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Pumpkin (Cucurbita maxima) seed oil extraction using supercritical carbon dioxide and physicochemical properties of the oil
Journal of Food Engineering 95 (2009) 208–213
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Pumpkin (Cucurbita maxima) seed oil extraction using supercritical carbon dioxide and physicochemical properties of the oil Pranabendu Mitra a, Hosahalli S. Ramaswamy b, Kyu Seob Chang a,* a b
Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Quebec, Canada H9X3V9
a r t i c l e
i n f o
Article history: Received 13 July 2008 Received in revised form 28 April 2009 Accepted 29 April 2009 Available online 10 May 2009 Keywords: Pumpkin seed oil Supercritical extraction Supercritical carbon dioxide, optimization Physicochemical properties
a b s t r a c t Pumpkin (Cucurbita maxima) seed oil was extracted using supercritical carbon dioxide and the physicochemical properties of the oil were determined. A central composite rotatable design was used to analyse the impact of extraction parameters (temperature, time and pressure) and a response surface methodology was used to obtain optimal extraction conditions for the maximum oil yield. All three variables studied were significant demonstrating quadratic effects. The maximum yield of the extracted oil was 30.7% and the optimum conditions were 32,140 kPa and 68.1 °C for 94.6 min which was within the experimental domain. Physicochemical properties of the oil showed that the extracted oil could be used as food oil supplement. Ó 2009 Published by Elsevier Ltd.
1. Introduction Pumpkin (Cucurbita maxima) seeds contain many valuable functional components and have been traditionally used for herbal, therapeutic as well as clinical applications. Pumpkin seeds have been used as safe deworming and diuretic agents, and the seed oil as a nerve tonic (Ghani, 2003; Younis et al., 2000). Pumpkin seed oil has strong antioxidant properties (Stevenson et al., 2007) and has been recognized for several health benefits such as prevention of the growth and reduction of the size of prostate, retardation of the progression of hypertension, mitigation of hypercholesterolemia and arthritis, reduction of bladder and urethral pressure and improving bladder compliance, alleviation of diabetes by promoting hypoglycemic activity, and lowering levels of gastric, breast, lung, and colorectal cancer (Caili et al., 2006; Stevenson et al., 2007). Organic solvents are frequently used to extract oil; for example, hexane has been used for decades to extract oil from cottonseed. However, environmental safety regulations and increased public health risk are necessitating the industry to consider alternatives to the organic solvents for use in oil extraction (Bhattacharjee et al., 2007). Toxic organic residues were considered a serious problem in the solvent extraction of pumpkin seed oil (Wenli et al., 2004). Lang and Wai (2001) reported that conventional
* Corresponding author. Tel.: +82 42 821 6727; fax: +82 42 821 8897. E-mail addresses: [email protected], [email protected] (P. Mitra), [email protected] (H.S. Ramaswamy), [email protected] (K.S. Chang). 0260-8774/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2009.04.033
extraction methods such as hydro-distillation and organic solvent extraction requires the control of several adjustable parameters to improve the extraction processes, and suggested developing alternate extraction techniques for better selectivity and efficiency. Supercritical extraction with carbon dioxide (SC-CO2) is getting popular as a cost effective and environmentally friendly method for extracting useful components such as oils from plant extracts. Supercritical fluids possess both gas-like and liquid-like qualities, and the dual role of such fluids provide ideal conditions for extracting compounds with a high degree of recovery in a short period of time (Reverchon, 1997; Wang and Weller, 2006). Supercritical carbon dioxide is an odourless, chemically inert, non-toxic, non-flammable, non-corrosive, and low cost solvent with high purity, and leaves no residue or contamination in the extract. SC-CO2 possesses moderately low critical temperature (31.1 °C) and pressure (7380 kPa) and the solvent can be removed by simple depressurization (Venkat and Kothandaraman, 1998; Lang and Wai, 2001; Wang and Weller, 2006). Supercritical carbon dioxide, with its particularly attractive properties, is the preferred solvent for many supercritical fluid extractions (Venkat and Kothandaraman, 1998). SC-CO2 possesses superior mass transfer properties with a higher diffusion coefficient and lower viscosity than liquid solvents. The absence of surface tension allows the rapid penetration of SC-CO2 into the pores of heterogeneous matrices and helps to enhance extraction efficiencies (Venkat and Kothandaraman, 1998; Lang and Wai, 2001). Food industry is a major user of the SC-CO2 extraction process for extraction and fractionation of fats, oils, essences, pigments and functional or bioactive compounds
P. Mitra et al. / Journal of Food Engineering 95 (2009) 208–213
(Hierro and Santa-Maria, 1992; Reverchon, 1997). Majority of the published work on SC-CO2 has focussed on oil extraction (Bernardo-Gil and Cardoso Lopes, 2004; Bhattacharjee et al., 2007; Hierro and Santa-Maria, 1992; Reverchon, 1997; Wenli et al., 2004) and it is not surprising the technique is becoming a standard method for oil extraction because of its high extraction efficiency, short extraction time and no residue problems (Bhattacharjee et al., 2007; Lang and Wai, 2001). However, the SC-CO2 technique has not been widely used for pumpkin (C. maxima) seed oil extraction. The efficiency of SC-CO2 extraction method depends on the process temperature and pressure. The selectivity or the dissolving power of SC-CO2 can be adjusted by properly selecting such variables. In order to get the best efficiency (maximum yield), the process requires optimization of the three independent variables, temperature, pressure and time. A statistical experimental design based on central composite rotatable design (CCRD) and a response surface methodology (RSM) are often used to characterize the influence of process variables and arrive at optimal processing conditions (Ge et al., 2002). Physical (refractive index, color, etc.) and chemical (acid value, iodine value, saponification value and peroxide value) properties of the oil, and the fatty acid composition are important quality attributes which will determine the oil’s acceptance as a food or medicinal supplement. The objective of this work were therefore to characterize and optimize of extraction parameters of SC-CO2 process for extraction pumpkin seed oil (C. maxima) and to evaluate the physicochemical properties of the extracted oil to highlight the potential for its food use.
