Production of enzymatic hydrolysates with in vitro antioxidant, antihypertensive, and antidiabetic properties from proteins derived from Arthrospira platensis.

The microalga Arthrospira platensis BEA 005B was produced using 80 m2 (9 m3) raceway photobioreactors achieving a biomass productivity of 28.2 g·m-2·day-1 when operating the reactors in semi-continuous mode (0.33 day-1). The produced biomass was rich in proteins (58.1 g·100 g-1) and carbohydrates (25.6 g·100 g-1); the content of phycocyanins and allophycocyanins was 115.4 and 36.9 mg·g-1, respectively. Ultrasounds and high-pressure homogenisation allowed recovering approximately 90% of the initial protein content of the biomass; however, the energetic requirements of the former (∼100 kJ·kg-1) were significantly lower than those of high-pressure homogenisation (∼200 kJ·kg-1). An in silico analysis revealed that papain and ficin would allow releasing a large number of bioactive peptides with antioxidant, antihypertensive (ACE-I and renin), and antidiabetic (DPP-IV, α-amylase, and α-glucosidase) properties. Both were assessed in vitro together with Alcalase and pepsin leading to the generation of enzymatic hydrolysates with in vitro bioactivity.

Biorefinery Approach Applied to the Production of Food Colourants and Biostimulants from Oscillatoria sp.

In this study, a biorefinery based on Oscillatoria sp. is developed to produce high-value compounds such as C-phycocyanin, used in food colourant applications, and biostimulants, used in agriculture-related applications. First, the Oscillatoria biomass production was optimized at a pilot scale in an open raceway reactor, with biomass productivities equivalent to 52 t/ha·year being achieved using regular fertilizers as the nutrient source. The biomass produced contained 0.5% C-phycocyanins, 95% of which were obtained after freeze-thawing and extraction at pH 6.5 and ionic strength (FI) 100 mM, with a purity ratio of 0.71 achieved in the final extract. This purity ratio allows for use of the extract directly as a food colourant. Then, the extract's colourant capacity on different beverages was evaluated. The results confirm that C-phycocyanin concentrations ranging from 22 to 106 mg/L produce colours similar to commercial products, thus avoiding the need for synthetic colourants. The colour remained stable for up to 12 days. Moreover, the safety of the extracted C-phycocyanin was confirmed through toxicity tests. The waste biomass was evaluated for use as a biostimulant, with the results confirming a relevant auxin-like positive effect. Finally, an economic analysis was conducted to evaluate different scenarios. The results confirm that the production of both C-phycocyanin and biostimulants is the best scenario from an economic standpoint. Therefore, the developed biomass processing scheme provides an opportunity to expand the range of commercial applications for microalgae-related processes.

Using a B-Phycoerythrin Extract as a Natural Colorant: Application in Milk-Based Products.

Nowadays, there is a growing interest in finding new coloring molecules of natural origin that can increase and diversify the offer of natural food dyes already present in the market. In the present work, a B-phycoerythrin extract from the microalgae Porphyridium cruentum was tested as a food colorant in milk-based products. Using spectroscopy and colorimetry, the extract was characterized and gave evidence of good properties and good stability in the pH range between 4.0 and 9.0. Coloring studies were conducted to demonstrate that samples carrying the pink extract could be used for simulating the pink color of marketed milk-based products. The staining factors, representing the amount of pink protein to be added to reproduce the color of strawberry commercial products, ranged between 1.6 mg/L and 49.5 mg/L, being sufficiently low in all samples. Additionally, color stability during a short period of cold storage was studied: it demonstrated that the three tested types of dairy products remained stable throughout the 11-day analysis period with no significant changes. These results prove the potential of the B-phycoerythrin extract as a natural colorant and alternative ingredient to synthetic coloring molecules.

Improvement of stability and carotenoids fraction of virgin olive oils by addition of microalgae Scenedesmus almeriensis extracts.

