Enrichment of Refined Olive Oil with Palmitic and Docosahexaenoic Acids to Produce a Human Milk Fat Analogue

Refined olive oil was enriched with palmitic acid (PA) and docosahexaenoic acid (DHA) via lipase-catalyzed acidolysis reaction using Novozym 435 in hexane. The enrichment reaction was optimized by response surface methodology. Three independent variables, reaction time (12, 18, and 24 h), temperature (55, 60, and 65 °C), and substrate molar ratio (refined olive oil:DHA single cell oil free fatty acid:PA 1:1:6, 1:1:9, and 1:1:12) and three responses, total PA and DHA incorporation, and PA content at the sn-2 position were investigated. Results showed that PA was incorporated into the triacylglycerols(TAGs) of refined olive oil at up to 55.79 mol % while incorporation of PA at the sn-2 position and total DHA were found to be up to 33.63 and 3.54 mol %, respectively. Second-order models were generated for each of the three responses. A Chi-square test verified that the predicted values from the models were not significantly different from the observed ones. The prediction power of the models was further confirmed by a solvent-free scale-up reaction. The produced structured lipids have the potential to be used in infant formula.     

Characterization of Terebinth Fruit Oil and Optimization of Acidolysis Reaction with Caprylic and Stearic Acids

Characterization of the fatty acid and triacylglycerol composition of terebinth fruit oil and the synthesis of structured lipids (SL) were performed in this study. Interesterification reaction of terebinth fruits oil (Pistacia terebinthus L.) with caprylic acid (CA) and stearic acid (SA) to produce a SL was performed in n-hexane using immobilized sn-1,3 specific lipase from Mucor miehei. The effect of reaction conditions and relationship among them were analyzed by response surface methodology (RSM) with a four-factors five-level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–30 wt% based on substrates), reaction time (7–18 h), reaction temperature (40–60 °C) and substrate mole ratio (terebinth oil:SA:CA 1:1:1–1:1:3). The best fitting quadratic model was determined by regression and backward elimination. Based on the fitted model, the optimal reaction conditions for the incorporation of CA and SA were found to be temperature 50 °C; time 18 h; enzyme load 30 wt%; substrate ratio 1:1:3. Under these optimum conditions, the incorporation of SA and CA could be obtained as 19 and 14%, respectively.     

Enzymatic Modification of Menhaden Oil to Incorporate Caprylic and/or Stearic Acid

Menhaden oil was enzymatically modified with caprylic (8:0) and/or stearic acid (18:0) to produce structured lipids (SL). The goal was to produce SL with high amounts of polyunsaturated fatty acids (PUFA), a low level of saturation, and a melting point of 25–35 °C. Substrate (menhaden oil to acyl donor) molar ratios were 1:1, 1:3, and 1:5 for 8:0, and 1:1, 1:2, and 1:3 for 18:0. Enzyme load was 10% of the total weight of substrates. Time course study determined optimal time for maximum acyl donor incorporation. Linear interpolation estimated molar ratios that yielded SL with 20 or 30 mol% incorporation of 8:0 or 18:0. Enzymatic reactions were also conducted with molar ratios of menhaden oil to acyl donors:8:0:18:0 (1:1:3, 1:2:2, and 1:3:1). Lipases from Candida antarctica, Lipozyme® 435, and Rhizomucor miehei, Lipozyme® RM IM (Novozymes North America, Inc., Franklinton, NC, USA), were compared for all reactions. Total and sn-2 fatty acid compositions, triacylglycerol (TAG) molecular species, thermal behavior, volatile lipid oxidation products, solid fat contents, and oxidative stability were compared. When 8:0 was the acyl donor, the 1:3.03 and 1:4.58 ratios resulted in incorporation of 20 and 30 mol% 8:0, respectively. With 18:0 as the acyl donor, the 1:1.32 and 1:2.41 ratios led to incorporation of 20 and 30 mol% 18:0, respectively. The 1:3:1 ratio SL had a crystallization onset (C₀) of 15.3 °C and a melting completion (M_c) of 33.1 °C. The physicochemical properties of these SL suggest that some may be useful in formulating food products such as margarines and spreads.     

