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Supercritical fluid extraction of carotenoids from Scenedesmus almeriensis
Food Chemistry 123 (2010) 928–935
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Analytical Methods
Supercritical fluid extraction of carotenoids from Scenedesmus almeriensis M.D. Macías-Sánchez *, J.M. Fernandez-Sevilla, F.G. Acién Fernández, M.C. Cerón García, E. Molina Grima Departamento de Ingeniería Química, Universidad de Almería, 04120 Almería, Spain
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 8 March 2010 Accepted 28 April 2010
Keywords: Supercritical fluid extraction Scenedesmus almeriensis Lutein b-Carotene
a b s t r a c t Scenedesmus almeriensis is a fast-growing highly productive new strain and is also a good producer of lutein. The aim of this study was to determine the influences of pressure and temperature on the supercritical fluid extraction of lutein and b-carotene from a freeze-dried powder of the marine microalga, S. almeriensis. The operating conditions were as follows: pressure in the range 200–600 bar and temperatures between 32 and 60 °C. The extracts were analysed by HPLC. Empirical correlations were also developed. The results demonstrate that it is necessary to work at a pressure of 400 bar and a temperature of 60 °C to obtain a significant yield in the extraction of pigments. In comparison with the reference extraction process used, the results show that better yields are obtained in the extraction of b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Carotenoids, or tetraterpenoids, are a class of terpenoid pigments with a 40 carbon backbone. These compounds are a relevant group of pigments because of their structural diversity and their numerous biological functions. They are essential for photosynthesis and in general for life in the presence of oxygen. Most carotenoids dissolve well in non-polar solvents (HorneroMéndez & Mínguez-Mosquera, 2000; Römer, 1999) and are therefore liposoluble, a property that gives fruits, plants, birds and marine fauna some characteristic yellow, orange and red colours. This colouring capacity comes from the presence of a long series of conjugated double bonds that is also responsible for the antioxidant properties of these materials (Baysal, Ersus, & Starmans, 2000). Hydrocarbon carotenoids are referred to as carotenes, while their oxygenated derivatives are known as xanthophylls. Within the latter group, the oxygen atom can be present in the form of hydroxyl groups (as in the case of lutein) or as oxy groups (as in the case of cantaxanthin) or as a combination of both (as in astaxanthin, Higuera-Ciapara, Félix-Valenzuela, & Goycoolea, 2006). Lutein is one of the most important carotenoids and is widely found in foods and also in human serum. Lutein, together with zeaxanthin, imparts colour to the macula lutea, the spot in the human retina that allows the appreciation of fine details, and it is believed
* Corresponding author. Address: Department of Chemical Engineering, University of Almería, Ctra. Sacramento s/n, La Cañada de San Urbano, 04120 Almería, Spain. Tel./fax: +34 950015484. E-mail addresses: [email protected], [email protected] (M.D. Macías-Sánchez). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.076
to have an important role as an antioxidant (Krinsky, 2002; Rapp, Maple, & Choi, 2000). Part of the retina is rich in polyunsaturated fatty acids that are easily attacked by oxygen free radicals, generated by the presence of light, and undergo an oxidative degradation that renders them dysfunctional (Connor & Neuringer, 1988). This compound also filters out blue light, thus protecting the eye from some of the damaging effects of sunlight. On the other hand, lutein is widely used as a food colouring agent, as well as in aquaculture and poultry breeding (Lorenz & Cysewski, 2000). During the past few years, lutein has attracted increasing attention with regard to its application to human health. In a number of studies, lutein has been found to play a role in the prevention of age-related macular degeneration (AMD), cataracts, amelioration of the first stages of atherosclerosis and even some types of cancer (Dwyer et al., 2001; Lu et al., 2005; Seddon et al., 1994). Although marigold is the current source of lutein, there are other alternate sources that can be considered. For example, the microalga (clorophycea), Scenedesmus almeriensis (CCAP-276/24), is a very rich source (up to 1% d.wt. of lutein under outdoor culture conditions) and has a very favourable carotenoid profile, which consists of up to 70% of free lutein/zeaxanthin (Granado-Lorencio et al., 2009), with the rest being b-carotene and violaxanthin and negligible amounts of other carotenoids. S. almeriensis is a fastgrowing highly productive strain (Sánchez et al., 2008) and is also an extraordinary producer of lutein. Carotenoids have been extracted using organic solvents (Aravantinos-Zafiris, Oreopoulou, Tzia, & Thomopoulos, 1992; O’Day and Rosenau, 1982) but the heightened public awareness of health issues and the application of more restrictive regulations have stimulated the technological development of supercritical
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fluids and brought about an increase in interest in the recovery of natural products using supercritical carbon dioxide. Mendes et al. (1995), Careri et al. (2001) and Macías-Sánchez et al. (2005,
2007, 2009) have all successfully used supercritical carbon dioxide (scCO2) to obtain carotenoid extracts from the microalgae Chlorella vulgaris, Spirulina platenses, Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina.
Table 1 Characteristics of reagents and solvents used in the SCF extraction procedure.
2. Materials and methods 2.1. Raw biomass
Name
Purity
Supplier
Characteristics
Carbon dioxide
99.995%
Methanol
HPLC grade
Supercritical fluid extraction HPLC analyses
Acetone
HPLC grade
Abello Linde J.T. Baker J.T. Baker
Acetone
Industrial
PQS
Acetone
PRS-CODEX
Panreac
Water Nitrogen
HPLC grade 99.99%
Potassium hydroxide 85% pellets Alumina
PRS-CODEX
Panreac Abello Linde Panreac
A-5
Sigma
Tetrabutylammonium acetate Ammonium acetate Ethyl ether
PA-ACS
Panreac
PA-ACS PA-ACS
Panreac Panreac
Sodium chloride
Purissimum
Panreac
Cryothermostatic Criothermostatic bath
Reference extraction solvent Grinding of the raw material HPLC analyses HPLC analyses Reference extraction solvent Reference extraction solvent
P
BPR1
600
500
400
T
HE2
300 MV-2 Mass flow meter
P P
MX-1
HE1
200
T Extractor
Pump P50
BPR2
T Separator
T
Collection solvent for the extracts HPLC analyses Cleaning of supercritical extraction equipment Reference extraction solvent HPLC analyses Evaporation of solvents
The microalga used in this study was the clorophycea, S. almeriensis, and this was isolated by the University of Almería, Chemical Engineering Department (Sánchez et al., 2008) and a sample is deposited at the CCAP (Oban, Scotland). This microalga averages a 3% d.wt. pigment content that consists largely of lutein (up to 1% d.wt. with an average of 6 mg/g), as well as significant amounts of other carotenoids, such as b-carotene (0.75 mg/g), violaxanthin (0.23 mg/g), asthaxanthin (0.22 mg/g), cantaxanthin (0.125 mg/g) and astaxanthin esters (0.20 mg/g). The biomass used was cultured in a large scale facility at ‘‘Estación Experimental de Las Palmerillas–Cajamar’’ (El Ejido, Almería, Spain), as described by Sánchez et al. (2008). The biomass was harvested using a RINA continuous centrifuge (Riera Nadeu S.A., Spain), then frozen, freeze-dried and finally milled to obtain a homogenised powder that was stored in the dark at 30 °C.
