Nannochloropsis oculata is a microalga widely recognized as a potential source of polyunsaturated fatty acids, namely the omega-3 eicosapentaenoic acid (EPA), which beneficial health effects have been demonstrated. In order to maximize the EPA amount that may be obtained from a culture, different strategies may be pursued, such as increasing the microalgas’ biomass EPA content and the efficiency of its extraction process. Based on the above rationale, the main goal of the current thesis was to apply different stress conditions to N. oculata culture, which would increase EPA content, but without hindering growth. The strategy employed entailed the application of stress in a modulated sinusoidal fashion, to stimulate the response, although providing the microalga the possibility to adapt and not cease its growth. Two abiotic factors were independently utilized (temperature and light intensity), and conditions were alternated between optimum and stress levels, in multiday cycles. The factors were applied at lower-than-optimum values (5 and 10 ºC; 30 and 50 μmol photons/m2/s for temperature and light intensity, respectively), and it was found that the approach was successful at achieving the desired goals. While temperature stress (10 ºC) presented the best results, with an 158% EPA content increase, light intensity (30 μmol photons/m2/s) also increased EPA content significantly by 126%. Moreover, the increases in EPA content originated from individual cells contents and not just from overall biomass increase, and these were obtained in a relatively short period of time, which is of utmost importance regarding its industrial viability. Eicosapentaenoic acid is conventionally extracted with hazardous organic solvents. Therefore, an additional line of research was undertaken, aiming at extracting EPA by using less hazardous solvent mixtures (SM), namely hexane:isopropanol (Hxn:2-PrOH; 3:2) and diethyl ether:ethanol (Et2O:EtOH; 2:1). The use of alternative technologies, namely high hydrostatic pressure (HHP) and moderate electric fields (MEF), was also assessed as adjuvants to increase the extraction efficiency of the SM. Concerning SM, Et2O:EtOH was able to increase 1.3-fold the EPA yield of the conventional Folch SM. Regarding the technologies, neither increased extraction yields when used independently; however their sequential combination was able to increase EPA yield by 162%. When associated, Et2O:EtOH and the combined technologies (HHP - 200 MPa, 21 °C, 15 min – followed by MEF processing at 40 °C, 15 min) enabled to extract higher amounts of EPA from N. oculata wet biomass, which is extremely relevant for the industries as no expensive drying step is required. Moreover, it is of dire importance to provide such alternatives to the “classical” extraction processes and solvents, as the industries require higher yields and lower environmental impact in their downstream processing. Since the biological potential of a microalga (or an extract thereof) is intrinsically dependent on its composition, the impact of the stress modulation and the SM utilized for extraction of lipid fraction were studied by characterization and assessment of biochemical and biological activities. Concerning the lipid extracts, it was revealed that the one obtained from the stressed culture presented the best results, namely in terms of anti-steatosis (hepatic lipid accumulation < 8% than that of steatosis-induced cells) and anti-inflammatory activities (decreased expression of interleukine (IL)-6, IL-10, and interferon- in LPS-stimulated RAW 264.7 macrophages). Moreover, both extracts presented oxygen radical absorbance capacity (within the range of 49- 50 μmolTrolox equivalent/mgextract), no metabolic inhibition of several cell lines (Caco-2, HT29-MTXE12, Hep G2, 3T3-L1, and RAW 264.7), and inhibition of triacylglycerols hepatic accumulation (glycerol release ca. 2-fold higher than in non-treated cells). Overall, the results showed that the modulated stress did indeed enhance the potential beneficial effects of the N. oculata lipid extracts on human health, as well as maintain their safety. Once the lipid fraction was extracted from the stressed culture, remaining biomass may still contain other compounds with interesting biological activities. Hence, by applying a biorefinery concept, the defatted biomass was enzymatically hydrolyzed with cellulase Celusoft Supreme for 3 h, followed by the protease NewPro, for 6 h, both at 53 ºC, after which the soluble fraction of hydrolysate (SFH) was characterized in terms of peptide and polysaccharide profiles, and assessed regarding its biochemical and biological activities. The results showed that the defatted biomass may still be utilized as a resource, since the SFH presented antioxidant capacity (3.2 μmolTrolox equivalent/mgFDB), antidiabetic (19.4% inhibition of α-glucosidase) and immunosuppression potential (NO production by LPS-stimulated Raw 264.7 cells decreased to < 10% that of the control), anti-inflammatory activity (decreased expression of IL-6, IL-8 and tumor necrosis factor-α by Caco-2 cells in LPS-induced inflammation) and antimicrobial activity, particularly against Gram-positive bacteria. In conclusion, it was demonstrated how modulated stress may enhance the production of bioactive compounds from N. oculata (specifically EPA) and new strategies to efficiently extract them were identified, as well as their biological potential was unveiled. The strategies presented herein for production of N. oculata with increased EPA content, and extraction thereof, may constitute solutions for the food and nutraceutical industries, allowing them to obtain EPA faster and with higher yields, concomitantly decreasing the process environmental impact. Ultimately, that will increase the economic viability of obtaining EPA from N. oculata.
Date of Award | 22 Jan 2024 |
---|
Original language | English |
---|
Awarding Institution | - Universidade Católica Portuguesa
- University of Aveiro
- University of Minho
|
---|
Sponsors | Fundação para a Ciência e a Tecnologia |
---|
Supervisor | Ana Maria Gomes (Supervisor) & Ana Paula Taboada da Costa Santos Carvalho (Co-Supervisor) |
---|