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Optimizations and comparison of two supercritical extractions of adlay oil
Innovative Food Science and Emerging Technologies 13 (2012) 128–133
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Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset
Optimizations and comparison of two supercritical extractions of adlay oil Aijun Hu ⁎, 1, Zhihua Zhang, Jie Zheng, Yiming Wang, Qiongxi Chen, Rong Liu, Xue Liu, Shujing Zhang Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
a r t i c l e
i n f o
Article history: Received 19 July 2011 Accepted 1 October 2011 Editor Proof Receive Date 1 November 2011 Keywords: Adlay oil Supercritical fluid Ultrasound Extraction GC-MS analysis
a b s t r a c t Oil is an important component of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In this work, a supercritical fluid extraction (SFE) and a novel ultrasonic-assisted supercritical fluid extraction (USFE) were studied and compared. Their operating conditions on the oil extraction, including extraction temperature (T), pressure (P), time (t), and CO2 flow rate (F), were optimized. Based on the yield of extraction, the favorable conditions for SFE were: T at 45 °C, P at 25 MPa, t at 4.0 h and F at 3.5 L/h. While ultrasound was applied as in USFE, the following parameters were preferred: T at 40 °C, P at 20 MPa, t at 3.5 h and F at 3.0 L/h, respectively. The results show that supercritical fluid extraction with the assistance of ultrasound could reduce the temperature, pressure, CO2 flow rate, as well as time used in the process. Compared with SFE, USFE could give a 14.0% increase in the yield for extracting oil from adlay seed with less severe operating conditions. According to the quality of adlay oil and the analysis of its fatty acids by GC-MS, there was no significant difference between USFE and SFE. Industrial relevance: Adlay oil is an important component of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In China, there is already this kind of product produced by factories. However, its production method is conventional solvent extraction. Although supercritical fluid extraction has obvious advantages compared to conventional extraction method, it has also some disadvantages such as low extraction efficiency for some components, very high operation pressure and safety problem. In our work, a novel ultrasonic-assisted supercritical fluid extraction (USFE) was studied and compared. As an emerging technology, it has huge potential and remarkable significance in its application in adlay oil and other relevant products' production at factory-scale. The experimental results and conclusion will provide a useful background to application of the novel technology in industrials. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Wild coix lachrymal-jobi L. (Coix) is native to and extensively grown in South Asia (Arora, 1977). The cultivated variety, coix lachrymal-jobi L. var. adlay (Adlay), is a soft-shelled seed crop cultivated in countries such as India (Arora, 1977), Brazil (Ottoboni, Leite, Targon, Crozier, & Arruda, 1990), Japan (Kondo, Nakajima, Nozoe, & Suzuki, 1998) and China (Kuo, Chiang, Liu, Chien, Chang, Lee, et. al., 2002). Adlay has long been used as an animal feed, as food for humans and in herbal medicine. It contains abundant active components such as adlay oil which has lots of important health and medical functions. Adlay oil can inhibit the growth of cancer cells with an efficiency of above 87%, and help prevent the decrease of white blood cells during chemical therapy (Lu, Zhang, & Zhang, 1999). ⁎ Corresponding author at: College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue, Tianjin EconomicalTechnological Development Area, Tanggu district, Tianjin 300457, P. R. China. Tel.: +86 13512056805; fax: +86 22 60601445. E-mail address: [email protected] (A. Hu). 1 Research orientation: application of physical field techniques in food science and engineering, has already published nearly 100 papers, 6 books and applied 12 patents. 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.10.002
Normally adlay oil is obtained using mechanical or chemical processes. Mechanical processes often associate with low yields, while chemical extraction methods often involve the use of organic solvents which can be harmful to human health and environment (Mustakas, 1987). Tough new regulatory requirements on the use of organic solvents have prompted active research on clean extraction technologies (Reverchon & Osseo, 1994). Supercritical fluid extraction (SFE) is one of the newly emerging clean and environmentally friendly technologies for food and pharmaceutical products (De Azevedo, Kopcak, & Mohamed, 2003). Among supercritical fluids, CO2 is the most commonly used solvent for the extraction of oils from natural products. However, the efficiency of SFE is hindered by the low solubility of the triglycerides in CO2, and the high pressure and long extraction time required (Reverchon & Osseo, 1994). Ultrasound, a kind of elastic mechanical wave, can produce thermal effect, mechanical fluctuant effect and cavitation effect. Cavitation effect would cause the formation, the growth, the compression and the explosion of bubbles in the solution, which can lead to the dispersion of the solid particles, the increase of the contact area between the particles and extraction solvent, and the improvement of mass transfer rate from solid phase to liquid phase (Mantysale & Mantysalo, 2000; Velickovic,
A. Hu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 128–133
Milenovic, & Ristic, 2006; Zheng & Hu, 2007). The ultrasound can effectively increase the extraction rate and speed up the extraction process. Recently, the application of ultrasound techniques in solvent extraction has attracted more attention. The solvent extraction of oil from oiltea camellia seed, soybean and flaxseed (Hu, Feng, & Zheng, 2009, 2002, Hu, Qiu, & Liu, 2002; Zhang & Zhao, 2006), as well as the extraction of functional ingredients from the Chinese herbal medicine(Ben & Qiu, 2006; Li, Pordesimo, & Weiss, 2004; Zeng & Qiu, 2005; Zhang, Wang, & Li, 2008), was found to be significantly improved by the introduction of ultrasound wave, and extensive researches on ultrasound assisted solvent extraction have been conducted. At present, a great deal of researches on supercritical fluid techniques have been done, including SFE, and its applications in preparation of microparticles, nanoparticles recrystallization and dissolution (Chattopadhyay & Gupta, 2001, 2002; Enokida, Abd EI-Fatah, & Wai, 2002; Han, Zhang, & Cheng, 2007; Jia, Lu, & Sun, 2007). However, the application of ultrasound in SFE is still in the developing era. Sethuraman studied ultrasound-assisted SFE (USFE) of capsaicin from peppers, the experimental results indicated the extraction amount and the loading amount of extraction vessel of USFE were higher than those of SFE (Sethuraman, 1997). Trofimov used ultrasound during the dissolution of uranium oxides in supercritical carbon dioxide. The results showed the dissolution of uranium trioxide with sonification was increased by approximately 100% at 60 °C and 150 atm (Trofimov, Samsonov, Lee, Smart, & Wai, 2001). More recently the use of power ultrasound in SFE to enhance the extraction yield of oil from almonds was reported by Riera, Golas, Blanco, Gallego, Blasco, & Mulet (2004), and he found that power ultrasound significantly accelerated the kinetics of the process and improved the final extraction yields by 20%, due to the effects produced by ultrasound, such as the compression, the decompression, radiation pressure and acoustic streaming. Till now, the optimal SFE and USFE conditions of adlay oil have not been reported, and the effects of ultrasound in SFE on the quality and components of adlay oil have also not been studied. The main objective of this work is to extract adlay oil using SFE with and without ultrasound. The results obtained from USFE will be compared with that obtained from the SFE to reveal the effects of ultrasound under supercritical conditions.
