Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor

Production of specific-structured lipids (interesterified lipids with a specific structure) by enzymatic interesterification was carried out in a continuous enzyme bed pilot scale reactor. Commercial immobilized lipase (Lipozyme IM) was used and investigations of acyl migration, pressure drop, water dependence, production efficiency, and other basic features of the process were performed. The extent of acyl migration (defined as a side reaction) occurring in the present enzyme bed reactor was compared to that in a pilot batch reactor. The continuous enzyme bed reactor was better than the batch reactor in minimizing acyl migration. Generally the former produced about one-fourth the acyl migration produced by the latter at a similar extent of incorporation. Pressure drop and production efficiency were evaluated in order to obtain a suitable yield in one reaction step. High incorporation was favored by high substrate ratios between acyl donors and oils, requiring long reaction times on the enzyme bed. Under these conditions, the pressure drop of the reactor was modeled statistically and theoretically. Residence time, water content, and effects of mass transfers were also investigated. Incorporation of medium-chain fatty acids increased with increased residence time. Approximately 40% of lipase activity was lost after a 4-wk run. External mass transfer was not a major problem in the linear flow range, but internal mass transfer did impose some transfer limitations.     

Production of specific-structured lipids by enzymatic interesterification: Elucidation of Acyl migration by response surface design

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of by-products. In this paper, the elucidation of acyl migration by response surface design was carried out in the Lipozyme IM (Rhizomucor miehei)-catalyzed interesterification between rapeseed oil and capric acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors, water content, reaction temperature, enzyme load, reaction time and substrate ratio, were varied at three levels together with two star points. All parameters besides substrate ratio had strong positive influences on acyl migration, and reaction temperature was most significant. The contour plots clearly show the interactions between the parameters. The migration rates of different fatty acids were also compared from three different sets of experiments during the lipase-catalyzed reaction. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficients of determination (R ²) of the model were 0.996 and 0.981 for Q ² value. The results show that the fitted quadratic model satisfactorily expresses acyl migration for the enzymatic interesterification in the batch reactor used.     

Production of specific-structured lipids by enzymatic interesterification: Elucidation of acyl migration by response surface design

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of byproducts. In this paper, the elucidation of acyl migration by response surface design was carried out in the Lipozyme IM (Rhizomucor miehei)-catalyzed interesterification between rapeseed oil and capric acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors, water content, reaction temperature, enzyme load, reaction time and substrate ratio, were varied at three levels together with two star points. All parameters besides substrate ratio had strong positive influences on acyl migration, and reaction temperature was most significant. The contour plots clearly show the interactions between the parameters. The migration rates of different fatty acids were also compared from three different sets of experiments during the lipase-catalyzed reaction. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficients of determination (R ²) of the model were 0.996 and 0.981 for Q ² value. The results show that the fitted quadratic model satisfactorily expresses acyl migration for the enzymatic interesterification in the batch reactor used.     

Enzymatic acidolysis in hexane to produce n−3 or n−6 FA-enriched structured lipids from coconut oil: Optimization of reactions by response surface methodology

Response surface methodology is a statistical design that helps one to determine optimal conditions for an enzyme-catalyzed reaction by performing a minimal number of experiments. This methodology was adapted for modifying coconut oil TAG by using lipase-catalyzed acidolysis in hexane to incorporate n−3 or n−6 PUFA. FFA obtained after hydrolysis of cod liver oil and safflower oil were used as acyl donors. Immobilized lipase, Lipozyme IM60, from Rhizomucor miehei was used for catalyzing the reaction. The reaction conditions—substrate molar ratio, incubation time, and temperature—were optimized. The experimental data were fitted to a response function based on the central composite rotatable design. The optimal conditions generated from models indicated that maximal incorporation of n−3 PUFA occurred at a 1∶4 molar ratio of TAG/FFA when incubation was carried out for 34 h at 54°C. Similarly, maximal incorporation of n−6 FA was predicted at a 1∶3 molar ratio of TAG/FFA when incubated for 48.5 h at 39°C. Experiments conducted at optimized conditions predicted by the equation obtained from response surface methodology yielded structured lipids with 13.65 and 45.5% of n−3 and n−6 FA, respectively. These values agreed well with that predicted by the model. The reactions were also scaled up to 100 g levels in batch reactors with the incorporation level of n−3 and n−6 fatty acids agreeing closely with that observed when the reactions were carried out at lab scale (100 mg). These studies indicated that response surface methodology is a useful tool in predicting the conditions for incorporating desired levels of specific FA during the synthesis of structured lipids.     

