Production of structured triacylglycerols from microalgae Tomáš R ˇ ezanka a,⇑ , Jaromír Lukavsky ´ b , Linda Nedbalová b,c , Karel Sigler a a Institute of Microbiology, Academy of Sciences of the Czech Republic, Víden ˇ ská 1083, CZ-142 20 Prague 4, Czech Republic b Institute of Botany, Academy of Sciences of the Czech Republic, Centre for Bioindication and Revitalization, Dukelská 135, 379 82 Tr ˇebon ˇ , Czech Republic c Charles University in Prague, Faculty of Science, Department of Ecology, Vinic ˇná 7, 128 44 Prague 2, Czech Republic article info Article history: Received 16 January 2014 Received in revised form 8 April 2014 Available online xxxx Keywords: Structured triacylglycerols Microalgae NARP-LC/MS-APCI Polyunsaturated fatty acids Polyunsaturated triacylglycerols Enantiomers Chiral LC abstract Structured triacylglycerols (TAGs) were isolated from nine cultivated strains of microalgae belonging to different taxonomic groups, i.e. Audouinella eugena, Balbiania investiens, Myrmecia bisecta, Nannochloropsis limnetica, Palmodictyon varium, Phaeodactylum tricornutum, Pseudochantransia sp., Thorea ramosissima, and Trachydiscus minutus. They were separated and isolated by means of NARP-LC/MS-APCI and chiral LC and the positional isomers and enantiomers of TAGs with two polyunsaturated, i.e. arachidonic (A) and eicosapentaenoic (E) acids and one saturated, i.e. palmitic acid (P) were identified. Algae that produce eicosapentaenoic acid were found to biosynthesize more asymmetrical TAGs, i.e. PPE or PEE, whereas algae which produced arachidonic acid give rise to symmetrical TAGs, i.e. PAP or APA, irrespective of their taxonomical classification. Nitrogen and phosphorus starvation consistently reversed the ratio of asym- metrical and symmetrical TAGs. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The nutritional value of n3 polyunsaturated fatty acids (PUFAs) found in algae has attracted increasing attention (Khozin-Goldberg et al., 2011). Polyunsaturated fatty acids have beneficial effects on human health; one of the best known of these acids is eicosapenta- enoic acid (E, 20:5n3) which has been used for years in the preven- tion of atherosclerosis and thrombosis (Simopoulos, 1991). Arachidonic acid (A, 20:4n6) does not belong to essential fatty acids but some mammals lack the ability, or possess it in a very limited degree, to biosynthesize arachidonic acid from linoleic acid. Since the amount of arachidonic acid in the diet is negligible, it is in fact an essential part of their diets (MacDonald et al., 1984). Arachidonic acid plays an important part in cellular signaling as a lipid messen- ger in the regulation of signaling enzymes or as a precursor of eico- sanoids (especially leucotrienes, prostaglandins, prostacyclins and thromboxanes) (Piomelli, 1993). The position of a fatty acid (FA) in the molecule of triacylglycerols (TAGs) has been described to affect many nutritional properties, oxi- dative stability, absorption and metabolism in the organism, as well as atherogenesis (Cubow, 1996; Cossignani et al., 1999; Mu and Porsgaard, 2005). FA bound in positions sn-1 and sn-3 are better hydrolyzed by pancreatic lipase, whereas FA in position sn-2 of the glycerol are much better absorbed in the form of monoacylglycerols; thus children absorb better palmitic acid bound in position sn-2 and contained in maternal milk than the same FA from plant oils that is bound in positions sn-1 or sn-3(Quinlan and Moore, 1993). The best nutritional properties were found with TAGs having PUFAs in posi- tion sn-2, especially arachidonic and eicosapentaenoic acids; these TAGs are better absorbed than TAGs with the same FA composition in which the FA are accidentally distributed. An interest is currently on the rise in the preparation of structured triacylglycerols (STAGs) containing saturated and short chain FAs in positions sn-1 and sn-3 and long chain PUFAs in position sn-2. It is worth noting that these structured TAGs can protect the organism against hypertriglyceride- mia and obesity caused by high dietary fat (Takeuchi et al., 2002). Structured TAGs could be potentially used for inducing weight loss and lower fat accumulation, and for serum cholesterol lowering (Kunesova et al., 2006). The simplest and most direct way to synthe- size these STAGs is acidolysis catalyzed by specific sn-1, sn-3 lipases (Hita et al., 2007). However, the process involves side reactions, e.g. a mutual migration of acyls which reduces the yield of the STAGs. To circumvent this problem, the reaction is performed in two steps. The first step is enzymatic hydrolysis yielding 2-MAGs (monoacyl- glycerols), which are then esterified in positions sn-1 and sn-3 by specific lipases. The weak point of the process is the deactivation of lipases caused by ethanol or the ensuing glycerol and an increased stability of esters on primary hydroxyl, i.e. in positions sn-1 and sn-3 (Soumanou et al., 1998). STAGs are most often prepared from fish oils (cod liver, tuna, anchovy/sardine or bonito oils). Unfortunately, these oils are a http://dx.doi.org/10.1016/j.phytochem.2014.04.013 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +420 241 062 300; fax: +420 241 062 347. E-mail address: rezanka@biomed.cas.cz (T. R ˇ ezanka). Phytochemistry xxx (2014) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: R ˇ ezanka, T., et al. Production of structured triacylglycerols from microalgae. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.04.013