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As showed in Fig. 1, the USFE system is made up of two units: SFE unit and ultrasonic unit. The major components of SFE include a positive displacement liquid pump, a 1000 mL pressurized extraction vessel, a separation column and a separation vessel. The extraction vessel and two separators are equipped with water jackets and temperature controllers. In order to ensure accurate and stable supercritical CO2 delivery, the pump head is cooled by circulating water. The temperatures in the system are controlled within ±0.1 °C and the pressures within ±0.5 MPa. The probe with Langevin type transducer is installed in the upper part of the extractor, and driven by electrical signals from an ultrasound generator, which gives adjustable continuous power outputs at fixed frequency of 20 kHz. The transducer can stand for a maximum temperature of 105 °C. The ultrasound generator consists of a power amplifier and a special electro circuit designed to justify the power outputs at a constant level during the USFE process. The electroacoustical efficiency of the ultrasonic transducer is 87%. 2.2. Materials Adlay seeds were bought from the local traditional Chinese medicine shop. The oil content of the adlay seed, as determined by extraction with acetone, was found to be 9.5%. CO2 of 99.9% purity was obtained from Guangzhou Gas Company, China. All chemicals were purchased from local chemical stores and at analytical grades. 2.3. Experimental methods In order to fully compare the effects of USFE and SFE, experiments were carried out at various extraction temperatures, pressures, times and CO2 flow rates. 100 g of grounded adlay seeds was placed into the extractor in a typical extraction experiment. Liquid CO2 was pumped into the extractor until the desired extraction pressure was reached. The extractor was heated to the extraction temperature, and pressure valves located downstream of the extractor were slowly opened while maintaining the pressure constant in the extractor. When the set conditions reached, the extraction process started to time. The adlay oil, which precipitated under a low pressure and temperature in the separation vessel, was recovered, when extraction time reached.
2. Materials and methods
2.4. Calculation of adlay oil extraction yield
2.1. SFE and USFE system
The adlay oil extraction yield (EY) is calculated using the following formula:
The SFE equipment was designed by Guangzhou Light Engineering Institute. The USFE system was self-designed based on the SFE equipment.
EY ¼ mt =m0 x0 100
ð1Þ
Fig. 1. Ultrasonic-assisted supercritical fluid extraction apparatus2 2 1. CO2 steel cylinder (vessel); 2. Cold trap; 3. Flowmeter; 4. CO2 deposition tank; 5. CO2 pressure pump; 6. Heat exchanger; 7. Extraction vessel; 8. Separation column; 9. Separation vessel; 10. Ultrasound generator; 11. Cooled engine; 12, 13, 14. Thermostatic water-bath heater; 15. Modifier pump; 16. Modifier vessel.
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A. Hu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 128–133
where, m0 is the initial mass of adlay seed put into the extractor (mg), Mt is the mass of extracted adlay oil (mg), and X0 the initial adlay oil content of raw adlay seed (mg/mg). 2.5. GC-MS analysis of adlay oil's fatty acids 10 mg of adlay oil and 2 mL of 0.5 M KOH–MeOH solution were put into a 20 mL test tube, then shaken fully in 60 °C water. After being fully saponified, adlay oil was cooled, added 2 mL of BF3-ether solution, shaken again in 60 °C water. After being cooled for 30 min, hexane was added and shaken well. Then the test tube was added with saturated NaCl solution, vertically shaken and layered. The above hexane phase was collected, dried with anhydrous sodium sulfate and filtered. The filtrate was diluted and analyzed on HP5890 gas chromatography-HP5973 mass spectrometer (made by U.S. Hewlett Packard Company). GC column was SE-30 (30 m ×0.25 mm ×0.25 μm). The oven temperature was programmed to 8 °C/min to 280 °C and kept for 10 min. The injector temperature was 250 °C. The carrier gas was highly pure He. MS ion source was EI. Electron energy was 70 eV with ion source temperature maintained at 230 °C. The full-scanning range of MS was 30–500 amu. The SI line temperature was 280 °C. 2.6. Adlay oil quality determination Light absorbency: the Appendix V of Pharmacopoeia of the People's Republic of China 2000. Acid value: the Appendix IX N of Pharmacopoeia of the People's Republic of China 2000. Iodine value: according to GB5534-85. Peroxide value: according to IUPAC. Heavy metal: the second method of the Appendix IX E, Pharmacopoeia of the People's Republic of China 2000. Arsenic content: the atom absorption method of Pharmacopoeia of the People's Republic of China 2000. 3. Results and discussion 3.1. Optimizations of SFE and USFE
Table 2 Analytical result of maximal difference among factor levels of SFE.
