Many food components have demonstrated biological activities useful in the prevention, management and treatment of deseases, and the use of food protein-derived bioactive peptides as functional ingredients in foods is a rapidly developing area of food innovation.
Microalgae are a group of unicellular photosynthetic microscopic organisms, widely found in aquatic and terrestrial habitats, and characterized by a particular versatility in adjusting to distinct growth conditions. Microalgae are currently cultivated on a large scale for biotechnological exploitation, and the potential for producing functional foods using microalgae derived peptides and other bioactive compounds is enormous.
The main goal of this research is the characterization and isolation of bioactive metabolites from microalgae of Chlorella and Scenedesmus species. First, the attention will be focused on bioactive peptides, due to their importance in the food industry, then on lipids. Due to the limited information on the detailed profiles and primary sequences of microalgae proteins, the primary aim of this study is the development of an analytical strategy for the recovery, separation and identification of bioactive peptides from microalgae. For this purpose, different extraction protocols and enzymes for protein hydrolysis will be tested. The peptide purification will be carried out by multidimensional liquid chromatography, preferably using environmental friendly solvents.
Lipid analysis will be performed by a shotgun lipidomic approach, coupling liquid chromatography to high resolution mass spectrometry to sequence eluting lipids. Data will be handled by freeware software for lipid identification. According to sample complexity, a fractionation step prior to the lipidomic analysis and lipid identification will be performed to identify the specific lipids responsible of the observed bioactivity, similarly to what previously described for peptides.
The potential for producing functional foods using microalgae derived peptides is enormous. High biomass, easy production, high levels of proteins and other bioactive compounds, and less competitiveness with other food sources as a raw material are key factors that make microalgae an effective peptide-based functional food source. The current market for microalgae is mainly focused
on biofuel production, niche nutritional supplements, alginates in food, and as fish feed. Cultivating microalgae as functional ingredients for the food, cosmetic, and pharmaceutical industries is expected to expand as a result of emerging evidence of their beneficial effects. However, a comprehensive knowledge about the identity and mechanism of bioactive microalgae metabolites is still missing, and this information is mandatory for a wider employment of microalgae in the food industry.
As already stated above, there is only a limited information on the detailed profiles and primary sequences of microalgae proteins, because the complete genome maps of these organisms are lacking,
therefore, the complete proteome characterization of microalgae cannot be realized. Nonetheless, within this project, we aim to enlarge the knowledge about the identity of bioactive peptides that could be recovered from microalgae, in particular from Chlorella and Scenedesmus species. Indeed, often, bioactive compounds are extracted from a matrix as a mixture and used as is, without investigating which compound is actually effective. In this research, we aim to assign an identity to each single bioactive peptide.
Analogously to peptides, also lipids from microalgae are compounds with high biotechnological potential for food and health applications. However, despite of the interesting bioactive properties of lipids, their structure and diversity are far from being entirely recognized, preventing the full exploitation of the biotechnological potential of microalgae. In fact, for lipids, separation of single components could be unfeasible, because of the presence of several isomers differing only in the double bond position. However, by means of a lipidomic approach, we could obtain the identification of each particular lipid signature of each distinct microalgae lineage, as well as the deviations induced by environmental and growth conditions fostering better understanding of the relation between the microalgae species/structure and associated bioactivity.
Lipidomics analysis is nowadays supported by LC-MS-based approaches that allow the profiling of the total lipid extract obtained from the algae material without chemical modification, requiring relatively small amounts of sample. However, the main limitations for LC-MS-based untargeted lipidomics reside in the lack of adequate computational and cheminformatics tools that are able to support the analysis of several thousands of species from biological samples, enabling data mining and automating lipid identification and external prediction processes. To overcome this issue, we will use and compare various freeware dedicated software, such as MZmine and a newly developed one, named ¿Lipostar¿, able to work also without the support of preformed lipid databases.
Besides, in order to improve the identification of isomer lipids, the potential of the Vanquish UHPLC systems will be fully exploited. The system, in fact, allows to be equipped also with two columns in an in series configuration. Thus, different types of columns or column combinations will be employed to improve the separation of a complex mixture of polar lipids. Finally, the performance of hypercarb column, i.e., packed with porous graphitic carbon material, in separation of polar lipids will be also exploited. This kind of stationary phase is not widely diffused because its retention mechanisms have not been fully understood yet.