During the project ‘NUVAPY-BLUE’, a multidisciplinary biorefinery strategy was applied to maximize the use of biomass from Porphyra species, targeting high-value bioactive compounds for nutraceutical and cosmeceutical applications.
The activities were structured into several technical and scientific workstreams:
Cultivation Optimization: A key focus was the optimization of cultivation conditions to enhance biomass quality and functional compound yield. A novel cultivation setup was tested, simulating tidal cycles to better mimic the natural environment of Porphyra/Pyropia. This simulation led to visibly more robust algae, with improved resistance to environmental stress. Additionally, controlled experiments were conducted using different nitrate concentrations to assess the impact on the endogenous production of mycosporine-like amino acids (MAAs). Parallel trials with varying doses of UV radiation were also performed, aiming to stimulate the biosynthesis of these photoprotective compounds. When testing different nitrate concentrations in combination with varying UV doses, nitrate availability proved to be the most critical factor in enhancing the natural production of MAAs, indicating its predominant role over UV exposure in optimizing the biosynthesis of these compounds. Furthermore, the feasibility of using fish effluent as a natural nitrate source was explored and compared with synthetic nitrate. The results showed that the use of fishpond effluents allowed for comparable MAA production with lower overall nitrate concentrations, indicating a more efficient and sustainable nutrient source that aligns with circular aquaculture and integrated multi-trophic aquaculture systems (IMTA).
Extraction Process Development: Extraction protocols were evaluated using both fresh and freeze-dried biomass of Porphyra. Initial comparisons showed that fresh biomass consistently yielded higher concentrations of bioactive compounds, guiding subsequent extraction trials. Based on this, different extraction strategies were tested, including alkaline and enzymatic hydrolysis. These approaches significantly increased the extraction yield of key antioxidant compounds, particularly polyphenols, phycobiliproteins, and carbohydrates. In contrast, for MAAs (HPLC), the best results were obtained using water-based extraction at elevated temperatures—higher than those typically reported in the literature—suggesting a more efficient release of these thermoresistant molecules under these conditions. Additionally, specific protocols were developed for the extraction and partial purification of the polysaccharide porphyran (GC-MS), a valuable compound with known bioactive and gelling properties.
Bioactivity Screening: The obtained extracts were tested in vitro for antioxidant activity using the ABTS assay and for cellular response in HCT116 human colorectal carcinoma cells. Additionally, the sun protection factors (SPF and UVAPF) of selected extracts were evaluated using PMMA plates, simulating topical application conditions and enabling quantification of photoprotective properties relevant to cosmeceutical applications.
Nanoencapsulation Development: A novel nanoencapsulation strategy is being developed using porphyran, as a natural cross-linking polymer. This approach is designed as a sustainable alternative to traditional encapsulating agents such as sodium tripolyphosphate (TPP). Porphyran-based nanoparticles are being formulated to encapsulate selected bioactive compounds, aiming to enhance their stability, bioavailability, and controlled release. Preliminary characterization of the nanoparticles (e.g. size distribution, zeta potential, encapsulation efficiency) supports the potential of porphyran as a functional biomaterial for drug delivery and cosmeceutical applications.
Bioactive Residue Valorization for Biostimulant Applications: In line with the biorefinery approach, the residual biomass remaining after primary extractions was further valorized through a secondary, simplified water-based extraction under controlled temperature. The resulting extracts were evaluated for potential plant biostimulant activity using bioassays targeting hormone-like effects. Specifically, cytokinin-like activity was assessed via the cucumber cotyledon expansion test, and gibberellin-like activity was evaluated through the germination index assay in watercress seed. The positive responses observed in both tests indicate that the residual biomass still contains bioactive compounds with plant-growth promoting potential, suggesting its applicability as a sustainable input in agricultural or horticultural formulations.
Outcomes of the actions:
• Demonstrated that simulating tidal cycles in cultivation systems improves the physiological robustness and stress resistance of Porphyra/Pyropia biomass.
• Identified nitrate concentration as the key factor in enhancing the endogenous production of mycosporine-like amino acids (MAAs), surpassing the influence of UV exposure.
• Proved the potential of fishpond effluents as an alternative nutrient source, achieving similar MAA yields with lower nitrate concentrations, supporting sustainable aquaculture integration.
• Developed efficient extraction protocols using alkaline and enzymatic hydrolysis, significantly increasing yields of phenolics, phycobiliproteins, and carbohydrates.
• Optimized MAA extraction using high-temperature, water-based methods, achieving superior results to previously reported techniques.
• Successfully extracted and partially purified porphyran, confirming its presence and biofunctional potential through GC-MS analysis.
• Validated the antioxidant potential of extracts through ABTS assay.
• Confirmed photoprotective potential by determining SPF values on PMMA plates, indicating suitability for cosmeceutical applications.
• Initiated development of a natural and sustainable nanoencapsulation system using porphyran as a cross-linking polymer. Replaced synthetic agents like TPP with porphyran, aligning with clean-label and eco-friendly formulation goals.
• Demonstrated that secondary extracts from residual biomass retain biological activity with plant hormone-like effects.
• Supported the zero-waste biorefinery concept by adding value to process residues, contributing to sustainability and circular economy in marine biomass valorization.