K1 K2 K3 K4 k1 k2 k3 k4 R
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
190.49 198.76 292.40 196.02 47.62 49.69 73.10 49.01 25.48
170.27 216.42 245.15 245.83 42.57 54.11 61.29 61.46 18.89
174.86 199.35 229.52 273.94 43.72 49.84 57.38 68.49 24.77
196.37 213.88 233.23 234.19 49.09 53.47 58.31 58.55 9.46
192.20 212.37 236.20 236.90 48.05 53.09 59.05 59.23 11.18
Based on Table 1, the analytical results of maximal difference of adlay oil contents, corresponding to factor levels of SFE, are written as Table 2, and the variance is listed in Table 3. As shown in Tables 2 and 3, for adlay oil extracted by SFE, the order of each factor affecting the extraction yield was: temperature > extraction pressure > CO2 flow rate > time > particle size. The optimal parameters were: temperature at 45 °C, CO2 flow rate at 3.5 L/h, extraction pressure at 25 MPa, particle size of adlay seeds was 40–60 mesh and extraction time at 4 h. In order to optimizate USFE, the orthogonal experiment of L16(54) was designed and done with factors of temperature, particle size of adlay seeds, CO2 flow rate, extraction pressure and time. The ultrasound of 110.5 W at 20 kHz was applied in USFE system, and the experimental result was obtained in Table 4. Based on Table 4, the analytical results of maximal difference among factor levels of USFE are written as Table 5, and analysis of variance (ANOVA) is listed in Table 6. As shown in Tables 5 and 6, for adlay oil extracted by USFE, the order of each factor affecting the extraction yield was: temperature > particle size> extraction pressure > extraction time> CO2 flow rate. The optimal parameters were: temperature at 40 °C, CO2 flow rate at 3.0 L/h, extraction pressure at 20 MPa, particle size of adlay seeds was 40–60 mesh, and time at 3.5 h. 3.2. Reconfirmation and comparison of the extraction yields of adlay oil by SFE and USFE
The orthogonal experiments were used to optimize the SFE and USFE processes based on our investigation of factors affecting above two extractions (Hu, Zhao, Liang, Qiu, & Chen, 2007). For SFE, L16(5 4) was designed with factors of temperature, particle size, CO2 flow rate, extraction pressure and time, and its experimental results were shown in Table 1.
To reconfirm the experimental results, the parallel confirmation runs were repeated twice at the optimal parameters from the above orthogonal experiments of SFE and USFE. The results and standard deviations are listed in Table 7. As showed in Table 7, values of all parameters were reduced when ultrasound was applied. The EYs of SFE
Table 1 The results of the orthogonal experiment of SFE. Experiment no.
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
Adlay oil/ %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 (35) 1 1 1 2 (40) 2 2 2 3 (45) 3 3 3 4 (50) 4 4 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 (10) 2 (15) 3 (20) 4 (25) 2 1 4 3 3 4 1 2 4 3 2 1
1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3
1 (3.0) 2 (3.5) 3 (4.0) 4 (4.5) 4 3 2 1 2 1 4 3 3 4 1 2
11.63 38.71 64.23 75.92 40.21 45.69 62.23 50.63 65.27 82.63 71.38 73.12 53.16 49.39 47.31 46.16
(2.5) (3.0) (3.5) (4.0)
(12–20) (20–40) (40–60) (60–80)
A. Hu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 128–133
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Table 3 The variance analysis of orthogonal experiment of SFE. Factors
Sum of deviation square
Degree of freedom
F ratio
F critical value (α = 0.10)
Significance
Extraction temperature CO2 flow rate Extraction pressure Particle size Extraction time Error
1784.355 946.061 1365.710 242.716 344.437 242.72
3 3 3 3 3 3
7.352 3.898 5.627 1.000 1.419
5.390 5.390 5.390 5.390 5.390
* *
Table 4 The results of orthogonal experiment of adlay oil extraction by USFE. Experiment no.
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
Adlay oil/ %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 (30) 1 1 1 2 (35) 2 2 2 3 (40) 3 3 3 4 (45) 4 4 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 (10) 2 (15) 3 (20) 4 (25) 2 1 4 3 3 4 1 2 4 3 2 1
1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3
1 (2.5) 2 (3.0) 3 (3.5) 4 (4.0) 4 3 2 1 2 1 4 3 3 4 1 2
20.82 49.71 82.13 85.53 67.98 70.79 63.27 66.13 93.01 95.76 81.72 85.49 70.37 73.56 67.34 70.52
(1.5) (2.0) (2.5) (3.0)
and USFE were 84.95% and 96.36% respectively, they were higher than those of any experiments in Tables 1 and 4, so the technical parameters optimized by the orthogonal experiments of SFE and USFE were adequate. By comparing to SFE operating at its own optimum conditions with 5% significant level, the EYs of USFE could be increased significantly 14%. Furthermore, the extraction temperature and pressure can be 5 °C and 5 MPa lower with ultrasound compared to that without it. The extraction time as well as CO2 flow rate can also be reduced at 0.5 h and 0.5 L/h with the use of ultrasound.
