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Ultrasound-assisted extraction of oil from flaxseed
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Separation and Purification Technology 62 (2008) 192–198
Ultrasound-assisted extraction of oil from flaxseed Zhen-Shan Zhang a , Li-Jun Wang b , Dong Li a,∗ , Shun-Shan Jiao a , Xiao Dong Chen a,∗ , Zhi-Huai Mao a b
a College of Engineering, China Agricultural University, P.O. Box 50, 17 Qinghua Donglu, Beijing 100083, China College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, China
Received 26 November 2007; received in revised form 12 January 2008; accepted 16 January 2008
Abstract Flaxseed oil is the main component of the flaxseed and has many beneficial functions to human health. In this paper, the ultrasound-assisted extraction of oil from flaxseed is reported. The effects of some operating parameters such as ultrasonic power, extraction time, extraction temperature and solvent to solid ratio on the yield of flaxseed oil have been investigated and some of the results have been compared with that of conventional method. It has been found that ultrasound-assisted extraction requires a shorter extraction time and a reduced solvent consumption. The yield of flaxseed oil has been found to increase with the increase of the ultrasonic power and to decrease as the temperature is increased. Scanning electronic microscopy analysis was carried out on the flaxseed powder after the extraction. The images are powerful evidences to show the effect of ultrasound. The fatty acid compositions of the oils extracted by the ultrasound-assisted method and the conventional method have been analyzed using gas chromatography. It has been shown that the compositions of the flaxseed oils were not affected significantly by the application of ultrasound (p > 0.05). The ultrasound-assisted extraction may be an effective method for lipid production. © 2008 Elsevier B.V. All rights reserved. Keywords: Extraction; Ultrasound-assisted; Oil; Flaxseed
1. Introduction Flaxseed, which is also called linseed, is an important oilseed in the world. It is mainly grown in Canada, Argentina, America, China and India [1]. Usually, flaxseed contains about 40% oil, 30% dietary fiber, 20% protein, 4% ash, and 6% moisture [2]. In the past, flaxseed was mainly used to provide flaxseed oil for industrial purposes such as the production of paints, linoleum, varnishes, inks and cosmetics [3,4]. However, flaxseed is playing an important role in functional foods for its nutritional and pharmaceutical values. The nutritional components of flaxseed are oil, protein, lignans, soluble fiber, minerals and vitamin, etc. [5]. Flaxseed is known as the richest source of the n-3 fatty acid, alpha linolenic acid (ALA), which comprises approximately 55% of the total fatty acids and this percentage is 5.5 times higher than that in the next-highest sources [6]. ALA can be metabolized to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in human intestine due to the action of some
∗
Corresponding authors. Tel.: +86 10 62737351; fax: +86 10 62737351. E-mail addresses: [email protected] (D. Li), [email protected] (X.D. Chen). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.01.014
kinds of enzyme [7]. It is well established, in human bodies, that an increase in the ingestion of long-chain poly-unsaturated fatty acids (LC-PUFA), especially EPA and DHA, in diet can reduce the risk of diseases [8]. Previous studies have proven that flaxseed oil has positive effect on the minimization of many diseases such as hyperlipidemia [9], colon tumor [10], mammary cancer [11–13] and atherosclerosis [14,15]. At present, flaxseed oil is mainly extracted from flaxseed by press extraction and solvent extraction. Press extraction is often associated with lower yield and more energy consumption, while solvent extraction often involves longer extraction time. The use of large quantities of organic solvents is not desirable and can be harmful to human and environment. Moreover, flaxseed oil is thermally unstable and may degrade at higher temperatures. For these reasons, an improved or better extraction technique is desirable. Thus, supercritical fluid extraction, aqueous enzymatic extraction and ultrasound-assisted extraction (UAE) techniques can be further developed. Recent studies have shown that the ultrasound-assisted extraction can enhance the extraction efficiency through acoustic cavitation and some mechanical effects [16–18]. Acoustic cavitation can disrupt cell walls facilitating solvent to penetrate into the plant material and allowing the intracellular product
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release. Another mechanical effect caused by ultrasound may also be the agitation of the solvent used for extraction, thus increasing the contact surface area between the solvent and targeted compounds by permitting greater penetration of solvent into the sample matrix. Therefore the main advantages of ultrasound-assisted extraction include the reduced extraction time and reduced solvent consumption [19]. In addition, ultrasound-assisted extraction can be carried out at a lower temperature which can avoid thermal damage to the extracts and minimize the loss of bioactive compounds [20]. So far, there seem to be no report about ultrasound-assisted extraction oil from flaxseed. The aim of this study was to determine the effects of some factors on the yield of flaxseed oil with or without ultrasound treatment. The results from UAE were compared with that obtained from the conventional solvent extraction, i.e. maceration extraction (ME). After the extraction of flaxseed oil, microscopic images of flaxseed powder were obtained by scanning electron microscope (SEM) to prove the existence of ultrasonic effects. The fatty acid compositions of flaxseed oils extracted by UAE and ME were measured using gas chromatography (GC) to investigate the effect of ultrasound on the quality of flaxseed oil extracted. 