Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Microalgae's polysaccharides, are they potent antioxidants? Critical review.

The scientific community continue to explore novel bioactive molecules by investigating natural origins; microalgae are photosynthetic organisms considered as a sustainable resource to use in many fields. They present a high diversity in species and richness in terms of attractive bio-compounds. The aim of this review is to (1) provide first an overview of current issues related to oxidative stress, and propose a natural metabolite derived from eukaryotic and prokaryotic microalgae; 'polysaccharides' as a powerful antioxidant agent, then, (2) organize the available data on the antioxidant potential of polysaccharides derived from the main microalgal groups (red microalgae, green microalgae, and cyanobacteria) and especially highlighted the key species of each group (Porphyridium sp., Chlorella sp., and Arthrospira sp., respectively), meanwhile, (3) we described the chemical composition of polysaccharides from each class, and (4) we cite briefly the most factors affecting the antioxidant activity of these molecules. Finally, we explored the major challenges and gaps found to require more investigation.

In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Sulfated exopolysaccharides from Porphyridium cruentum: A useful strategy to extend the shelf life of minced beef meat.

Sulfated exopolysaccharides (EPS) from Porphyridium cruentum strain were extracted and their antioxidant and anti-bacterial potentials were evaluated based on DPPH free radical, ABTS•+ radical cation and DNA nicking assays, and against four foodborne pathogenic bacteria, respectively. They showed also interesting functional, foaming and emulsion properties. Moreover, microbiological and chemical effects of EPS at 0.5, 1 and 2% on refrigerated minced beef meat were undertaken. Chemical analyses revealed that the treated meat underwent significant decrease (P < 0.05) of primary and secondary lipid oxidation. By the end of the storage period, exopolysaccharides at 2% reduced the metmyoglobin and carbonyl group accumulation compared to control samples and were more efficient (P < 0.05) against microflora proliferation. Furthermore, two multivariate exploratory techniques namely Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were applied successfully to all obtained data describing the main characteristics attributed to refrigerated meat samples. Overall, these findings indicated that EPS from P. cruentum are worthy being developed as functional and bioactive components for the meat industry.

Biochemical changes in microalga Porphyridium cruentum associated with silver nanoparticles biosynthesis.

For the first time, the microalga Porphyridium cruentum was tested for its ability to produce silver nanoparticles. To characterize formed silver nanoparticles UV-vis Spectrometry, Scanning Electron Microscopy, Energy-dispersive analysis of X-rays and X-ray diffraction were used. It was shown that after biomass exposure to silver nitrate solution the extracellular formation of spherical-like nanoparticles took place. Functional groups responsible for metal binding were determined by Fourier-transform infrared spectroscopy. The complex of biochemical tests was used for biomass characterization and assessment of the changes of its main components (proteins, lipids, carbohydrates, and phycobilin) during nanoparticle formation. Obtained data indicate a significant decrease of proteins, carbohydrates, phycobiliproteins, and lipids content as well as antiradical activity of biomass. The obtained results show the necessity of determination of optimal conditions for obtaining Porphyridium cruentum biomass enriched with silver nanoparticles for its further application in the pharmaceuticals industry.

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.

Rheological characterization of Porphyridium sordidum and Porphyridium purpureum exopolysaccharides.

Porphyridium exopolysaccharides (EPSs), which contain sulfate and methyl groups, have a similar potential for use in multiple industrial applications as macroalgae counterparts but lack detailed characterization. For this reason, we produced 0.21 g L-1 of P. sordidum EPS and 0.17 g L-1P. purpureum EPS, followed by a thorough rheological characterization in respect to their differences in monomer composition, sulfate concentrations and methyl patterns. Furthermore, the effect of NaCl and CaCl2 was evaluated, and the effect of high salinity media on the rheological properties of the biopolymers was analyzed. Both Porphyridium EPSs show a remarkable stability at high temperature and under the effect of mono- and divalent cations, and high salinity cultivation medium, which was evidenced by the rheological properties of the EPS. This feature is not displayed by many carbohydrate polymers, making it possible to enrich current applications in which EPS are used.

Effects of Different Environmental Factors on the Growth and Bioactive Substance Accumulation of Porphyridium purpureum.