32 S1
T
MV-3
MV-1
T
Fig. 1. Diagram showing the flow in the extraction process with supercritical carbon dioxide. MV, manual valve; BPR, back pressure regulator valve; HE, heat exchanger; S, cyclonic separator.
46
53
60
Fig. 2. User-defined factorial set of experiments using a response surface method (RSM).
Table 2 Lutein and b-carotene yields obtained for an extraction time of 300 min. Pressure (bar)
Temperature (°C)
mg Lutein/g dry weight of microalgae
mg b-Carotene/g dry weight of microalgae
200
32 46 60
0.0013 0.0000 0.0109
0.0631 0.000 0.582
300
39 53
0.0236 0.0090
0.210 0.310
400
32 46 60
0.0299 0.0327 0.0466
0.807 0.303 1.50
500
39 53
0.0459 0.0084
0.300 0.230
600
32 46 60
0.0000 0.0147 0.0294
0.0307 0.595 1.22
2.33
3.07
CO22
Controller
39
Reference extraction process
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tract was analysed by HPLC, as described by Del Campo et al. (2004) and the content of the different carotenoids was quantified.
2.2. Biomass pre-treatment Preliminary scCO2 extraction experiments (not shown), with the biomass prepared as described in the previous section, resulted in extremely low recovery yields. Thus, a cell breakage step was included prior to the extraction as a standard part of the extraction procedure. This disgregation step was carried out by molturation in a ball mill for 10 min of a 50:50 wt. mixture of freeze-dried biomass and a disrupting agent (Alumina A-5 for cell disruption, SIGMA, St. Louis, MO, USA). The choice of operating variables and other details of the procedure are described in detail elsewhere (Cerón et al., 2008).
2.5. Analytical methods
The chemicals, solvents and reagents used are summarised in Table 1.
The recovery of lutein and b-carotene, attained in the scCO2 extraction process, was measured by HPLC (Del Campo et al., 2004) with a Shimadzu SPD-M10AV chromatograph, using a diode array detector. The column used was a 5 lm XTerra MSC18 (4.6 150 mm). The sample was eluted at 1 ml/min with the following solvents: (A) water/ion pair reagent/methanol (1:1:8, v/v) and (B) acetone/methanol (1:1, v/v). The ion pair reagent was a solution of tetrabutylammonium acetate (0.05 M) and ammonium acetate (1 M) in water. Absorbance was monitored in the range 360–700 nm. Lutein and b-carotene were quantified by integration at 450 nm. Lutein and b-carotene standards were obtained from Sigma Chemical Co., St Louis, MO, USA.
2.4. Reference extraction process
2.6. Supercritical extraction
A reference extraction process under non-supercritical conditions was used as a comparison in order to evaluate the performance of the scCO2 extraction process. This procedure led to the extraction of almost 100% of the carotenoids and is described elsewhere (García-Malea, Acién, Fernández, Cerón, & Molina, 2006). The procedure consisted of a disgregation step in which a biomass sample was milled in a mortar with the same weight of alumina as a disrupting agent. The sample was then repeatedly extracted with acetone until all of the carotenoids were recovered. The final ex-
The experiments were carried out in a Thar SFE 10 extractor (Thar Technologies Inc., Pittsburgh, PA, USA) equipped with a thermostatted 100 ml sample cell, a pump and a backpressure regulator capable of operating at pressures of up to 600 bar (Fig. 1). All of the components of the extractor (scCO2 preheater, extraction cell, backpressure regulator and SCF pump) were automatic and were controlled by computer. The extractor was also equipped with temperature sensors during the extraction in the inlet and in the jacket of the extraction cell, pressure sensors in the back-
2.3. Reagents and solvents
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05
T= 32ºC
0.04 0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
0.04 0.03 0.02 0.01 0.00 150
650
T= 39ºC
200
250
300
350
400
450
500
550
600
650
500
550
600
650
pressure (bar)
0.05 mg lutein/g dry weight of microalgae
0.05
T= 46ºC
0.04 0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
650
0.04
T= 53ºC
0.03 0.02 0.01 0.00 150
200
250
300
pressure (bar)
350
400
450
pressure (bar)
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
pressure (bar)
0.04
T= 60ºC
0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
650
pressure (bar)
Fig. 3. Effect of pressure on the yield of lutein for an extraction time of 300 min using supercritical carbon dioxide.
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the number of randomized experiments was 13 plus two repetitions of the central points. The experimental data were processed with the software package ‘‘STATGRAPHICS Plus 5.1 (1994–2001, Statistical Graphics Corp.) in order to obtain empirical equations that allow the prediction of the recovery yields of the major pigments present in S. almeriensis, namely lutein and b-carotene, and recovered in the SCF extraction process, as a function of operating pressure and temperature. This possibility is the result of the application of a 2-parameter 5-level surface response analysis to the SCF extraction procedure totalling 15 randomized experiments.
pressure regulator and mass flow and density sensors for the extraction solvent. This configuration allowed a completely stable operation in the temperature and pressure ranges selected. The carotenoids extracted during the process were recovered in a cyclonic separator. After a given extraction time, the separator was opened and the pigments were recovered by washing with HPLC grade acetone and then quantified as described above. Pressure was tested in the range 200–600 bar and temperature from 32 to 60 °C. Lower pressures were not used as these were reported elsewhere to give negligible yields (Macías-Sánchez et al., 2005, 2007). In each run, a 38 g sample of a 50:50 w/w biomass and alumina mixture was disrupted and homogenised as described above. The residue was loaded into the extraction cell and the flow of CO2 was started at 1 g/min under the desired conditions. The extraction time was 5 h, which is relatively long, in order to ensure complete recovery under the given conditions. The extracted product was dissolved in acetone and stored at 30 °C with the exclusion of light prior to subsequent analysis.
3. Experimental results The yields of lutein and b-carotene extractions from S. almeriensis under the different conditions of the proposed experimental set are shown in Table 2. The results are expressed as mg of pigment per g dry weight of microalga. These values were obtained for an extraction time of 300 min under the different extraction conditions studied. The yields of the reference extraction process are indicated at the end of Table 2.
2.7. Experimental design The influences of pressure and temperature in the recovery of lutein and b-carotene were measured using a response surface method (RSM). The two factors tested were pressure, in the range 200–600 bar, and temperature, from 32 to 60 °C. The experimental design was proposed to take into account quadratic responses and interactions. For this purpose a specific user-defined factorial set of experiments (shown in Fig. 2) was proposed. The design included the centre, vertices, centre of edges and inner points. In general
4. Discussion of the results 4.1. Lutein extraction The experimental yields for the extraction of lutein are shown in Figs. 3 and 4. These yields were obtained at different pressures and temperatures for an extraction time of 300 min.