2. Materials and methods 2.1. Samples and reagents The flaxseed with a moisture content of 6.2 wt% was purchased from the market of Hebei Province of China. The flaxseed was cleaned by hand carefully to remove the foreign materials such as other seeds, stones and small stalks. The cleaned flaxseed was dried for 12 h at 105 ◦ C in an oven, and then crushed into powder in a grinder (B-400, B¨uchi Labortechnik AG, Switzerland) with a size range of 0.45–1.2 mm. The resulted powder was kept in a vacuum dryer until use. n-Hexane used in the extraction was analytical grade and was purchased from Beijing Beihua Chemical Company. 2.2. Conventional extraction The crushed flaxseed powder (10 g) was mixed with n-hexane in a flask. The flask was put into the water bath with a controlled temperature. After each extraction experiment, the extracts were filtered through the Whatman No. 1 filter paper under vacuum, and then the solution was collected and concentrated with a rotary evaporator (RE-2S, Beijing Jing Zhi Jie Laboratory Apparatus Co. Ltd., China) to acquire the flaxseed oil. The acquired flaxseed oil was further dried in a vacuum dryer to remove the residual n-hexane. 2.3. Ultrasound-assisted extraction For the UAE experiments, a 250 W, 20 kHz ultrasonic emulsifier (88-1, Institute of Acoustics, Chinese Academy of Sciences, China) with a 2.00 cm flat tip probe was used (Fig. 1). The ultrasonic output power could be set to a desired level ranging from 0 to 100% of the nominal power by the amplitude controller.
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Ultrasonic output powers were determined calorimetrically and ranged from 10 to 50 W according to the method described by Li et al. [16]. The ultrasound-assisted extraction used in this study was similar to that described by Zhao et al. [21], with a little modification. The flaxseed powder was mixed in 100 mL n-hexane contained in a 200 mL plastic beaker. The ultrasonic probe was inserted into the mixture directly. The samples were extracted under continuous ultrasonic waves at 20 kHz at different levels of power output. During extraction, the temperature was controlled at a desired level within ±1 ◦ C. The post-treatment of the extracts was the same as that mentioned in the conventional extraction. In this study, all the experiments were performed in triplicates, and the results reported here are the means of the three trials. 2.4. Yield determination The yield of flaxseed oil was calculated using the following formula: We yield (%) = × 100 Wt where We is the mass of flaxseed oil extracted from the sample (g) and Wt is the mass of total oil in the sample (g). Wt was obtained by Soxhlet standard extraction mode of the Buchi extraction system (B-811, B¨uchi Labortechnik AG, Switzerland) after hydrolyzing for 30 min in 4 M HCl in a hydrolysis Unit (B-411, B¨uchi Labortechnik AG, Switzerland). 2.5. Scanning electron microscopy (SEM) analysis SEM can be used to reveal the microstructure of the material [22]. In order to investigate the influence of ultrasound during extraction on the structure of the materials and to understand the extraction mechanism, the solid remained was collected and dried in air after the extraction of flaxseed oil for the SEM analysis. Sample particles were fixed on the silicon wafer and sputtered with gold to a thickness of about 100 nm. The shape and the surface characters of the samples were observed and recorded on the scanning electron microscope (KYKY-2800, KYKY Technology Development Ltd., China). 2.6. Gas chromatographic analysis Fatty acid compositions of the oils extracted by UAE and ME were determined using GC after derivatization to fatty acid methyl esters (FAME). The preparation of FAME was performed via saponification in 0.5 M NaOH–MeOH solution and methylation with 14% BF3 –MeOH (Sigma, USA), according to the 5509 ISO method [23]. FAME separation and identification were carried out on the gas chromatograph (6890N, Agilent Technologies, USA) equipped with a flame ionization detector and capillary column HP-Innowax (30 m × 0.32 mm × 0.25 m). The amount of each sample injected was 1.0 L. Nitrogen, at a constant flow 1.0 mL/min, was used as the carrier gas and a spilt/spiltless injector was used with a split ratio of 50:1. The injector temperature was 250 ◦ C and the detector temperature
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Fig. 1. The schematic diagram of the apparatus of the ultrasound-assisted extraction.
was 280 ◦ C. The column temperature program was as follow: the initial column temperature was 40 ◦ C, which was maintained for 0.5 min, from 40 ◦ C to 195 ◦ C at 25 ◦ C/min, from 195 ◦ C to 205 ◦ C at 3 ◦ C/min, from 205 ◦ C to 230 ◦ C at 8 ◦ C/min and held for 4 min at 230 ◦ C, and then to a final temperature of 240 ◦ C at a rate of 5 ◦ C/min, and held for another 2 min. Fatty acid methyl esters were identified by comparison with the standard fatty acid methyl esters (Sigma, USA). Fatty acid methyl esters were quantified as percentages of the total methyl ester peak areas.
ated and collapsed [24]. Since the temperature and pressure were very high inside the bubbles and the collapse of bubbles occurred over very short time, the violent shock wave and high-speed jet were generated which could enhance the penetration of the sol-
3. Results and discussion 3.1. Effect of ultrasonic power The effect of ultrasonic power on the yield of flaxseed oil is shown in Fig. 2. It can be seen that the yield of flaxseed oil increases almost linearly with increasing ultrasonic power. When the power was increased from 20 to 50 W (1.5 folds increases), the yield of flaxseed oil was increased from 66.7% to 84.9% (18.2% increases). As the larger amplitude ultrasonic wave traveled though a liquid medium, more bubbles were cre-
Fig. 2. The effect of ultrasonic output power on the yield of flaxseed oil (extraction time: 30 min; solid to liquid ratio: 1:10; extraction temperature: 25 ◦ C).
