Considerations on the use of enzyme-assisted extraction in combination with pressurized liquids to recover bioactive compounds from algae.

Pressurized liquids, PLE, and enzyme-assisted extraction, EAE, have been tested to improve the extraction of phlorotannins from the seaweed Sargassum muticum. Enzymatic treatment with proteases and carbohydrases, alkaline hydrolysis and PLE with ethanol:water as extracting solvent have been studied in terms of extraction yield, total phenolic content and antioxidant activity (TEAC assay). Results demonstrated that the application of PLE alone provided the highest yields and relevant antioxidant activity. An experimental design was employed to further optimize the PLE extraction conditions; optimum parameters included the use of 160 °C and 95% ethanol. Under these conditions, values of 21.9%, 94.0mg gallic acid equivalents g(-1), 5.018 mg phloroglucinol equivalents g(-1) and 1.275 mmol trolox equivalents g(-1) were obtained for extraction yield, total phenols, total phlorotannins and TEAC, respectively. A preliminary chemical characterization by liquid chromatography coupled to mass spectrometry provided insight in terms of the mechanisms involved in the different processes.

Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum muticum collected on North-Atlantic coasts.

In the present work, the phlorotannin composition of different Sargassum muticum samples collected at different locations along the North Atlantic coasts as well as the bioactivities related to these components were investigated. After pressurized liquid extraction, the samples collected at the extreme locations of a latitudinal gradient from Portugal and Norway, were found to be the richest on total phenols and, particularly, on phlorotannins, containing up to 148.97 and 5.12mg phloroglucinol equivalents g(-1), respectively. The extracts obtained from these locations were further purified and chemically characterized using a modified HILIC×RP-DAD-MS/MS method. The application of this methodology allowed the tentative identification of a great variability of phlorotannins with different degrees of polymerization (from 3 to 11) and structures, determined for the first time in S. muticum. The most-abundant phlorotannins on these samples were fuhalols, hydroxyfuhalols and phlorethols, showing also particularities and important differences depending on the geographical location. Afterwards, the antiproliferative activity of these extracts against HT-29 adenocarcinoma colon cancer cells was studied. Results revealed that the richest S. muticum samples in terms of total phlorotannins, i.e., those from Norway, presented the highest activity, showing a good cytotoxic potential at concentrations in the medium micromolar range.

Rapid geographical differentiation of the European spread brown macroalga Sargassum muticum using HRMAS NMR and Fourier-Transform Infrared spectroscopy.

Two recent techniques based on chemical footprinting analysis, HRMAS NMR and FTIR spectroscopy, were tested on a brown macroalgal model. These powerful and easily-to-use techniques allowed us to discriminate Sargassum muticum specimens collected in five different countries along Atlantic coasts, from Portugal to Norway. HRMAS NMR and FTIR permitted the obtaining of an overview of metabolites produced by the alga. Based on spectra analysis, results allowed us to successfully group the samples according to their geographical origin. HRMAS NMR and FTIR spectroscopy respectively point out the relation between the geographical localization and the chemical composition and demonstrated macromolecules variations regarding to environmental stress. Then, our results are discussed in regard of the powerful of these techniques together with the variability of the main molecules produced by Sargassum muticum along the Atlantic coasts.

Comparison of cryopreservation methods for the long term storage of the marine diatom Haslea ostrearia (simonsen).

Long term maintenance of microalgal strains by serial subculturing is often expensive and time-consuming. Alternative methods, such as cryopreservation, present several benefits and thus seem more relevant. Our study aimed at comparing two cryopreservation procedures applied to the marine diatom Haslea ostrearia (Simonsen): (1) a two-step freezing method in liquid media using 5%, 10% and 20% MeOH, Me2SO or Glycerol, and (2) an immobilization-dehydration method consisting in an algal cell entrapped in 0.7 M sucrose dehydrated and air-flow desiccated calcium alginate beads before "direct" or "two-step" freezing. Our results showed that the cryopreservation of H. ostrearia was feasible. With the two-step freezing protocol only Me2SO maintained cell viability without contamination but the low percentage of viability (<10%) prevents its use. Conversely, the immobilization-dehydration methods tested in this study were effective. Average viability of 57% and 77% were obtained with the "direct" and the "two step" cooling assays respectively, ensuring preservation of the genetic traits of H. ostrearia.