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Journal of Nutrition & Food Sciences DOI: 10.4172/2155-9600.1000305

ciences
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ISSN: 2155-9600

Research Article Open Access

Wet Extraction of Lipids and Astaxanthin from Haematococcus pluvialis

by Liquefied Dimethyl Ether
Panatpong Boonnoun1,2, Yuko Kurita1, Yuichi Kamo1, Wahyudiono1, Siti Machmudah1,3, Yuji Okita4, Eiji Ohashi4, Hideki Kanda1,5* and
Motonobu Goto1
1
Department of Chemical Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan
2
Department of Industrial engineering, Faculty of Engineering, Naresuan University, Phitsanulok 65000, Thailand
3
Department of Chemical Engineering, Sepuluh Nopember Institute of Technology, Kampus ITS Sukolilo, Surabaya 60111, Indonesia
4
Nippon Suisan Kaisha, Ltd., Hachioji, Tokyo 192-0991, Japan
5
Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Abstract
Recently, a simple method for the extraction of lipids from wet cyanobacterial microalgae using liquefied dimethyl
ether (DME) without drying, cell disruption, or heating was proposed. Herein, the versatility of this method was
evaluated using Haematococcus pluvialis at 0.59 MPa and 25°C. Direct extraction of lipids from H. pluvialis of
moisture-rich microalgae was successfully achieved, in a lipid extraction yield of 30.0% of the dry weight of the
microalgae. The astaxanthin concentration in the extracted lipid was 0.33%, which was lower than the 1.82%
achieved by the commonly-used acetone extraction, which incorporates drying and cell disruption. The carbon and
hydrogen content were improved after DME extraction. In addition, 92% of water in the wet H. pluvialis was removed.
Compared with other extractions, liquefied DME combines drying, cell description, and solvent extraction into one
step.

Keywords: Haematococcus pluvialis; Astaxanthin; Fatty acid; toxicity that it has been examined as a prospective solvent for use in
Dimethyl ether; Wet extraction food processing [18]. Additionally, it is resistant towards autoxidation,
unlike other alkyl ethers [19]. In the previous study, In addition to the
Abbreviations: DME: Dimethyl Ether; SC-CO2,: Supercritical aforementioned advantages of DME, lipids were successfully extracted
Carbon dioxide; FAMEs: Fatty acid methyl esters; HPLC: High from seven wet cyanobacteria microalgae by DME without the need
Performance Liquid Chromatography for cell drying, cell disruption, or the use of a high-power heat source
for solvent evaporation. The extraction yields were over 97% compared
Introduction to those obtained using the Bligh Dyer method, with only minor
The energy crisis, in addition to the lack of fossil fuels and global differences in the elemental analysis [9]. However, the validity of DME
warming, represents one of the most important challenges faced by extraction was confirmed only for the cyanobacteria. In addition to
human society. Microalgal biomass-based biofuels are considered one cyanobacteria, there exist other microalgae, such as green algae and
of the most promising substitutes for fossil fuels, since microalgae diatoms. While green algae and diatoms are eukaryotes, cyanobacteria
have high growth rates and high lipid contents and require smaller are prokaryotes. The cell wall of cyanobacteria is mainly composed
cultivation areas, which do not compete with food or feed stocks [1]. of peptidoglycan, whereas the main component is cellulose in some
However, the net energy obtained from the production of microalgal green algae and silica in diatoms. Because of such differences in the
biofuel could be negative [2,3] because of the energy required for components of the cell walls of microalgae, the findings related to
removing water by drying, dewatering, and flocculation processes previous study from cyanobacteria obtained to the present may not be
prior to oil extraction [4-6]. In addition, microalgal lipids are typically applicable to green algae or diatoms.
encapsulated within cellular structures. Cell disruption is imperative in
Green algae Haematococcus pluvialis is an interesting strain for lipid
lipid extraction as it liberates microalgal lipids from the cellular matrix
extraction since it has been reported to contain about 17-46% of lipids
into the surrounding media [7]. Recently, the successful use of liquefied
(dry weight) [20]. Moreover, the co-production of valuable products such
dimethyl ether (DME) for the extraction of organic components of
as astaxanthin is necessary for economic bio fuel processes. It is widely
wet cyanobacterial microalgae was reported [8-9]. DME has been
considered to accumulate the highest amount of astaxanthin compared
developed as a synthetic fuel for use in both liquid and gaseous forms.
with other sources such as salmon, red fish, shrimp, krill, and lobster at
In China, DME is synthesized using small-scale coalfields of low
1.5-3% by weight [21-23]. The astaxanthin concentration in cells is 500
commercial value and is produced as a fuel at a cost equivalent to that
of imported liquefied petroleum gas. Because most previous studies
regarding DME focused on its synthesis and combustion [10-12], only
*Corresponding author: Hideki Kanda, Department of Chemical Engineering,
our group and a group in New Zealand have examined DME as an Nagoya University, Chikusa, Nagoya 464-8603, Japan, Tel: +81 52 789 5484; Fax:
extraction solvent [8,9,13,14]. In the previous study, liquefied DME can +81 52 789 5484; E-mail: [email protected]
extract lipids from cyanobacterial microalgae without drying because
Received July 17, 2014; Accepted August 25, 2014; Published August 27, 2014
DME has a high affinity towards oily chemicals and is partially miscible
with water [15]. Moreover, the use of liquefied DME does not require Citation: Boonnoun P, Kurita Y, Kamo Y, Wahyudiono, Machmudah S, et al.
(2014) Wet Extraction of Lipids and Astaxanthin from Haematococcus pluvialis by
cell disruption in cyanobacterial microalgae. In addition, DME can be Liquefied Dimethyl Ether. J Nutr Food Sci 4: 305. doi: 10.4172/2155-9600.1000305
easily separated from the lipids and other residues and re-used because
Copyright: © 2014 Boonnoun P, et al. This is an open-access article distributed
of its low boiling point (-24.8°C) [16]. Therefore, DME lipid extraction under the terms of the Creative Commons Attribution License, which permits
consumes less energy and emits less greenhouse gases than extractions unrestricted use, distribution, and reproduction in any medium, provided the
with other solvents [17]. Furthermore, liquefied DME has such a low original author and source are credited.