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vent into the cell tissues and accelerate the intracellular product release into the solvent by disrupting the cell walls. Moreover, the violent shock wave and high-speed jet might have caused the molecules to mix better enhancing the mass transfer rate. Due to the presence of the hard cell walls which are not so permeable, the large increase in ultrasonic power resulted in a moderate rise in yield. Similar results were obtained by Li et al. [16] and Sivakumar et al. [25], respectively, in the ultrasound-assisted extraction of oil from soybean and tannin from myrobalan nut. Based on the current results, the maximum ultrasonic power of 50 W was chosen as the output power. 3.2. Effect of extraction time Fig. 3 shows the effect of experimental duration for extraction on the yield of flaxseed oil. It can be seen that the yield of flaxseed oil increases with the extraction time in both methods. The yield of flaxseed oil in UAE was higher than that in ME at all the time here. The difference was about 12% between ME and UAE during the first 15 min and increases gradually to 30 min to about 18%. The difference then became very gradual and for the rest of the extraction up to 180 min the difference levels increased to about 16%. This process indicates that the effect of ultrasound is more effective in the first 30 min. It may be that ultrasonic wave could disrupt the cell walls, so larger contact area between solvent and material was created and more oil was appeared on the surface in UAE than that in ME. However, this effect would be increasingly weak on the inner cell walls as the distance is increased. It should be noted that both processes occur very rapidly at the beginning of the extractions. UAE has the greatest rise in yield. Perhaps the ultrasonic waves affect the mass transfer rate mainly in the solvent penetration stage. It should of course also enhance the dissolved oil to transfer out of the solid structure. F-Test showed that the extraction time had a significant effect on the yield of flaxseed oil (p < 0.005), and the effect of ultrasound on the yield also was significant (p < 0.001). Similar results were obtained by Zhao et al. [21], Hemwimol et al. [24] and Balachandran et al. [26] in their researches. In the research of Balachandran et al. [26], it had been confirmed that
Fig. 3. The effect of ultrasonic extraction time on the yield of flaxseed oil (ultrasonic output power: 50 W; solid to liquid ratio: 1:10; extraction temperature: 25 ◦ C).
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the ultrasound continued to be effective even during the later stage of extraction through an enhancement to the internal diffusivity, although the effects were smaller. In any case, based on the results so far, 30 min was found to be an optimum operating time for UAE. 3.3. Effect of temperature As shown in Fig. 4, the yield of flaxseed oil does not change with increasing temperature in ME. When the temperature changed from 30 ◦ C to 50 ◦ C, the yield of flaxseed oil increased only about 0.1% in ME. This is somewhat surprising, as temperature rise should increase the solubility and diffusivities. It may be that the limiting process for extraction (as expected usually) is dissolution and diffusion within the intact solid matrix. The temperature dependence is not that sensitive at this lower range of temperature. In the UAE, the yield of flaxseed oil was found to decrease with the increase of temperature. The yield of flaxseed oil was about 83% at 30 ◦ C and decreased by about 6% to about 77% at 50 ◦ C. This is surprising as well, one reason for this may be that the vapor pressure of solvent increased with the increase of temperature and the vapor pressure had a great influence on the occurrence and the intensity of acoustic cavitation. At lower temperature, the vapor pressure is lower. Ultrasound produces a few cavitational bubbles as a result of high acoustic cavitation threshold. However, the bubbles explodes with relatively greater force, which enhanced cell tissues disruption during extraction. At higher temperature, the vapor pressure was higher and more bubbles were created, but they collapsed with less intensity due to a smaller pressure difference between inside and outside of bubbles [20]. Another reason may be due to the surface tension which decreased with the increase of temperature affecting the bubble formation and collapse. The bubbles may be so easily collapsed at higher temperature thus reducing the intensity of the mass transfer enhancement. F-Test showed that the effect of extraction temperature was insignificant (p > 0.05), but the ultrasound had a significant effect on the yield of flaxseed oil (p < 0.025). Finally, 30 ◦ C or room temperature may be seen as an optimum condition for the extraction of flaxseed oil in UAE.
Fig. 4. The effect of solvent temperature on the yield of flaxseed oil (extraction time: 30 min; ultrasonic output power: 50 W; solid to liquid ratio: 1:10).
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Fig. 5. The effect of solvent to solid ratio on the yield of flaxseed oil (extraction time: 30 min; ultrasonic output power: 50 W; extraction temperature: 25 ◦ C).
