# Two-Step Enzymatic Synthesis of Structured Triacylglycerglycerols from ARASCO+
## 3 Results and Discussion  
### Ethanolysis of the ARA-Enriched ARASCO(r) Fraction  
The immobilized lipase B from _C. antarctica_, commercially known as Novozym 435, was selected as the biocatalyst for the ethanolysis reaction of the ARA-enriched TAG fraction from ARASCO (containing 76.5% ARA of total FA) to yield _sn_-2 MAG because of its high versatility in the food and pharmaceutical industries, as well as its high stability and stereoselectivity, making it a more suitable choice than other commercial lipases for biocatalytic processing [25]. Furthermore, it is known that Novozym 435 shows 1,3-reigospecific activity in the presence of polar solvents such as ethanol [26; 27]. Different reaction times (0.5 to 5 h) and two molar ratios between the ARA-rich oil and ethanol (1:5 or 1:10 mol/mol) were assayed, and the resulting yields of _sn_-2 MAG are depicted in Figure 2. The highest yields of _sn_-2 MAG were obtained at 1 and 2 h for the 1:5 molar ratio between oil and ethanol (23.3 and 23.1 mol% of all reaction products, respectively) and at 1 h for the 1:10 molar ratio (22.1 mol%), with values significantly higher than the rest of the assayed reaction times. Reactions were carried out at 25 \({}^{\circ}\)C to prevent acyl migration, which promoted the shift of the FA esterified in the _sn_-2 position to the more thermodynamically stable outer positions (_sn_-1,3) of the glycerol backbone and was enhanced when higher temperatures were used during the reaction [20]. Attempting to purify the _sn_-2 MAG from the reaction medium involved time-consuming and laborious steps that may have led to increased rates of undesired acyl migration, which decreased the production yield of _sn_-2 MAG [28]. Due to this, the ethanolysis in the current work aimed to obtain the highest possible yield of _sn_-2 MAG, but no further purification was attempted to prevent acyl migration phenomena. According to the results shown in Figure 2, the ethanolysis of the ARA-rich oil at 1 h using a 1:5 molar ratio between substrates provided the highest yield of _sn_-2 MAG (23.3 mol%, containing 68.6% ARA of total FA), and therefore such conditions were selected to produce _sn_-2 MAG as the substrate for the enzymatic synthesis of STAG. Longer reaction times led to lower yields of _sn_-2 MAG, probably due to increased acyl migration rates [21]. Novozym 435 has been used in previous works for the alcoholysis of PUFA-rich oils to yield _sn_-2 MAG. For example, [PERSON] et al. performed the alcoholysis of cod liver oil using Novozym 435 with an oil/ethanol ratio of 1:4 \(w/w\) and a lipase load of 10% of the total substrate weight. The highest yield of _sn_-2 MAG was achieved after 1 h of reaction, and the yield decreased at longer times, as observed in the current study [29]. The purification of _sn_-2 MAG from the alcoholysis medium has been reported using a liquid-liquid separation with _n_-hexane and mixtures of ethanol and water [26,29]. However, this process implies the use of organic solvents, it is time-consuming, and it may be complex to separate _sn_-2 MAG from water, with the possibility of increasing acyl migration and therefore reducing the yield of _sn_-2 MAG during the process [27]. Novozym 435 was also used as a biocatalyst for the alcoholysis of tuna oil performed at room temperature, achieving a 23.9% yield of _sn_-2 MAG with a molar ratio between oil and ethanol of 1:59, a lipase load of 10% of the total substrate weight, and a 3 h reaction time, which was identified as the optimal time to maximize the yield of _sn_-2 MAG [26]. These authors found lower yields when using the immobilized lipases from _T. lanuginosus_ (Lipozyme TL-IM) (14.4%) and from _R. miehei_ (Lipozyme RM-IM) (2.7%), and only Lipozyme 435 showed a slightly but not significantly higher yield (27.7%) for _sn_-2 MAG production. Another study focused on the synthesis of ARA-rich _sn_-2 MAG through ethanolysis catalyzed by Lipozyme 435 using the oil from _M.