7th Drug hypersensitivity meeting: part two

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Astaxanthin extraction from Haematococcus pluvialis using CO2-expanded ethanol

Microalgae represent diverse branch of microorganism that can produce a wide range of unique functional ingredients that can be used in food, cosmetics, pharmaceuticals, and energy. Among them, Haematococcus pluvialis is known for accumulating the highest levels of a potent natural antioxidant, astaxanthin, which has demonstrated positive health effects. Therefore, the aim of numerous studies has been to develop novel and efficient extraction techniques to produce high-quality (purity and antioxidant activity) extracts, while complying with the Green Chemistry Principles. Supercritical CO2 (scCO2) emerges as an alternative to organic solvents because of its high selectivity and bioactivity-preserving qualities. Nevertheless, astaxanthin is a large molecule with low solubility in scCO2 that usually requires long extractions at high pressures. Ethanol has been used as co-solvent to increase astaxanthin solubility in scCO2. In this work, a Box-Behnken experimental design was used to study the effects of operating pressure (20-35 MPa), temperature (40-70 °C), and ethanol content in scCO2 (0-13%, w/w) on the yield, astaxanthin content, and antioxidant activity of H. pluvialis extract. Results showed that ethanol content in CO2 has a more significant effect on all responses than pressure and temperature. These results lead us to investigate the effect of a further increase in ethanol content, up to the region of gas-expanded liquids. We studied the effects of temperature (30-60 °C) and ethanol content (50-70%, w/w) at a fixed pressure (7 MPa) on the same response variables using CO2-expanded ethanol (CXE). Results showed that temperature and ethanol content had a significant influence on astaxanthin yield and antioxidant activity. Also, the overall responses of CXE surpassed scCO2 extractions to match conventional extraction with acetone, maintaining high quality extracts, thus validating the use of this new type of green technology for extraction of high-value compounds. © 2014 Elsevier B.V.The authors want to thank Spanish Projects AGL2011-29857-C03-01 and CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Economía y Competitividad) and the European project MIRACLES (KBBE.2013.3.2-02: The CO2 algae biorefinery). Fabián A. Reyes would like to thank CONICYT-Chile for supporting his fellowship in CIAL-CSIC, Spain.Peer Reviewe

Gas expanded liquids (GXLs) as new solvents for bioactives extraction

Resumen del trabajo presentado al XIII Scientific Meeting of the Spanish Society of Chromatography and Related Techniques celebrado en Tenerife (España) del 8 al 11 de octubre de 2013.Haematococcus pluvialis is a microalgae known for accumulating the highest levels of a potent natural antioxidant, astaxanthin, that has demonstrated positive health effects. Therefore, numerous studies have focus on the development of novel and efficient extraction techniques that are in agreement with the strong demands in terms of quality of the extracts (purity and antioxidant activity), while complying with the Green Chemistry Principles. Supercritical CO2 (scCO2) emerges as an alternative to organic solvents because of its high selectivity and bioactivity-preserving qualities. Nevertheless, astaxanthin is a large molecule with low solubility in scCO2 that usually requires high pressures, long extraction times and a necessary pretreatment (i.e grinding) of the microalgae. Ethanol has been used as a green co-solvent for greatly improving astaxanthin yield by avoiding the problems faced by the use of pure scCO2. Nonetheless, it is not clear the relationship that exists between other extraction factors such as pressure and temperature. In this study we used a Box Behnken experimental design (BBD) that investigated the effects of operating pressure (200-350 bar), temperature (40-70 ºC) and modifier (0-13 %w/w Ethanol) on scCO2 extraction of oleoresin yield, astaxanthin yield and antioxidant activity of the extracts. The experimental design showed that for all response variables the modifier effect was the most significant factor over pressure and temperature. These results lead us to investigate the effects of a further increase in the modifier content, up to the region of gas-expanded liquids (GXL). GXLs have shown to have improved mass transfer through reduced viscosity, increased solute diffusivity and decreased interfacial tension.We studied the effect of temperature (30-60 ºC) and modifier (50-70%) at a fixed pressure (70 bar) on the same response variables. Results showed that increasing temperature generally worsened the recovery of astaxanthin and the antioxidant activity of the extracts. Still, low temperatures and 50% w/w of modifier could achieve more than 90% of recovery of astaxanthin within 2 hours of extraction. Furthermore, in the case of 45ºC and 50% w/w of modifier the recovery of astaxanthin surpassed the recovery achieved by a conventional acetone extraction, hence, validating the use of this new type of green technology for extraction of high valued compounds.Peer Reviewe