0.05
P= 200 bar
0.04
mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05
0.03 0.02 0.01 0.00 30
35
40
45
50
55
60
0.03 0.02 0.01 0.00
65
P= 300 bar
0.04
30
35
40
temperature (ºC)
50
55
60
65
55
60
65
0.05 mg lutein/g dry weight of microalgae
P= 400 bar
0.03 0.02 0.01 0.00
P= 500 bar
0.04 0.03 0.02 0.01 0.00
30
35
40
45
50
55
60
65
30
35
40
temperature (ºC)
45
50
temperature (ºC)
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05 0.04
45
temperature (ºC)
P= 600 bar
0.04 0.03 0.02 0.01 0.00 30
35
40
45
50
55
60
65
temperature (ºC)
Fig. 4. Effect of temperature on the yield of lutein for an extraction time of 300 min using supercritical carbon dioxide.
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extraction at 46 °C where the maximum was attained at 600 bar. This can be attributed to the two effects that the increase in pressure has on CO2. On the one hand, the increased density enhances the solvating power of CO2, which favours the solubilisation of the products. On the other hand, the decreased diffusivity hampers the ability of the supercritical fluid to enter the matrix, thus decreasing the interaction between solvent and solute – hence the decrease in the extraction yield (Mantell, 2000). The predominance of one or other effect explains the trend observed in the experimental data.
The results from the analysis of the experimental design are given in Table 3. The estimated effects and interactions within the range of variables studied and the analysis of variance of the extraction process are also shown. The sign associated with each of the effects indicates a positive or negative influence on the yield of the dependent variable. The degree of significance for each factor is represented in the table by its p-value; when a factor has a pvalue less than 0.05 it influences the process in a significant way with a confidence level of 0.95. The results obtained show that temperature, pressure and the interaction of these two variables have positive influences on the yield of lutein extraction, albeit not in significant ways (pvalue > 0.05).
4.1.2. Temperature effect Analysis of Fig. 4 indicates that, at 200 and 400 bar, an increase in the temperature up to 46 °C increases the lutein extraction yield until 60 °C. A similar trend is observed at 600 bar, although in this case the recovery yield increases continuously from 32 °C. At 300 and 500 bar the maximum yield was found at 39 °C and no further improvement was observed on increasing the temperature to 53 °C. The changes in the recovery yield caused by increases in the temperature are the result of a complex interaction between the change in density, which decreases and leads to a poorer solvating power, and variations in the vapour pressure of the pigments, which increase, thus favouring pigment solubility. The predominance of one or other effect explains the trend observed in the experimental data. Similar behaviour in the extraction yields of carotenoids, according to the effect of pressure and temperature, has been
4.1.1. Pressure effect It can be seen from Fig. 3 that, in general, the maximum extraction yield is obtained at intermediate pressures, except for the
Table 3 Estimated effects and the analysis of variance of the process for the lutein and b-carotene extraction with supercritical carbon dioxide. Lutein
mg beta carotene/g dry weight of microalgae
Pressure (P) Temperature (T) PP PT TT
b-Carotene
Effects
p-Value
Effects
p-Value
0.012 ± 0.013 0.008 ± 0.013 0.035 ± 0.020 0.007 ± 0.016 0.026 ± 0.020
0.355 0.515 0.116 0.689 0.226
0.492 ± 0.279 0.541 ± 0.279 0.764 ± 0.452 0.540 ± 0.358 0.945 ± 0.452
0.111 0.084 0.125 0.166 0.066
1.60
1.60 mg beta carotene/g dry weight of microalgae
Variable
T= 32ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
T= 39ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00
250
350
450
550
150
650
250
mg beta carotene/g dry weight of microalgae
T= 46ºC
1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
450
550
650
1.60 T=53ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00
250
350
450
550
650
150
250
350
450
550
pressure (bar)
pressure (bar)
mg beta carotene/g dry weight of microalgae
mg beta carotene/ g dry weight of microalgae
1.60 1.40
350
pressure (bar)
pressure (bar)
1.60 1.40
T=60ºC
1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
250
350
450
550
650
pressure (bar)
Fig. 5. Effect of pressure on the yield of b-carotene for an extraction time of 300 min using supercritical carbon dioxide.
650
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M.D. Macías-Sánchez et al. / Food Chemistry 123 (2010) 928–935 1.60
P=200 bar
1.40
mg beta carotene/g dry weight of microalgae
mg beta carotene/g dry weight of microalgae
1.60 1.20 1.00 0.80 0.60 0.40 0.20 0.00
1.20 1.00 0.80 0.60 0.40 0.20 0.00
30
35
40
45
50
55
60
P=300 bar
1.40
65
30
35
40
45
1.60
55
60
65
55
60
65
1.60
P=400 bar
1.40
mg beta carotene/g dry weight of microalgae
mg beta carotene/g dry weight of microalgae
50
temperature (ºC)
temperature (ºC)
1.20 1.00 0.80 0.60 0.40 0.20 0.00 30
35
40
45
50
55
60
1.20 1.00 0.80 0.60 0.40 0.20 0.00
65
P=500 bar
1.40
30
35
40
45
50
temperature (ºC)
temperature (ºC)
mg beta carotene/g dry weight of microalgae
1.60
P=600 bar
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 30
35
40
45
50
55
60
65
temperature (ºC)
Fig. 6. Effect of temperature on the yield of b-carotene for an extraction time of 300 min using supercritical carbon dioxide.
reported in the literature (Macías-Sánchez et al., 2005, 2007, 2009), although in these studies the raw material used did not undergo pre-treatment, such as cell disruption, which was used in the case of S. almeriensis. 4.2. b-Carotene extraction The yields obtained in the extraction of b-carotene from the microalga, S. almeriensis, are represented in Figs. 5 and 6 for different pressures and operating temperatures. All of the results correspond to an extraction time of 300 min.
mglutein/g dryweightof microalgae
The results of the experimental design analysis are gathered in Table 3. An estimation of the effects and interactions between the range of studied variables and the analysis of variance of the process are also presented. The results obtained show that temperature, pressure and crossed interactions have a positive influence on the yield of b-carotene extraction, albeit not in a significant way (p-value > 0.05). 4.2.1. Effect of pressure It can be seen (Fig. 5) that the yields obtained in the extraction of b-carotene follow the same trend as the yields obtained in the
0.05
1.60
0.04 0.04
1.40 1.20
0.03 0.03 0.02 0.02 0.01 0.01 0.00
mg beta-carotene/g 1.00 dry weight of 0.80 microalgae 0.60
600 500 pressure (bar)
400
300
200
32
37
42
47
52
62 57
temperature (ºC)
Fig. 7. Estimated yields of lutein extraction with supercritical carbon dioxide using the empirical correlation.
0.40 0.20 0.00 600
500
pressure (bar)
400
300
200
32
37
42
47
62 57 52
temperature (ºC)
Fig. 8. Estimated yields of b-carotene extraction with supercritical carbon dioxide using the empirical correlation.