3.4. Effect of liquid to solid ratio As shown in Fig. 5, the ratio of liquid to solid in both methods had similar effect on the yield of flaxseed oil. Both the yields of flaxseed oil extracted by UAE and ME were found to increase with the increase of the ratio of liquid to solid and then level off at the high ratios. The larger liquid (solvent) to solid ratio means a larger concentration difference which favors mass transfer. When the liquid to solid ratio is small, this effect is more obvious. Too much liquid would not change much of the driving force any more as the limitation to mass transfer is more confined to the solid interior. Again, UAE outperformed ME at all liquid–solid ratios tested here. Similar result has also been shown by Zhao et al. [21]. F-Test showed that both the liquid to solid ratio and ultrasound had a significant effect on the yield of flaxseed oil (p < 0.001). The liquid to solid ratio of 6:1 seems to be appropriate for UAE. 3.5. Effect of ultrasound on the physical structure The SEM images of the flaxseed powder at a magnification factor of 1000× are shown in Fig. 6. There was a significant flaxseed oil on the surface of material (Fig. 6(a)). After 30 min maceration extraction, the majority of flaxseed oil on the surface of material had gone (yield = 66.7%) (Fig. 6(b)). After 30 min ultrasound-assisted extraction, most of the oil from the flaxseed had been extracted (yield = 84.9%) (Fig. 6(c)). Fig. 6(b) shows significant amount of the flaxseed oil is still present in the sample (in contrast to the UAE extracted sample shown in Fig. 6(c), which has clearly much less oil content). This visual observation demonstrated that the application of ultrasound in the UAE accelerated the flaxseed oil removal. 3.6. Effect of UAE on the composition of flaxseed oil The fatty acid compositions of flaxseed oil extracted by UAE and ME are given in Table 1. These results are consistent with that reported by Choo et al. [27]. The variance analysis and hypothesis testing were carried out between the data of fatty acid
Fig. 6. Scanning electron microscopy images of flaxseed powder. (a) Untreated. (b) After 30 min maceration extraction. (c) After 30 min ultrasound-assisted extraction.
compositions extracted by UAE and ME. The results showed that there were no remarkable differences (p > 0.05) in the oil profiles extracted by both methods. This indicates that the compositions of flaxseed oil are hardly affected by the application of the ultrasound. Luque-Garc´ıa and Luque de Castro [28] investigated the influence of ultrasound on the fatty acid composition of other oilseeds such as soybean, sunflower and rapeseeds and the results are also compatible with ours. In this work, the physical properties that include density, colour, refractive index, transparency, moisture and volatile matter, etc. were not studied in detail. Similarly, the chem-
Z.-S. Zhang et al. / Separation and Purification Technology 62 (2008) 192–198 Table 1 Fatty acid composition (% of total fatty acids) of flaxseed oil extracted by different methods Fatty acid
UAE
ME
C14:0 C15:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:2 C20:4 C22:0 C22:1 C22:2 C23:0 C24:0 C24:1 Other SFA MUFA PUFA n-3/n-6 ratio
0.038 0.018 5.529 0.053 0.059 0.042 3.267 16.919 15.835 56.006 0.216 0.315 0.061 0.144 0.021 0.092 0.026 0.094 0.017 1.248 9.391 17.052 72.309 3.523
0.050 0.019 5.613 0.058 0.063 0.045 3.245 16.762 15.810 55.776 0.138 0.320 0.072 0.130 0.020 0.114 0.017 0.085 0.014 1.649 9.360 16.899 72.092 3.512
UAE, ultrasound-assisted extraction; ME, maceration extraction; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-3, omega-3 fatty acid; n-6, omega-6 fatty acid.
ical properties which consist of acid value, iodine value, peroxide value, saponification value, etc. were left for future study. 4. Conclusions In the study presented in this paper, two extraction techniques for oil in flaxseed were investigated and compared. The yield of flaxseed oil increased with increasing extraction time, ultrasonic power and ratio of liquid to solid. Ultrasound-assisted extraction showed a higher yield and a lower solvent consumption. It is important to note that using ultrasound, the extraction time is significantly shortened. Thus, the ultrasound-assisted extraction technique was shown to be very efficient in the extraction of oil from flaxseed. The good conditions for UAE were found to be ultrasonic power: 50 W, extraction temperature: 30 ◦ C, extraction time: 30 min and liquid to solid ratio: 6:1 (v/w). The SEM images have provided an excellent evidence of the effectiveness of UAE. The GC analysis has shown that the compositions of the flaxseed oil extracted were hardly affected by the application of ultrasound. As such, it may be said that UAE is an effective and indeed feasible method for the production of the plant oil. Acknowledgements Research support was provided by the Research and Development Fund for University’s Doctoral Discipline of the Chinese
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Ministry of Education (No. 20050019029), the Science and Technology Research Key Program of Chinese Ministry of Education (No. 105014), the High Technology Research and Development Program of Chinese Ministry of Science and Technology (No. 2006AA10256-02), and the Science and Technology Development Planning Program of Beijing Municipal Education Committee (No. KM200710011005).