Adsorbent-assisted supercritical CO2 extraction of carotenoids from Neochloris oleoabundans paste

Neochloris oleoabundans emerges as alternative source of bioactives that complies with the algae-based biorefinery concept, which consists of a platform that offers a multitude of algae bioproducts. The development of an integrated extractive processes in line with the green chemistry principles have motivated the use of Supercritical CO (scCO), as an alternative to toxic organic solvents, for the extraction of bioactives. However, process integration and optimization is challenging because microalgae are grown in liquid cultures, therefore is often necessary a drying step prior to scCO extraction. Moreover, this step is usually energy intensive and risks damaging the compound's bioactivity. An alternative is the simultaneous extraction process of the microalgae paste (containing around 70-80% water), nevertheless little information is available that explores this type of extraction. This work aims to explore the direct extraction of microalgae paste and to evaluate the effect of water on carotenoid extractions of N. oleoabundans. To study the extraction under a batch-wise system, an indirect extraction system was developed by mixing the microalgae paste with low cost adsorbents as support medium. Two types of silica gels, two different chitosans and active carbon were tested as adsorbents; sea sand was used as inert control. All of the materials showed different adsorbent capacity, being chitosan adsorbents those with higher capacity. However, oleoresin yield and recovery was negligible in a system with only scCO as a solvent and ethanol as co-solvent was required to improve the extraction yield. Although the overall oleoresin recoveries were low for all adsorbents (ranging from 2 to 10%), chitosan-assisted extraction showed the highest carotenoid recoveries (60-140% g/g) surpassing acetone benchmark extraction in case of chitosan microspheres. These results are interesting for the development of low energy consumption processes, since there is no need to dry the microalgal paste.Spanish Projects AGL2011-29857-C03-01, AGL2014-53609-P (MINECO, Spain) and ALIBIRD-CM, S2013/ABI-2728 (Comunidad de Madrid) and the European project MIRACLES (KBBE.2013.3.2-02: The CO2 algae biorefinery) are acknowledged for their financial support. Fabián A. Reyes would like to thank CONICYT-Chile for supporting his fellowship in CIAL-CSIC, Spain.Peer Reviewe

Isthmin 1 is Expressed by Progenitor-Like Cells in the Lung: Phenotypical Analysis of Isthmin 1+ Hematopoietic Stem-Like Cells in Homeostasis and during Infection

The process by which blood cells are generated has been widely studied in homeostasis and during pathogen-triggered inflammatory response. Recently, murine lungs have been shown to be a significant source of hematopoietic progenitors in a process known as extramedullary hematopoiesis. Using multiparametric flow cytometry, we have identified mesenchymal, endothelial, and hematopoietic progenitor cells that express the secreted small protein Isthmin 1 (ISM1). Further characterization of hematopoietic progenitor cells indicated that ISM1+ Lineage- Sca-1+ c-kit+ (ISM1+ LSK) cells are enriched in short-term hematopoietic stem cells (ST-HSCs). Moreover, most Sca-1+ ISM1+ cells express the residence marker CD49a, and this correlated with their localization in the extravascular region of the lung, indicating that ISM1+ cells are lung-resident cells. We also observed that ISM1+ cells express TLR4, TLR5, and TLR9, and, in a mouse model of sepsis induced by P. aeruginosa, we observed that all the LSK and ISM1+LSK cells were affected. We conclude that ISM1 is a novel biomarker associated with progenitor-like cells. ISM1+ cells are involved in the response to a bacterial challenge, suggesting an association between ISM1-producing cells and dangerous inflammatory responses like sepsis