2. Materials and methods 2.1. Materials Pumpkin (C. maxima) seeds were obtained from the local market (Jung-Ang, Daejeon, Korea). The seeds were dried in a vacuum drier (Lab companion OV-02, Jeio Tech. Co. Ltd., Korea) at 75 °C and 25 kPa for 24 h to a moisture content of 2%. The dried seeds were then ground in a food grinder (Hanil Science Industrial Co. Ltd., Korea) to reduce the particle size to a maximum diameter of 500 lm as measured by a sieve (Chung Gye Sang Gong Sa, Korea), sealed in a plastic container and stored in a refrigerator until extraction. Carbon dioxide gas (99.99%) used for SC-CO2 extraction was supplied by the SAMO Gas Kungsa (Daejeon, Korea). Chemicals used for seed oil analysis were hexane, ethanol, chloroform and phenolphthalein indicator, and wijs (iodine monochloride in acetic acid glacial) solution (Daejung Chemicals and Metals Co. Ltd., Korea); acetone and ethyl acetate (DC Chemicals Co. Ltd., Korea); acetic acid, phosphoric acid, potassium phosphate and sulfuric acid (Duskan chemical Co. Ltd., Korea); carbon tetrachloride and diethyl ether (Oriental chemical Industrial Co. Ltd., Korea), and heptadecanoic acid (Sigma, USA) and other materials such as TLC plate of 0.25 mm silica gel 60F254 (MERCK, Germany). 2.2. Pumpkin seed oil extraction using SC-CO2 A laboratory assembled SC-CO2 extraction apparatus (Fig. 1) consisted of an extractor vessel (HIP Inc. Erie, PA), compression pump (Haskel Co. Barbank, CA), pressure regulator (HIP Inc. Erie, PA), and pressure gauge (MacDaniel Controls Co.). Twenty-five grams of pumpkin seed powder was placed in the stainless steel extractor with filters at the top and bottom to retain the small particles. The liquid carbon dioxide was cooled through a glycol chiller, compressed (using the liquid pump) and allowed to enter the extractor at the bottom through a hot water bath (for temperature control). The flow rate of CO2 was kept constant at 0.25 L/min for
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Fig. 1. Schematic diagram of supercritical carbon dioxide extraction equipment.
all treatments by manually adjusting the gas flow valve and monitoring the rate through a gas flowmeter. The extractor was brought up to the desired supercritical pressure and temperature. SC-CO2 was then pushed upward through the sample powder. Oil enriched SC-CO2 was then discharged into the separator where carbon dioxide was depressurized. The gaseous carbon dioxide moved out through the gas meter leaving the extracted oil in the separator. The pressures in both the extractor and separator were controlled manually using a back-pressure regulator. After extraction, the oil was collected at the bottom of the separator into 300 mL hexane placed there prior to the extraction process, and then stored in a refrigerator for analysis. Hexane was removed from the extracted oil and concentrated using a rotary vacuum evaporator at 45 °C. During this process, a gentle stream of nitrogen was passed though the oil for complete removal of hexane and to remove oxygen. The yield of the oil was then measured gravimetrically. In order to optimize the extraction process, extraction was carried out at different conditions using a CCRD design and an RSM model. 2.3. Fatty acid composition of pumpkin seed oil Methylated injection samples were prepared prior to gas chromatographic (GC) analysis. Thin layer chromatography (TLC) was carried out by eluting a mobile phase (hexane:diethyl ether:acetic acid = 50:50:1) to separate the triglycerol from the pumpkin seed oil. The spot visible in upper most part of TLC plate corresponding to triglycerol was scratched off and taken into a test tube. Three milli-litres of 6% 1 N H2SO4 in methanol was added followed by 50 lL (1 mg/1 mL hexane) of hepta-decanoic acid (standard) and mixed with a vortex mixer. The sample was heated in an oven at 70 °C for 1 h and cooled; 2 mL hexane (HPLC grade) was then added and mixed in a vortex mixer and finally passed through the Na2SO4 column to remove the residual moisture. The concentrated sample was subjected to gas chromatography (GC) analysis. An HP 6890 Series GC unit (Hewlett-Packard, Avondale, PA) with a flame-ionization detector and auto-injection system and a fusedsilica capillary column (SP-wax, 60 m 0.25 mm Supelco, Bellefonte, PA) were used for the fatty acid analysis. The column was held at 150 °C for 5 min and raised to 220 °C at a rate of 4 °C/ min. The injector and detector temperatures were held at 250 and 260 °C, respectively. The carrier gas was nitrogen with a split ratio of 60:1. 2.4. Physicochemical properties of pumpkin seed oil Acid value, iodine value, peroxide value and saponification value of the extracted pumpkin seed oil were determined using
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AOAC (1995) methods. The Hunterlab color coordinates (L, a, b) of the oil were measured with a colorimeter (MINOLTA, CR-300, Japan). Refractive index of the oil was determined using a hand held refractometer (ATAGO N3, Atago Co. Ltd., Japan) at 20 °C. All the measurements were made in duplicate. 2.5. Statistical analysis 2.5.1. Experimental design A CCRD was used to study the effect of extraction variables on the oil yield. The three input variables were temperature (35–75 °C, X1), time (30–150 min, X2) and pressure (15,160–34,450 kPa, X3) and the five levels chosen were 2, 1, 0, +1 and +2 as shown in Table 1. The input ranges and levels were selected based on preliminary experiments (Kim and Chang, 2005). The experimental design consisted 16 runs (n = 2k + 2k + m, where, n = total experimental points, input variables, k = 3 and central point, m = 2), which included eight factorial points, six axial points and two replicated central points (Mason et al., 2003) as shown in Table 2. 2.5.2. Response surface modeling A second order polynomial equation was used to fit the coded variables:
Table 1 Treatments levels and coded values for each of the independent variables used in developing the experimental data to optimize the pumpkin seed oil extraction using supercritical carbon dioxide. Independent variable
Symbols
Extraction temperature (°C)
Extraction time (min)
Extraction pressure (kPa)
Coded
Uncoded
Coded
T
X1
35 45 55 65 75 30 60 90 120 150 15,160 19,980 24,805 29,630 34,450
2 1 0 1 2 2 1 0 1 2 2 1 0 1 2
P
þ B12 X 1 X 2 þ B23 X 2 X 3 þ B13 X 1 X 3
ð1Þ
where, Y represented the experimental response, B0, B1, B2, B3, B11, B22, B33, B12, B23 and B13 were constants and regression coefficients, respectively, of the model, and X1, X2 and X3 were the independent variables. The model thus included linear, quadratic and crossproduct terms to determine the effect of process variables on the response. The CCRD experimental design was combined with RSM to solve the regression equation and investigate the effects of three independent input variables of temperature, time and pressure to arrive at the optimal conditions giving the maximum oil yield extraction. Statistical analyses were done by a computer program in SAS 8.1. 3. Results and discussion 3.1. Experimental data Temperature, pressure and time were used as the input process variables in the SC-CO2 extraction process as has been commonly done in other studies. Bhattacharjee et al. (2007) used the same factors to extract cotton seed oil using SC-CO2 and Kim and Chang (2005) used them to optimize falcarinol extraction. Results obtained under the different testing conditions are shown in Table 2. The highest yield of oil was 31.5% and was obtained after 120 min extraction at 65 °C and 29,630 kPa. Overall observation of the results showed that the oil yield generally increased with the quadratic effects of time, temperature and pressure of the SC-CO2 extraction process.
