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

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