How to us Nile Red, a selective fluorescent stain for microalgal neutral lipids

The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125 mg/mL), or EDTA (3.0 to 3.8 mg/mL). Temperatures between 30 and 40 °C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25 μg/mL and 2.0 μg/mL, and the cell concentration > 5 × 104 cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.</p

Supercritical Carbon Dioxide and Microwave-Assisted Extraction of Functional Lipophilic Compounds from Arthrospira platensis

Arthrospira platensis biomass was used in order to obtain functional lipophilic compounds through green extraction technologies such as supercritical carbon dioxide fluid extraction (SFE) and microwave-assisted extraction (MAE). The temperature (T) factor was evaluated for MAE, while for SFE, pressure (P), temperature (T), and co-solvent (ethanol) (CS) were evaluated. The maximum extraction yield of the obtained oleoresin was (4.07% ± 0.14%) and (4.27% ± 0.10%) for SFE and MAE, respectively. Extracts were characterized by gas chromatography mass spectrometry (GC-MS) and gas chromatography flame ionization detector (GC-FID). The maximum contents of functional lipophilic compounds in the SFE and MAE extracts were: for carotenoids 283 ± 0.10 μg/g and 629 ± 0.13 μg/g, respectively; for tocopherols 5.01 ± 0.05 μg/g and 2.46 ± 0.09 μg/g, respectively; and for fatty acids 34.76 ± 0.08 mg/g and 15.88 ± 0.06 mg/g, respectively. In conclusion, the SFE process at P 450 bar, T 60 °C and CS 53.33% of CO2 produced the highest yield of tocopherols, carotenoids and fatty acids. The MAE process at 400 W and 50 °C gives the best extracts in terms of tocopherols and carotenoids. For yield and fatty acids, the MAE process at 400 W and 70 °C produced the highest values. Both SFE and MAE showed to be suitable green extraction technologies for obtaining functional lipophilic compounds from Arthrospira platensis

Laccase-based biosensors for detection of phenolic compounds

Monitoring of phenolic compounds in the food industry and for environmental and medical applications has become more relevant in recent years. Conventional methods for detection and quantification of these compounds, such as spectrophotometry and chromatography, are time consuming and expensive. However, laccase biosensors represent a fast method for on-line and in situ monitoring of these compounds. We discuss the main transduction principles. We divide the electrochemical principle into amperometric, voltammetric, potentiometric and conductometric sensors. We divide optical transducers into fluorescence and absorption. The amperometric transducer method is the most widely studied and used for laccase biosensors. Optical biosensors present higher sensitivity than the other biosensors. Laccase production is dominated by a few fungus genera: Trametes, Aspergillus, and Ganoderma. We present an overview of laccase biosensors used for the determination of phenolic compounds in industrial application