J Nutr Food Sci, an open access journal

ISSN: 2155-9600 Volume 4 • Issue 5 • 1000305
Citation: Boonnoun P, Kurita Y, Kamo Y, Wahyudiono, Machmudah S, et al. (2014) Wet Extraction of Lipids and Astaxanthin from Haematococcus
pluvialis by Liquefied Dimethyl Ether. J Nutr Food Sci 4: 305. doi: 10.4172/2155-9600.1000305

Page 2 of 4

pg/cell [21]. Astaxanthin is one the most valuable caroteniods since it solution was then transferred to a centrifuge tube and was centrifuged
has many biological activities and beneficial health effects, including the at 3000 rpm at room temperature for 5 min. The cell residue was then
protection of skin from radiation injuries caused by sunlight, blocking separated from the supernatant, homogenized, then centrifuged by the
reactions induced by other chemicals or toxins, deceleration of age- aforementioned method two additional times. Each supernatant was
related macular degeneration [24], favorable effects on blood pressure combined, filtrated, and evaporated to remove acetone.
[25,26], in addition to cardio-protective effects [27]. It also shows higher
antioxidant activities than other carotenoids such as β-carotene, lutein, Analysis of organic components
canthaxanthin, zeaxanthin, and vitamin E [28,29,30]. Therefore, it has For elemental analysis, the original H. pluvialis microalgae, lipids,
been widely used for pharmaceutical and other applications such as in and residues were pretreated by continual drying at 107°C until
cosmetics and food additives. However, astaxanthin is accumulated in constant weights were obtained. The carbon, hydrogen, and nitrogen
the thick-walled cells of H. pluvialis. The thick-walled cell hinders the content were determined by a CHN corder (MT-6 Elemental analyzer,
extraction of lipids and astaxanthin [31]. Therefore, cell disruption can Yanaco New Science Inc., Kyoto, Japan) based on flash combustion,
effectively increase the extraction amount of lipids and astaxanthin which converts all organic substances into combustion gases. The
from H. pluvialis [32]. Therefore, various extraction methods of lipids oxygen content was calculated using the difference.
and astaxanthin from H. pluvialis including organic acetone extraction
and supercritical carbon dioxide (SC-CO2) extraction have been studied To analyze the fatty acid content, the extracted lipids were esterified
[33-35]. Nevertheless, certain limitations including the toxicity of the at 80°C for 3 h with a 10% HCl-methanol solution in a dry thermo unit
organic solvents, high-pressure operations, and required drying process (DTU-2 C, TAITEC Co., Ltd, Koshigaya, Japan). The analysis of fatty
prior to SC-CO2 extraction are concerns. Therefore, this work aims to acid methyl esters (FAMEs) was performed with a gas chromatograph
study the extraction of lipids and astaxanthin from wet H. pluvialis using (GC-2014, Shimadzu, Kyoto, Japan). An injection port and a flame-
liquefied DME at room temperature and a mild pressure. In addition, ionization detector were maintained at 250°C. The column temperature
the extracted organic components including certain elements and fatty was initially held at 150°C and programmed to increase to 220°C at
acids were characterized. 2°C/min. Helium and hydrogen were used as the carrier gases.