Levels
Uncoded
t
Y ¼ B0 þ B1 X 1 þ B2 X 2 þ B3 X 3 þ B11 X 21 þ B22 X 22 þ B33 X 23
X2
X3
X1 = (T 55)/10, X2 = (t90)/30, X3 = (P 24,805)/4825.
3.2. Regression model The developed regression model for the relationship between oil yield (Y) and the coded values of independent variables of temperature (X1), time (X2) and pressure (X3) and their interaction is shown in the following equation:
Y ¼ 24:79 þ 4:23X 1 þ 1:63X 2 þ 4:00X 3 4:11X 21 2:48X 22 3:03X 23 þ 1:09X 1 X 2 1:51X 2 X 3 þ 4:17X 1 X 3
ð2Þ
The significance of each coefficient and their interactions was determined using the t-test (t value). The linear terms of extraction temperature (X1, absolute t value = 3.98), extraction time (X2, absolute t value = 2.34) and extraction pressure (X3, absolute t value = 3.77), the second order terms of X 21 (absolute t value = 3.88) and X 23 (absolute t value = 2.85) and the cross-product term
Table 2 Experimental design and results of pumpkin seed oil extraction using supercritical carbon dioxide. Treatment
Extraction temperature, T (°C) X1
Extraction time, t (min) X2
Extraction Pressure, P (kPa) X3
Amounts of oil (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
45 45 45 45 65 65 65 65 55 55 35 75 55 55 55 55
60 (1) 60 (1) 120 (1) 120 (1) 60 (1) 60 (1) 120 (1) 120 (1) 90 (0) 90 (0) 90 (0) 90 (0) 30 (2) 150 (2) 90 (0) 90 (0)
19,980 29,630 19,980 29,630 19,980 29,630 19,980 29,630 24,805 24,805 24,805 24,805 24,805 24,805 15,160 34,450
06.48 09.08 09.40 10.20 06.72 30.24 18.24 31.48 25.00 24.48 04.28 12.32 12.52 17.12 06.68 18.60
(1) (1) (1) (1) (1) (1) (1) (1) (0) (0) (2) (2) (0) (0) (0) (0)
(1) (1) (1) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (2) (2)
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P. Mitra et al. / Journal of Food Engineering 95 (2009) 208–213 Table 3 Analysis of variance (ANOVA) of the regression parameters for the response surface model of pumpkin seed oil yield. Regression
DF
Type I sum of squares
R-square (R2)
F Value
Pr > F
Linear Quadratic Cross-product Total model
3 3 3 9
583.86 290.59 166.86 1041.31
0.51 0.25 0.15 0.91
10.82 5.39 3.09 6.43
0.0078 0.0388 0.1112 0.0172
between extraction temperature and pressure (X1X3, absolute t value = 2.78) gave highly significant effects on oil yield having higher absolute t value than critical t value of 2.26 (at p value <0.05 and degree of freedom = 9). The coefficients were highly significant with an absolute t value higher than critical t value. Mason et al. (2003) emphasized that in order to reject the null hypothesis the model t value needs to exceed the critical t value. The second order term of X 22 (time) and cross-product terms of X1X2 (temperature–time) and X2X3 (time–pressure) did not show a significant effect on oil yield (p > 0.05) while X1X3 (temperature–pressure) interaction was significant. Analysis of variance (ANOVA) results of the model are shown in Table 3 indicating a good model performance with an R2 value of 0.91 and an F value of 6.43 among linear, quadratic, cross-product and total model. Interaction among the variables was not negligible. R2 value is one of the measures of degree of fit and Guan and Yao (2008) reported that an R2 should be at least 0.80 for the good fit of a model. The model R2 value of 0.91 implies that 91% of the variations associated with the pumpkin seed oil extraction yield can be attributed to the three independent variables (temperature, time and pressure). A plot of the experimental values of oil yield versus predicted values (Eq. (2)) is shown in Fig. 2. The plot showed a close fit and a uniform distribution of the observed values around the predicted ones. Thus, a statistically significant multiple regression relationship between the independent variables (X1, X2 and X3) and the response variable (Y) could be established. The second order polynomial model could therefore be effectively used to represent the relationship among the parameters selected. 3.3. Response surface analysis The 3-dimentional plots of the response surfaces are presented in Figs. 3–5 by presenting the response as function of two factors and keeping the third one at the constant mid-level. Fig. 3 shows that the extraction temperature and the extraction time have the quadratic effects on oil yield. Quadratic effects on oil yield can also be observed in Fig. 4 (for the extraction time and pressure) and in Fig. 5 (for the extraction temperature and pressure). In general the
Fig. 2. Comparison between experimental and predicted yields of oil (%).
Fig. 3. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of temperature and time.
trend indicates that the extracted oil yield increases up to about 68 °C, 120 min and 32,140 kPa, followed by a decrease in the oil yield with a further increase in the level of process variables. As shown in these figures, pressure and temperature had larger effects on the oil yield than time, and their combination had the most significant effect than other combination of extraction parameters. 3.4. Optimum operating conditions for maximizing the oil yield Regression model Eq. (2) was used to determine the optimum processing conditions for SC-CO2 extraction of oil from pumpkin seed. The values of independent variables (X1, X2, X3) were determined and response was calculated at the optimum point. In order to get these optimum values, first the partial derivatives of the regression Eq. (2) were derived with respect to X1, X2, and X3, respectively, and they were set to zero (Ge et al., 2002; Quanhong and Caili, 2005) to get the three equations given below:
8:22X 1 1:09X 2 4:17X 3 ¼ 4:23
ð3Þ
1:09X 1 þ 4:96X 2 þ 1:51X 3 ¼ 1:63
ð4Þ
4:17X 1 þ 1:51X 2 þ 6:06X 3 ¼ 4:00
ð5Þ
By solving Eqs. (3)–(5), the optimum extraction point, called stationary point, was obtained for the oil yield, and the corresponding values of independent variables at this point in coded form were: X1 = 1.3065, X2 = 0.1527 and X3 = 1.5210 [the corresponding experimental parametric values were 68.1 °C (optimum temperature), 94.6 min (optimum time) and 32,140 kPa (optimum pressure), respectively], and the predicted oil yield at the optimum point was 30.7%. The stationary point as obtained above can be a point of maximum response, minimum response or a saddle point. In order to determine the nature of the stationary point, the regression equation was transformed to the canonical form and the eigenvalues were determined using a computer program in SAS 8.1. The eigenvalues obtained were 1.34, 2.32 and 5.95. The stationary point was a maximum response as all the eigenvalues were negative (Bhattacharjee et al., 2007; Mason et al., 2003).