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Ultrasound-assisted extraction of oil from flaxseed Zhen-Shan Zhang a , Li-Jun Wang b , Dong Li a,∗ , Shun-Shan Jiao a , Xiao Dong Chen a,∗ , Zhi-Huai Mao a b
a College of Engineering, China Agricultural University, P.O. Box 50, 17 Qinghua Donglu, Beijing 100083, China College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, China
Received 26 November 2007; received in revised form 12 January 2008; accepted 16 January 2008
Abstract Flaxseed oil is the main component of the flaxseed and has many beneficial functions to human health. In this paper, the ultrasound-assisted extraction of oil from flaxseed is reported. The effects of some operating parameters such as ultrasonic power, extraction time, extraction temperature and solvent to solid ratio on the yield of flaxseed oil have been investigated and some of the results have been compared with that of conventional method. It has been found that ultrasound-assisted extraction requires a shorter extraction time and a reduced solvent consumption. The yield of flaxseed oil has been found to increase with the increase of the ultrasonic power and to decrease as the temperature is increased. Scanning electronic microscopy analysis was carried out on the flaxseed powder after the extraction. The images are powerful evidences to show the effect of ultrasound. The fatty acid compositions of the oils extracted by the ultrasound-assisted method and the conventional method have been analyzed using gas chromatography. It has been shown that the compositions of the flaxseed oils were not affected significantly by the application of ultrasound (p > 0.05). The ultrasound-assisted extraction may be an effective method for lipid production. © 2008 Elsevier B.V. All rights reserved. Keywords: Extraction; Ultrasound-assisted; Oil; Flaxseed
1. Introduction Flaxseed, which is also called linseed, is an important oilseed in the world. It is mainly grown in Canada, Argentina, America, China and India [1]. Usually, flaxseed contains about 40% oil, 30% dietary fiber, 20% protein, 4% ash, and 6% moisture [2]. In the past, flaxseed was mainly used to provide flaxseed oil for industrial purposes such as the production of paints, linoleum, varnishes, inks and cosmetics [3,4]. However, flaxseed is playing an important role in functional foods for its nutritional and pharmaceutical values. The nutritional components of flaxseed are oil, protein, lignans, soluble fiber, minerals and vitamin, etc. [5]. Flaxseed is known as the richest source of the n-3 fatty acid, alpha linolenic acid (ALA), which comprises approximately 55% of the total fatty acids and this percentage is 5.5 times higher than that in the next-highest sources [6]. ALA can be metabolized to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in human intestine due to the action of some
∗
Corresponding authors. Tel.: +86 10 62737351; fax: +86 10 62737351. E-mail addresses: [email protected] (D. Li), [email protected] (X.D. Chen). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.01.014
kinds of enzyme [7]. It is well established, in human bodies, that an increase in the ingestion of long-chain poly-unsaturated fatty acids (LC-PUFA), especially EPA and DHA, in diet can reduce the risk of diseases [8]. Previous studies have proven that flaxseed oil has positive effect on the minimization of many diseases such as hyperlipidemia [9], colon tumor [10], mammary cancer [11–13] and atherosclerosis [14,15]. At present, flaxseed oil is mainly extracted from flaxseed by press extraction and solvent extraction. Press extraction is often associated with lower yield and more energy consumption, while solvent extraction often involves longer extraction time. The use of large quantities of organic solvents is not desirable and can be harmful to human and environment. Moreover, flaxseed oil is thermally unstable and may degrade at higher temperatures. For these reasons, an improved or better extraction technique is desirable. Thus, supercritical fluid extraction, aqueous enzymatic extraction and ultrasound-assisted extraction (UAE) techniques can be further developed. Recent studies have shown that the ultrasound-assisted extraction can enhance the extraction efficiency through acoustic cavitation and some mechanical effects [16–18]. Acoustic cavitation can disrupt cell walls facilitating solvent to penetrate into the plant material and allowing the intracellular product
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release. Another mechanical effect caused by ultrasound may also be the agitation of the solvent used for extraction, thus increasing the contact surface area between the solvent and targeted compounds by permitting greater penetration of solvent into the sample matrix. Therefore the main advantages of ultrasound-assisted extraction include the reduced extraction time and reduced solvent consumption [19]. In addition, ultrasound-assisted extraction can be carried out at a lower temperature which can avoid thermal damage to the extracts and minimize the loss of bioactive compounds [20]. So far, there seem to be no report about ultrasound-assisted extraction oil from flaxseed. The aim of this study was to determine the effects of some factors on the yield of flaxseed oil with or without ultrasound treatment. The results from UAE were compared with that obtained from the conventional solvent extraction, i.e. maceration extraction (ME). After the extraction of flaxseed oil, microscopic images of flaxseed powder were obtained by scanning electron microscope (SEM) to prove the existence of ultrasonic effects. The fatty acid compositions of flaxseed oils extracted by UAE and ME were measured using gas chromatography (GC) to investigate the effect of ultrasound on the quality of flaxseed oil extracted. 2. Materials and methods 2.1. Samples and reagents The flaxseed with a moisture content of 6.2 wt% was purchased from the market of Hebei Province of China. The flaxseed was cleaned by hand carefully to remove the foreign materials such as other seeds, stones and small stalks. The cleaned flaxseed was dried for 12 h at 105 ◦ C in an oven, and then crushed into powder in a grinder (B-400, B¨uchi Labortechnik AG, Switzerland) with a size range of 0.45–1.2 mm. The resulted powder was kept in a vacuum dryer until use. n-Hexane used in the extraction was analytical grade and was purchased from Beijing Beihua Chemical Company. 2.2. Conventional extraction The crushed flaxseed powder (10 g) was mixed with n-hexane in a flask. The flask was put into the water bath with a controlled temperature. After each extraction experiment, the extracts were filtered through the Whatman No. 1 filter paper under vacuum, and then the solution was collected and concentrated with a rotary evaporator (RE-2S, Beijing Jing Zhi Jie Laboratory Apparatus Co. Ltd., China) to acquire the flaxseed oil. The acquired flaxseed oil was further dried in a vacuum dryer to remove the residual n-hexane. 2.3. Ultrasound-assisted extraction For the UAE experiments, a 250 W, 20 kHz ultrasonic emulsifier (88-1, Institute of Acoustics, Chinese Academy of Sciences, China) with a 2.00 cm flat tip probe was used (Fig. 1). The ultrasonic output power could be set to a desired level ranging from 0 to 100% of the nominal power by the amplitude controller.