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A detailed analysis of the graph indicates that the highest yield was obtained at a temperature of 60 °C, which is the same conclusion as deduced previously. In relation to the pressure, experimentally the maximum was obtained at 400 bar while the proposed correlation provides a value of 453 bar. Empirical correlations were obtained using the experimental data and the programme STATGRAPHICS Plus 4.0 (1994–2001, Statistical Graphics Corp.). The expression in the case of b-carotene is Eq. (2):
1.00 Fraction of carotenoids yield compared to reference extraction process
0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20
R ¼ 4:337 þ 4:440e3 P 0:241 T 9:551e6 P2
0.10
þ 9:635e5 P T þ 2:410e3 T 2
0.00 Reference extraction process
lutein
β carotene
Fig. 9. Fraction of carotenoid yields compared with a reference extraction process.
lutein extraction. The only difference concerns the trend at 53 °C; the yield diminished as the pressure increased from 300 to 500 bar. On increasing the pressure for each temperature studied, the solvating power of the carbon dioxide increased. This increase favours the extraction process but is offset, to some degree, by a decrease in the diffusion coefficient, which reduces the penetration capacity of the solvent and diminishes the yield at higher pressures. 4.2.2. Effect of temperature Analysis of Fig. 6 shows that the yields obtained in the extraction of b-carotene follow the same trend as the yields obtained in the lutein extraction. A modification is even observed at 300 bar, in which the maximum extraction yield is obtained at 53 °C. The increase in the extraction yield for a constant pressure is due to the enhancement in the solubility of the pigment in the solvent as an increase is produced in the vapour pressure of this pigment. In contrast, the decrease in the yield is due to the prevalence of the effect of the reduction of the carbon dioxide density as the pressure is increased. 4.3. Empirical correlations Starting from the experimental data, and with the help of the programme STATGRAPHICS Plus 4.0 (1994–2001, Statistical Graphics Corp.), an empirical correlation was developed that predicted the yields obtained in the extraction of lutein and b-carotene from the microalga, S. almeriensis, with scCO2. In the case of lutein, Eq. (1) relates the variables that influence the process (pressure and temperature): (Fig. 7).
R ¼ 8:660e2 þ 3:291e4 P 6:374e3 T 4:416e7 P2 þ 1:189e6 P T þ 6:741e5 T 2
ð1Þ
where R is the yield of extracted lutein (mg/g dry weight of microalga), T is the temperature (°C) and P is the pressure (bar). The resulting correlation coefficient is 0.60.
ð2Þ
where R is the yield of extracted b-carotene expressed in mg pigment per g dry weight of microalga, T is the temperature (°C) and P the pressure (bar). The resulting correlation coefficient is 0.79. Eq. (2) is represented graphically in Fig. 8 for the different operating conditions. A detailed analysis of the graph leads to the same conclusions as deduced previously for temperature; the highest yield is obtained at 60 °C. As far as pressure is concerned, experimentally the maximum yield was obtained at 400 bar and the empirical correlation determined this value to be 535 bar. 4.4. Comparative analysis between supercritical fluid extraction and reference extraction process The data for lutein and b-carotene yields from S. almeriensis, using supercritical carbon dioxide, can be compared to extraction yields using a reference extraction process (Fig. 9). This reference procedure (in non-supercritical conditions) led to the extraction of almost 100% of carotenoids. The comparison is expressed in as per one of lutein and b-carotene yields compared with the reference extraction process described elsewhere (García-Malea et al., 2006). The maximum extraction yields of the two carotenoids represented in Fig. 9 were obtained at 400 bar and 60 °C. An HPLC profile of this supercritical fluid extract is presented in Fig. 10. Chlorophylls are not extracted using scCO2 without cosolvent. On the one hand, the results show that the best yields are obtained in the extraction of b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. On the other hand, the percentage of lutein obtained is very poor. 5. Conclusions Within the pressure range selected in these experiments, the highest extraction yields of lutein and b-carotene were obtained on operating at 400 bar. The most appropriate operating temperature to obtain the best yields in the extraction of these pigments is 60 °C. It is not advisable to increase the temperature beyond this point as thermal degradation of the resulting extracts may occur. In comparison with the reference extraction process used, the results show that the best yields are obtained in the extraction of
Fig. 10. Chromatogram of the supercritical fluid extract under the optimum extraction conditions (400 bar and 60 °C).
M.D. Macías-Sánchez et al. / Food Chemistry 123 (2010) 928–935
b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. The percentage of lutein obtained is very low.
Acknowledgements This research was supported by the Ministerio de Educación y Ciencia (CTQ2005-00335/PPQ), Spain.
References Aravantinos-Zafiris, G., Oreopoulou, V., Tzia, C., & Thomopoulos, C. D. (1992). Utilisation of orange by-products orange peel carotenoids. Journal of Science and Food Agriculture, 59, 77–79. Baysal, T., Ersus, S., & Starmans, D. A. J. (2000). Supercritical carbon dioxide extraction of beta carotene and lycopene from tomato paste waste. Journal of Agricultural and Food Chemistry, 48, 5507–5511. Careri, M., Furlattini, L., Mangia, A., Musci, M., Anklam, E., & Theobald, A. (2001). Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina pacifica algae: A chemometric approach. Journal of Chromatography A, 912, 61–71. Cerón, M. C., Campos, I., Sánchez, J. F., Acién, F. G., Molina, E., & Fernández-Sevilla, J. M. (2008). Recovery of lutein from microalgae biomass: Development of a process for Scenedesmus almeriensis biomass. Journal of Agricultural and Food Chemistry, 56, 11761–11766. Connor, W. E., & Neuringer, M. (1988). The effects of n-3 fatty acid deficiency and repletion upon the fatty acid composition and function of the brain and retina. Progress in Clinical and Biological Research, 282, 275–294. Del Campo, J. A., Rodríguez, H., Moreno, J., Vargas, M. A., Rivas, J., & Guerrero, M. G. (2004). Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Applied Microbiology Biotechnology, 64, 848–854. Dwyer, J. H., Navab, M., Dwyer, K. M., Hassan, K., Sun, P., Shircore, A., et al. (2001). Oxygenated carotenoid lutein and the progression of early atherosclerosis. The Los Angeles atherosclerosis study. Circulation, 103, 2922–2927. García-Malea, M. C., Acién, F. C., Fernández, J. M., Cerón, M. C., & Molina, E. (2006). Continuous production of green cells of Haematococcus pluviales: Modeling of the irradiance effect. Enzyme and Microbial Technology, 38, 981–989. Granado-Lorencio, F., Herrero-Barbudo, C., Acién-Fernández, G., Molina-Grima, E., Fernández-Sevilla, J. M., Pérez-Sacristán, B., et al. (2009). In vitro bioaccesibility of lutein and zeaxanthin from the microalgae Scenedesmus almeriensis. Food Chemistry, 114, 747–752.