Experimental Design HPLC analysis

For the determination of astaxanthin, the extract was analyzed
DME extraction
using HPLC (Jasco, Hachioji, Japan). The astaxanthin standard and
The DME extraction apparatus and procedure are described in the sample extract were diluted with mixture of acetone before being
detail in a previous study [8]. Briefly, a liquefied DME storage tank injected with a 20 μL loop. The separation column was a 5 C18-MS-PAQ
(TVS-1-100, Taiatsu Techno Corp., Saitama, Japan), an extractor (HPG- COSMOSIL 250 × 4.6 mm (i.d.) (Nacalai Tesque Inc, Kyoto, Japan)
10-5; Taiatsu Techno Corp., Saitama, Japan; 190 mm×11.6 mm internal equipped with an intelligent UV/VIS detector UV-2075 plus (Jasco,
diameter, volume 10 cm3), liquefied DME, and extract storage vessels Hachioji, Japan). The isocractic elution was provided at 1.5 mL min–1
(HPG-96-3; Taiatsu Techno Corp.) made of pressure-resistant glass with a mobile phase consisting of methanol and THF at the volume
coated with polycarbonate were connected in series. The water content ratio of 9: 1. Astaxanthin was detected at 470 nm. The amount of
of the wet and unbroken cyst H. pluvialis paste provided by Biogenic astaxanthin in the extract was analyzed on the basis of the peak area
Co., Ltd, Tokyo, Japan was 82.1%, as determined by drying at 107°C to compared to that of standard astaxanthin.
a constant weight. An average of 1.187 g of wet H. pluvialis was loaded
into the lower part of the extraction column, and the upper part of the Results and Discussion
column was loaded with glass wool and glass beads ranging in diameter The amount of extracted lipids from wet unbroken H. pluvialis by
from 1.5 mm to 2.5 mm. Liquefied DME was allowed to flow into the liquefied DME is shown in Figure 1. With 195.5 g of liquefied DME, the
extraction column at a flow rate of 10 ± 2 cm3 min–1. The temperature lipid extraction yield based on the dry weight of H. pluvialis was 30.0%.
of liquefied DME in the extractor was 20 ± 2°C, as determined by an On the other hand, with dry unbroken H. pluvialis, acetone could not
infrared thermometer. The operation pressure was 0.51 ± 0.03 MPa, extract lipids at all. In the case of acetone extraction with cell disruption,
the saturated vapor pressure of liquefied DME [14]. After passing the lipid extraction yield was 43.0%. Therefore, an advantage of the
liquefied DME through the extractor for different time intervals, DME use of liquefied DME was evident in that it could extract lipids from
was evaporated by opening the reducing valve of the storage vessel. The unbroken H. pluvialis, whereas acetone could not.
weights of the samples were measured before and after they were placed
in the oven at 40°C, to determine the amount of extracted organic
components and water. In the case of the astaxanthin extraction, the
extracts were diluted with ethanol without drying and then the amount
of extracted astaxanthin was analyzed by high performance liquid
chromatography (HPLC) as described herein below.
Acetone extraction
Acetone is toxic and is typically used for the astaxanthin extraction
from H. pluvialis [36]. The results by liquefied DME were compared
with those of the conventional acetone extraction. Acetone (1 mL),
1 g of glass beads (1 mm diameter), and 16.4 mg of dry H. pluvialis
were put into a disruption tube and the solution was homogenized by
a Disruptor genie digital (Scientific Industries, Inc., New York, USA) at
Figure 1: Extraction of lipid as function of liquefied DME amount.
8000 rpm at room temperature for 10 min to disrupt the cell wall. The

J Nutr Food Sci, an open access journal

ISSN: 2155-9600 Volume 4 • Issue 5 • 1000305
Citation: Boonnoun P, Kurita Y, Kamo Y, Wahyudiono, Machmudah S, et al. (2014) Wet Extraction of Lipids and Astaxanthin from Haematococcus
pluvialis by Liquefied Dimethyl Ether. J Nutr Food Sci 4: 305. doi: 10.4172/2155-9600.1000305