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Fig. 4. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of time and pressure.
ize the oil. The refractive index of the oil was 1.46 ± 0.01. Hunterlab coordinates were: a (1.51 ± 0.52), b (19.10 ± 1.19) and L (40.15 ± 1.72) and indicated the oil to possess green to yellowish color. Peroxide value (3.46 ± 0.42, milli-equivalent of peroxide/kg of oil) of the pumpkin seed oil was very low, which was indicative of a higher oxidative stability for the seed oil. The iodine value was high (115 ± 4.55 g of I2/100 g of oil) indicating a high degree of unsaturation. The combination of high iodine value and low peroxide value demonstrate that the pumpkin seed oil possesses the desirable qualities of an edible oil. Saponification number, 200 ± 2.21 (mg of KOH/g of oil), observed for the pumpkin seed oil was within range 175–250 normally found in other seed oils such as raspberry seed, safflower, sunflower and corn (Yong and Salimon, 2006). The acid value of the pumpkin seed oil was 5.57 ± 0.59 (mg of NaOH/g of oil). Therefore, both physical and chemical characteristics of the oil demonstrate that the pumpkin seed oil is a good candidate for use as an edible oil or supplement.
4. Conclusions
Fig. 5. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of temperature and pressure.
3.5. Fatty acid composition of pumpkin seed oil The fatty acid analysis of the SC-CO2 extracted pumpkin seed oil as determined by gas chromatography (GC) analysis indicated the four leading fatty acids to be palmitic C16:0 (13.8%), stearic C18:0 (11.2%), oleic C18:1 (29.5%) and linoleic C18:2 (45.5%). Younis et al. (2000) reported that the dominant fatty acids found in the pumpkin (Cucurbita pepo) seed oil were: palmitic C16:0 (11.2–14%), stearic C18:0 (8.0–8.2%), oleic C18:1 (28.2–34.0%) and linoleic C18:2 (43.0–53.0%), which were similar to those found in this work. This work shows that the pumpkin seed oil is a rich source of linoleic acid (C18:2) accounting for up to 45% of the total oil. The essential fatty acids like linoleic acid are not easily synthesized in the human system and must be supplied externally through the diet, and pumpkin seed oil can be a good nutritional supplement as a source of linoleic acid. The oil has also been suggested to be used as a cooking or salad oil, or used in the preparation of margarine (El-Adawy and Taha, 2001). 3.6. Physicochemical properties of pumpkin seed oil Refractive index and color (physical properties), and acid value, iodine value, peroxide value and saponification value (chemical properties) of the pumpkin seed oil were determined to character-
Pumpkin (C. maxima) seed oil was extracted by supercritical carbon dioxide and evaluated for quality. The technique was simple and gave a high oil yield. Statistical analysis showed temperature and pressure to be the most important parameters for SC-CO2 extraction of the oil. The oil yield was effectively modelled as a function of the independent variables (temperature, time and pressure) and the response surface model suggested the parametric optimum conditions to be at 68.1 °C for 94.6 min under 32,140 kPa with a predicted oil yield of 30.7% which was within the experimental domain, and accounted for almost 98% of the real maximum yield which was obtained after a much longer extraction time. The refractive index of the oil was 1.46 and the color of the oil was green to yellowish. The acid value, iodine value, peroxide value and saponification value of the pumpkin seed oil were 5.57 (mg of NaOH/g of oil), 115 (g of I2/100 g of oil), 3.46 (milli-equivalent of peroxide/kg of oil) and 200 (mg of KOH/g of oil), respectively. Palmitic C16:0 (13.8%), stearic C18:0 (11.2%), oleic C18:1 (29.5%) and linoleic C18:2 (45.5%) were the major fatty acids found in the pumpkin seed oil. Physicochemical properties of the oil showed that the oil can be a rich source of linoleic acid and has the potential to be used as a nutrient rich food oil. Acknowledgement We gratefully acknowledge the Korea Research Foundation (KRF), Republic of Korea for financial supporting. References AOAC, 1995. Official Method of Analysis, 16th ed. Association of official Agricultural Chemists, Washington, DC. Bernardo-Gil, M.G., Cardoso Lopes, L.M., 2004. Supercritical fluid extraction of Cucurbita ficifolia seed oil. European Food Research Technology 219, 593–597. Bhattacharjee, P., Singhal, R.S., Tiwari, S.R., 2007. Supercritical carbon dioxide extraction of cottonseed oil. Journal of Food Engineering 79, 892–898. Caili, F., Huan, S., Quanhong, L., 2006. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods for Human Nutrition 61, 73–80. El-Adawy, T.A., Taha, K.M., 2001. Characteristics and composition of watermelon, pumpkin, and paprika seed oils and flours. Journal of Agricultural and Food Chemistry 49, 1253–1259. Ge, Y., Ni, Y., Yan, H., Chen, Y., Cai, T., 2002. Optimization of the supercritical fluid extraction of natural vitamin E from wheat germ using response surface methodology. Journal of Food Science 67 (1), 239–243. Ghani, A., 2003. Medicinal Plants of Bangladesh. Asiatic Society of Bangladesh, Dhaka. Guan, X., Yao, H., 2008. Optimization of viscozyme L-assisted extraction of oat bran protein using response surface methodology. Food Chemistry 106, 345–351.
