Effect of microalga preconditioning on supercritical CO2 ... - ISSF 2012

<strong>Effect</strong> <strong>of</strong> <strong>microalga</strong> <strong>preconditioning</strong> on supercritical CO2
extraction <strong>of</strong> astaxanthin from Haematoccus pluvialis
Raúl I. Aravena* & José M. del Valle
Pontificia Universidad Católica de Chile, Departamento de Ingeniería Química y Bioprocesos, Santiago, Chile
* Corresponding autor: riaraven@uc.cl; Phone: (+56) 998220688
ABSTRACT
Haematoccocus pluvialis is the main natural source <strong>of</strong> astaxanthin, a carotenoid with high antioxidant power.
We used supercritical CO2 (scCO2) to extract H. pluvialis to minimize the thermal damage <strong>of</strong> astaxanthin and
avoid its contamination with traces <strong>of</strong> undesirable organic solvents. In addition, because use <strong>of</strong> a liquid substrate
may improve the selectivity <strong>of</strong> scCO2 extraction and allow continuous processing, we attempted also the
extraction <strong>of</strong> aqueous homogenate samples containing 25% w/w H. pluvialis, and compared it with the
extraction <strong>of</strong> dry powder counterparts. We used cysts <strong>of</strong> H. pluvialis cultivated in Northern Chile that were
disrupted and spray dried by the producer. Results showed that temperature (40 or 70 °C) and pressure (35, 45,
or 55 MPa) had both positive effects on astaxanthin recovery from dry powder samples because <strong>of</strong> the increase
in solute volatility with temperature, and the increase in density and solvent power <strong>of</strong> scCO2 with pressure.
Astaxanthin recovery reached a top value <strong>of</strong> 61% at 70 ºC and 55 MPa when extracting dry powder samples for
4.5 h. The effects <strong>of</strong> extraction temperature and pressure were not as clear when extracting aqueous H. pluvialis’
homogenate samples, case where top recovery was only 54% at optimal conditions <strong>of</strong> 70 ºC and 45 MPa
following a 10-h treatment. The extraction <strong>of</strong> aqueous homogenate samples was much slower than that <strong>of</strong> the dry
powder counterparts, because extraction <strong>of</strong> aqueous samples was solubility-controlled and negatively affected by
water; being the solubility <strong>of</strong> water in scCO2 low, water in aqueous homogenate samples acted as a barrier to
mass transfer. However, using aqueous samples as the substrate increased slightly the concentration <strong>of</strong>
astaxanthin in the extract, which in this study reached a maximal value <strong>of</strong> 7% when extracting homogenate
samples at 40 ºC and 45 MPa.
INRODUCTION
Astaxanthin is a ketocarotenoid that belongs to the xantophyll class and has potent antioxidant activity, higher
than that <strong>of</strong> lutein, β-carotene, or α-tocophero [1]. The potent antioxidant activity <strong>of</strong> astaxanthin benefits human
health, and this has stirred industrial interest in its use as ingredient in nutraceutical and pharmaceutical products
[2-4]. Astaxanthin can be chemically synthesized or isolated from natural (biological) sources, but only natural
astaxanthin is allowed for human consumption. The main natural source <strong>of</strong> astaxanthin is the <strong>microalga</strong>e
Haematococcus pluvialis that contains up to 5% astaxanthin in a dry basis [5]. There is consequently a need to
recover, isolate and concentrate astaxanthin from H. pluvialis to take full advantage <strong>of</strong> its bioactivity in the
formulation <strong>of</strong> nutraceutical and pharmaceutical products.
Supercritical CO2 (scCO2) extraction is an alternative for the recovery <strong>of</strong> high-value natural compounds such as
H. pluvialis’ astaxanthin. scCO2 has been amply studied as an alternative <strong>of</strong> organic solvents for the extraction <strong>of</strong>
natural products due to its high selectivity, nontoxicity (GRAS status), and reduced thermal damage <strong>of</strong> the
substrate or extract [6]. scCO2 extraction can be applied to either solid or liquid samples. Particularly, the use <strong>of</strong>
a liquid sample as substrate may improve selectivity and allow continuous processing, thus reducing solvent
requirements. There are reports in literature on the scCO2 extraction <strong>of</strong> astaxanthin from H. pluvialis’ powder [7-
10] but not on the recovery <strong>of</strong> astaxanthin from (liquid) H. pluvialis’ homogenate. These previous studies
evaluated the effect on astaxanthin extraction <strong>of</strong> system temperature (40-80 °C), system pressure (20-55 MPa),
and extraction time (1-4 h), that influence positively extraction rate and yield, but had not always a positive
effect on extraction selectivity.