Page 3 of 4

Six samples were collected with different amounts of liquefied

DME (Figure 1) and their elemental and fatty acid compositions were
analyzed. The results of the elemental analysis are shown in Table 1. The
distribution of elements in the compounds extracted from H. pluvialis
via the two extraction methods was as follows: 71.7% carbon (C),
10.8% hydrogen (H), 0.3% nitrogen (N), and 17.2% oxygen (O). The
differences between the distribution of compounds from either DME or
acetone extraction were similar to the measurement errors, indicating
that both extraction methods led to similar results. However, extracted
lipids contained more carbon and hydrogen compared with original H.
pluvialis and its residues. Therefore, extracted lipids are more suitable
for use as biofuel than original H. pluvialis as biomass.
For fatty acid analysis, the samples were separately analyzed and the
results are shown in Table 2. The other components were detected as Figure 2: Extraction of astaxanthin as function of liquefied DME amount.
numerous peaks after the C18 fatty acids in the HPLC analysis. Because
each peak after the C18 fatty acids was very small, correlated materials
could not be identified. However, summation of the small peak areas in the extracted lipid. On the other hand, 7.84 mg/g of astaxanthin
was not negligible. Lipid sample number 1shown in Figure 1 mainly was extracted by acetone, which corresponded to 1.82%. These results
contained the saturated fatty acid C16:0 (palmitic acid). A significant indicate that liquefied DME can extract compounds with a narrower
amount of C18:2n-6, C18:3n-6, and C18: 3n-3 (linoleic acid, γ-linolenic range of polarity from H. pluvialis because of its lower polarity than that
acid, and α-linolenic acid) were obtained from sample 2 and 3. Moreover, of acetone. Therefore, the residues extracted by liquefied DME were
the total amount of free fatty acids extracted by DME (calculated by extracted again with acetone, because acetone extraction led to a higher
the summation of sample number 1 to 6) contained less unsaturated concentration of astaxanthin. The astaxanthin concentration in the
fatty acids compared to the acetone extraction. Saturated fatty acids lipids extracted by acetone from the liquefied DME residue was 6.56%.
often have non-advantageous cold flow properties (a high cloud point The extracted lipid and astaxanthin amounts based on the dry weight
and a high pour point), while biodiesel made from unsaturated fatty of H. pluvialis were 10.2% and 6.69 mg/g, respectively. The residue of
acids (C18: 1, C18: 2, C18: 3) tends to be volatile and is susceptible H. pluvialis therefore could potentially be used as valuable materials
to low oxidation stability [7]. The organic components obtained from with a high concentration of astaxanthin. In other words, low-value-
H. pluvialis should therefore be modified prior to use as biofuel. In added lipid should be extracted for fuel by liquefied DME at first, and
addition, 92% of the initial water content was removed from the wet H. then valuable astaxanthin should be extracted from the liquefied DME
pluvialis, and the residue was almost entirely dried. Therefore, because residue by acetone.
the residue of H. pluvialis contained low water content, its viability for
use as a biomass fuel without drying was evident. Conclusion
The extracted samples were analyzed by HPLC to determine the This study clearly demonstrated that liquefied DME can be used
concomitant amount of astaxanthin. As shown in Figure 2, with 295.5 to extract lipids directly from H. pluvialis with high moisture content.
g of liquefied DME, the amount of astaxanthin based on the dry weight Overall, we showed that astaxanthin and lipids can be extracted from
of H. pluvialis was 1.00 mg/g, or 0.33% of astaxanthin concentration H. pluvialis without drying or cell disruption in an energy saving
fashion with the use of liquefied DME as the extraction solvent. The
extraction yields of astaxanthin by liquefied DME were clearly lower
Original Liquefied DME Acetone than that obtained by the commonly-used acetone extraction. A high
H. pluvialis Lipid Residue Lipid Residue concentration of astaxanthin can be obtained from the residue of H.
C (%) 51.5 71.7 54.0 71.4 53.0 pluvialis by liquefied DME without drying. Thus, the extraction of H.
H (%) 7.9 10.8 8.2 10.2 6.9 pluvialis with liquefied DME does not require drying, cell disruption, or
N (%) 2.4 0.3 2.2 0.3 3.0 heating and can save energy, as compared to other extraction methods.
O (%) 38.2 17.2 35.6 18.1 37.1
Acknowledgements
Table 1: Elemental compositions during the liquefied DME extraction and
acetone extraction. This research was supported by a grant from the Precursory
Research for Embryonic Science and Technology Program (PRESTO;
Fatty acid Liquefied DME
Acetone H. Kanda) of Japan Science and Technology Agency (JST).
[%] 1 2 3 4 5 6 1-6
C14:0 0.7 1.2 0.6 0.7 0.9 0.8 0.7 0.4 References
C16:0 39.4 22.0 18.0 9.2 10.9 8.5 15.9 31.1 1. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25: 294-306.
C18:0 2.7 6.1 4.9 4.7 6.7 4.0 5.1 1.4 2. Lardon L, Hélias A, Sialve B, Steyer JP, Bernard O (2009) Life-cycle assessment
C18:1n-9 15.2 10.8 9.4 4.0 4.3 3.5 7.2 14.6 of biodiesel production from microalgae. Environ Sci Technol 43: 6475-6481.
C18:2n-6 9.5 9.0 8.7 3.0 2.9 2.5 5.7 18.7 3. Reijnders L (2008) Do biofuels from microalgae beat biofuels from terrestrial
C18:3n-6 1.2 4.6 4.2 3.9 5.5 7.9 4.3 1.8 plants? Trends Biotechnol 26: 349-350.
C18:3n-3 2.6 2.8 2.9 1.0 1.1 0.9 1.9 7.1 4. Sander K, Murthy GS (2010) Life cycle analysis of algae biodiesel. Int J Life
Others 28.8 43.5 51.3 73.5 67.7 71.9 59.3 24.8 Cycle Assess 15: 704-714.