(12–20) (20–40) (40–60) (60–80)
3.3. Quality comparison of adlay oil extracted by SFE and USFE In ordinary circumstances, slightly higher acid value and peroxide value will not be harmful to human health. But in the event of a serious deterioration, the aldehyde, the alkone, the acid produced by oil can destroy the fat soluble vitamin, and may have an adverse effect on human health. Heavy metals such as arsenic and lead are stable elements and bio-accumulative, they have no function in the body and can be highly toxic. In our work, the quality of adlay oil extracted by
Table 5 Analytical results of maximal difference among factor levels of USFE.
K1 K2 K3 K4 k1 k2 k3 k4 R
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
238.19 268.17 355.98 281.79 59.55 67.04 89.00 70.45 29.45
252.18 289.82 294.46 307.67 63.05 72.46 73.62 76.92 13.87
243.85 270.52 314.83 314.93 60.96 67.63 78.71 78.73 17.77
243.14 267.93 316.39 316.67 60.79 66.98 79.10 79.17 18.38
250.05 276.51 308.78 308.79 62.51 69.13 77.20 77.20 14.69
Table 6 ANOVA of orthogonal experiment of USFE. Factors
Sum of deviation square
Degree of freedom
F ratio
F critical value (α = 0.10)
Extraction temperature CO2 flow rate Extraction pressure Particle size Extraction time Error
1879.656 424.885 921.091 1006.925 605.193 424.880
3 3 3 3 3 3
4.424 1.000 2.168 2.370 1.424
5.390 5.390 5.390 5.390 5.390
Significance
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A. Hu et al. / Innovative Food Science and Emerging Technologies 13 (2012) 128–133
Table 7 Vertification and comparison between adlay oil extraction yields by SFE and USFE a. Extraction methods
Extraction mass
T/ °C
P/ MPa
F/ L/h− 1
I/ W
f/ kHz
t/ h
EY/ %b(S.D.)
SFE USFE
Adlay oil Adlay oil
45 40
25 20
3.5 3.0
0 110.5
0 20
4 3.5
84.95 (1.22) 96.36 (0.09)
a T represents extraction temperature; P represents extraction pressure; F represents CO2 flow rate; I represents ultrasound power; f represents ultrasound frequency; t represents extraction time; EY represents extraction yield. b Difference between SFE and USFE is significant at 95% confidence interval.
Table 8 Quality comparison of adlay oil extracted by different methodsa. Extraction method
OD/ 450 nm
AV a
SFE USFE
0.241 ± 0.007 0.242 ± 0.008a
PV/ mgeq/kg− 1
IV a
a
1.92 ± 0.04 1.91 ± 0.02a
102.30 ± 2.05 103.10 ± 2.07a
a
HM/ μg/g− 1
US/ % a
7.10 ± 0.08 7.14 ± 0.09a
a
1.30 ± 0.03 1.50 ± 0.04a
6.30 ± 0.06 6.6 ± 0.07a
AC/ μg/g− 1 0.012 ± 0.001a 0.012 ± 0.001a
a OD represents light absorbency; AV represents acid value; IV represents iodine value; PV represents peroxide value; US represents unsaponifiable substance; HM represents heavy metal; AC represents arsenic content; a represents no significant difference between two values in the same column (n = 6, α = 0.1).
Table 9 Fatty acids analysis of adlay oil extracted by different methods (%)a. Methods
1
SFE USFE
0.03±0.01a 0.02±0.01a 0.40±0.03a 13.34±0.12a 1.43±0.09a 32.20±0.19a 47.52±0.26a 1.22±0.06a 0.97±0.04a 0.97±0.04a 0.56±0.03a 0.26±0.01a 0.05±0.02a 0.03±0.01a 0.37±0.02a 13.50±0.13a 1.06±0.08a 32.88±0.20a 47.58±0.25a 1.31±0.07a 1.00±0.05a 0.99±0.05a 0.51±0.02a 0.25±0.01a
2
3
4
5
6
7
8
9
10
11
12
a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 represent myristic acid, pentadecyl acid, palmitoleic acid, palmitic acid, heptadecanoic acid, linoleic acid, oleic acid, stearic acid, eicosanoid, eicosanoic acid, docosanoic acid and hexacosanoic acid respectively; a represents no significant difference between two values in the same column (n = 6, α = 0.1).