Genus Porphyridium is a primitive single-celled red algae widely distributed in seawater, freshwater, and moist soil. It can synthesize bioactive substances such as phycoerythrin, extracellular polysaccharides and polyunsaturated fatty acids during the growth process. In this paper, the culture and bioactive substance yield of Porphyridium purpureum were studied by setting salinity, nitrogen-to-phosphorus ratio, and pH at different gradient levels. The results showed that the optimal conditions for the growth of P. purpureum were salinity 34 ppt, nitrogen-to-phosphorus ratio 169:1, and pH 8; the optimal conditions for obtaining the polysaccharides were salinity 17 ppt, nitrogen-to-phosphorus ratio 14:1, and pH 8; the optimal conditions for obtaining phycoerythrin were salinity 17 ppt, nitrogen-to-phosphorus ratio 68:1, and pH 8; the optimal conditions for obtaining the lipids were salinity 34 ppt, nitrogen-to-phosphorus ratio 1:1, and pH 8. In actual production applications, culture conditions should be set according to different product accumulation purposes in order to achieve the optimal production efficiency.

Light-diffractive patterning of Porphyridium purpureum.

Light guidance is a convenient and versatile way to control the positions of phototactic microorganisms. However, the illumination strategies require adaption to the respective organism. We report on the generation of structures composed of the gliding and exopolysaccharide-secreting algae Porphyridium purpureum via their photomovement. Light patterns from a two-dimensional computer-generated hologram were projected onto inoculated agar plates. The obtained pixelated algae patterns were evaluated with regard to the illuminated intensity, contrast and pixel size. Upper and lower thresholds for algae accumulation were determined, allowing to enhance future manipulation of phototactic microorganisms.

Structural basis of energy transfer in Porphyridium purpureum phycobilisome.

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

Physico-chemical characteristics of the sulfated polysaccharides of the red microalgae Dixoniella grisea and Porphyridium aerugineum.

The sulfated polysaccharides of red microalgae have attracted increasing attention in recent years due to their unique rheological and bioactivities. Todate, most studies are devoted to the polysaccharide of the marine species Porphyridium sp., with limited information about that of the brackish water- Dixoniella grisea and the freshwater- Porphyridium aerugineum. We therefore conducted a comparative study of the two less explored sulfated polysaccharides, emphasizing their similarities and differences in composition, physical properties and biocompatibility. Both polysaccharides were found to be composed of 6-8 monosaccharides, predominantly xylose. Sulfur content was 0.8% for P. aerugineum and 1.6% for D. grisea. Solutions of both polysaccharides were highly viscous and exhibited shear thinning and weak gel behavior. Both were found to be stable in an alkaline environment, whereas in an acidic environment the viscosity of the polysaccharide of the brackish water species increased while that of the freshwater species decreased. Both exhibited a similar morphology, having a porous fibrous structure with a rough amorphous surface. By complementing previous studies on the Porphyridium sp. polysaccharide, we have established a sound basis for elucidating the structure/function relationships that in turn, will promote the development of innovative applications for the biotech industries for pharmaceutics, food and drug-delivery.

Subunit pI Can Influence Protein Complex Dissociation Characteristics.

Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.

Development of a Green Downstream Process for the Valorization of Porphyridium cruentum Biomass.

As the interest in biorefinery approaches is continuously increasing, new alternatives for the downstream valorization of biomasses are sought. Porphyridium cruentum microalga is a good natural source for a variety of interesting bioactive compounds, including carotenoids, phycoerythrin, and sulfated polysaccharides. In the present contribution, the use of compressed fluids-based techniques is explored towards the efficient and green extraction of bioactive compounds to valorize microalgal biomass. The extraction of carotenoids was first optimized using pressurized ethanol. The best extraction conditions involved the use of 125 °C for 20 min at 10.5 MPa. Subsequently, a sequential valorization process was devised based on the application of different steps directed towards the extraction of phycoerythrin, sulfated polysaccharides, and carotenoids, respectively. The applied pressurized conditions allowed the attainment of a good recovery of polar components without compromising the stability and extraction of carotenoids. Therefore, the proposed approach could be employed to obtain different bioactives from P. cruentum microalgal biomass employing green extraction processes.

Atheroprotective effect of novel peptides from Porphyridium purpureum in RAW 264.7 macrophage cell line and its molecular docking study.

