Ultrasound-Based Recovery of Anti-Inflammatory and Antimicrobial Extracts of the Acidophilic Microalga Coccomyxa onubensis.

In the present study, the recovery of valuable molecules of proven anti-inflammatory and antimicrobial activity of the acidophilic microalga Coccomyxa onubensis (C. onubensis) were evaluated using green technologies based on ultrasound-assisted extraction (UAE). Using a factorial design (3 × 2) based on response surface methodology and Pareto charts, two types of ultrasonic equipment (bath and probe) were evaluated to recover valuable compounds, including the major terpenoid of C. onubensis, lutein, and the antimicrobial activity of the microalgal extracts obtained under optimal ultrasound conditions (desirability function) was evaluated versus conventional extraction. Significant differences in lutein recovery were observed between ultrasonic bath and ultrasonic probe and conventional extraction. Furthermore, the antimicrobial activity displayed by C. onubensis UAE-based extracts was greater than that obtained in solvent-based extracts, highlighting the effects of the extracts against pathogens such as Enterococcus hirae and Bacillus subtilis, followed by Staphylococcus aureus and Escherichia coli. In addition, gas chromatography-mass spectrometry was performed to detect valuable anti-inflammatory and antimicrobial biomolecules present in the optimal C. onubensis extracts, which revealed that phytol, sterol-like, terpenoid, and even fatty acid structures could also be responsible for the antibacterial activities of the extracts. Moreover, UAE displayed a positive effect on the recovery of valuable molecules, improving biocidal effects. Our study results facilitate the use of green technology as a good tool in algal bioprocess engineering, improving energy consumption and minimizing environmental impacts and process costs, as well as provide a valuable product for applications in the field of biotechnology.

Methodological Optimization of Supercritical Fluid Extraction of Valuable Bioactive Compounds from the Acidophilic Microalga Coccomyxa onubensis.

Microalgae grow in diverse environments and possess a great biotechnological potential as they contain useful bioactive compounds. These bioactive compounds can be obtained by selective and energy-efficient extraction methods. Various industries are using the supercritical fluid extraction (SFE) method to extract these valuable bioactive compounds. Hence, for the first time, we evaluated the effects of SFE on the recovery of bioactive and antioxidant compounds using Coccomyxa onubensis, a eukaryotic acidophilic microalga of potential relevance which can be used in the field of nutraceutical and functional foods. It was isolated from the Tinto River (Pyritic Belt, Huelva, Spain), a mining region in Spain. Variables such as extraction yield, lutein purity (LP) and recovery (LR), total phenols, and antioxidant capacity (Trolox equivalents antioxidant capacity method) were studied using a Box-Behnken design based on a response surface methodology along with the overall extraction curve fitted to a spline linear model. The effects of temperature (30, 50, and 70 °C), pressure (25, 40, and 55 MPa), and the percentage of co-solvent (0, 25%, and 50% v/v ethanol) on SFE were analyzed, resulting in the co-solvent and temperature as the most significant factors followed by the pressure. Under 70 °C, 40 MPa, and 50% v/v ethanol, C. onubensis reached a maximum of 66.98% of LR. The extracts were richest in total phenols and showed the maximum antioxidant activity (36.08 mg GAEs/g extracts and 2.237 mmol TE/g extracts, respectively) under similar pressure and co-solvent percentage values and different temperatures (30 and 70 °C, respectively). The extracts obtained in this study may have potential applications in the food, nutraceutical, and cosmetic industries. SFE is a highly efficient method to valorize microorganisms living in extreme environments, which are so far unexplored using green extraction methods.

Supercritical Fluid Extraction of Fucoxanthin from the Diatom Phaeodactylum tricornutum and Biogas Production through Anaerobic Digestion.

Phaeodactylum tricornutum is the marine diatom best known for high-value compounds that are useful in aquaculture and food area. In this study, fucoxanthin was first extracted from the diatom using supercritical fluid extraction (SFE) and then using the extracted diatom-like substrate to produce bioenergy through anaerobic digestion (AD) processes. Factors such as temperature (30 °C and 50 °C), pressure (20, 30, and 40 MPa), and ethanol (co-solvent concentration from 10% to 50% v/v) were optimized for improving the yield, purity, and recovery of fucoxanthin extracted using SFE. The highest yield (24.41% w/w) was obtained at 30 MPa, 30 °C, and 30% ethanol but the highest fucoxanthin purity and recovery (85.03mg/g extract and 66.60% w/w, respectively) were obtained at 30 MPa, 30 °C, and 40%ethanol. Furthermore, ethanol as a factor had the most significant effect on the overall process of SFE. Subsequently, P.tricornutum biomass and SFE-extracted diatom were used as substrates for biogas production through AD. The effect of fucoxanthin was studied on the yield of AD, which resulted in 77.15 ± 3.85 LSTP CH4/kg volatile solids (VS) and 56.66 ± 1.90 LSTP CH4/kg VS for the whole diatom and the extracted P.tricornutum, respectively. Therefore, P.tricornutuman can be considered a potential source of fucoxanthin and methane and both productions will contribute to the sustainability of the algae-biorefinery processes.

Variability of Omega-3/6 Fatty Acid Obtained Through Extraction-Transesterification Processes from Phaeodactylum tricornutum.