Table 2: Fatty acid analysis during the liquefied DME extraction and acetone 5. Kadam KL (2002) Environmental implications of power generation via coal-
extraction. microalgae cofiring. Energy 27: 905-922.

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Citation: Boonnoun P, Kurita Y, Kamo Y, Wahyudiono, Machmudah S, et al. (2014) Wet Extraction of Lipids and Astaxanthin from Haematococcus
pluvialis by Liquefied Dimethyl Ether. J Nutr Food Sci 4: 305. doi: 10.4172/2155-9600.1000305

Page 4 of 4

6. Stephenson AL, Kazamia E, Dennis JS, Howe CJ, Scott SA, et al. (2010) Life- formation in the Chlorophyte Haematococcus pluvialis. Bioresour Technol 55:
cycle assessment of potential algal biodiesel production in the United Kingdom: 207-214.
a comparison of raceways and air-lift tubular bioreactors. Energ Fuel 24: 4062-
4077. 22. Gong X, Chen F (1998) Influence of medium components on astaxanthin
content and production of Haematococcus pluvialis. Process Biochem 55: 385-
7. Halim R1, Rupasinghe TW, Tull DL, Webley PA (2013) Mechanical cell 391.
disruption for lipid extraction from microalgal biomass. Bioresour Technol 140:
53-63. 23. Machmudah S, Shotipruk A, Goto M, Sasaki M, Hirose T (2006) Extraction of
astaxanthin from Haematococcus pluvialis using supercritical CO2 and ethanol
8. Kanda H Li P (2011) Simple extraction method of green crude from natural as entrainer. Ind Eng Chem Res 45: 3652-3657.
blue-green microalgae by dimethyl ether. Fuel 90: 1264-1266.
24. Miao F, Lu D, Li Y, Zeng M (2006) Characterization of astaxanthin esters in
9. Kanda H, Li P, Ikehara T, Yasumoto-Hirose M (2012) Lipids extracted from Haematococcus pluvialis by liquid chromatography-atmospheric pressure
several species of natural blue–green microalgae by dimethyl ether: extraction chemical ionization mass spectrometry. Anal Biochem 352: 176-181.
yield and properties. Fuel 95: 88-92.
25. Hussein G, Nakamura M, Zhao Q, Iguchi T, Goto H, et al. (2005) Antihypertensive
10. Fast G, Kuhn D, Class AG, Maas U (2009) Auto-ignition during in stationary and neuroprotective effects of astaxanthin in experimental animals. Biol Pharm
jet evolution of dimethyl ether (DME) in a high-pressure atmosphere. Combust Bull 28: 47-52.
Flame 156: 200-213.
26. Hussein G, Goto H, Oda S, Iguchi T, Sankawa U, et al. (2005) Antihypertensive
11. Lee MC, Seo SB, Chung JH, Joo YJ, Ahn DH (2009) Industrial gas turbine potential and mechanism of action of astaxanthin: II. Vascular reactivity and
combustion performance test of DME to use as an alternative fuel for power hemorheology in spontaneously hypertensive rats. Biol Pharm Bull 28: 967-
generation. Fuel 88: 657-662. 971.