The objective <strong>of</strong> this work was to compare the scCO2 extraction <strong>of</strong> astaxanthin from an aqueous H. pluvialis
homogenate with that from a dry powder counterpart.
MATERIALS and METHODS
Sample and sample analysis
Disrupted dried cysts <strong>of</strong> H. pluvialis containing 4% water were supplied by Atacama BioNatural Products S.A.
(Iquique, Chile). They were vacuum-packed and stored in a freezer at -15 ºC in the dark. The astaxanthin content
in H. pluvialis’ powder was measured by extraction with acetone <strong>of</strong> 10 mg samples to exhaustion in several
stages (up to the point where they were discolored). Prior to further analysis, the acetone contained in extract
samples was removed in a nitrogen atmosphere.
Supercritical CO2 extraction
The extraction process was carried out in a one-pass, laboratory device (Thar Designs SFE-1L, Pittsburgh, PA)
using food-grade (99.8% pure) CO2 (AGA S.A, Santiago, Chile). Dry powder H. pluvialis’ samples (1 g) mixed
with 1.5 g <strong>of</strong> celite (Merck, Darmstadt, Germany) or aqueous homogenate samples (4 g suspensions containing
25% w/w <strong>of</strong> H. pluvialis’ powder) were loaded in a extraction vessel (50 cm 3 ) that was filled with glass spheres.
Extractions were carried out using 10 g/min <strong>of</strong> CO2 at 40 or 70 °C and 35, 45, or 55 MPa. Cumulative yields <strong>of</strong>
total extract and astaxanthin were determined as a function <strong>of</strong> process time by collecting, weighing, and
analyzing extract samples in 1 h intervals up to a total extraction time <strong>of</strong> 10 h in the case <strong>of</strong> aqueous homogenate
samples. Extract aliquots were collected in variable-time intervals during 4.5 h in scCO2 extractions <strong>of</strong> powder
samples. Prior to further analyses, extracts were dried in a nitrogen atmosphere as done with acetone extract
samples.
Astaxanthin quantification
Astaxanthin content in acetone or scCO2 extracts was determined in a UV/VIS spectrophotometer (Hach
dr/2000, Loveland, CO) after dissolving them in acetone. The concentration <strong>of</strong> astaxanthin in the solutions was
estimated using its optical extinction coefficient at λ = 470 nm in acetone (E 1% 1cm=2100) using eq. 1 [11]:
x=Ay/ E 1% 1cm x 100
where x is the amount <strong>of</strong> pigment (g), A is the absorbance, and y the added amount <strong>of</strong> acetone (cm 3 ).
RESULTS AND DISCUSSION
Figure 1 shows cumulative extraction curves for astaxanthin as a function <strong>of</strong> specific CO2 consumption for the
extraction <strong>of</strong> powder samples, and Table 1 summarizes values <strong>of</strong> astaxanthin recovery and concentration in
extract samples for the 4.5-h extractions. Disrupted H. pluvialis’ cysts contained 32% w/w acetone extract, and
1.92% w/w astaxanthin. Results indicate a positive effect <strong>of</strong> system temperature and pressure on astaxanthin
recovery that reached a maximum <strong>of</strong> ca. 61% at 70°C and 55 MPa. The positive effect <strong>of</strong> temperature can be
explained by an increase in the vapor pressure <strong>of</strong> the solute with temperature, which facilitates its transfer to the
scCO2 phase. On the other hand, the positive effect <strong>of</strong> pressure is due possibly to the increase in density and
solvent power <strong>of</strong> CO2 that increases the solubility <strong>of</strong> oleoresin and astaxanthin in it. The effect <strong>of</strong> a 30 ºC
increase in temperature outweighs the effect <strong>of</strong> a 20 MPa increase in pressure within our experimental region.
The positive effects <strong>of</strong> temperature and pressure are consistent with those reported by others [7-10], although the
magnitude <strong>of</strong> these effects varies from study to study. For example, Machmudah et al. [9] reported that an
increase in temperature from 40 to 70 °C at 55 MPa imcreases astaxanthin recovery from ca. 15 to 78% (a 5-fold
increase), a much larger effect that observed by us (an increase <strong>of</strong> only ca. 5%). On the other hand, an increase
in pressure from 40 to 55 MPa at 70 °C increases astaxanthin recovery from ca. 25 to 78% (a 3-fold increase)
[9], which is also much higher than the 7-17% increase observed in this work. The differences between studies