The effect of direct transesterification methods on the omega-3/6 composition of extracts from Phaeodactylum tricornutum was studied. The aim of this work was to identify an extraction method which allowed to obtain the most suitable profile of fatty acids in terms of its potential benefits to health, particularly if further used in the food industry. Seven methods using acids, alkalis, and heterogeneous-catalysts, (namely methods from 1 to 7, abbreviated as M1-M7) were performed to determine α-linolenic (ALA), linoleic (LA), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids. The composition of fatty acids was in all cases characterized by the major abundance of palmitic (23.95-34.08%), palmitoleic (30.94-35.56%), oleic acids (3.00-7.41%), and EPA (0.5-6.45%). Unsaturated fatty acids extraction yield was higher with a two-step transesterification process (M6, 63.65%). The total fatty acid methyl ester content (FAME) obtained with acid-transesterification (M1) reached about 21% wt, and 60% w/w total lipids. ALA higher relative content (ALA/LA ratio) was obtained when a lipid pre-extraction step was performed prior to acid-catalysis (M4). The transesterification method based on alkali-catalyst (M3, KOH catalyst) led to obtain higher DHA relative contents (DHA/EPA ratio up to 0.11), although its FAME content was 3.75-fold lower than that obtained with acid-transesterification (M1). Overall, this study shows that direct transesterification with alkali-catalyst (M3) improves the determination of PUFA content from the diatom through a more efficient transesterification-based extraction process, and thus allow to assess the value of the biomass more accurately for application in the food industry.

Effect of CO2 Flow Rate on the Extraction of Astaxanthin and Fatty Acids from Haematococcus pluvialis Using Supercritical Fluid Technology.

Haematococcus pluvialis is the largest producer of natural astaxanthin in the world. Astaxanthin is a bioactive compound used in food, feed, nutraceutics, and cosmetics. In this study, astaxanthin extraction from H. pluvialis by supercritical fluid extraction was evaluated. The effects of temperature (40 and 50 °C), pressure (40 and 50 MPa), and CO2 flow rate (2 and 4 L/min) were investigated. The results showed that the highest astaxanthin recovery was obtained at 50 °C/50 MPa and the CO2 flow rates evaluated had no significant effect. It was possible to achieve astaxanthin recoveries of 95% after 175 min for a CO2 flow rate of 2 L/min, and 95 min for CO2 flow rate of 4 L/min. The ω-6/ω-3 ratios obtained were similar in all conditions, reaching 0.87, demonstrating that the extracts from H. pluvialis by SFE are rich in unsaturated fatty acids (UFA) which increases their positive effects when used as a functional ingredient in food.

Effect of Drying Methods on Lutein Content and Recovery by Supercritical Extraction from the Microalga Muriellopsis sp. (MCH35) Cultivated in the Arid North of Chile.

In this study, we determined the effect of drying on extraction kinetics, yield, and lutein content and recovery of the microalga Muriellopsis sp. (MCH35) using the supercritical fluid extraction (SFE) process. The strain was cultivated in an open-raceways reactor in the presence of seawater culture media and arid outdoor conditions in the north of Chile. Spray-drying (SD) and freeze-drying (FD) techniques were used for dehydrating the microalgal biomass. Extraction experiments were performed by using Box-Behnken designs, and the parameters were studied: pressure (30-50 MPa), temperature (40-70 °C), and co-solvent (0-30% ethanol), with a CO2 flow rate of 3.62 g/min for 60 min. Spline linear model was applied in the central point of the experimental design to obtain an overall extraction curve and to reveal extraction kinetics involved in the SFE process. A significant increase in all variables was observed when the level of ethanol (15-30% v/v) was increased. However, temperature and pressure were non-significant parameters in the SFE process. The FD method showed an increase in lutein content and recovery by 0.3-2.5-fold more than the SD method. Overall, Muriellopsis sp. (MCH35) is a potential candidate for cost-effective lutein production, especially in desert areas and for different biotechnological applications.

Determining the Potential of Haematococcus pluvialis Oleoresin as a Rich Source of Antioxidants.

Haematococcus pluvialis is known to be a natural source of antioxidants for numerous applications. In this study, an oleoresin rich in carotenoids extracted by supercritical CO2 treatment of H. pluvialis was extensively characterized for its antioxidant capacity. Carotenoid content, fatty acid profile, total phenol content, antioxidant capacity, and viscosity of the oleoresin were determined with the aim of ascertaining the potential of the oleoresin in terms of its antioxidant content for food applications. The oleoresin contained 96.22 mg/g of total astaxanthin (which includes free astaxanthin and astaxanthin esters) and mostly included unsaturated fatty acids (~78% of total fatty acids). High total phenol content and ferric reducing antioxidant potential indicated high antioxidant capacity, but oxygen radical absorbance capacity was lower compared to the oleoresin samples obtained from other species. The oleoresin was a non-Newtonian fluid since it had shear-thinning (pseudoplastic) and shear-thickening (dilatant) flow. Therefore, the H. pluvialis oleoresin is a potential alternative in developing functional ingredients for designing healthy food products. To the best of our knowledge, this is the first study that has reported an extensive characterization of the antioxidant properties of a microalgal oleoresin obtained by means of supercritical CO2 fluid extraction.