Modification of Stearidonic Acid Soybean Oil by Immobilized Rhizomucor miehei Lipase to Incorporate Caprylic Acid

Immobilized sn-1,3 specific Rhizomucor miehei lipase (RML) was used to catalyze the incorporation of caprylic acid (C8:0) into high stearidonic acid (SDA, C18:4ω3) soybean oil (SDASO) to form structured lipids (SL). The effects of type of biocatalyst (Celite-, octyl-Sepharose-, and Duolite-immobilized RML) and reaction temperature (30, 40, 50, and 60 °C) on acidolysis and acyl migration were studied. Celite-immobilized RML (C-RML) at 50 °C maximized C8:0 incorporation and minimized acyl migration compared to other treatments. Optimal levels of substrate molar ratio (C8:0 to SDASO), incubation time, and enzyme load for SL synthesis by C-RML at 50 °C was determined by response surface methodology to be 6:1, 24 h, and 20 % weight of substrates, respectively. This optimum treatment was scaled-up in hexane or solvent-free reaction media using SDASO or an SDA-enriched acylglycerol mixture as substrate. This yielded various SL with C8:0 contents ranging from 17.0 to 32.5 mol% and SDA contents ranging from 20.6 to 42.3 mol%. When digested, these SL may deliver C8:0 for quick energy and SDA for heart health making them potentially valuable for medical and nutraceutical applications.     

Structured phosphatidylcholine with elevated content of conjugated linoleic acid: Optimization by response surface methodology

Structured phosphatidylcholine was successfully produced by immobilized phospholipase A₁ catalyzed acidolysis of phosphatidylcholine and CLA. Response surface methodology was applied to optimize the reaction system using three process parameters: enzyme load, temperature, and substrates molar ratio. Optimal conditions obtained from the model were 15% enzyme load at 55°C for a 1:4 (PC/CLA) substrates molar ratio, which produced a yield of 85.8% of CLA incorporation. The total correlation coefficient (R²) was 0.92 and no lack of fit was detected. This suggests the fitness of the model obtained and it suggests that the model is sufficiently accurate to estimate the incorporation of CLA into PC. Practical applications: This study could contribute to process development for enzyme catalyzed phospholipid modification. By changing the fatty acid composition of lecithin, delivery of desired (beneficial) fatty acids could be better achieved. Additionally, emulsification properties of the phospholipids could open a wide area of applications in the food and cosmetic industries.     

Optimization of Lipase-Catalyzed Interesterification of Flaxseed Oil and Tricaprylin Using Response Surface Methodology

The biosynthesis of structured lipids (SL) in organic solvent media was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC), using Novozym 435. The bioconversion yield (BY, %) of medium-long-medium type SL, including C-caprylic and Ln-linolenic acids (CLnC), La-linoleic acid (CLaC) and O-oleic acid (COC), was monitored. Response surface methodology was used to obtain significant models for the responses, on the basis of a five level, five variable central composite rotatable design. In the experimental preliminary trials significant reaction parameters, including reaction temperature (T _r), TC/FO molar ratio (M _r), enzyme concentration (E _c), reaction time (R _t) and initial water activity (a _w), were considered for optimization. Significant models for CLnC, CLaC and COC were determined after regression analysis with backward elimination. The optimal conditions, generated for a maximum CLnC, CLaC and COC, were found to be 54.50–56.25 °C for T _r, 6.23–6.25 mol/mol for M _r, 2.68–3.13 % for E _c, 36.58–37.50 h for R _t and 0.15–0.33 for a _w. Under these optimum conditions, the BY of CLnC, CLaC and COC was predicted to be 32.48–36.67, 3.26–3.38 and 5.79–6.16 %, respectively.     

Combination of Urea Complexation and Molecular Distillation to Purify DHA and EPA from Sardine Oil Ethyl Esters

Urea complexation (UC) and the molecular distillation (MD) technique were applied jointly to purify eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from sardine oil ethyl esters (SOEE). Response surface methodology (RSM) was used to measure the influences of the variables to the responses and the optimal conditions. Regression analysis and variance analysis of the models demonstrated that each multinomial correctly represented the relationships between the responses and the variables. The urea‐to‐SOEE ratio was much more significant than crystallization temperature in UC, and the quadratic term of rotation speed of swept‐surface scrapers was the most significant variable in MD. Optimal UC conditions were 1.9:1 urea‐to‐SOEE ratio and ?1 °C crystallization temperature at which the purity and total recovery of EPA and DHA were 65.6 % and 46.8 %, respectively. The best conditions predicted for MD were 75 °C distillation temperature, 54.8 °C preheat temperature, 4.5 °C condensation temperature, and 307 rpm rotation speed at which the purity of EPA and DHA was 83.5 %. The predicted values were verified to be reasonably close to the experimentally observed values.