SFE and USFE was determined and compared including acid value, peroxide value, heavy metals and unsaponifiable substance, which is shown in Table 8. As shown in Table 8, the quality of adlay oil extracted by SFE and USFE is not completely the same. The unsaponifiable substance, heavy metal, arsenic content and peroxide value of adlay oil extracted by USFE are higher than those extracted by SFE. However, the difference between SFE and USFE is not significant. They all meet the requirements of adlay oil quality standard. 3.4. Fatty acids comparison of adlay oil extracted by SFE and USFE It is seen from Table 9 that fatty acids content of adlay oil extracted by SFE and USFE had no significant difference. Ultrasound did not change the selectivity of supercritical CO2 to fatty acids. 4. Conclusions Our study indicated that ultrasonic irradiation in supercritical CO2 fluid promotes the extraction of adlay oil from adlay seeds, and the EYs increased 14% with sonication. There are optimum temperatures which gives the maximum EYs in both USFE and SFE. Compared with SFE, USFE had a lower extraction pressure, temperature, CO2 flow rate and shorter extraction time. The differences between the quality and fatty acids of adlay oil extracted by SFE and USFE were slight. Ultrasound did not change the selectivity of supercritical CO2 fluid to fatty acids. Acknowledgments The authors acknowledge the financial support from the National Natural Science Foundation of China (Project No.: 31071608), the China Postdoctoral Science Foundation funded project (Grant No.: 20100470777), the District Key Sci. and Tech. Project of Fujian province (Project No.: 2010N3026), and the National Key Project of ‘Eleventh
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Optimizations and comparison of two supercritical extractions of adlay oil Aijun Hu ⁎, 1, Zhihua Zhang, Jie Zheng, Yiming Wang, Qiongxi Chen, Rong Liu, Xue Liu, Shujing Zhang Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
a r t i c l e
i n f o
Article history: Received 19 July 2011 Accepted 1 October 2011 Editor Proof Receive Date 1 November 2011 Keywords: Adlay oil Supercritical fluid Ultrasound Extraction GC-MS analysis
a b s t r a c t Oil is an important component of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In this work, a supercritical fluid extraction (SFE) and a novel ultrasonic-assisted supercritical fluid extraction (USFE) were studied and compared. Their operating conditions on the oil extraction, including extraction temperature (T), pressure (P), time (t), and CO2 flow rate (F), were optimized. Based on the yield of extraction, the favorable conditions for SFE were: T at 45 °C, P at 25 MPa, t at 4.0 h and F at 3.5 L/h. While ultrasound was applied as in USFE, the following parameters were preferred: T at 40 °C, P at 20 MPa, t at 3.5 h and F at 3.0 L/h, respectively. The results show that supercritical fluid extraction with the assistance of ultrasound could reduce the temperature, pressure, CO2 flow rate, as well as time used in the process. Compared with SFE, USFE could give a 14.0% increase in the yield for extracting oil from adlay seed with less severe operating conditions. According to the quality of adlay oil and the analysis of its fatty acids by GC-MS, there was no significant difference between USFE and SFE. Industrial relevance: Adlay oil is an important component of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In China, there is already this kind of product produced by factories. However, its production method is conventional solvent extraction. Although supercritical fluid extraction has obvious advantages compared to conventional extraction method, it has also some disadvantages such as low extraction efficiency for some components, very high operation pressure and safety problem. In our work, a novel ultrasonic-assisted supercritical fluid extraction (USFE) was studied and compared. As an emerging technology, it has huge potential and remarkable significance in its application in adlay oil and other relevant products' production at factory-scale. The experimental results and conclusion will provide a useful background to application of the novel technology in industrials. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Wild coix lachrymal-jobi L. (Coix) is native to and extensively grown in South Asia (Arora, 1977). The cultivated variety, coix lachrymal-jobi L. var. adlay (Adlay), is a soft-shelled seed crop cultivated in countries such as India (Arora, 1977), Brazil (Ottoboni, Leite, Targon, Crozier, & Arruda, 1990), Japan (Kondo, Nakajima, Nozoe, & Suzuki, 1998) and China (Kuo, Chiang, Liu, Chien, Chang, Lee, et. al., 2002). Adlay has long been used as an animal feed, as food for humans and in herbal medicine. It contains abundant active components such as adlay oil which has lots of important health and medical functions. Adlay oil can inhibit the growth of cancer cells with an efficiency of above 87%, and help prevent the decrease of white blood cells during chemical therapy (Lu, Zhang, & Zhang, 1999). ⁎ Corresponding author at: College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue, Tianjin EconomicalTechnological Development Area, Tanggu district, Tianjin 300457, P. R. China. Tel.: +86 13512056805; fax: +86 22 60601445. E-mail address: [email protected] (A. Hu). 1 Research orientation: application of physical field techniques in food science and engineering, has already published nearly 100 papers, 6 books and applied 12 patents. 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.10.002
Normally adlay oil is obtained using mechanical or chemical processes. Mechanical processes often associate with low yields, while chemical extraction methods often involve the use of organic solvents which can be harmful to human health and environment (Mustakas, 1987). Tough new regulatory requirements on the use of organic solvents have prompted active research on clean extraction technologies (Reverchon & Osseo, 1994). Supercritical fluid extraction (SFE) is one of the newly emerging clean and environmentally friendly technologies for food and pharmaceutical products (De Azevedo, Kopcak, & Mohamed, 2003). Among supercritical fluids, CO2 is the most commonly used solvent for the extraction of oils from natural products. However, the efficiency of SFE is hindered by the low solubility of the triglycerides in CO2, and the high pressure and long extraction time required (Reverchon & Osseo, 1994). Ultrasound, a kind of elastic mechanical wave, can produce thermal effect, mechanical fluctuant effect and cavitation effect. Cavitation effect would cause the formation, the growth, the compression and the explosion of bubbles in the solution, which can lead to the dispersion of the solid particles, the increase of the contact area between the particles and extraction solvent, and the improvement of mass transfer rate from solid phase to liquid phase (Mantysale & Mantysalo, 2000; Velickovic,
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Milenovic, & Ristic, 2006; Zheng & Hu, 2007). The ultrasound can effectively increase the extraction rate and speed up the extraction process. Recently, the application of ultrasound techniques in solvent extraction has attracted more attention. The solvent extraction of oil from oiltea camellia seed, soybean and flaxseed (Hu, Feng, & Zheng, 2009, 2002, Hu, Qiu, & Liu, 2002; Zhang & Zhao, 2006), as well as the extraction of functional ingredients from the Chinese herbal medicine(Ben & Qiu, 2006; Li, Pordesimo, & Weiss, 2004; Zeng & Qiu, 2005; Zhang, Wang, & Li, 2008), was found to be significantly improved by the introduction of ultrasound wave, and extensive researches on ultrasound assisted solvent extraction have been conducted. At present, a great deal of researches on supercritical fluid techniques have been done, including SFE, and its applications in preparation of microparticles, nanoparticles recrystallization and dissolution (Chattopadhyay & Gupta, 2001, 2002; Enokida, Abd EI-Fatah, & Wai, 2002; Han, Zhang, & Cheng, 2007; Jia, Lu, & Sun, 2007). However, the application of ultrasound in SFE is still in the developing era. Sethuraman studied ultrasound-assisted SFE (USFE) of capsaicin from peppers, the experimental results indicated the extraction amount and the loading amount of extraction vessel of USFE were higher than those of SFE (Sethuraman, 1997). Trofimov used ultrasound during the dissolution of uranium oxides in supercritical carbon dioxide. The results showed the dissolution of uranium trioxide with sonification was increased by approximately 100% at 60 °C and 150 atm (Trofimov, Samsonov, Lee, Smart, & Wai, 2001). More recently the use of power ultrasound in SFE to enhance the extraction yield of oil from almonds was reported by Riera, Golas, Blanco, Gallego, Blasco, & Mulet (2004), and he found that power ultrasound significantly accelerated the kinetics of the process and improved the final extraction yields by 20%, due to the effects produced by ultrasound, such as the compression, the decompression, radiation pressure and acoustic streaming. Till now, the optimal SFE and USFE conditions of adlay oil have not been reported, and the effects of ultrasound in SFE on the quality and components of adlay oil have also not been studied. The main objective of this work is to extract adlay oil using SFE with and without ultrasound. The results obtained from USFE will be compared with that obtained from the SFE to reveal the effects of ultrasound under supercritical conditions.