P. Mitra et al. / Journal of Food Engineering 95 (2009) 208–213 Hierro, M.T.G., Santa-Maria, G., 1992. Supercritical fluid extraction of vegetable and animal fats with CO2 – a mini review. Food Chemistry 45, 189–192. Kim, S.J., Chang, K.S., 2005. Optimization of the falcarinol extraction conditions from Acanthopanax senticosus using supercritical fluid carbon dioxide. Food Engineering Progress 9 (1), 52–58. Lang, Q., Wai, C.M., 2001. Supercritical fluid extraction in herbal and natural product studies – a practical review. Talanta 53, 771–782. Mason, R.L., Richard, F., Gunst, R.F., Hess, J.L., 2003. Statistical Design and Analysis of Experiments with Applications to Engineering and Science. John Wiley & Sons, Inc, Hoboken, New Jersey. Quanhong, L., Caili, F., 2005. Application of response surface methodology for extraction optimization of germinant pumpkin seeds protein. Food Chemistry 92, 701–706. Reverchon, E., 1997. Supercritical fluid extraction and fractionation of essential oils and related products. Journal of Supercritical Fluids 10, l–37.
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Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Pumpkin (Cucurbita maxima) seed oil extraction using supercritical carbon dioxide and physicochemical properties of the oil Pranabendu Mitra a, Hosahalli S. Ramaswamy b, Kyu Seob Chang a,* a b
Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Quebec, Canada H9X3V9
a r t i c l e
i n f o
Article history: Received 13 July 2008 Received in revised form 28 April 2009 Accepted 29 April 2009 Available online 10 May 2009 Keywords: Pumpkin seed oil Supercritical extraction Supercritical carbon dioxide, optimization Physicochemical properties
a b s t r a c t Pumpkin (Cucurbita maxima) seed oil was extracted using supercritical carbon dioxide and the physicochemical properties of the oil were determined. A central composite rotatable design was used to analyse the impact of extraction parameters (temperature, time and pressure) and a response surface methodology was used to obtain optimal extraction conditions for the maximum oil yield. All three variables studied were significant demonstrating quadratic effects. The maximum yield of the extracted oil was 30.7% and the optimum conditions were 32,140 kPa and 68.1 °C for 94.6 min which was within the experimental domain. Physicochemical properties of the oil showed that the extracted oil could be used as food oil supplement. Ó 2009 Published by Elsevier Ltd.
1. Introduction Pumpkin (Cucurbita maxima) seeds contain many valuable functional components and have been traditionally used for herbal, therapeutic as well as clinical applications. Pumpkin seeds have been used as safe deworming and diuretic agents, and the seed oil as a nerve tonic (Ghani, 2003; Younis et al., 2000). Pumpkin seed oil has strong antioxidant properties (Stevenson et al., 2007) and has been recognized for several health benefits such as prevention of the growth and reduction of the size of prostate, retardation of the progression of hypertension, mitigation of hypercholesterolemia and arthritis, reduction of bladder and urethral pressure and improving bladder compliance, alleviation of diabetes by promoting hypoglycemic activity, and lowering levels of gastric, breast, lung, and colorectal cancer (Caili et al., 2006; Stevenson et al., 2007). Organic solvents are frequently used to extract oil; for example, hexane has been used for decades to extract oil from cottonseed. However, environmental safety regulations and increased public health risk are necessitating the industry to consider alternatives to the organic solvents for use in oil extraction (Bhattacharjee et al., 2007). Toxic organic residues were considered a serious problem in the solvent extraction of pumpkin seed oil (Wenli et al., 2004). Lang and Wai (2001) reported that conventional
* Corresponding author. Tel.: +82 42 821 6727; fax: +82 42 821 8897. E-mail addresses: [email protected], [email protected] (P. Mitra), [email protected] (H.S. Ramaswamy), [email protected] (K.S. Chang). 0260-8774/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2009.04.033
extraction methods such as hydro-distillation and organic solvent extraction requires the control of several adjustable parameters to improve the extraction processes, and suggested developing alternate extraction techniques for better selectivity and efficiency. Supercritical extraction with carbon dioxide (SC-CO2) is getting popular as a cost effective and environmentally friendly method for extracting useful components such as oils from plant extracts. Supercritical fluids possess both gas-like and liquid-like qualities, and the dual role of such fluids provide ideal conditions for extracting compounds with a high degree of recovery in a short period of time (Reverchon, 1997; Wang and Weller, 2006). Supercritical carbon dioxide is an odourless, chemically inert, non-toxic, non-flammable, non-corrosive, and low cost solvent with high purity, and leaves no residue or contamination in the extract. SC-CO2 possesses moderately low critical temperature (31.1 °C) and pressure (7380 kPa) and the solvent can be removed by simple depressurization (Venkat and Kothandaraman, 1998; Lang and Wai, 2001; Wang and Weller, 2006). Supercritical carbon dioxide, with its particularly attractive properties, is the preferred solvent for many supercritical fluid extractions (Venkat and Kothandaraman, 1998). SC-CO2 possesses superior mass transfer properties with a higher diffusion coefficient and lower viscosity than liquid solvents. The absence of surface tension allows the rapid penetration of SC-CO2 into the pores of heterogeneous matrices and helps to enhance extraction efficiencies (Venkat and Kothandaraman, 1998; Lang and Wai, 2001). Food industry is a major user of the SC-CO2 extraction process for extraction and fractionation of fats, oils, essences, pigments and functional or bioactive compounds
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(Hierro and Santa-Maria, 1992; Reverchon, 1997). Majority of the published work on SC-CO2 has focussed on oil extraction (Bernardo-Gil and Cardoso Lopes, 2004; Bhattacharjee et al., 2007; Hierro and Santa-Maria, 1992; Reverchon, 1997; Wenli et al., 2004) and it is not surprising the technique is becoming a standard method for oil extraction because of its high extraction efficiency, short extraction time and no residue problems (Bhattacharjee et al., 2007; Lang and Wai, 2001). However, the SC-CO2 technique has not been widely used for pumpkin (C. maxima) seed oil extraction. The efficiency of SC-CO2 extraction method depends on the process temperature and pressure. The selectivity or the dissolving power of SC-CO2 can be adjusted by properly selecting such variables. In order to get the best efficiency (maximum yield), the process requires optimization of the three independent variables, temperature, pressure and time. A statistical experimental design based on central composite rotatable design (CCRD) and a response surface methodology (RSM) are often used to characterize the influence of process variables and arrive at optimal processing conditions (Ge et al., 2002). Physical (refractive index, color, etc.) and chemical (acid value, iodine value, saponification value and peroxide value) properties of the oil, and the fatty acid composition are important quality attributes which will determine the oil’s acceptance as a food or medicinal supplement. The objective of this work were therefore to characterize and optimize of extraction parameters of SC-CO2 process for extraction pumpkin seed oil (C. maxima) and to evaluate the physicochemical properties of the extracted oil to highlight the potential for its food use.