y (mg astaxantina/gr <strong>microalga</strong>)
12
10
8
6
4
2
550, 70
450,70
350, 70
550,40
450, 40
350,40
0
0 0,5 1 1,5 2 2,5
F (kg CO2/gr <strong>microalga</strong>)
Figure 1. Cumulative extraction curves <strong>of</strong> dry Haematococcus pluvialis powder samples as a function <strong>of</strong> extraction
temperature and pressure.
Table 1. Recovery and concentration in extract <strong>of</strong> astaxanthin from Haematococcus pluvialis as a function <strong>of</strong> extraction
conditions.
Temperature Pressure Astaxanthin recovery (%) Astaxanthin concentration (%)
(ºC) (MPa) Dry powder Homogenate Dry powder Homogenate
40 35 51.72 39.16 5.95 5.65
40 45 51.99 50.68 5.6 6.77
40 75 58.11 48.84 5.24 6.54
70 35 51.72 54.84 5.31 4.73
70 45 56.86 54.19 5.96 4.88
70 75 60.75 48.45 5.34 3.87
can be explained by differences between samples in cyst rupture degree, which affects extraction performance
strongly [7].
Other important factor to consider is the selectivity <strong>of</strong> the extraction, characterized by the concentration <strong>of</strong>
astaxanthin in extract samples (Table 1) or purity. In our experiments on dry H. pluvialis powder extraction, the
purity <strong>of</strong> extracts samples changed little, being the highest (ca. 6%) at an intermediate condition (70°C and 45
MPa) that did not coincided with that for highest astaxanthin recovery. Unlike in this study, Machmudah et al.
[9] reported a strong dependence <strong>of</strong> in extract purity on extraction conditions; as an example, it increases from
ca. 3.5 to 12.5% (a 3.5-fold increase) when increasing temperature from 40 to 70°C at 55 MPa.
Figure 2 shows cumulative extraction curves for astaxanthin as a function <strong>of</strong> specific CO2 consumption for the
extraction <strong>of</strong> aqueous homogenate samples, and Table 1 summarizes values <strong>of</strong> astaxanthin recovery and
concentration in extract samples for the 10-h extractions. In this case, the effect <strong>of</strong> extraction temperature and
pressure on extraction rate and yield is not as evident or as easily explainable as in the extraction <strong>of</strong> powder
samples (Fig. 1). Initial extraction rates <strong>of</strong> homogenates were smaller than those <strong>of</strong> dry powder samples, and
extraction curves were S-shaped. The lag-period when extracting homogenates may be associated to a negative

y (mg astaxantina/gr <strong>microalga</strong>)
12
10
8
6
4
2
550, 70
450, 70
350,70
550, 40
450,40
350, 40
0
0 1 2 3 4 5 6
F (kg de CO2/ g <strong>microalga</strong>)
Figure 2. Cumulative extraction curves <strong>of</strong> aqueous Haematococcus pluvialis homogenate samples as a function <strong>of</strong> extraction
temperature and pressure.
effect <strong>of</strong> the water layer that is not very soluble in scCO2 and may retard extraction. Water is extracted, however,
and its removal can explain the subsequent increase in extraction rate following the initial lag period. Dry
pockets may have developed during extraction that exposed H. pluvialis to scCO2 and facilitated removal <strong>of</strong>
astaxanthin and other components <strong>of</strong> the extract [12]. Following the initial lag period, as water is being removed
from homogenates, the extraction rate remains approximately constant, which suggest that this process is
solubility-controlled. A comparison <strong>of</strong> operational solubilities (Table 2), defined as the slope <strong>of</strong> cumulative
extraction curves in Figure 1 and Figure 2 in the zones where the extraction rate is constant, shows that this is
much higher when extracting dried powder than aqueous homogenate samples. This highlights the negative
effect <strong>of</strong> water on the solubility <strong>of</strong> the lipidic (hydrophobic) components <strong>of</strong> H. pluvialis extract.
Table 2. Operational solubility <strong>of</strong> astaxanthin in supercritical CO 2 (mg/kg) as a function <strong>of</strong> extraction conditions.
Temperature Pressure Operational solubility
(ºC) (MPa) Dry powder Homogenate
40 35 14 1,3
40 45 19 1,6
40 75 36 1,7
70 35 17 1,6
70 45 33 1,8
70 75 65 1,5
Astaxanthin concentration in the extracts <strong>of</strong> aqueous homogenate samples depended also strongly on extraction
conditions (Table 1). In this case, the purity <strong>of</strong> extracts decreased with extraction temperature and was the
highest at an intermediate pressure <strong>of</strong> 45 MPa. Furthermore, the extraction <strong>of</strong> homogenates allowed higher

purities than the extraction <strong>of</strong> dry powder samples. Consequently, astaxanthin concentration reached a top value
<strong>of</strong> ca. 7% when extracting an homogenate sample at 40°C and 45 MPa.
CONCLUSIONS AND PERSPECTIVES
Results showed that scCO2 extraction <strong>of</strong> astaxanthin from a H. pluvialis’ cysts is slower when using aqueous
homogenate than dry powder samples, possibly due to an increase in the mass transfer resistance and a reduction
in the operational solubility <strong>of</strong> astaxanthin in the presence <strong>of</strong> water. Nevertheless, scCO2 extraction <strong>of</strong>
homogenates permits a small increase in purity. So, the challenge remains to overcome the disadvantage <strong>of</strong> slow
extraction to take advantage <strong>of</strong> increased selectivity <strong>of</strong> the extraction <strong>of</strong> aqueous homogenate samples. An
alternative may be feeding homogenates continually to a countercurrent contact column so as to improve the
CO2 / homogenate contact and increase productivity as compared to the batch extraction process <strong>of</strong> static
samples.
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