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Higuera-Ciapara, I., Félix-Valenzuela, L., & Goycoolea, F. M. (2006). Astaxanthin: A review of its chemistry and applications. Critical Reviews in Food Science and Nutrition, 46, 185–196. Hornero-Méndez, D., & Mínguez-Mosquera, M. I. (2000). Carotenoid pigments in Rosa mosqueta Hips, an alternative carotenoid source for foods. Journal of Agricultural and Food Chemistry, 48, 825–828. Krinsky, N. I. (2002). Possible biologic mechanisms for a protective role of xanthophylls. Journal of Nutrition, 132, 540–542. Lorenz, R. T., & Cysewski, G. R. (2000). Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends in Biotechnology, 18, 160–167. Lu, Q. Y., Arteaga, J. R., Zhang, Q., Huerta, S., Go, V. L., & Heber, D. (2005). Inhibition of prostate cancer cell growth by an avocado extract: Role of lipid-soluble bioactive substances. Journal of Nutrition and Biochemistry, 16, 23–30. Macías-Sánchez, M. D., Mantell, C., Rodríguez, M., Martínez de la Ossa, E., Lubián, L. M., & Montero, O. (2005). Supercritical fluid extraction of carotenoids and chlorophyll a from Nannochloropsis gaditana. Journal of Food Engineering, 66, 245–251. Macías-Sánchez, M. D., Mantell, C., Rodríguez, M., Martínez de la Ossa, E., Lubián, L. M., & Montero, O. (2007). Supercritical fluid extraction of carotenoids and chlorophyll a from Synechococcus sp. Journal of Supercritical Fluids, 39, 323–329. Macías-Sánchez, M. D., Mantell, C., Rodríguez, M., Martínez de la Ossa, E., Lubián, L. M., & Montero, O. (2009). Comparison of supercritical fluid and ultrasoundassisted extraction of carotenoids and chlorophyll a from Dunaliella salina. Talanta, 77, 948–952. Mantell, C. (2000). Tesis doctoral: Estudio de un proceso de extracción de antocianos, a partir de hollejo de uva tinta, utilizando dióxido de carbono alta presión y codisolventes. University of Cádiz, Department of Chemical Engineering. Mendes, R. L., Fernandes, H. L., Coelho, J. P., Reis, E. C., Cabral, J. M. S., Novais, J. M., et al. (1995). Supercritical CO2 extraction of carotenoids and other lipids from Chlorella vulgaris. Food Chemistry, 53, 99–103. O’Day, D. M., & Rosenau, J. R. (1982). Solvent extraction of carotenoides from alfalfa. Transactions of ASAE, 25, 515–519. Rapp, L. M., Maple, S. S., & Choi, J. H. (2000). Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina. Investigative Ophthalmology and Visual Science, 41, 1200–1209. Römer, S. (1999). Carotenoids in higher plants and algae. The Chloroplast: From Molecular Biology to Biotechnology, 64, 217–223. Sánchez, J. F., Fernández, J. M., Acién, F. G., Rueda, A., Pérez-Parra, J., & Molina, E. (2008). Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis. Process Biochemistry, 43, 398–405. Seddon, J. M., Ajani, U. A., Sperduto, R. D., Hiller, R., Blair, N., Burton, T. C., et al. (1994). Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Eye disease case-control study group. JAMA, 272, 1413–1420.
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Analytical Methods
Supercritical fluid extraction of carotenoids from Scenedesmus almeriensis M.D. Macías-Sánchez *, J.M. Fernandez-Sevilla, F.G. Acién Fernández, M.C. Cerón García, E. Molina Grima Departamento de Ingeniería Química, Universidad de Almería, 04120 Almería, Spain
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 8 March 2010 Accepted 28 April 2010
Keywords: Supercritical fluid extraction Scenedesmus almeriensis Lutein b-Carotene
a b s t r a c t Scenedesmus almeriensis is a fast-growing highly productive new strain and is also a good producer of lutein. The aim of this study was to determine the influences of pressure and temperature on the supercritical fluid extraction of lutein and b-carotene from a freeze-dried powder of the marine microalga, S. almeriensis. The operating conditions were as follows: pressure in the range 200–600 bar and temperatures between 32 and 60 °C. The extracts were analysed by HPLC. Empirical correlations were also developed. The results demonstrate that it is necessary to work at a pressure of 400 bar and a temperature of 60 °C to obtain a significant yield in the extraction of pigments. In comparison with the reference extraction process used, the results show that better yields are obtained in the extraction of b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Carotenoids, or tetraterpenoids, are a class of terpenoid pigments with a 40 carbon backbone. These compounds are a relevant group of pigments because of their structural diversity and their numerous biological functions. They are essential for photosynthesis and in general for life in the presence of oxygen. Most carotenoids dissolve well in non-polar solvents (HorneroMéndez & Mínguez-Mosquera, 2000; Römer, 1999) and are therefore liposoluble, a property that gives fruits, plants, birds and marine fauna some characteristic yellow, orange and red colours. This colouring capacity comes from the presence of a long series of conjugated double bonds that is also responsible for the antioxidant properties of these materials (Baysal, Ersus, & Starmans, 2000). Hydrocarbon carotenoids are referred to as carotenes, while their oxygenated derivatives are known as xanthophylls. Within the latter group, the oxygen atom can be present in the form of hydroxyl groups (as in the case of lutein) or as oxy groups (as in the case of cantaxanthin) or as a combination of both (as in astaxanthin, Higuera-Ciapara, Félix-Valenzuela, & Goycoolea, 2006). Lutein is one of the most important carotenoids and is widely found in foods and also in human serum. Lutein, together with zeaxanthin, imparts colour to the macula lutea, the spot in the human retina that allows the appreciation of fine details, and it is believed
* Corresponding author. Address: Department of Chemical Engineering, University of Almería, Ctra. Sacramento s/n, La Cañada de San Urbano, 04120 Almería, Spain. Tel./fax: +34 950015484. E-mail addresses: [email protected], [email protected] (M.D. Macías-Sánchez). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.076
to have an important role as an antioxidant (Krinsky, 2002; Rapp, Maple, & Choi, 2000). Part of the retina is rich in polyunsaturated fatty acids that are easily attacked by oxygen free radicals, generated by the presence of light, and undergo an oxidative degradation that renders them dysfunctional (Connor & Neuringer, 1988). This compound also filters out blue light, thus protecting the eye from some of the damaging effects of sunlight. On the other hand, lutein is widely used as a food colouring agent, as well as in aquaculture and poultry breeding (Lorenz & Cysewski, 2000). During the past few years, lutein has attracted increasing attention with regard to its application to human health. In a number of studies, lutein has been found to play a role in the prevention of age-related macular degeneration (AMD), cataracts, amelioration of the first stages of atherosclerosis and even some types of cancer (Dwyer et al., 2001; Lu et al., 2005; Seddon et al., 1994). Although marigold is the current source of lutein, there are other alternate sources that can be considered. For example, the microalga (clorophycea), Scenedesmus almeriensis (CCAP-276/24), is a very rich source (up to 1% d.wt. of lutein under outdoor culture conditions) and has a very favourable carotenoid profile, which consists of up to 70% of free lutein/zeaxanthin (Granado-Lorencio et al., 2009), with the rest being b-carotene and violaxanthin and negligible amounts of other carotenoids. S. almeriensis is a fastgrowing highly productive strain (Sánchez et al., 2008) and is also an extraordinary producer of lutein. Carotenoids have been extracted using organic solvents (Aravantinos-Zafiris, Oreopoulou, Tzia, & Thomopoulos, 1992; O’Day and Rosenau, 1982) but the heightened public awareness of health issues and the application of more restrictive regulations have stimulated the technological development of supercritical
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fluids and brought about an increase in interest in the recovery of natural products using supercritical carbon dioxide. Mendes et al. (1995), Careri et al. (2001) and Macías-Sánchez et al. (2005,
2007, 2009) have all successfully used supercritical carbon dioxide (scCO2) to obtain carotenoid extracts from the microalgae Chlorella vulgaris, Spirulina platenses, Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina.