OBJECTIVE: To explore the atherogenic foam cell prevention efficiency of two dipeptides purified from Porphyridium purpureum on RAW 264.7 cell line and to study its molecular interaction through molecular docking. RESULT: P. purpureum consists of 29.9% protein and 2.98% phycoerythrin on a dry weight basis. The two dipeptides namely of Histidine-Glutamic acid (HE) and Glycine-Proline (GP) isolated from the total protein and purified phycoerythrin of P. purpureum respectively, were evaluated for atherogenic foam cell prevention capacity in RAW 264.7 cell line. The IC5O values of peptides were found to be 91.2 ± 1.81 µg/ml (GP), 103.3 ± 4.8 µg/ml (HE) in MTT assay. The two peptides reduce the foam cell formation, intracellular lipid accumulation (cholesterol and triglycerides) and the secretion of TNF-α and IL-6 which are inflammatory cytokines in RAW 264.7 cell line at non-cytotoxic concentrations. A molecular interaction study proposed the binding pose for GP and HE peptides targeting the scavenging receptors CD36, SRA1, and Map Kinase p38 (a protein mediator). CONCLUSIONS: The cell line and molecular docking study indicated that among the two dipeptides, peptide GP has the highest atherogenic foam cell prevention efficiency.

Impact of different sequences of mechanical and thermal processing on the rheological properties of Porphyridium cruentum and Chlorella vulgaris as functional food ingredients.

Microalgae are a promising and sustainable source for enhancing the nutritional value of food products. Moreover, incorporation of the total biomass might contribute to the structural properties of the enriched food product. Our previous study demonstrated the potential of Porphyridium cruentum and Chlorella vulgaris as multifunctional food ingredients, as they displayed interesting rheological properties after applying a specific combination of mechanical and thermal processing. The aim of the current study was to investigate the impact of a different sequence of high pressure homogenization (HPH) and thermal processing on the thickening and gelling potential of these microalgal biomasses in aqueous suspensions. Thermal processing largely increased the gel strength and viscosity of both microalgae, which was ascribed to larger and stronger aggregates as a result of partial solubilization of polymers, while subsequent HPH generally reduced the rheological properties. Interestingly, large amounts of intact cells were still observed for both microalgae when HPH was performed after a thermal treatment, irrespective of the applied homogenization pressure, implying that cell disruption was hindered by the preceding thermal treatment. Although thermal processing was regarded as the most effective processing technique to obtain increased rheological properties, the combination with a preceding HPH treatment should still be considered when cell disruption is desired, for instance to increase the bioavailability of intracellular components. Finally, biomass of P. cruentum showed the largest potential for use as a structuring agent, as the gel strength and viscosity in thermally treated suspensions of this microalga were about 10 times higher than for C. vulgaris.

One-stage immobilization of the microalga Porphyridium purpureum using a biocompatible silica precursor and study of the fluorescence of its pigments.

The biocompatible silica precursor tetrakis(2-hydroxyethyl)orthosilicate with ethylene glycol residues was used instead of the common alcohol-containing tetraethoxysilane for the first time to prepare a biorecognition element by entrapping the marine microalga Porphyridium purpureum into a silica matrix by a one-stage sol-gel procedure at conditions (pH, ionic strength, and temperature) appropriate for living cells. We show that the microalga immobilized in this way fully maintains its viability and functionality. We furthermore show that the silica matrix had a stabilizing effect, providing microalgal survival and functionality at increased temperature. The high optical transparency of the silica matrix allowed us to study the optical properties of Porphyridium purpureum thoroughly. When irradiated by a laser, intense fluorescence of chlorophyll-a and phycoerythrin of the photosynthetic system was observed. The characteristics of this fluorescence differed notably from that observed with P. purpureum in suspension before immobilization; possible reasons for this and an underlying mechanism are discussed.

Economic analysis of pilot-scale production of B-phycoerythrin.

ß-Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B-phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two-phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two-phase systems in a scaled-up process. This study analyzed the production of B-Phycoerythrin using real data obtained during the scale-up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two-phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10-year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B-phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472-1479, 2016.

One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum.

B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and ß and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.

Optimization of bead milling parameters for the cell disruption of microalgae: process modeling and application to Porphyridium cruentum and Nannochloropsis oculata.

A study of cell disruption by bead milling for two microalgae, Nannochloropsis oculata and Porphyridium cruentum, was performed. Strains robustness was quantified by high-pressure disruption assays. The hydrodynamics in the bead mill grinding chamber was studied by Residence Time Distribution modeling. Operating parameters effects were analyzed and modeled in terms of stress intensities and stress number. RTD corresponded to a 2 CSTR in series model. First order kinetics cell disruption was modeled in consequence. Continuous bead milling was efficient for both strains disruption. SI-SN modeling was successfully adapted to microalgae. As predicted by high pressure assays, N. oculata was more resistant than P. cruentum. The critical stress intensity was twice more important for N. oculata than for P. cruentum. SI-SN modeling allows the determination of operating parameters minimizing energy consumption and gives a scalable approach to develop and optimize microalgal disruption by bead milling.