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As showed in Fig. 1, the USFE system is made up of two units: SFE unit and ultrasonic unit. The major components of SFE include a positive displacement liquid pump, a 1000 mL pressurized extraction vessel, a separation column and a separation vessel. The extraction vessel and two separators are equipped with water jackets and temperature controllers. In order to ensure accurate and stable supercritical CO2 delivery, the pump head is cooled by circulating water. The temperatures in the system are controlled within ±0.1 °C and the pressures within ±0.5 MPa. The probe with Langevin type transducer is installed in the upper part of the extractor, and driven by electrical signals from an ultrasound generator, which gives adjustable continuous power outputs at fixed frequency of 20 kHz. The transducer can stand for a maximum temperature of 105 °C. The ultrasound generator consists of a power amplifier and a special electro circuit designed to justify the power outputs at a constant level during the USFE process. The electroacoustical efficiency of the ultrasonic transducer is 87%. 2.2. Materials Adlay seeds were bought from the local traditional Chinese medicine shop. The oil content of the adlay seed, as determined by extraction with acetone, was found to be 9.5%. CO2 of 99.9% purity was obtained from Guangzhou Gas Company, China. All chemicals were purchased from local chemical stores and at analytical grades. 2.3. Experimental methods In order to fully compare the effects of USFE and SFE, experiments were carried out at various extraction temperatures, pressures, times and CO2 flow rates. 100 g of grounded adlay seeds was placed into the extractor in a typical extraction experiment. Liquid CO2 was pumped into the extractor until the desired extraction pressure was reached. The extractor was heated to the extraction temperature, and pressure valves located downstream of the extractor were slowly opened while maintaining the pressure constant in the extractor. When the set conditions reached, the extraction process started to time. The adlay oil, which precipitated under a low pressure and temperature in the separation vessel, was recovered, when extraction time reached.
2. Materials and methods
2.4. Calculation of adlay oil extraction yield
2.1. SFE and USFE system
The adlay oil extraction yield (EY) is calculated using the following formula:
The SFE equipment was designed by Guangzhou Light Engineering Institute. The USFE system was self-designed based on the SFE equipment.
EY ¼ mt =m0 x0 100
ð1Þ
Fig. 1. Ultrasonic-assisted supercritical fluid extraction apparatus2 2 1. CO2 steel cylinder (vessel); 2. Cold trap; 3. Flowmeter; 4. CO2 deposition tank; 5. CO2 pressure pump; 6. Heat exchanger; 7. Extraction vessel; 8. Separation column; 9. Separation vessel; 10. Ultrasound generator; 11. Cooled engine; 12, 13, 14. Thermostatic water-bath heater; 15. Modifier pump; 16. Modifier vessel.
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where, m0 is the initial mass of adlay seed put into the extractor (mg), Mt is the mass of extracted adlay oil (mg), and X0 the initial adlay oil content of raw adlay seed (mg/mg). 2.5. GC-MS analysis of adlay oil's fatty acids 10 mg of adlay oil and 2 mL of 0.5 M KOH–MeOH solution were put into a 20 mL test tube, then shaken fully in 60 °C water. After being fully saponified, adlay oil was cooled, added 2 mL of BF3-ether solution, shaken again in 60 °C water. After being cooled for 30 min, hexane was added and shaken well. Then the test tube was added with saturated NaCl solution, vertically shaken and layered. The above hexane phase was collected, dried with anhydrous sodium sulfate and filtered. The filtrate was diluted and analyzed on HP5890 gas chromatography-HP5973 mass spectrometer (made by U.S. Hewlett Packard Company). GC column was SE-30 (30 m ×0.25 mm ×0.25 μm). The oven temperature was programmed to 8 °C/min to 280 °C and kept for 10 min. The injector temperature was 250 °C. The carrier gas was highly pure He. MS ion source was EI. Electron energy was 70 eV with ion source temperature maintained at 230 °C. The full-scanning range of MS was 30–500 amu. The SI line temperature was 280 °C. 2.6. Adlay oil quality determination Light absorbency: the Appendix V of Pharmacopoeia of the People's Republic of China 2000. Acid value: the Appendix IX N of Pharmacopoeia of the People's Republic of China 2000. Iodine value: according to GB5534-85. Peroxide value: according to IUPAC. Heavy metal: the second method of the Appendix IX E, Pharmacopoeia of the People's Republic of China 2000. Arsenic content: the atom absorption method of Pharmacopoeia of the People's Republic of China 2000. 3. Results and discussion 3.1. Optimizations of SFE and USFE
Table 2 Analytical result of maximal difference among factor levels of SFE.
