Exploring the Microalga Euglena cantabrica by Pressurized Liquid Extraction to Obtain Bioactive Compounds

Abstract

In the present study, the chemical composition of the microalga Euglena cantabrica was investigated. The extraction of bioactive compounds was done using pressurized liquid extraction (PLE) at different temperatures (40-180 °C) and using green solvents (ethanol-water mixtures). A statistical design of experiments was used to optimize the maximum antioxidant capacity of the extracts by response surface methodology. The antioxidant capacity was determined through the inhibition of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, while the chemical analyses of the extracts were carried out using different chromatographic techniques. Chlorophylls and carotenoids were analyzed by high-performance liquid chromatography coupled to a diode array detector and mass spectrometry (HPLC-DAD-MS/MS) and carbohydrates by gas chromatography with flame ionization detection (GC-FID) and high-pressure size-exclusion chromatography coupled to an evaporative light-scattering detector (HPSEC-ELSD). The results showed different possibilities for the extraction conditions, depending on the desired bioactivity or chemical composition. Briefly, (i) mixtures of ethanol-water containing around 40% ethanol at 180 °C gave the best antioxidant capacity, (ii) mixtures containing around 50% ethanol at 110 °C gave the best yield of β-glucan paramylon, and (iii) the use of pure ethanol at a low temperature (40 °C) is the best choice for the recovery of carotenoids such as diatoxanthin. Summing up, E. cantabrica seems to be a good candidate to be used in biorefinery to obtain different bioactive compounds.

Keywords: Euglena cantabrica; carbohydrates; carotenoid; microalga; paramylon; pressurized liquid extraction; response surface.

Figure 1

Microscopic view of Euglena cantabrica. N (nucleus), CP (chloroplast), ES (eyespot), PC (paramylon center), and M (mucocysts). Image from Shin and Triemer, 2004 [6] with permission of John Wiley and Sons, Ltd.

Figure 2

Pareto’s diagrams and estimated response surfaces for (a) extraction yield, (b) total phenols, and antioxidant capacity measured by (c) ABTS test and (d) DPPH test. Vertical line of Pareto’s diagrams shows a confidence range of 95%, and the color shows if the influence of the factors is positive (increasing the value of the response variable, grey color) or negative (blue color).

Figure 3

High-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) chromatographic profile obtained at 450 nm from the PLE extracts carried out at (a) 180 °C, 100% EtOH (solvent composition); (b) 110 °C, 100% EtOH; (c) 40 °C, 100% EtOH; and (d) 110°C, 50% EtOH.

Figure 4

HPLC-DAD profile corresponding to the analysis of pigments obtained at 450 nm from an extract done at 110 °C and 100% ethanol. Carotenoids are represented with an underlined number and chlorophylls without underline.

Figure 5

HPSEC-ELSD profile of the carbohydrate fraction corresponding to bioactive component extractions at 110 °C with 50% of EtOH. Peaks labeled as * and ** were out of the range of estimation of the method.

Figure 6

Distribution and abundance of the different carbohydrate fractions according to the estimations of the molecular weight (Mw) obtained by HPSEC-ELSD of the extractions carried out at 40 (a), 110 (b), and 180 °C (c).

Figure 7

Chromatographic profile obtained by gas chromatography with flame ionization detection (GC-FID) of trimethylsilyl (TMS) oxymes of monosaccharides after hydrolysis with 2-N trifluoroacetic acid (TFA) of extracted pectin from Euglena cantabrica by PLE (110 °C with 50% of EtOH). (1) Xylose (Xyl), (2) xylose+arabinose (Xyl+Ara), (3) rhamnose (Rha), (4) fucose (Fuc), (5) galactose (Gal), (6) mannose (Man), (7) glucose (Glc), (8) galacturonic acid (Gal A), and (9) internal standard.

Figure 8

Distribution and abundance of the monomeric compositions (% over total carbohydrates) obtained by GC-FID after the hydrolysis with TFA of the extractions carried out at 40 (a), 110 (b), and 180 °C (c).