Lanh Van Nguyen

The objective of this study was to produce structured lipids (SLs) enriched in docosahexaenoic acid (DHA) by enzymatic interesterification of a medium-chain triacylglycerol (MCT) oil and docosahexaenoic acid single cell oil (DHASCO), using immobilized lipases from Thermomyces lanuginosus (TL) and Candida rugosa (CR). The enzymes were screened for their effectiveness, and optimal conditions were employed to evaluate their ability to incorporate DHA or medium-chain fatty acids (MCFAs) into the triacylglycerol backbone. Fatty acid composition, regiospecific distribution, and oxidative stability were assessed in both the original and structured lipids. Structured lipids were prepared under ultrasonic-assisted enzymatic interesterification, and their oxidative stability was evaluated using conjugated dienes (CD) and 2-thiobarbituric acid reactive substances (TBARS) assays. The results indicated that after 1 h of incubation, DHA content in the resultant product was reduced to 31.22% and 32.66% with TL and CR lipases, respectively. For MCFAs, long chain saturated fatty acids (LC-SFAs) and long chain polyunsaturated fatty acids (LC-PUFAs), the amounts were 35.29%, 15.91% and 15.06% (TL: Thermomyces lanuginosus ), and 25.92%, 20.04% and 17.83% (CR: Candida rugosa ), respectively. In DHASCO, DHA was predominantly esterified to the sn-2 position, while MCFAs occupied mainly the sn-1 and sn-3 positions. In MCT, MCFAs were esterified to all positions, but as the chain length increased from C8 to C16, saturated FAs decreased in the sn-1 and sn-2 positions but not in the sn-3 position. In novel structured lipids so produced, DHA was mainly attached to the sn-2 position, with C8:0 to C16:0 esterified to sn-1,3 positions. Enzymatically modified oil generally had higher CD and TBARS values compared to their unmodified counterparts. These findings demonstrate potential for rational design of structured lipids, offering a venue to tailor their composition and enhanced potential nutritional value. Graphical Abstract

Novel structured lipids (SLs) were successfully produced from virgin coconut oil (VCO) and docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Stereospecific analysis results indicated that both DHA and EPA were predominantly esterified at the sn‐1 and sn‐3 positions. For DHA‐enriched VCO, caprylic acid (CyA, C8:0) was mainly esterified at the sn‐2 position, while capric acid (CaA, C10:0) and lauric acid (LaA, C12:0) were primarily located at the sn‐3 position. In EPA and DHA + EPA‐enriched VCO, C8:0 and C10:0 were almost randomly distributed across all three positions, except for C12:0, which was mostly found at the sn‐2 position. The oxidative stability results of the SLs showed higher levels of conjugated dienes (CD) and 2‐thiobarbituric acid reactive substances (TBARS) after 12 days of storage. Furthermore, the primary volatile compounds identified in the SLs using headspace gas chromatography–mass spectrometry (HS GC–MS) included acetaldehyde, propanal, and 2‐propenal (acrolein), which were absent in the unmodified oils. These results indicate that the modified oils exhibited a higher susceptibility to oxidation compared to their unmodified counterparts. Oils high in polyunsaturated fatty acids (PUFAs) require protection from oxidation to mitigate the detrimental effects of oxidation products and preserve their nutritional and health benefits.

Structured lipids (SLs) containing docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and DHA + EPA were synthesized via enzymatic acidolysis using virgin coconut oil (VCO) as the substrate in n‐hexane. Commercially available enzymes Lipozyme TL IM (produced from Thermomyces lanuginosus, a 1,3‐specific lipase), Lipozyme IM60 (produced from Rhizomucor miehei, a 1,3‐specific lipase), and non‐specific lipase from Candida rugosa (powder) were used as biocatalysts. The T. lanuginosus lipase was chosen to evaluate the effects of various parameters on the incorporation of PUFAs into VCO and to optimize the process. As the enzyme load increased from 1% to 4%, the incorporation of omega‐3 PUFAs also increased; however, it decreased when the enzyme load was further increased to 6%. The incorporation of these fatty acids increased with reaction time from 12 to 36 h but decreased at 48 h. Similarly, the incorporation increased with temperature from 35 to 45 °C, but decreased at 55 and 65 °C. The highest incorporation rates of DHA (18.91%), EPA (30.38%), and DHA + EPA (34.64%) were achieved at a mole ratio of 1:3 (VCO to DHA or EPA) or 1:3:3 (VCO to DHA + EPA), with a 4% enzyme load, 36 h incubation time, and a temperature of 45 °C. A central composite design (CCD) with three levels and three factors—reaction temperature (35, 45, and 55 °C), enzyme amount (2%, 4%, and 6%), and reaction time (24, 36, and 48 h)—was used to model and optimize the reaction conditions via response surface methodology (RSM). Under optimal conditions of 3.3% T. lanuginosus enzyme, 42.22 °C, and 33.38 h, the incorporation rates were 32.92% for DHA, 44.48% for EPA, and 47.04% for DHA + EPA in VCO.

Structured lipids (SLs) containing DHA, EPA, and DHA+EPA were synthesized via enzymatic acidolysis using VCO as the substrate in n -hexane. Commercially available enzymes Lipozyme TL IM (produced from Thermomyces lanuginosus , a 1,3-specific lipase), Lipozyme IM60 (produced from Rhizomucor miehei , a 1,3-specific lipase), and non-specific lipase from Candida rugosa (powder) were used as biocatalysts. The T. lanuginosus lipase was chosen to evaluate the effects of various parameters on the incorporation of PUFAs into VCO and to optimize the process. As the enzyme load increased from 1 to 4%, the incorporation of omega-3 PUFAs also increased; however, it decreased when the enzyme load was further increased to 6%. The incorporation of these fatty acids increased with reaction time from 12 to 36h but decreased at 48h. Similarly, the incorporation increased with temperature from 35 to 45 ^o C, but decreased at 55 and 65 ^o C. The highest incorporation rates of DHA (18.91%), EPA (30.38%), and DHA+EPA (34.64%) were achieved at a mole ratio of 1:3 (VCO to DHA or EPA) or 1:3:3 (VCO to DHA+EPA), with a 4% enzyme load, 36h incubation time, and a temperature of 45 ^o C. A central composite design (CCD) with three levels and three factors—reaction temperature (35, 45, 55 ^o C), enzyme amount (2, 4, 6%), and reaction time (24, 36, 48h)—was used to model and optimize the reaction conditions via response surface methodology (RSM). Under optimal conditions of 3.3% T. lanuginosus enzyme, 42.22°C, and 33.38h, the incorporation rates were 32.92% for DHA, 44.48% for EPA, and 47.04% for DHA+EPA in VCO.