# Two-Step Enzymatic Synthesis of Structured Triacylglycerglycerols from ARASCO+
## 3 Results and Discussion
### STAG Synthesis and Purification
\) \\ \hline \multicolumn{1}{c}{**STAG using**} & & & & & & & & & & \\ \multicolumn{1}{c}{_T. lanuginess_ lipase} & & & & & & & & & \\ \multicolumn{1}{c}{_Whole STAG_} & \( \begin{array}{c}2.77\pm 0.45\\ \ast\\ \end{array} \) & \( \begin{array}{c}4.03\pm 0.45\\ \ast\\ \end{array} \) & \( \begin{array}{c}0.45\pm 0.30\\ \ast\\ \end{array} \) & \( \begin{array}{c}18.3\pm 1.3\\ \ast\\ \end{array} \) & \(\begin{array}{c}13.  
also contained a significant amount of MMM-type STAGs, mainly with LaA. Concerning MLM-type STAGs, the lipase from _R. oryzae_ gave a better yield, with 34.8% of ARA found at the _sn_-2 position and 35.1% of CpA and LaA esterified at the _sn_-1,3 positions. The proportion of MLM-type STAGs produced by the lipase from _R. miehei_ was rather low because it preferentially esterified ARAs at the _sn_-1,3 positions of the glycerol backbone (63.8%) instead of MCFAs. Overall, the STAGs obtained after the enzymatic synthesis with the lipase from _R. oryzae_ simultaneously contained higher proportions of ARAs at the _sn_-2 position and MCFAs at the _sn_-1,3 positions than the other two tested lipases, making this mixture more interesting from a nutritional view. The lipase from _R. miehei_ showed the lowest affinity for the FAEEs from coconut oil. This may be attributed to the substrate specificity of lipases, which is influenced by factors such as the size, shape, depth, and physicochemical properties of their substrate binding pockets [38]. Another relevant factor to consider is the optimal operating temperature of each lipase when catalyzing the synthesis reaction of structured lipids. It has been stated that the optimal operating temperature of _R. oryzae_ lipase is in the range of 30-45 \({}^{\circ}\)C [39], whereas for lipases from _T. lunuginosus_ and _R. miehei_, most reactions are performed at temperatures between 50 and 60 \({}^{\circ}\)C, showing a higher optimal temperature range [17; 40; 41; 42]. In this study, the synthesis of MLM-STAGs was carried out at 35 \({}^{\circ}\)C to decrease the acyl migration rate, which is within the optimal temperature range for the lipase from _R. oryzae_. In a recent study, the enzymatic synthesis of ARA-rich MLM-STAG was attempted in a two-step process by first obtaining _sn_-2 MAGs from _Schizochytrium_ sp. oil (58.8% ARA of total FA) by ethanolysis catalyzed by Lipozyme 435 (10% lipase loading) at 25 \({}^{\circ}\)C, with a maximum yield of 21.2% after 3 h of reaction [43]. MAGs were then extracted from the reaction medium by multi-step solvent extraction with acetonitrile, water, hexane, chloroform, and ethanol and purified by low-temperature crystallization in acetonitrile (\(-\)40 \({}^{\circ}\)C, 12 h). Finally, the purified MAG was reacted with CpA (1:3 molar ratio) under a vacuum to produce MLM-STAGs by biocatalysis with the RM-IM lipase (8% load) at 25 \({}^{\circ}\)C for 5 h. The authors reported a 40.4% yield of STAG containing two CpA chains and one ARA chain (CpA-ARA-CpA and its isomers), with 32.3 and 51.1% ARA in total STAGs and at the _sn_-2 position, respectively [43]. However, several steps involving the extraction and purification of _sn_-2 MAG were performed, making this process time-consuming and expensive, which constitute undesirable characteristics if STAG production is intended on a larger scale. In contrast, in the current study, entire ethanolysis products of the ARA-rich TAG fraction from ARASCO and coconut oil were used as substrates for the reaction catalyzed by the lipase from R. oryzae, and although the yield of the target MLM-STAG was somewhat lower, the mixture was still interesting from a nutritional point of view, and direct synthesis would allow it to be reproduced on a larger scale without the need for _sn_-2 MAG purification.