49.54
Σ MUFA 21.17 17.77 18.21
Σ PUFA 13.71 32.05 32.20
Σ n-6 FA 4.51 6.51 6.18
Σ n-3 FA 9.20 25.54 26.02
Σ n-3/n-6 2.03 3.92 4.21
Σ DHA/EPA 2.98 3.41 3.44
Values are means + S.D; n=3; means within each row with different lower case superscripts are significantly (p<0.05) different; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: poly unsaturated fatty acids; n-6 FA: omega-6 fatty acids; n-3FA: omega-3 fatty acids; Σ: Total; MKM: modified Kinsella method; RT: retention time; SC-CO2: supercritical carbon dioxide; BSC-CO2 parameters: pressure=321 bars; temperature=54 °C; time=80 min; CO2 flow rate=18 g/min.


Among the three extraction methods (Soxhlet, MKM and SC-CO2) compared for their extraction efficiency and their recovery of PUFAs particularly EPA and DHA, MKM and SFE (32.05 and 32.2%, respectively) exhibited similar results in this regard, nevertheless, certain points in SC-CO2 showed higher but not significant yield and/or PUFAs than MKM, depending on pressure and/or temperature. However, a slightly lower yield and remarkably decreased ratios of PUFAs (yield=5.1±0.1% and PUFAs=13.7%) compared to those of MKM and SC-CO2 (yield=6.83±0.15 and 6.46±2.36%, respectively) were found with Soxhlet extraction. Although good yields could be found using MKM, criticism about safety aspects due to the harmful extraction solvents has been raised, thus turning to SC-CO2 which is safe, residue free, non-flammable and uses inexpensive CO2 seems a more attractive choice recently for the extraction of lipids, nutraceuticals and bioactive compounds from diverse sources.


This project was partly supported by the research grant from the Department of Fishery, Ministry of Agriculture, Malaysia.



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