2. Materials and methods 2.1. Materials Pumpkin (C. maxima) seeds were obtained from the local market (Jung-Ang, Daejeon, Korea). The seeds were dried in a vacuum drier (Lab companion OV-02, Jeio Tech. Co. Ltd., Korea) at 75 °C and 25 kPa for 24 h to a moisture content of 2%. The dried seeds were then ground in a food grinder (Hanil Science Industrial Co. Ltd., Korea) to reduce the particle size to a maximum diameter of 500 lm as measured by a sieve (Chung Gye Sang Gong Sa, Korea), sealed in a plastic container and stored in a refrigerator until extraction. Carbon dioxide gas (99.99%) used for SC-CO2 extraction was supplied by the SAMO Gas Kungsa (Daejeon, Korea). Chemicals used for seed oil analysis were hexane, ethanol, chloroform and phenolphthalein indicator, and wijs (iodine monochloride in acetic acid glacial) solution (Daejung Chemicals and Metals Co. Ltd., Korea); acetone and ethyl acetate (DC Chemicals Co. Ltd., Korea); acetic acid, phosphoric acid, potassium phosphate and sulfuric acid (Duskan chemical Co. Ltd., Korea); carbon tetrachloride and diethyl ether (Oriental chemical Industrial Co. Ltd., Korea), and heptadecanoic acid (Sigma, USA) and other materials such as TLC plate of 0.25 mm silica gel 60F254 (MERCK, Germany). 2.2. Pumpkin seed oil extraction using SC-CO2 A laboratory assembled SC-CO2 extraction apparatus (Fig. 1) consisted of an extractor vessel (HIP Inc. Erie, PA), compression pump (Haskel Co. Barbank, CA), pressure regulator (HIP Inc. Erie, PA), and pressure gauge (MacDaniel Controls Co.). Twenty-five grams of pumpkin seed powder was placed in the stainless steel extractor with filters at the top and bottom to retain the small particles. The liquid carbon dioxide was cooled through a glycol chiller, compressed (using the liquid pump) and allowed to enter the extractor at the bottom through a hot water bath (for temperature control). The flow rate of CO2 was kept constant at 0.25 L/min for
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Fig. 1. Schematic diagram of supercritical carbon dioxide extraction equipment.
all treatments by manually adjusting the gas flow valve and monitoring the rate through a gas flowmeter. The extractor was brought up to the desired supercritical pressure and temperature. SC-CO2 was then pushed upward through the sample powder. Oil enriched SC-CO2 was then discharged into the separator where carbon dioxide was depressurized. The gaseous carbon dioxide moved out through the gas meter leaving the extracted oil in the separator. The pressures in both the extractor and separator were controlled manually using a back-pressure regulator. After extraction, the oil was collected at the bottom of the separator into 300 mL hexane placed there prior to the extraction process, and then stored in a refrigerator for analysis. Hexane was removed from the extracted oil and concentrated using a rotary vacuum evaporator at 45 °C. During this process, a gentle stream of nitrogen was passed though the oil for complete removal of hexane and to remove oxygen. The yield of the oil was then measured gravimetrically. In order to optimize the extraction process, extraction was carried out at different conditions using a CCRD design and an RSM model. 2.3. Fatty acid composition of pumpkin seed oil Methylated injection samples were prepared prior to gas chromatographic (GC) analysis. Thin layer chromatography (TLC) was carried out by eluting a mobile phase (hexane:diethyl ether:acetic acid = 50:50:1) to separate the triglycerol from the pumpkin seed oil. The spot visible in upper most part of TLC plate corresponding to triglycerol was scratched off and taken into a test tube. Three milli-litres of 6% 1 N H2SO4 in methanol was added followed by 50 lL (1 mg/1 mL hexane) of hepta-decanoic acid (standard) and mixed with a vortex mixer. The sample was heated in an oven at 70 °C for 1 h and cooled; 2 mL hexane (HPLC grade) was then added and mixed in a vortex mixer and finally passed through the Na2SO4 column to remove the residual moisture. The concentrated sample was subjected to gas chromatography (GC) analysis. An HP 6890 Series GC unit (Hewlett-Packard, Avondale, PA) with a flame-ionization detector and auto-injection system and a fusedsilica capillary column (SP-wax, 60 m 0.25 mm Supelco, Bellefonte, PA) were used for the fatty acid analysis. The column was held at 150 °C for 5 min and raised to 220 °C at a rate of 4 °C/ min. The injector and detector temperatures were held at 250 and 260 °C, respectively. The carrier gas was nitrogen with a split ratio of 60:1. 2.4. Physicochemical properties of pumpkin seed oil Acid value, iodine value, peroxide value and saponification value of the extracted pumpkin seed oil were determined using
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AOAC (1995) methods. The Hunterlab color coordinates (L, a, b) of the oil were measured with a colorimeter (MINOLTA, CR-300, Japan). Refractive index of the oil was determined using a hand held refractometer (ATAGO N3, Atago Co. Ltd., Japan) at 20 °C. All the measurements were made in duplicate. 2.5. Statistical analysis 2.5.1. Experimental design A CCRD was used to study the effect of extraction variables on the oil yield. The three input variables were temperature (35–75 °C, X1), time (30–150 min, X2) and pressure (15,160–34,450 kPa, X3) and the five levels chosen were 2, 1, 0, +1 and +2 as shown in Table 1. The input ranges and levels were selected based on preliminary experiments (Kim and Chang, 2005). The experimental design consisted 16 runs (n = 2k + 2k + m, where, n = total experimental points, input variables, k = 3 and central point, m = 2), which included eight factorial points, six axial points and two replicated central points (Mason et al., 2003) as shown in Table 2. 2.5.2. Response surface modeling A second order polynomial equation was used to fit the coded variables:
Table 1 Treatments levels and coded values for each of the independent variables used in developing the experimental data to optimize the pumpkin seed oil extraction using supercritical carbon dioxide. Independent variable
Symbols
Extraction temperature (°C)
Extraction time (min)
Extraction pressure (kPa)
Coded
Uncoded
Coded
T
X1
35 45 55 65 75 30 60 90 120 150 15,160 19,980 24,805 29,630 34,450
2 1 0 1 2 2 1 0 1 2 2 1 0 1 2
P
þ B12 X 1 X 2 þ B23 X 2 X 3 þ B13 X 1 X 3
ð1Þ
where, Y represented the experimental response, B0, B1, B2, B3, B11, B22, B33, B12, B23 and B13 were constants and regression coefficients, respectively, of the model, and X1, X2 and X3 were the independent variables. The model thus included linear, quadratic and crossproduct terms to determine the effect of process variables on the response. The CCRD experimental design was combined with RSM to solve the regression equation and investigate the effects of three independent input variables of temperature, time and pressure to arrive at the optimal conditions giving the maximum oil yield extraction. Statistical analyses were done by a computer program in SAS 8.1. 3. Results and discussion 3.1. Experimental data Temperature, pressure and time were used as the input process variables in the SC-CO2 extraction process as has been commonly done in other studies. Bhattacharjee et al. (2007) used the same factors to extract cotton seed oil using SC-CO2 and Kim and Chang (2005) used them to optimize falcarinol extraction. Results obtained under the different testing conditions are shown in Table 2. The highest yield of oil was 31.5% and was obtained after 120 min extraction at 65 °C and 29,630 kPa. Overall observation of the results showed that the oil yield generally increased with the quadratic effects of time, temperature and pressure of the SC-CO2 extraction process.
