Green processes based on the extraction with pressurized fluids to obtain potent antimicrobials from Haematococcus pluvialis microalgae
Introduction
Nowadays, the growing interest in the formulation of new functional foods continues to raise the demand for new food ingredients. Along with this trend, the possibility of replacing synthetic preservatives with natural ones is receiving much attention. These natural preservatives are preferred to come from plants, algae and microalgae. To obtain these natural food ingredients and/or additives, environmentally clean extraction techniques are desired.
Pressurized liquid extraction (PLE) is an emerging technique that presents important advantages over traditional extraction ones. Traditional solvent extraction techniques use large quantities of toxic organic solvents, are labor intense, need long extraction times, possess low selectivity and/or low extraction yields, and can expose the extracts to excessive heat, light and oxygen. In contrast, PLE uses less solvent in a shorter period of time, is automated and involves retaining the sample in an oxygen- and light-free environment (King, 2000, Denery et al., 2004). Whereas other environmentally-friendly techniques, such as supercritical fluid extraction (SFE), are frequently used to obtain functional compound from natural sources, PLE has not been widely applied as a routine tool in natural product extraction. However, recent studies have used PLE for the extraction of compounds from natural sources (Benthin et al., 1999, Dunford and Zhang, 2003, Breithaupt, 2004; Herrero, Ibañez, Señorans, & Cifuentes, 2004; Piñeiro, Palma, & Barroso, 2004).
Algae have long been used for therapeutic purposes and their systematic evaluation for biologically active substances began in the 1950s. However, in the last decade microalgae have become the focus for extensive screening of novel compounds with interesting biological activities, which may lead to therapeutically useful agents (Mendes et al., 2003, Mayer and Hamann, 2005, Cardozo et al., 2006). The fact that microalgae may produce antibiotics is already well known, since a large number of microalgae extracts and/or extracellular products have been shown to have antimicrobial (antibacterial, antifungal, antiprotozoal) activity (Kellan and Walker, 1989, Ozemir et al., 2004, Herrero et al., 2006). Although several studies have tried to identify the compounds responsible for this antimicrobial activity (Boriwitzka, 1999, Tringali, 1997), the identity of most of them still remains unknown.
Haematococcus pluvialis is a green microalgae whose cells showed morphological changes from green motile, flagellated cells (green phase) to red hematocysts without flagella (red phase) during cultivation in stress conditions. This microalga has been extensively studied as a natural source of the carotenoid pigment astaxanthin, since astaxanthin is a strong coloring agent and a potent antioxidant (Guerin et al., 2003, Dufossé et al., 2005, Higuera-Ciapara et al., 2006). However, no studies have evaluated its potential to produce antimicrobial agents.
Therefore, the goal of the present study was to obtain and characterize PLE extracts of different composition from Haematococcus pluvialis and evaluate them as antimicrobial agents for use as preservatives in the food industry.
Section snippets
Samples and chemicals
Haematococcus pluvialis (BNA 10/024, National Bank of Algae, Canary Islands, Spain), were grown in modified Bold's Basal Medium (Nichols & Bold, 1964) enriched with NaNO3 (0.75 g/l). Cells (green phase) were cultured phototrophically in 20 l Carboys bubbled with air, at 25 °C, in light/dark cycles (16:8 h) with white fluorescent lamps providing 80 μmol m−2 s−1. To induce astaxanthin biosynthesis (red phase) exponentially grown cultures were transferred to nitrogen deprived medium and continuously
Pressurized liquid extraction
The two solvents (hexane and ethanol) were selected in order to evaluate the influence of the solvent polarity on the extraction of antimicrobial compounds from the microalgae. Additionally, different extraction temperatures were also tested, since it is the primary variable involved in the PLE process, as has been previously shown with different microalgae (Santoyo et al., 2006, Herrero et al., 2006).
The extraction conditions employed as well as the extraction yields obtained in all cases are
Conclusions
In the present study we have demonstrated the ability of pressurized liquid extraction to produce extracts with antimicrobial activity from a natural source such as the microalga Haemotococcus pluvialis. Results showed that most active extracts, in terms of antimicrobial activity, were those obtained from the red phase of the microalga, using ethanol as extraction solvent at 100 or 150 °C. After GC–MS characterization of the different samples, it could be deduced that the main compounds
Acknowledgments
This work has been financed by Spanish Ministry of Education (AGL2005-06726-C04-01/02/04) and Comunidad Autónoma de Madrid (S-0505/AGR/000153) projects. I.R. thanks the Comunidad Autónoma de Madrid for her grant.
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