12. Cho W, Song T, Mitsos A, McKinnon JT, Ko GH, et al. (2009) Optimal design 27. Gross GJ, Lockwood SF (2004) Cardioprotection and myocardial salvage by
and operation of a natural gas tri-reforming reactor for DME synthesis. Catal a disodium disuccinate astaxanthin derivative (Cardax). Life Sci 75: 215-224.
Today 139: 261-267.
28. Miki W (1991) Biological functions and activities of animal carotenoids. Pure
13. Kanda H, Shirai H (2002) Method for removing water contained in solid using Appl Chem 63: 141-146.
liquefied material. Patent number – JP 4 291 772 B2 WO2003/101579, Japan.
29. Naguib YM (2000) Antioxidant activities of astaxanthin and related carotenoids.
14. Catchpole OJ, Grey JB, Perry NB, Burgess EJ, Redmond WA, et al. (2003) J Agric Food Chem 48: 1150-1154.
Extraction of chili, black pepper, and ginger with near-critical CO2, propane,
and dimethyl ether: analysis of the extracts by quantitative nuclear magnetic 30. Shimidzu N, Goto M, Miki W (1996) Carotenoids as singlet oxygen quenchers
resonance. J Agric Food Chem 51: 4853-4860. in marine organisms. Fish Sci 61: 134-137.

15. Holldorff H, Knapp H (1988) Binary vapor-liquid-liquid equilibrium of dimethyl 31. Sarada R, Vidhyavathi R, Usha D, Ravishankar GA (2006) An efficient method
ether-water and mutual solubilities of methyl chloride and water: experimental for extraction of astaxanthin from green alga Haematococcus pluvialis. J Agric
results and data reduction. Fluid Phase Equilib 44: 195-209. Food Chem 54: 7585-7588.

16. Wu J, Zhou Y, Lemmon EW (2011) An equation of state for the thermodynamic 32. Mendes-Pinto MM, Raposo MFJ, Bowen J, Young AJ, Morais R (2001)
properties of dimethyl ether. J Phys Chem Ref Data 40: art. no. 023104. Evaluation of different cell disruption processes on encysted cells of
Haematococcus pluvialis: effects on astaxanthin recovery and implications for
17. Delrue F, Setier PA, Sahut C, Cournac L, Roubaud A, et al. (2012) An economic, bio-availability. J Appl Phycol 13: 19-24.
sustainability, and energetic model of biodiesel production from microalgae.
Bioresour Technol 111: 191-200. 33. Krichnavaruk S, Shotipruk A, Goto M, Pavasant P (2008) Supercritical carbon
dioxide extraction of astaxanthin from Haematococcus pluvialis with vegetable
18. Varlet V, Smith F, Augsburger M (2014) New trends in the kitchen: propellants oils as co-solvent. Bioresour Technol 99: 5556-5560.
assessment of edible food aerosol sprays used on food. Food Chem 142: 311-
317. 34. Damiani MC, Popovich CA, Constenla D, Leonardi PI (2010) Lipid analysis in
Haematococcuspluvialis to assess its potential use as a biodiesel feedstock.
19. Naito M, Radcliffe C, Wada Y, Hoshino T, Liu X, et al. (2005) A comparative Bioresour Technol 101: 3801-3807.
study on the autoxidation of dimethyl ether (DME) comparison with diethyl ether
(DEE) and diisopropyl ether (DIPE). J Loss Prev Process Ind 18: 469-473. 35. Wang L, Yang B, Yan B, Yao X (2012) Supercritical fluid extraction of astaxanthin
from Haematococcus pluvialis and its antioxidant potential in sunflower oil.
20. Saha SK, McHugh E, Hayes J, Moane S, Walsh D, et al. (2013) Effect of Innov Food Sci Emerg Technol 13: 120-127.
various stress-regulatory factors on biomass and lipid production in microalga
Haematococcus pluvialis. Bioresour Technol 128: 118-124. 36. Lababpour A, Lee C-G (2006) Simultaneous measurement of chlorophyll and
astaxanthin in Haematococcus pluvialis cells by first-order derivative ultraviolet-
21. Harker M, Tsavalos AJ, Young AJ (1996) Factors responsible for astaxanthin visible spectrophotometry. J Biosci Bioeng 101: 104-110.

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