Levels
Uncoded
t
Y ¼ B0 þ B1 X 1 þ B2 X 2 þ B3 X 3 þ B11 X 21 þ B22 X 22 þ B33 X 23
X2
X3
X1 = (T 55)/10, X2 = (t90)/30, X3 = (P 24,805)/4825.
3.2. Regression model The developed regression model for the relationship between oil yield (Y) and the coded values of independent variables of temperature (X1), time (X2) and pressure (X3) and their interaction is shown in the following equation:
Y ¼ 24:79 þ 4:23X 1 þ 1:63X 2 þ 4:00X 3 4:11X 21 2:48X 22 3:03X 23 þ 1:09X 1 X 2 1:51X 2 X 3 þ 4:17X 1 X 3
ð2Þ
The significance of each coefficient and their interactions was determined using the t-test (t value). The linear terms of extraction temperature (X1, absolute t value = 3.98), extraction time (X2, absolute t value = 2.34) and extraction pressure (X3, absolute t value = 3.77), the second order terms of X 21 (absolute t value = 3.88) and X 23 (absolute t value = 2.85) and the cross-product term
Table 2 Experimental design and results of pumpkin seed oil extraction using supercritical carbon dioxide. Treatment
Extraction temperature, T (°C) X1
Extraction time, t (min) X2
Extraction Pressure, P (kPa) X3
Amounts of oil (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
45 45 45 45 65 65 65 65 55 55 35 75 55 55 55 55
60 (1) 60 (1) 120 (1) 120 (1) 60 (1) 60 (1) 120 (1) 120 (1) 90 (0) 90 (0) 90 (0) 90 (0) 30 (2) 150 (2) 90 (0) 90 (0)
19,980 29,630 19,980 29,630 19,980 29,630 19,980 29,630 24,805 24,805 24,805 24,805 24,805 24,805 15,160 34,450
06.48 09.08 09.40 10.20 06.72 30.24 18.24 31.48 25.00 24.48 04.28 12.32 12.52 17.12 06.68 18.60
(1) (1) (1) (1) (1) (1) (1) (1) (0) (0) (2) (2) (0) (0) (0) (0)
(1) (1) (1) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (2) (2)
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P. Mitra et al. / Journal of Food Engineering 95 (2009) 208–213 Table 3 Analysis of variance (ANOVA) of the regression parameters for the response surface model of pumpkin seed oil yield. Regression
DF
Type I sum of squares
R-square (R2)
F Value
Pr > F
Linear Quadratic Cross-product Total model
3 3 3 9
583.86 290.59 166.86 1041.31
0.51 0.25 0.15 0.91
10.82 5.39 3.09 6.43
0.0078 0.0388 0.1112 0.0172
between extraction temperature and pressure (X1X3, absolute t value = 2.78) gave highly significant effects on oil yield having higher absolute t value than critical t value of 2.26 (at p value <0.05 and degree of freedom = 9). The coefficients were highly significant with an absolute t value higher than critical t value. Mason et al. (2003) emphasized that in order to reject the null hypothesis the model t value needs to exceed the critical t value. The second order term of X 22 (time) and cross-product terms of X1X2 (temperature–time) and X2X3 (time–pressure) did not show a significant effect on oil yield (p > 0.05) while X1X3 (temperature–pressure) interaction was significant. Analysis of variance (ANOVA) results of the model are shown in Table 3 indicating a good model performance with an R2 value of 0.91 and an F value of 6.43 among linear, quadratic, cross-product and total model. Interaction among the variables was not negligible. R2 value is one of the measures of degree of fit and Guan and Yao (2008) reported that an R2 should be at least 0.80 for the good fit of a model. The model R2 value of 0.91 implies that 91% of the variations associated with the pumpkin seed oil extraction yield can be attributed to the three independent variables (temperature, time and pressure). A plot of the experimental values of oil yield versus predicted values (Eq. (2)) is shown in Fig. 2. The plot showed a close fit and a uniform distribution of the observed values around the predicted ones. Thus, a statistically significant multiple regression relationship between the independent variables (X1, X2 and X3) and the response variable (Y) could be established. The second order polynomial model could therefore be effectively used to represent the relationship among the parameters selected. 3.3. Response surface analysis The 3-dimentional plots of the response surfaces are presented in Figs. 3–5 by presenting the response as function of two factors and keeping the third one at the constant mid-level. Fig. 3 shows that the extraction temperature and the extraction time have the quadratic effects on oil yield. Quadratic effects on oil yield can also be observed in Fig. 4 (for the extraction time and pressure) and in Fig. 5 (for the extraction temperature and pressure). In general the
Fig. 2. Comparison between experimental and predicted yields of oil (%).