Table 1 Characteristics of reagents and solvents used in the SCF extraction procedure.
2. Materials and methods 2.1. Raw biomass
Name
Purity
Supplier
Characteristics
Carbon dioxide
99.995%
Methanol
HPLC grade
Supercritical fluid extraction HPLC analyses
Acetone
HPLC grade
Abello Linde J.T. Baker J.T. Baker
Acetone
Industrial
PQS
Acetone
PRS-CODEX
Panreac
Water Nitrogen
HPLC grade 99.99%
Potassium hydroxide 85% pellets Alumina
PRS-CODEX
Panreac Abello Linde Panreac
A-5
Sigma
Tetrabutylammonium acetate Ammonium acetate Ethyl ether
PA-ACS
Panreac
PA-ACS PA-ACS
Panreac Panreac
Sodium chloride
Purissimum
Panreac
Cryothermostatic Criothermostatic bath
Reference extraction solvent Grinding of the raw material HPLC analyses HPLC analyses Reference extraction solvent Reference extraction solvent
P
BPR1
600
500
400
T
HE2
300 MV-2 Mass flow meter
P P
MX-1
HE1
200
T Extractor
Pump P50
BPR2
T Separator
T
Collection solvent for the extracts HPLC analyses Cleaning of supercritical extraction equipment Reference extraction solvent HPLC analyses Evaporation of solvents
The microalga used in this study was the clorophycea, S. almeriensis, and this was isolated by the University of Almería, Chemical Engineering Department (Sánchez et al., 2008) and a sample is deposited at the CCAP (Oban, Scotland). This microalga averages a 3% d.wt. pigment content that consists largely of lutein (up to 1% d.wt. with an average of 6 mg/g), as well as significant amounts of other carotenoids, such as b-carotene (0.75 mg/g), violaxanthin (0.23 mg/g), asthaxanthin (0.22 mg/g), cantaxanthin (0.125 mg/g) and astaxanthin esters (0.20 mg/g). The biomass used was cultured in a large scale facility at ‘‘Estación Experimental de Las Palmerillas–Cajamar’’ (El Ejido, Almería, Spain), as described by Sánchez et al. (2008). The biomass was harvested using a RINA continuous centrifuge (Riera Nadeu S.A., Spain), then frozen, freeze-dried and finally milled to obtain a homogenised powder that was stored in the dark at 30 °C.
32 S1
T
MV-3
MV-1
T
Fig. 1. Diagram showing the flow in the extraction process with supercritical carbon dioxide. MV, manual valve; BPR, back pressure regulator valve; HE, heat exchanger; S, cyclonic separator.
46
53
60
Fig. 2. User-defined factorial set of experiments using a response surface method (RSM).
Table 2 Lutein and b-carotene yields obtained for an extraction time of 300 min. Pressure (bar)
Temperature (°C)
mg Lutein/g dry weight of microalgae
mg b-Carotene/g dry weight of microalgae
200
32 46 60
0.0013 0.0000 0.0109
0.0631 0.000 0.582
300
39 53
0.0236 0.0090
0.210 0.310
400
32 46 60
0.0299 0.0327 0.0466
0.807 0.303 1.50
500
39 53
0.0459 0.0084
0.300 0.230
600
32 46 60
0.0000 0.0147 0.0294
0.0307 0.595 1.22
2.33
3.07
CO22
Controller
39
Reference extraction process
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tract was analysed by HPLC, as described by Del Campo et al. (2004) and the content of the different carotenoids was quantified.
2.2. Biomass pre-treatment Preliminary scCO2 extraction experiments (not shown), with the biomass prepared as described in the previous section, resulted in extremely low recovery yields. Thus, a cell breakage step was included prior to the extraction as a standard part of the extraction procedure. This disgregation step was carried out by molturation in a ball mill for 10 min of a 50:50 wt. mixture of freeze-dried biomass and a disrupting agent (Alumina A-5 for cell disruption, SIGMA, St. Louis, MO, USA). The choice of operating variables and other details of the procedure are described in detail elsewhere (Cerón et al., 2008).
2.5. Analytical methods
The chemicals, solvents and reagents used are summarised in Table 1.
The recovery of lutein and b-carotene, attained in the scCO2 extraction process, was measured by HPLC (Del Campo et al., 2004) with a Shimadzu SPD-M10AV chromatograph, using a diode array detector. The column used was a 5 lm XTerra MSC18 (4.6 150 mm). The sample was eluted at 1 ml/min with the following solvents: (A) water/ion pair reagent/methanol (1:1:8, v/v) and (B) acetone/methanol (1:1, v/v). The ion pair reagent was a solution of tetrabutylammonium acetate (0.05 M) and ammonium acetate (1 M) in water. Absorbance was monitored in the range 360–700 nm. Lutein and b-carotene were quantified by integration at 450 nm. Lutein and b-carotene standards were obtained from Sigma Chemical Co., St Louis, MO, USA.
2.4. Reference extraction process
2.6. Supercritical extraction
A reference extraction process under non-supercritical conditions was used as a comparison in order to evaluate the performance of the scCO2 extraction process. This procedure led to the extraction of almost 100% of the carotenoids and is described elsewhere (García-Malea, Acién, Fernández, Cerón, & Molina, 2006). The procedure consisted of a disgregation step in which a biomass sample was milled in a mortar with the same weight of alumina as a disrupting agent. The sample was then repeatedly extracted with acetone until all of the carotenoids were recovered. The final ex-
The experiments were carried out in a Thar SFE 10 extractor (Thar Technologies Inc., Pittsburgh, PA, USA) equipped with a thermostatted 100 ml sample cell, a pump and a backpressure regulator capable of operating at pressures of up to 600 bar (Fig. 1). All of the components of the extractor (scCO2 preheater, extraction cell, backpressure regulator and SCF pump) were automatic and were controlled by computer. The extractor was also equipped with temperature sensors during the extraction in the inlet and in the jacket of the extraction cell, pressure sensors in the back-
2.3. Reagents and solvents
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05
T= 32ºC
0.04 0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
0.04 0.03 0.02 0.01 0.00 150
650
T= 39ºC
200
250
300
350
400
450
500
550
600
650
500
550
600
650
pressure (bar)
0.05 mg lutein/g dry weight of microalgae
0.05
T= 46ºC
0.04 0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
650
0.04
T= 53ºC
0.03 0.02 0.01 0.00 150
200
250
300
pressure (bar)
350
400
450
pressure (bar)
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
pressure (bar)
0.04
T= 60ºC
0.03 0.02 0.01 0.00 150
200
250
300
350
400
450
500
550
600
650
pressure (bar)
Fig. 3. Effect of pressure on the yield of lutein for an extraction time of 300 min using supercritical carbon dioxide.
