Lipase-catalysed incorporation of EPA into emu oil: Formation and characterisation of new structured lipids

Abstract

Partial hydrolysis of emu oil was performed using Thermomyces lanuginosus lipase to remove some shorter chain fatty acids. Then eicosapentaenoic acid (EPA) was incorporated into the modified emu oil using either Lipozyme RMIM or Lipozyme TLIM to produce new EPA enriched structured lipids. Using Isooctane as a reaction solvent increased the level of EPA incorporation, which was higher with RMIM than with TLIM. TLIM incorporated EPA almost exclusively into the sn-1,3 positions, whereas RMIM incorporated EPA at sn-1,3 and sn-2 positions in an almost statistical ratio. Both structured lipids were less oxidatively stable than emu oil.

Introduction

Emu oil is extracted from the subcutaneous and retroperitoneal fat of emu and has been widely used by some indigenous Australians and the early white settlers for wound healing, pain alleviation and the treatment of inflamed joints (Abimosleh et al, 2012, Abimosleh et al, 2012, Snowden, Whitehouse, 1997). Today emu oil is used in cosmetics and as a therapeutic oil, primarily due to its anti-inflammatory effects (Abimosleh et al, 2012, Abimosleh et al, 2012, Abimosleh et al, 2012, Caputo et al, 1995). Emu oil has been shown to significantly reduce the levels of pro-inflammatory cytokines TNF-α and IL-1α (Yoganathan et al., 2003), and has been shown in some cases to reduce inflammation to an extent comparable with oral ibuprofen, a non-steroidal anti-inflammatory drug (Snowden & Whitehouse, 1997). The anti-inflammatory potency of emu oil was reported to be greater than that of flaxseed oil, olive oil or chicken fats (Yoganathan et al., 2003). Surprisingly, the fatty acid composition of emu oil (Abimosleh et al, 2012, Abimosleh et al, 2012, Abimosleh et al, 2012) does not contain known major anti-inflammatory lipid mediators, such as EPA (Wall, Ross, Fitzgerald, & Stanton, 2010). Therefore, the potential exists to increase the anti-inflammatory properties of emu oil through the incorporation on EPA to produce structured lipids with improved properties.

The omega-3 fatty acid EPA (C20:5n3) has been reported to aid in the prevention of inflammatory diseases by modulating different stages of the immune response, as well as through suppressing the production of omega-6 (n-6) pro-inflammatory eicosanoids (Simopoulos, 1999, Wall et al, 2010). EPA is a major substrate for both cyclooxygenase and 5-lipoxygenase enzymes and a precursor to potent anti-inflammatory eicosanoids such as lipoxins and resolvins (Calder, 2009). Studies have also shown that dietary EPA can decrease inflammation in vivo (Belch et al, 1988, Lau et al, 1993). Therefore, incorporation of EPA into emu oil could help boost its anti-inflammatory properties and nutritional quality.

Lipid modifications via lipase-catalysed reactions have been widely reported (Akanbi et al, 2013, Akanbi et al, 2012, Akanbi et al, 2014, Akoh et al, 1992, Akoh, Moussata, 1998, Olusesan et al, 2011, Senanayake, Shahidi, 2004, Wanasundara, Shahidi, 1998), and conditions tend to be milder with less side reactions compared with non-enzymatic methods (Akanbi et al, 2013, Senanayake, Shahidi, 2004). Lipase-catalysed incorporation of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) into vegetable oils has been widely studied (Akoh, Sista, 1995, Fajardo et al, 2003, Senanayake, Shahidi, 2004). The incorporation of n-3 PUFAs into these oils is normally achieved by transesterification of n-3 PUFA ethyl esters (EEs), or by acidolysis of their free fatty acid (FFA), using immobilised lipases. However, FFA forms of n-3 PUFAs are preferred, since lipase action on ethyl esters is poor (Yang, Kuksis, & Myher, 1990). An advantage of using lipases rather than conventional chemical catalysts is the ability of lipases to incorporate fatty acids at specific positions on the glycerol backbone and thereby produce more specifically tailored lipids with improved functional properties or bioavailability.

In this study, the fatty acid profiles of emu oil and their stereospecific distributions were modified to contain EPA using three different immobilised lipases. A combination of Iatroscan-FID and GC-FID was used to determine the rate and amount of EPA incorporation. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, ¹H and ¹³C NMR spectroscopy were used to characterise the oils before and after EPA incorporation, including the fatty acid positional distributions. The oxidative stability of the resultant structured lipids was investigated by comparing the conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) values with those of emu oil at different storage times.