Microalgae are a rich source of organic macromolecules, such as lipids, proteins, carbohydrates, and pigments. Due to the high content of lipids in microalgae, they have been attracting increasing attention as a source of lipids for various commercial applications, for example in the manufacturing of food supplements or in the chemical, pharmaceutical and cosmetic industries. In the literature, investigations of the lipid profile of microalgae are mainly focused on free fatty acids and triacylglycerols, whereas information on the occurrence of glyco-, sulpho- and phospholipids is rather scarce. As the amount of lipids in microalgae is relatively small (15¿30 % on average depending on the species), the extraction procedure must be as efficient as possible to maximize lipids recovery. For this reason, in this project different lipids extraction procedure will be tested to extract polar lipids from spirulina microalgae, also using different solvents (methanol, tert-butyl-methyl-ether, chloroform). Lipids extract will be analyzed by Ultra High Performance Liquid Chromatography coupled to high resolution tandem mass spectrometry (MS) followed by a bioinformatics analysis by Lipostar, a comprehensive platform-neutral cheminformatics tool for lipidomics. Before analyzing real samples, a chromatographic optimization, based on the type and concentration of mobile phase additives, gradients and pH of mobile phase, will be carried out in order to separate the largest number of individual lipid classes with emphasis on glyco-, sulpho- and phospho- lipids under MS-compatible conditions. The optimized procedure will be applied to the analysis of spirulina microalgae for identification of possible new polar lipid species
The goal of the project is that of applying new and improved lipidomics technologies to the characterization of polar lipids in spirulina microalgae, as they represent an important class of bioactive lipids. In particular, with respect to the current state of the art.
1) Optimization of chromatographic conditions of polar lipids on stationary phases which have not been used for such purpose, before. A systematic evaluation of reversed phase and hydrophilic interaction chromatography stationary phases will be performed, to find the most suited for separation of polar lipids and coupling with high resolution mass spectrometry.
2) First comprehensive characterization of polar lipids in spirulina microalgae, a common food supplement. This part will take into consideration both polar lipid extract preparation and lipid identification. Firstly, a sample preparation procedure will be devised by comparison of different extraction procedures and adaptation to the spirulina matrix, to maximize lipid recovery, and lipid identification. This step coupled with the optimized lipid separation will help achieve a larger coverage of polar lipid species in spirulina.
3) Improvement of current Lipostar database with lipid classes belonging to microalgae, more specifically with spectral data on sulfolipids. Sulfolipids are a lipid class which is typical of microalgae and less represented in different matrices. Such class is not included in Lipostar database, thus exact precursor masses and fragmentation data will be collected and added to the database, to increase the probability of lipid identification in microalgae.
4) Lipids profile characterization of different microalgae species, focusing in particular on the fosfo-, sulfo- and glycolipid species, and qualitative comparison among them will be carried out.
5) In addition, bioactive polar lipids will be quantified throughout an optimized extraction procedure by using green solvents. Microalgae species will be characterized both from a qualitative point of view and from a quantitative point of view.