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the number of randomized experiments was 13 plus two repetitions of the central points. The experimental data were processed with the software package ‘‘STATGRAPHICS Plus 5.1 (1994–2001, Statistical Graphics Corp.) in order to obtain empirical equations that allow the prediction of the recovery yields of the major pigments present in S. almeriensis, namely lutein and b-carotene, and recovered in the SCF extraction process, as a function of operating pressure and temperature. This possibility is the result of the application of a 2-parameter 5-level surface response analysis to the SCF extraction procedure totalling 15 randomized experiments.
pressure regulator and mass flow and density sensors for the extraction solvent. This configuration allowed a completely stable operation in the temperature and pressure ranges selected. The carotenoids extracted during the process were recovered in a cyclonic separator. After a given extraction time, the separator was opened and the pigments were recovered by washing with HPLC grade acetone and then quantified as described above. Pressure was tested in the range 200–600 bar and temperature from 32 to 60 °C. Lower pressures were not used as these were reported elsewhere to give negligible yields (Macías-Sánchez et al., 2005, 2007). In each run, a 38 g sample of a 50:50 w/w biomass and alumina mixture was disrupted and homogenised as described above. The residue was loaded into the extraction cell and the flow of CO2 was started at 1 g/min under the desired conditions. The extraction time was 5 h, which is relatively long, in order to ensure complete recovery under the given conditions. The extracted product was dissolved in acetone and stored at 30 °C with the exclusion of light prior to subsequent analysis.
3. Experimental results The yields of lutein and b-carotene extractions from S. almeriensis under the different conditions of the proposed experimental set are shown in Table 2. The results are expressed as mg of pigment per g dry weight of microalga. These values were obtained for an extraction time of 300 min under the different extraction conditions studied. The yields of the reference extraction process are indicated at the end of Table 2.
2.7. Experimental design The influences of pressure and temperature in the recovery of lutein and b-carotene were measured using a response surface method (RSM). The two factors tested were pressure, in the range 200–600 bar, and temperature, from 32 to 60 °C. The experimental design was proposed to take into account quadratic responses and interactions. For this purpose a specific user-defined factorial set of experiments (shown in Fig. 2) was proposed. The design included the centre, vertices, centre of edges and inner points. In general
4. Discussion of the results 4.1. Lutein extraction The experimental yields for the extraction of lutein are shown in Figs. 3 and 4. These yields were obtained at different pressures and temperatures for an extraction time of 300 min.
0.05
P= 200 bar
0.04
mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05
0.03 0.02 0.01 0.00 30
35
40
45
50
55
60
0.03 0.02 0.01 0.00
65
P= 300 bar
0.04
30
35
40
temperature (ºC)
50
55
60
65
55
60
65
0.05 mg lutein/g dry weight of microalgae
P= 400 bar
0.03 0.02 0.01 0.00
P= 500 bar
0.04 0.03 0.02 0.01 0.00
30
35
40
45
50
55
60
65
30
35
40
temperature (ºC)
45
50
temperature (ºC)
0.05 mg lutein/g dry weight of microalgae
mg lutein/g dry weight of microalgae
0.05 0.04
45
temperature (ºC)
P= 600 bar
0.04 0.03 0.02 0.01 0.00 30
35
40
45
50
55
60
65
temperature (ºC)
Fig. 4. Effect of temperature on the yield of lutein for an extraction time of 300 min using supercritical carbon dioxide.
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extraction at 46 °C where the maximum was attained at 600 bar. This can be attributed to the two effects that the increase in pressure has on CO2. On the one hand, the increased density enhances the solvating power of CO2, which favours the solubilisation of the products. On the other hand, the decreased diffusivity hampers the ability of the supercritical fluid to enter the matrix, thus decreasing the interaction between solvent and solute – hence the decrease in the extraction yield (Mantell, 2000). The predominance of one or other effect explains the trend observed in the experimental data.
The results from the analysis of the experimental design are given in Table 3. The estimated effects and interactions within the range of variables studied and the analysis of variance of the extraction process are also shown. The sign associated with each of the effects indicates a positive or negative influence on the yield of the dependent variable. The degree of significance for each factor is represented in the table by its p-value; when a factor has a pvalue less than 0.05 it influences the process in a significant way with a confidence level of 0.95. The results obtained show that temperature, pressure and the interaction of these two variables have positive influences on the yield of lutein extraction, albeit not in significant ways (pvalue > 0.05).
4.1.2. Temperature effect Analysis of Fig. 4 indicates that, at 200 and 400 bar, an increase in the temperature up to 46 °C increases the lutein extraction yield until 60 °C. A similar trend is observed at 600 bar, although in this case the recovery yield increases continuously from 32 °C. At 300 and 500 bar the maximum yield was found at 39 °C and no further improvement was observed on increasing the temperature to 53 °C. The changes in the recovery yield caused by increases in the temperature are the result of a complex interaction between the change in density, which decreases and leads to a poorer solvating power, and variations in the vapour pressure of the pigments, which increase, thus favouring pigment solubility. The predominance of one or other effect explains the trend observed in the experimental data. Similar behaviour in the extraction yields of carotenoids, according to the effect of pressure and temperature, has been
4.1.1. Pressure effect It can be seen from Fig. 3 that, in general, the maximum extraction yield is obtained at intermediate pressures, except for the
Table 3 Estimated effects and the analysis of variance of the process for the lutein and b-carotene extraction with supercritical carbon dioxide. Lutein
mg beta carotene/g dry weight of microalgae
Pressure (P) Temperature (T) PP PT TT
b-Carotene
Effects
p-Value
Effects
p-Value
0.012 ± 0.013 0.008 ± 0.013 0.035 ± 0.020 0.007 ± 0.016 0.026 ± 0.020
0.355 0.515 0.116 0.689 0.226
0.492 ± 0.279 0.541 ± 0.279 0.764 ± 0.452 0.540 ± 0.358 0.945 ± 0.452
0.111 0.084 0.125 0.166 0.066
1.60
1.60 mg beta carotene/g dry weight of microalgae
Variable
T= 32ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
T= 39ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00
250
350
450
550
150
650
250
mg beta carotene/g dry weight of microalgae
T= 46ºC
1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
450
550
650
1.60 T=53ºC
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00
250
350
450
550
650
150
250
350
450
550
pressure (bar)
pressure (bar)
mg beta carotene/g dry weight of microalgae
mg beta carotene/ g dry weight of microalgae
1.60 1.40
350
pressure (bar)
pressure (bar)
1.60 1.40
T=60ºC
1.20 1.00 0.80 0.60 0.40 0.20 0.00 150
250
350
450
550
650
pressure (bar)
Fig. 5. Effect of pressure on the yield of b-carotene for an extraction time of 300 min using supercritical carbon dioxide.