Fig. 3. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of temperature and time.
trend indicates that the extracted oil yield increases up to about 68 °C, 120 min and 32,140 kPa, followed by a decrease in the oil yield with a further increase in the level of process variables. As shown in these figures, pressure and temperature had larger effects on the oil yield than time, and their combination had the most significant effect than other combination of extraction parameters. 3.4. Optimum operating conditions for maximizing the oil yield Regression model Eq. (2) was used to determine the optimum processing conditions for SC-CO2 extraction of oil from pumpkin seed. The values of independent variables (X1, X2, X3) were determined and response was calculated at the optimum point. In order to get these optimum values, first the partial derivatives of the regression Eq. (2) were derived with respect to X1, X2, and X3, respectively, and they were set to zero (Ge et al., 2002; Quanhong and Caili, 2005) to get the three equations given below:
8:22X 1 1:09X 2 4:17X 3 ¼ 4:23
ð3Þ
1:09X 1 þ 4:96X 2 þ 1:51X 3 ¼ 1:63
ð4Þ
4:17X 1 þ 1:51X 2 þ 6:06X 3 ¼ 4:00
ð5Þ
By solving Eqs. (3)–(5), the optimum extraction point, called stationary point, was obtained for the oil yield, and the corresponding values of independent variables at this point in coded form were: X1 = 1.3065, X2 = 0.1527 and X3 = 1.5210 [the corresponding experimental parametric values were 68.1 °C (optimum temperature), 94.6 min (optimum time) and 32,140 kPa (optimum pressure), respectively], and the predicted oil yield at the optimum point was 30.7%. The stationary point as obtained above can be a point of maximum response, minimum response or a saddle point. In order to determine the nature of the stationary point, the regression equation was transformed to the canonical form and the eigenvalues were determined using a computer program in SAS 8.1. The eigenvalues obtained were 1.34, 2.32 and 5.95. The stationary point was a maximum response as all the eigenvalues were negative (Bhattacharjee et al., 2007; Mason et al., 2003).
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Fig. 4. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of time and pressure.
ize the oil. The refractive index of the oil was 1.46 ± 0.01. Hunterlab coordinates were: a (1.51 ± 0.52), b (19.10 ± 1.19) and L (40.15 ± 1.72) and indicated the oil to possess green to yellowish color. Peroxide value (3.46 ± 0.42, milli-equivalent of peroxide/kg of oil) of the pumpkin seed oil was very low, which was indicative of a higher oxidative stability for the seed oil. The iodine value was high (115 ± 4.55 g of I2/100 g of oil) indicating a high degree of unsaturation. The combination of high iodine value and low peroxide value demonstrate that the pumpkin seed oil possesses the desirable qualities of an edible oil. Saponification number, 200 ± 2.21 (mg of KOH/g of oil), observed for the pumpkin seed oil was within range 175–250 normally found in other seed oils such as raspberry seed, safflower, sunflower and corn (Yong and Salimon, 2006). The acid value of the pumpkin seed oil was 5.57 ± 0.59 (mg of NaOH/g of oil). Therefore, both physical and chemical characteristics of the oil demonstrate that the pumpkin seed oil is a good candidate for use as an edible oil or supplement.
4. Conclusions
Fig. 5. Response surface plot of the supercritical carbon dioxide extraction of the pumpkin seed oil for the effect of temperature and pressure.
3.5. Fatty acid composition of pumpkin seed oil The fatty acid analysis of the SC-CO2 extracted pumpkin seed oil as determined by gas chromatography (GC) analysis indicated the four leading fatty acids to be palmitic C16:0 (13.8%), stearic C18:0 (11.2%), oleic C18:1 (29.5%) and linoleic C18:2 (45.5%). Younis et al. (2000) reported that the dominant fatty acids found in the pumpkin (Cucurbita pepo) seed oil were: palmitic C16:0 (11.2–14%), stearic C18:0 (8.0–8.2%), oleic C18:1 (28.2–34.0%) and linoleic C18:2 (43.0–53.0%), which were similar to those found in this work. This work shows that the pumpkin seed oil is a rich source of linoleic acid (C18:2) accounting for up to 45% of the total oil. The essential fatty acids like linoleic acid are not easily synthesized in the human system and must be supplied externally through the diet, and pumpkin seed oil can be a good nutritional supplement as a source of linoleic acid. The oil has also been suggested to be used as a cooking or salad oil, or used in the preparation of margarine (El-Adawy and Taha, 2001). 3.6. Physicochemical properties of pumpkin seed oil Refractive index and color (physical properties), and acid value, iodine value, peroxide value and saponification value (chemical properties) of the pumpkin seed oil were determined to character-
Pumpkin (C. maxima) seed oil was extracted by supercritical carbon dioxide and evaluated for quality. The technique was simple and gave a high oil yield. Statistical analysis showed temperature and pressure to be the most important parameters for SC-CO2 extraction of the oil. The oil yield was effectively modelled as a function of the independent variables (temperature, time and pressure) and the response surface model suggested the parametric optimum conditions to be at 68.1 °C for 94.6 min under 32,140 kPa with a predicted oil yield of 30.7% which was within the experimental domain, and accounted for almost 98% of the real maximum yield which was obtained after a much longer extraction time. The refractive index of the oil was 1.46 and the color of the oil was green to yellowish. The acid value, iodine value, peroxide value and saponification value of the pumpkin seed oil were 5.57 (mg of NaOH/g of oil), 115 (g of I2/100 g of oil), 3.46 (milli-equivalent of peroxide/kg of oil) and 200 (mg of KOH/g of oil), respectively. Palmitic C16:0 (13.8%), stearic C18:0 (11.2%), oleic C18:1 (29.5%) and linoleic C18:2 (45.5%) were the major fatty acids found in the pumpkin seed oil. Physicochemical properties of the oil showed that the oil can be a rich source of linoleic acid and has the potential to be used as a nutrient rich food oil. Acknowledgement We gratefully acknowledge the Korea Research Foundation (KRF), Republic of Korea for financial supporting. References AOAC, 1995. Official Method of Analysis, 16th ed. Association of official Agricultural Chemists, Washington, DC. Bernardo-Gil, M.G., Cardoso Lopes, L.M., 2004. Supercritical fluid extraction of Cucurbita ficifolia seed oil. European Food Research Technology 219, 593–597. Bhattacharjee, P., Singhal, R.S., Tiwari, S.R., 2007. Supercritical carbon dioxide extraction of cottonseed oil. Journal of Food Engineering 79, 892–898. Caili, F., Huan, S., Quanhong, L., 2006. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods for Human Nutrition 61, 73–80. El-Adawy, T.A., Taha, K.M., 2001. Characteristics and composition of watermelon, pumpkin, and paprika seed oils and flours. Journal of Agricultural and Food Chemistry 49, 1253–1259. Ge, Y., Ni, Y., Yan, H., Chen, Y., Cai, T., 2002. Optimization of the supercritical fluid extraction of natural vitamin E from wheat germ using response surface methodology. Journal of Food Science 67 (1), 239–243. Ghani, A., 2003. Medicinal Plants of Bangladesh. Asiatic Society of Bangladesh, Dhaka. Guan, X., Yao, H., 2008. Optimization of viscozyme L-assisted extraction of oat bran protein using response surface methodology. Food Chemistry 106, 345–351.
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