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Ultrasonic output powers were determined calorimetrically and ranged from 10 to 50 W according to the method described by Li et al. [16]. The ultrasound-assisted extraction used in this study was similar to that described by Zhao et al. [21], with a little modification. The flaxseed powder was mixed in 100 mL n-hexane contained in a 200 mL plastic beaker. The ultrasonic probe was inserted into the mixture directly. The samples were extracted under continuous ultrasonic waves at 20 kHz at different levels of power output. During extraction, the temperature was controlled at a desired level within ±1 ◦ C. The post-treatment of the extracts was the same as that mentioned in the conventional extraction. In this study, all the experiments were performed in triplicates, and the results reported here are the means of the three trials. 2.4. Yield determination The yield of flaxseed oil was calculated using the following formula: We yield (%) = × 100 Wt where We is the mass of flaxseed oil extracted from the sample (g) and Wt is the mass of total oil in the sample (g). Wt was obtained by Soxhlet standard extraction mode of the Buchi extraction system (B-811, B¨uchi Labortechnik AG, Switzerland) after hydrolyzing for 30 min in 4 M HCl in a hydrolysis Unit (B-411, B¨uchi Labortechnik AG, Switzerland). 2.5. Scanning electron microscopy (SEM) analysis SEM can be used to reveal the microstructure of the material [22]. In order to investigate the influence of ultrasound during extraction on the structure of the materials and to understand the extraction mechanism, the solid remained was collected and dried in air after the extraction of flaxseed oil for the SEM analysis. Sample particles were fixed on the silicon wafer and sputtered with gold to a thickness of about 100 nm. The shape and the surface characters of the samples were observed and recorded on the scanning electron microscope (KYKY-2800, KYKY Technology Development Ltd., China). 2.6. Gas chromatographic analysis Fatty acid compositions of the oils extracted by UAE and ME were determined using GC after derivatization to fatty acid methyl esters (FAME). The preparation of FAME was performed via saponification in 0.5 M NaOH–MeOH solution and methylation with 14% BF3 –MeOH (Sigma, USA), according to the 5509 ISO method [23]. FAME separation and identification were carried out on the gas chromatograph (6890N, Agilent Technologies, USA) equipped with a flame ionization detector and capillary column HP-Innowax (30 m × 0.32 mm × 0.25 m). The amount of each sample injected was 1.0 L. Nitrogen, at a constant flow 1.0 mL/min, was used as the carrier gas and a spilt/spiltless injector was used with a split ratio of 50:1. The injector temperature was 250 ◦ C and the detector temperature
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Fig. 1. The schematic diagram of the apparatus of the ultrasound-assisted extraction.
was 280 ◦ C. The column temperature program was as follow: the initial column temperature was 40 ◦ C, which was maintained for 0.5 min, from 40 ◦ C to 195 ◦ C at 25 ◦ C/min, from 195 ◦ C to 205 ◦ C at 3 ◦ C/min, from 205 ◦ C to 230 ◦ C at 8 ◦ C/min and held for 4 min at 230 ◦ C, and then to a final temperature of 240 ◦ C at a rate of 5 ◦ C/min, and held for another 2 min. Fatty acid methyl esters were identified by comparison with the standard fatty acid methyl esters (Sigma, USA). Fatty acid methyl esters were quantified as percentages of the total methyl ester peak areas.
ated and collapsed [24]. Since the temperature and pressure were very high inside the bubbles and the collapse of bubbles occurred over very short time, the violent shock wave and high-speed jet were generated which could enhance the penetration of the sol-
3. Results and discussion 3.1. Effect of ultrasonic power The effect of ultrasonic power on the yield of flaxseed oil is shown in Fig. 2. It can be seen that the yield of flaxseed oil increases almost linearly with increasing ultrasonic power. When the power was increased from 20 to 50 W (1.5 folds increases), the yield of flaxseed oil was increased from 66.7% to 84.9% (18.2% increases). As the larger amplitude ultrasonic wave traveled though a liquid medium, more bubbles were cre-
Fig. 2. The effect of ultrasonic output power on the yield of flaxseed oil (extraction time: 30 min; solid to liquid ratio: 1:10; extraction temperature: 25 ◦ C).
