Background

Synthesis of fatty acids is important for the storage of metabolic energy. The increasing population and energy cost have emphasized the need to produce sustainable renewable fuels. The source of second-generation biofuels is non-food oilseed crops or lignocellulosic biomass mainly comprising of wastes of crop plants like perennial grasses including switchgrass, husks, straw and forest residue (Hadar, 2013). In this context, plants can serve as an excellent system to study fatty acid for nutraceutical and biodiesel aspects. Further, in biodiesel production, clean burn properties of the fuel are influenced by structural features of FAs including chain length and the degree of unsaturation (Knothe, 2005). Lignocellulosic biomass is a greener alternative to produce these products directly from cost-effective resources. FA profiling using GC-MS permits the normalization, annotation and quantification of a relatively wide variety of fatty acids in a single plant extract. The efficiency of lipid extraction depends on the polarity of the solvent. Polar lipids (such as glycolipids or phospholipids) are more soluble in polar solvents (such as alcohols) and non-polar lipids (such as triacylglycerols) are more soluble in non-polar solvents (such as chloroform). Thus, the total lipid extraction depends on the nature of the organic solvent. Bligh and Dyer (1959) established a method for total lipid extraction using the mixture of chloroform and methanol as a solvent. The total lipids were converted to fatty acid methyl ester by transmethylation (Carreau and Dubacq, 1978). In one study, it was observed that the solvent system of chloroform/methanol is very effective for lipid extraction (Sheng et al., 2011). Moreover, lipid recovery in terms of total lipid content, lipid class and FA composition of different microalgal extracts are also affected by different types of pretreatment and cell disruption techniques and solvents (Ryckebosch et al., 2012). The chloroform/methanol mixture was also found to be useful for the extraction of lipids from microalgae (Ryckebosch et al., 2012). Similarly, there are several other reports suggest that chloroform/methanol/phosphate buffer-based solvent system has a higher efficiency for the extraction of lipids from different microalgae and plants (Kumari et al., 2013; Mishra et al., 2015; Pandey et al., 2015; Patel et al., 2016). Chloroform/methanol mixture exhibits strong dissolving power for the entire range of polarity found in lipids. This mixture is also able to break up membranes and denature the proteins (Schreiner, 2006). The buffer helps to overcome the ionic adsorption effects of salt that may hinder lipid extraction in plant tissue. In most of the studies, methanolic NaOH and methanolic HCl were found to be appropriate derivatizing agents for the profiling of FAs in plants.

Based on this information, here we provide a detailed protocol for extraction of lipids, identification, and quantification of fatty acid methyl esters. We also provide a detailed formula to estimate the total saturated fatty acids (SFA), unsaturated fatty acids (MUFAs and PUFAs), unsaturation index (Poerschmann et al., 2004), degree of unsaturation (Ramos et al., 2009), atherogenic and thrombogenic indices (Simat et al., 2015). Analyses of FAs are done by GC-MS. This protocol provides a relatively rapid and reproducible method. Moreover, this method can be used to profile fatty acids from different types of plant tissues. The produced information can be useful in several contexts of nutraceutical, pharmacological and industrial purposes.