# Two-Step Enzymatic Synthesis of Structured Triacylglycerglycerols from ARASCO+
## 1 Introduction
An ARA-rich oil (54% ARA) obtained after selective enzymatic hydrolysis of a commercial oil (TGA40, 39% ARA) using the lipase from _Candidia rugosa_ was used as a substrate with CA (1:2 weight ratio) to produce MLM-STAG by acidosis using an immobilized lipase from _Rhizopus oryzae_ (5%) at 35 \({}^{\circ}\)C in continuous flow, yielding 46% of the target STAG containing CA at the _sn_-1,3 positions and ARA at the _sn_-2 position [12]. ARASCO (ARA Single-Cell Oil) is an ARA-rich oil (40-50% ARA of total FA) extracted from the fungus _Mortierella alpina_ and used as an ingredient in infant formula [13; 14]. This oil was also used as a substrate for the synthesis of STAG via enzymatic biocatalysis. Lipase PS-30 from _Pseudomonas_ sp. catalyzed the acidosis reaction between ARASCO (39% ARA of total FA) and capric acid (CpA, 10:0) at a molar ratio of 1:3, 45 \({}^{\circ}\)C and 24 h, with a lipase load of 4% by weight of substrates and the addition of water (2% by weight of the weight of substrates and lipase) to give a STAG with 19.3% CpA and 36.5% ARA, with 89.7% of the CpA esterified found at the _sn_-1,3 positions [15]. Other authors reported the transesterification of ARASCO (49% ARA of total FA) and commercial medium-chain TAG (60% CA, 40% CpA) using Lipozyme 435 (8 wt%) at 90 \({}^{\circ}\)C for 3 h and a substrate molar ratio of 1:1, yielding 53.75% of the STAG containing 17.3% CA, 12.7% CpA, and 34.5% ARA [16]. A synthetic pathway commonly used to produce MLM-STAG is a two-step process where long-chain TAGs (LLL-TAG) are hydrolyzed to _sn_-2 MAG, and these intermediates are further purified and used as substrates with MCFA to yield MLM-STAG through an esterification reaction [17]. One of the most challenging issues when attempting to synthesize MLM-STAG is the acyl migration phenomenon, which consists of a displacement of the FA esterified at the _sn_-2 position of the intermediate MAG to _sn_-1,3 positions because they are more thermodynamically stable at the extreme positions than at the central position of the glycerol backbone, thus reducing the yield of the target MLM-STAG [18]. The reaction temperature is one of the most influential factors in acyl migration, and the higher the temperature, the higher the acyl migration rate [18]. In this work, STAGs containing medium- and long-chain FA have been synthesized using an ARA-rich fraction purified from ARASCO and coconut oil as starting sources in a two-step process: (i) enzymatic ethanolysis of the ARA-rich oil from ARASCO to produce _sn_-2 MAG; (ii) the esterification of the _sn_-2 MAG with FA ethyl esters (FAEEs) obtained from coconut oil to yield STAG. Several reaction conditions were tested to optimize the yield of the substrates (_sn_-2 MAGs and FAEEs from coconut oil). As the main target was to obtain a STAG with ARA at the _sn_-2 position and a MCFA at the _sn_-1,3 positions, an ARA-rich TAG fraction was purified from ARASCO to maximize the proportion of ARA-containing _sn_-2 MAG after the ethanolysis reaction of TAG. Coconut oil was selected because it is a naturally rich source of MCFAs such as caprylic, capric and lauric acids [19]. Unlike previous works, _sn_-2 MAGs were not isolated after the ethanolysis of the ARA-rich oil from ARASCO to minimize acyl migration rates and increase the yield of the target STAG.