Humans are not capable of synthesizing carotenoids de novo and thus, their presence in human tissues is entirely of dietary origin. Consumption of essential carotenoids is reduced due to the lower intake of fruits and vegetables. Microalgae are a good source of carotenoids that can be exploited. In the present work, carotenoids rich extracts from Scenedesmus almeriensis were added to extra-virgin olive oils at different concentrations (0.1 and 0.21 mg/mL) in order to enhance the consumption of these bioactives. Extracts brought changes in olive oils color, turning them orange-reddish. Quality of olive oils was improved, since peroxidation was inhibited. Olive oils fatty acids and tocopherols were not affected. ß-carotene and lutein contents increase considerably, as well as oxidative stability, improving olive oils shelf-life and nutritional value. Inclusion of S. almeriensis extracts is a good strategy to improve and enhance the consumption of carotenoids, since olive oil consumption is increasing.

Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography.

In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.

Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes.

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores label the same ferritin subunit, fluorescence resonance energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and the ratio of labeling fluorophores, we can modulate FRET such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect the properties of the metallic core.

Development of a method for the determination of ibafloxacin in plasma by HPLC with flourescence detection and its application to a pharmacokinetic study.

A simple, rapid, and sensitive high-performance liquid chromatographic method is developed for the determination of ibafloxacin in rabbit plasma. Plasma proteins are precipitated with acetonitrile, and after extraction with methylene chloride followed by desecation, ibafloxacin is determined by reversed-phase chromatography with fluorescence detection exciting at 330 nm and emission at 368 nm. Peaks corresponding to ibafloxacin and the internal standard (salycilic acid) are obtained at 9.8 and 5.2 min, respectively. The method is validated for a limit of quantitation of 10 ng/mL. The intraday relative standard deviation ranges from 4.78-7.15%, and the interday precision ranges from 1.32-4.03%. The method shows linearity for the two calibration curves used (10-100 ng/mL and 100-2000 ng/mL). The procedure described is applied successfully to a pharmacokinetics study of ibafloxacin in rabbits.

2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity.

The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in solution. Since biomolecules in general have some chemical groups that can act as proton acceptors/donors and are usually dissolved in buffer solutions which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (J. Phys. Chem. A 2005, 109, 734-747), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concentrations, analyzed by means of global compartmental analysis. In this article we use the rate constants and the corrected pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and associated pre-exponentials at different acetate buffer concentrations and constant pH and compare these theoretically calculated values with new experimental data. We also calculate the steady-state fluorescence intensity vs pH and vs acetate buffer concentration (at constant pH) and compare these calculated emission values with the experimental data previously published. The agreement between the experimental and simulated data is excellent.

Fluorescence energy transfer between fluorescein label and DNA intercalators to detect nucleic acids hybridization in homogeneous media.

A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5'), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5'), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.

Fluorescent behavior of B-phycoerythrin in microemulsions of aerosol OT/water/isooctane.

Taking advantage of its unusual fluorescent properties, the incorporation of B-phycoerythrin (B-PE) in aerosol OT (AOT, sodium bis-(2-ethylhexyl) sulphosuccinate)/water/isooctane microemulsions was investigated by following their steady-state and time-resolved fluorescence as a function of the water-to-surfactant molar ratio, w(0). The fluorescent intensity at 575 nm increased continuously with increasing water content, saturating at a w(0) around 35 and staying practically constant at w(0)> or =40. The steady-state anisotropy showed an initial increase with increasing water content until w(0)=23 and then decreased strongly, staying practically constant when w(0)> or =40. The values of the fluorescent parameters, anisotropy and fluorescent intensity, were unchanged when the water content of the system increased in the range between w(0)=40 to 50. This implies the effective incorporation of B-PE in the microemulsion droplets with w(0)> or =40, as well as the equilibrium of the dispersion at these water/surfactant ratios, since higher water content does not affect the main surrounding microenvironment of the protein. The overall incorporation in the microemulsion droplets caused minor spectroscopic changes with respect to biliprotein in aqueous solution of 20 mM sodium phosphate buffer, pH 7.0, such as a blue absorption shift of 3 nm and an emission shift of 1.5 nm, as well as a slight increase in excitation anisotropy spectrum mainly caused by a decrease in protein mobility. Therefore, there are no important interactions between the chromophores and the AOT sulfonate head groups. Emission intensity decays followed complex kinetics in both aqueous and dispersion media. The stability with time and temperature of the biliprotein in the microemulsion was higher than in the aqueous solution. All the results can be explained in terms of B-PE inclusion in the water droplets of AOT microemulsions where the protein has similar configuration and conformation to that in aqueous solution but with the chromophores more protected.

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography.

B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.