K1 K2 K3 K4 k1 k2 k3 k4 R
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
190.49 198.76 292.40 196.02 47.62 49.69 73.10 49.01 25.48
170.27 216.42 245.15 245.83 42.57 54.11 61.29 61.46 18.89
174.86 199.35 229.52 273.94 43.72 49.84 57.38 68.49 24.77
196.37 213.88 233.23 234.19 49.09 53.47 58.31 58.55 9.46
192.20 212.37 236.20 236.90 48.05 53.09 59.05 59.23 11.18
Based on Table 1, the analytical results of maximal difference of adlay oil contents, corresponding to factor levels of SFE, are written as Table 2, and the variance is listed in Table 3. As shown in Tables 2 and 3, for adlay oil extracted by SFE, the order of each factor affecting the extraction yield was: temperature > extraction pressure > CO2 flow rate > time > particle size. The optimal parameters were: temperature at 45 °C, CO2 flow rate at 3.5 L/h, extraction pressure at 25 MPa, particle size of adlay seeds was 40–60 mesh and extraction time at 4 h. In order to optimizate USFE, the orthogonal experiment of L16(54) was designed and done with factors of temperature, particle size of adlay seeds, CO2 flow rate, extraction pressure and time. The ultrasound of 110.5 W at 20 kHz was applied in USFE system, and the experimental result was obtained in Table 4. Based on Table 4, the analytical results of maximal difference among factor levels of USFE are written as Table 5, and analysis of variance (ANOVA) is listed in Table 6. As shown in Tables 5 and 6, for adlay oil extracted by USFE, the order of each factor affecting the extraction yield was: temperature > particle size> extraction pressure > extraction time> CO2 flow rate. The optimal parameters were: temperature at 40 °C, CO2 flow rate at 3.0 L/h, extraction pressure at 20 MPa, particle size of adlay seeds was 40–60 mesh, and time at 3.5 h. 3.2. Reconfirmation and comparison of the extraction yields of adlay oil by SFE and USFE
The orthogonal experiments were used to optimize the SFE and USFE processes based on our investigation of factors affecting above two extractions (Hu, Zhao, Liang, Qiu, & Chen, 2007). For SFE, L16(5 4) was designed with factors of temperature, particle size, CO2 flow rate, extraction pressure and time, and its experimental results were shown in Table 1.
To reconfirm the experimental results, the parallel confirmation runs were repeated twice at the optimal parameters from the above orthogonal experiments of SFE and USFE. The results and standard deviations are listed in Table 7. As showed in Table 7, values of all parameters were reduced when ultrasound was applied. The EYs of SFE
Table 1 The results of the orthogonal experiment of SFE. Experiment no.
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
Adlay oil/ %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 (35) 1 1 1 2 (40) 2 2 2 3 (45) 3 3 3 4 (50) 4 4 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 (10) 2 (15) 3 (20) 4 (25) 2 1 4 3 3 4 1 2 4 3 2 1
1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3
1 (3.0) 2 (3.5) 3 (4.0) 4 (4.5) 4 3 2 1 2 1 4 3 3 4 1 2
11.63 38.71 64.23 75.92 40.21 45.69 62.23 50.63 65.27 82.63 71.38 73.12 53.16 49.39 47.31 46.16
(2.5) (3.0) (3.5) (4.0)
(12–20) (20–40) (40–60) (60–80)
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Table 3 The variance analysis of orthogonal experiment of SFE. Factors
Sum of deviation square
Degree of freedom
F ratio
F critical value (α = 0.10)
Significance
Extraction temperature CO2 flow rate Extraction pressure Particle size Extraction time Error
1784.355 946.061 1365.710 242.716 344.437 242.72
3 3 3 3 3 3
7.352 3.898 5.627 1.000 1.419
5.390 5.390 5.390 5.390 5.390
* *
Table 4 The results of orthogonal experiment of adlay oil extraction by USFE. Experiment no.
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
Adlay oil/ %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 (30) 1 1 1 2 (35) 2 2 2 3 (40) 3 3 3 4 (45) 4 4 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 (10) 2 (15) 3 (20) 4 (25) 2 1 4 3 3 4 1 2 4 3 2 1
1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3
1 (2.5) 2 (3.0) 3 (3.5) 4 (4.0) 4 3 2 1 2 1 4 3 3 4 1 2
20.82 49.71 82.13 85.53 67.98 70.79 63.27 66.13 93.01 95.76 81.72 85.49 70.37 73.56 67.34 70.52
(1.5) (2.0) (2.5) (3.0)
and USFE were 84.95% and 96.36% respectively, they were higher than those of any experiments in Tables 1 and 4, so the technical parameters optimized by the orthogonal experiments of SFE and USFE were adequate. By comparing to SFE operating at its own optimum conditions with 5% significant level, the EYs of USFE could be increased significantly 14%. Furthermore, the extraction temperature and pressure can be 5 °C and 5 MPa lower with ultrasound compared to that without it. The extraction time as well as CO2 flow rate can also be reduced at 0.5 h and 0.5 L/h with the use of ultrasound.