650
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M.D. Macías-Sánchez et al. / Food Chemistry 123 (2010) 928–935 1.60
P=200 bar
1.40
mg beta carotene/g dry weight of microalgae
mg beta carotene/g dry weight of microalgae
1.60 1.20 1.00 0.80 0.60 0.40 0.20 0.00
1.20 1.00 0.80 0.60 0.40 0.20 0.00
30
35
40
45
50
55
60
P=300 bar
1.40
65
30
35
40
45
1.60
55
60
65
55
60
65
1.60
P=400 bar
1.40
mg beta carotene/g dry weight of microalgae
mg beta carotene/g dry weight of microalgae
50
temperature (ºC)
temperature (ºC)
1.20 1.00 0.80 0.60 0.40 0.20 0.00 30
35
40
45
50
55
60
1.20 1.00 0.80 0.60 0.40 0.20 0.00
65
P=500 bar
1.40
30
35
40
45
50
temperature (ºC)
temperature (ºC)
mg beta carotene/g dry weight of microalgae
1.60
P=600 bar
1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 30
35
40
45
50
55
60
65
temperature (ºC)
Fig. 6. Effect of temperature on the yield of b-carotene for an extraction time of 300 min using supercritical carbon dioxide.
reported in the literature (Macías-Sánchez et al., 2005, 2007, 2009), although in these studies the raw material used did not undergo pre-treatment, such as cell disruption, which was used in the case of S. almeriensis. 4.2. b-Carotene extraction The yields obtained in the extraction of b-carotene from the microalga, S. almeriensis, are represented in Figs. 5 and 6 for different pressures and operating temperatures. All of the results correspond to an extraction time of 300 min.
mglutein/g dryweightof microalgae
The results of the experimental design analysis are gathered in Table 3. An estimation of the effects and interactions between the range of studied variables and the analysis of variance of the process are also presented. The results obtained show that temperature, pressure and crossed interactions have a positive influence on the yield of b-carotene extraction, albeit not in a significant way (p-value > 0.05). 4.2.1. Effect of pressure It can be seen (Fig. 5) that the yields obtained in the extraction of b-carotene follow the same trend as the yields obtained in the
0.05
1.60
0.04 0.04
1.40 1.20
0.03 0.03 0.02 0.02 0.01 0.01 0.00
mg beta-carotene/g 1.00 dry weight of 0.80 microalgae 0.60
600 500 pressure (bar)
400
300
200
32
37
42
47
52
62 57
temperature (ºC)
Fig. 7. Estimated yields of lutein extraction with supercritical carbon dioxide using the empirical correlation.
0.40 0.20 0.00 600
500
pressure (bar)
400
300
200
32
37
42
47
62 57 52
temperature (ºC)
Fig. 8. Estimated yields of b-carotene extraction with supercritical carbon dioxide using the empirical correlation.
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A detailed analysis of the graph indicates that the highest yield was obtained at a temperature of 60 °C, which is the same conclusion as deduced previously. In relation to the pressure, experimentally the maximum was obtained at 400 bar while the proposed correlation provides a value of 453 bar. Empirical correlations were obtained using the experimental data and the programme STATGRAPHICS Plus 4.0 (1994–2001, Statistical Graphics Corp.). The expression in the case of b-carotene is Eq. (2):
1.00 Fraction of carotenoids yield compared to reference extraction process
0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20
R ¼ 4:337 þ 4:440e3 P 0:241 T 9:551e6 P2
0.10
þ 9:635e5 P T þ 2:410e3 T 2
0.00 Reference extraction process
lutein
β carotene
Fig. 9. Fraction of carotenoid yields compared with a reference extraction process.
lutein extraction. The only difference concerns the trend at 53 °C; the yield diminished as the pressure increased from 300 to 500 bar. On increasing the pressure for each temperature studied, the solvating power of the carbon dioxide increased. This increase favours the extraction process but is offset, to some degree, by a decrease in the diffusion coefficient, which reduces the penetration capacity of the solvent and diminishes the yield at higher pressures. 4.2.2. Effect of temperature Analysis of Fig. 6 shows that the yields obtained in the extraction of b-carotene follow the same trend as the yields obtained in the lutein extraction. A modification is even observed at 300 bar, in which the maximum extraction yield is obtained at 53 °C. The increase in the extraction yield for a constant pressure is due to the enhancement in the solubility of the pigment in the solvent as an increase is produced in the vapour pressure of this pigment. In contrast, the decrease in the yield is due to the prevalence of the effect of the reduction of the carbon dioxide density as the pressure is increased. 4.3. Empirical correlations Starting from the experimental data, and with the help of the programme STATGRAPHICS Plus 4.0 (1994–2001, Statistical Graphics Corp.), an empirical correlation was developed that predicted the yields obtained in the extraction of lutein and b-carotene from the microalga, S. almeriensis, with scCO2. In the case of lutein, Eq. (1) relates the variables that influence the process (pressure and temperature): (Fig. 7).
R ¼ 8:660e2 þ 3:291e4 P 6:374e3 T 4:416e7 P2 þ 1:189e6 P T þ 6:741e5 T 2
ð1Þ
where R is the yield of extracted lutein (mg/g dry weight of microalga), T is the temperature (°C) and P is the pressure (bar). The resulting correlation coefficient is 0.60.
ð2Þ
where R is the yield of extracted b-carotene expressed in mg pigment per g dry weight of microalga, T is the temperature (°C) and P the pressure (bar). The resulting correlation coefficient is 0.79. Eq. (2) is represented graphically in Fig. 8 for the different operating conditions. A detailed analysis of the graph leads to the same conclusions as deduced previously for temperature; the highest yield is obtained at 60 °C. As far as pressure is concerned, experimentally the maximum yield was obtained at 400 bar and the empirical correlation determined this value to be 535 bar. 4.4. Comparative analysis between supercritical fluid extraction and reference extraction process The data for lutein and b-carotene yields from S. almeriensis, using supercritical carbon dioxide, can be compared to extraction yields using a reference extraction process (Fig. 9). This reference procedure (in non-supercritical conditions) led to the extraction of almost 100% of carotenoids. The comparison is expressed in as per one of lutein and b-carotene yields compared with the reference extraction process described elsewhere (García-Malea et al., 2006). The maximum extraction yields of the two carotenoids represented in Fig. 9 were obtained at 400 bar and 60 °C. An HPLC profile of this supercritical fluid extract is presented in Fig. 10. Chlorophylls are not extracted using scCO2 without cosolvent. On the one hand, the results show that the best yields are obtained in the extraction of b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. On the other hand, the percentage of lutein obtained is very poor. 5. Conclusions Within the pressure range selected in these experiments, the highest extraction yields of lutein and b-carotene were obtained on operating at 400 bar. The most appropriate operating temperature to obtain the best yields in the extraction of these pigments is 60 °C. It is not advisable to increase the temperature beyond this point as thermal degradation of the resulting extracts may occur. In comparison with the reference extraction process used, the results show that the best yields are obtained in the extraction of
Fig. 10. Chromatogram of the supercritical fluid extract under the optimum extraction conditions (400 bar and 60 °C).
M.D. Macías-Sánchez et al. / Food Chemistry 123 (2010) 928–935
b-carotene; it is possible to extract 50% of the total of this pigment contained in the microalga studied. The percentage of lutein obtained is very low.
Acknowledgements This research was supported by the Ministerio de Educación y Ciencia (CTQ2005-00335/PPQ), Spain.
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