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vent into the cell tissues and accelerate the intracellular product release into the solvent by disrupting the cell walls. Moreover, the violent shock wave and high-speed jet might have caused the molecules to mix better enhancing the mass transfer rate. Due to the presence of the hard cell walls which are not so permeable, the large increase in ultrasonic power resulted in a moderate rise in yield. Similar results were obtained by Li et al. [16] and Sivakumar et al. [25], respectively, in the ultrasound-assisted extraction of oil from soybean and tannin from myrobalan nut. Based on the current results, the maximum ultrasonic power of 50 W was chosen as the output power. 3.2. Effect of extraction time Fig. 3 shows the effect of experimental duration for extraction on the yield of flaxseed oil. It can be seen that the yield of flaxseed oil increases with the extraction time in both methods. The yield of flaxseed oil in UAE was higher than that in ME at all the time here. The difference was about 12% between ME and UAE during the first 15 min and increases gradually to 30 min to about 18%. The difference then became very gradual and for the rest of the extraction up to 180 min the difference levels increased to about 16%. This process indicates that the effect of ultrasound is more effective in the first 30 min. It may be that ultrasonic wave could disrupt the cell walls, so larger contact area between solvent and material was created and more oil was appeared on the surface in UAE than that in ME. However, this effect would be increasingly weak on the inner cell walls as the distance is increased. It should be noted that both processes occur very rapidly at the beginning of the extractions. UAE has the greatest rise in yield. Perhaps the ultrasonic waves affect the mass transfer rate mainly in the solvent penetration stage. It should of course also enhance the dissolved oil to transfer out of the solid structure. F-Test showed that the extraction time had a significant effect on the yield of flaxseed oil (p < 0.005), and the effect of ultrasound on the yield also was significant (p < 0.001). Similar results were obtained by Zhao et al. [21], Hemwimol et al. [24] and Balachandran et al. [26] in their researches. In the research of Balachandran et al. [26], it had been confirmed that
Fig. 3. The effect of ultrasonic extraction time on the yield of flaxseed oil (ultrasonic output power: 50 W; solid to liquid ratio: 1:10; extraction temperature: 25 ◦ C).
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the ultrasound continued to be effective even during the later stage of extraction through an enhancement to the internal diffusivity, although the effects were smaller. In any case, based on the results so far, 30 min was found to be an optimum operating time for UAE. 3.3. Effect of temperature As shown in Fig. 4, the yield of flaxseed oil does not change with increasing temperature in ME. When the temperature changed from 30 ◦ C to 50 ◦ C, the yield of flaxseed oil increased only about 0.1% in ME. This is somewhat surprising, as temperature rise should increase the solubility and diffusivities. It may be that the limiting process for extraction (as expected usually) is dissolution and diffusion within the intact solid matrix. The temperature dependence is not that sensitive at this lower range of temperature. In the UAE, the yield of flaxseed oil was found to decrease with the increase of temperature. The yield of flaxseed oil was about 83% at 30 ◦ C and decreased by about 6% to about 77% at 50 ◦ C. This is surprising as well, one reason for this may be that the vapor pressure of solvent increased with the increase of temperature and the vapor pressure had a great influence on the occurrence and the intensity of acoustic cavitation. At lower temperature, the vapor pressure is lower. Ultrasound produces a few cavitational bubbles as a result of high acoustic cavitation threshold. However, the bubbles explodes with relatively greater force, which enhanced cell tissues disruption during extraction. At higher temperature, the vapor pressure was higher and more bubbles were created, but they collapsed with less intensity due to a smaller pressure difference between inside and outside of bubbles [20]. Another reason may be due to the surface tension which decreased with the increase of temperature affecting the bubble formation and collapse. The bubbles may be so easily collapsed at higher temperature thus reducing the intensity of the mass transfer enhancement. F-Test showed that the effect of extraction temperature was insignificant (p > 0.05), but the ultrasound had a significant effect on the yield of flaxseed oil (p < 0.025). Finally, 30 ◦ C or room temperature may be seen as an optimum condition for the extraction of flaxseed oil in UAE.
Fig. 4. The effect of solvent temperature on the yield of flaxseed oil (extraction time: 30 min; ultrasonic output power: 50 W; solid to liquid ratio: 1:10).
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Fig. 5. The effect of solvent to solid ratio on the yield of flaxseed oil (extraction time: 30 min; ultrasonic output power: 50 W; extraction temperature: 25 ◦ C).
