THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Fractionation of lipids and proteins from the microalga Nannochloropsis oculata

To feed Earth’s growing population, microalgae have been proposed as a source of protein and long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs). Currently, microalgae are mainly sold either as whole biomass, or the economically valuable LC n-3 PUFA fraction is extracted with organic solvents. As an alternative to solvent extraction, pH-driven protein solubilization in water followed by isoelectric precipitation, known as the pH-shift process, has been proposed to recover both lipids (i.e. oils) and functional proteins, while removing insoluble material. In this project, the pH-shift process was applied to Nannochloropsis oculata, a marine microalga containing LC n-3 PUFA and essential amino acids in more than adequate amounts for human nutrition. It was hypothesized that the pH-shift process would render separate lipid and protein fractions. A pH-shift process for Nannochloropsis was developed based on documentation of its pH-dependent protein solubility: Nannochloropsis proteins were solubilized at pH 7, insoluble material was removed by centrifugation at 4 000×g and the proteins were then recovered by precipitation at pH 3. By using seawater, a process in which algal culture medium was used directly in the pH-shift process was simulated, thus reducing freshwater consumption. Contrary to the hypothesis, the developed process did not result in two fractions, but in a combined product of 2.3% LC n-3 PUFA and 23% protein per dry weight, compared to 1.9% and 19% respectively in the initial material, suggesting that the product had potential as a functional food ingredient. However, nutrients need to also be accessible for uptake by the gastrointestinal tract. To assess the link between processing and accessibility of Nannochloropsis fatty acids and proteins, a static in vitro digestion model was applied to whole Nannochloropsis, and various products of the pH-shift process. The results indicated that whole Nannochloropsis cannot be digested by mammalian enzymes at all, hence the lipids and proteins cannot be absorbed. However, in celldisrupted Nannochloropsis ca. 35% fatty acids and protein were hydrolyzed, with hydrolysis somewhat increased with further pH-shift processing. This project indicates that the nutritional profile of Nannochloropsis oculata is favorable for human consumption, but that cell disruption is paramount to make the lipids and proteins accessible to the digestive enzymes. The pH-shift process applied here provided such cell disruption, slightly increased the concentration of proteins and lipid digestibility, and demonstrated a scalable process.