(12–20) (20–40) (40–60) (60–80)
3.3. Quality comparison of adlay oil extracted by SFE and USFE In ordinary circumstances, slightly higher acid value and peroxide value will not be harmful to human health. But in the event of a serious deterioration, the aldehyde, the alkone, the acid produced by oil can destroy the fat soluble vitamin, and may have an adverse effect on human health. Heavy metals such as arsenic and lead are stable elements and bio-accumulative, they have no function in the body and can be highly toxic. In our work, the quality of adlay oil extracted by
Table 5 Analytical results of maximal difference among factor levels of USFE.
K1 K2 K3 K4 k1 k2 k3 k4 R
Extraction temperature/ °C
CO2 flow rate/ (L/h)
Extraction pressure/ MPa
Particle size/ mesh
Extraction time/ h
238.19 268.17 355.98 281.79 59.55 67.04 89.00 70.45 29.45
252.18 289.82 294.46 307.67 63.05 72.46 73.62 76.92 13.87
243.85 270.52 314.83 314.93 60.96 67.63 78.71 78.73 17.77
243.14 267.93 316.39 316.67 60.79 66.98 79.10 79.17 18.38
250.05 276.51 308.78 308.79 62.51 69.13 77.20 77.20 14.69
Table 6 ANOVA of orthogonal experiment of USFE. Factors
Sum of deviation square
Degree of freedom
F ratio
F critical value (α = 0.10)
Extraction temperature CO2 flow rate Extraction pressure Particle size Extraction time Error
1879.656 424.885 921.091 1006.925 605.193 424.880
3 3 3 3 3 3
4.424 1.000 2.168 2.370 1.424
5.390 5.390 5.390 5.390 5.390
Significance
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Table 7 Vertification and comparison between adlay oil extraction yields by SFE and USFE a. Extraction methods
Extraction mass
T/ °C
P/ MPa
F/ L/h− 1
I/ W
f/ kHz
t/ h
EY/ %b(S.D.)
SFE USFE
Adlay oil Adlay oil
45 40
25 20
3.5 3.0
0 110.5
0 20
4 3.5
84.95 (1.22) 96.36 (0.09)
a T represents extraction temperature; P represents extraction pressure; F represents CO2 flow rate; I represents ultrasound power; f represents ultrasound frequency; t represents extraction time; EY represents extraction yield. b Difference between SFE and USFE is significant at 95% confidence interval.
Table 8 Quality comparison of adlay oil extracted by different methodsa. Extraction method
OD/ 450 nm
AV a
SFE USFE
0.241 ± 0.007 0.242 ± 0.008a
PV/ mgeq/kg− 1
IV a
a
1.92 ± 0.04 1.91 ± 0.02a
102.30 ± 2.05 103.10 ± 2.07a
a
HM/ μg/g− 1
US/ % a
7.10 ± 0.08 7.14 ± 0.09a
a
1.30 ± 0.03 1.50 ± 0.04a
6.30 ± 0.06 6.6 ± 0.07a
AC/ μg/g− 1 0.012 ± 0.001a 0.012 ± 0.001a
a OD represents light absorbency; AV represents acid value; IV represents iodine value; PV represents peroxide value; US represents unsaponifiable substance; HM represents heavy metal; AC represents arsenic content; a represents no significant difference between two values in the same column (n = 6, α = 0.1).
Table 9 Fatty acids analysis of adlay oil extracted by different methods (%)a. Methods
1
SFE USFE
0.03±0.01a 0.02±0.01a 0.40±0.03a 13.34±0.12a 1.43±0.09a 32.20±0.19a 47.52±0.26a 1.22±0.06a 0.97±0.04a 0.97±0.04a 0.56±0.03a 0.26±0.01a 0.05±0.02a 0.03±0.01a 0.37±0.02a 13.50±0.13a 1.06±0.08a 32.88±0.20a 47.58±0.25a 1.31±0.07a 1.00±0.05a 0.99±0.05a 0.51±0.02a 0.25±0.01a
2
3
4
5
6
7
8
9
10
11
12
a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 represent myristic acid, pentadecyl acid, palmitoleic acid, palmitic acid, heptadecanoic acid, linoleic acid, oleic acid, stearic acid, eicosanoid, eicosanoic acid, docosanoic acid and hexacosanoic acid respectively; a represents no significant difference between two values in the same column (n = 6, α = 0.1).
SFE and USFE was determined and compared including acid value, peroxide value, heavy metals and unsaponifiable substance, which is shown in Table 8. As shown in Table 8, the quality of adlay oil extracted by SFE and USFE is not completely the same. The unsaponifiable substance, heavy metal, arsenic content and peroxide value of adlay oil extracted by USFE are higher than those extracted by SFE. However, the difference between SFE and USFE is not significant. They all meet the requirements of adlay oil quality standard. 3.4. Fatty acids comparison of adlay oil extracted by SFE and USFE It is seen from Table 9 that fatty acids content of adlay oil extracted by SFE and USFE had no significant difference. Ultrasound did not change the selectivity of supercritical CO2 to fatty acids. 4. Conclusions Our study indicated that ultrasonic irradiation in supercritical CO2 fluid promotes the extraction of adlay oil from adlay seeds, and the EYs increased 14% with sonication. There are optimum temperatures which gives the maximum EYs in both USFE and SFE. Compared with SFE, USFE had a lower extraction pressure, temperature, CO2 flow rate and shorter extraction time. The differences between the quality and fatty acids of adlay oil extracted by SFE and USFE were slight. Ultrasound did not change the selectivity of supercritical CO2 fluid to fatty acids. Acknowledgments The authors acknowledge the financial support from the National Natural Science Foundation of China (Project No.: 31071608), the China Postdoctoral Science Foundation funded project (Grant No.: 20100470777), the District Key Sci. and Tech. Project of Fujian province (Project No.: 2010N3026), and the National Key Project of ‘Eleventh
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