3.4. Effect of liquid to solid ratio As shown in Fig. 5, the ratio of liquid to solid in both methods had similar effect on the yield of flaxseed oil. Both the yields of flaxseed oil extracted by UAE and ME were found to increase with the increase of the ratio of liquid to solid and then level off at the high ratios. The larger liquid (solvent) to solid ratio means a larger concentration difference which favors mass transfer. When the liquid to solid ratio is small, this effect is more obvious. Too much liquid would not change much of the driving force any more as the limitation to mass transfer is more confined to the solid interior. Again, UAE outperformed ME at all liquid–solid ratios tested here. Similar result has also been shown by Zhao et al. [21]. F-Test showed that both the liquid to solid ratio and ultrasound had a significant effect on the yield of flaxseed oil (p < 0.001). The liquid to solid ratio of 6:1 seems to be appropriate for UAE. 3.5. Effect of ultrasound on the physical structure The SEM images of the flaxseed powder at a magnification factor of 1000× are shown in Fig. 6. There was a significant flaxseed oil on the surface of material (Fig. 6(a)). After 30 min maceration extraction, the majority of flaxseed oil on the surface of material had gone (yield = 66.7%) (Fig. 6(b)). After 30 min ultrasound-assisted extraction, most of the oil from the flaxseed had been extracted (yield = 84.9%) (Fig. 6(c)). Fig. 6(b) shows significant amount of the flaxseed oil is still present in the sample (in contrast to the UAE extracted sample shown in Fig. 6(c), which has clearly much less oil content). This visual observation demonstrated that the application of ultrasound in the UAE accelerated the flaxseed oil removal. 3.6. Effect of UAE on the composition of flaxseed oil The fatty acid compositions of flaxseed oil extracted by UAE and ME are given in Table 1. These results are consistent with that reported by Choo et al. [27]. The variance analysis and hypothesis testing were carried out between the data of fatty acid
Fig. 6. Scanning electron microscopy images of flaxseed powder. (a) Untreated. (b) After 30 min maceration extraction. (c) After 30 min ultrasound-assisted extraction.
compositions extracted by UAE and ME. The results showed that there were no remarkable differences (p > 0.05) in the oil profiles extracted by both methods. This indicates that the compositions of flaxseed oil are hardly affected by the application of the ultrasound. Luque-Garc´ıa and Luque de Castro [28] investigated the influence of ultrasound on the fatty acid composition of other oilseeds such as soybean, sunflower and rapeseeds and the results are also compatible with ours. In this work, the physical properties that include density, colour, refractive index, transparency, moisture and volatile matter, etc. were not studied in detail. Similarly, the chem-
Z.-S. Zhang et al. / Separation and Purification Technology 62 (2008) 192–198 Table 1 Fatty acid composition (% of total fatty acids) of flaxseed oil extracted by different methods Fatty acid
UAE
ME
C14:0 C15:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:2 C20:4 C22:0 C22:1 C22:2 C23:0 C24:0 C24:1 Other SFA MUFA PUFA n-3/n-6 ratio
0.038 0.018 5.529 0.053 0.059 0.042 3.267 16.919 15.835 56.006 0.216 0.315 0.061 0.144 0.021 0.092 0.026 0.094 0.017 1.248 9.391 17.052 72.309 3.523
0.050 0.019 5.613 0.058 0.063 0.045 3.245 16.762 15.810 55.776 0.138 0.320 0.072 0.130 0.020 0.114 0.017 0.085 0.014 1.649 9.360 16.899 72.092 3.512
UAE, ultrasound-assisted extraction; ME, maceration extraction; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-3, omega-3 fatty acid; n-6, omega-6 fatty acid.
ical properties which consist of acid value, iodine value, peroxide value, saponification value, etc. were left for future study. 4. Conclusions In the study presented in this paper, two extraction techniques for oil in flaxseed were investigated and compared. The yield of flaxseed oil increased with increasing extraction time, ultrasonic power and ratio of liquid to solid. Ultrasound-assisted extraction showed a higher yield and a lower solvent consumption. It is important to note that using ultrasound, the extraction time is significantly shortened. Thus, the ultrasound-assisted extraction technique was shown to be very efficient in the extraction of oil from flaxseed. The good conditions for UAE were found to be ultrasonic power: 50 W, extraction temperature: 30 ◦ C, extraction time: 30 min and liquid to solid ratio: 6:1 (v/w). The SEM images have provided an excellent evidence of the effectiveness of UAE. The GC analysis has shown that the compositions of the flaxseed oil extracted were hardly affected by the application of ultrasound. As such, it may be said that UAE is an effective and indeed feasible method for the production of the plant oil. Acknowledgements Research support was provided by the Research and Development Fund for University’s Doctoral Discipline of the Chinese
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Ministry of Education (No. 20050019029), the Science and Technology Research Key Program of Chinese Ministry of Education (No. 105014), the High Technology Research and Development Program of Chinese Ministry of Science and Technology (No. 2006AA10256-02), and the Science and Technology Development Planning Program of Beijing Municipal Education Committee (No. KM200710011005).
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