Continuous production of structured phospholipids in a packed bed reactor with lipase from <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis>

The possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase-catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase-catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent-free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent-free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent. Presented at the 95th American Oil Chemists' Society Annual Meeting and Expo in Cincinnati, Ohio, May 10, 2004.     

Lipase-catalyzed acidolysis of menhaden oil with CLA: Optimization by factorial design

Menhaden oil was interesterified with CLA in a packed-bed reactor containing an immobilized lipase from Mucor miehei (L9) as the biocatalyst. Process optimization was studied using a sequence of 2²×3 factorial designs involving the mole ratio of reactants, the reaction temperature, and the space-time of the reactor as experimental parameters. Three different responses—percentage of incorporation of CLA, level of n−3 residues remaining, and conversion of CLA—were considered as objective functions. The parameters studied showed opposite effects for incorporation of CLA and the retention level of n−3 residues. A desirability function was constructed to describe a desirable balance of the conflicting response variables. Optimal conditions correspond to a molar ratio of CLA to fish oil of 0.8 to 1, a temperature of 60°C, and a space-time of 5 h.     

Optimization of enzymatic methanolysis of soybean oil by response surface methodology

Enzymatic methanolysis of refined soybean oil with methanol was investigated using Rhizomucor miehei lipase, Lipozyme RM IM, in n-hexane for reaction times of 30 min. Response surface methodology (RSM) based on three-level, three-factor (variable) face-centered cube design was used for the optimization of methanolysis. The independent variables that affect the methanolysis reaction conducted in n-hexane are temperature (°C), enzyme/oil weight ratio, and oil/methanol molar ratio. A good quadratic model was obtained for the methyl ester production by multiple regression and backward elimination. A linear relationship was observed between the observed and predicted values (R²−0.9635). The effects of temperature and enzyme amount, which affected methyl ester content of the product (response) positively, were significant (P<0.01). The quadratic term of temperature and the interaction term of enzyme amount with temperature affected the response negatively (P<0.01). The interaction term of enzyme amount with substrate mole ratio had a positive effect on the response (P<0.05). Critical conditions for the response at which methyl ester content of the product was 76.9% were determined to be 50°C, 2.37 methanol/oil mole ratio, and 0.09 enzyme/oil weight ratio.     

Four-factor response surface optimization of the enzymatic modification of triolein to structured lipids

The ability of an immobilized lipase to modify the fatty acid composition of (88.8% C_18:1, 4.3% C_16:0, 3.1% C_18:0, and 3.8% C_18:2 as determined by gas chromatography, and approximately 90% triolein) in hexane by incorporation of a medium-chain fatty acid, capric acid (C₁₀), to form structured triacylglycerol was studied. Response surface methodology was used to evaluate the effect of synthesis variables, such as reaction time (12–36 h), temperature (25–65°C), molar substrate ratio of capric acid to triolein (2:1–6:1), and enzyme amount (10–30% wt% of triacylglycerol), on the yield of structured lipid. Optimization of the transesterification was attempted to obtain maximum yield of structured lipid while using the minimum molar substrate ratio and enzyme amount as much as possible. Computer-generated contour plot interpretation revealed that a relatively high molar substrate ratio (6:1) combined with low enzyme amount (10%) after 30 h of reaction at 25°C gave optimum incorporation of capric acid. A total yield for combined monoand dicaproolein of up to 100% was obtained.     

Production of structured lipids in a packed-bed reactor with <Emphasis Type="Italic">thermomyces lanuginosa</Emphasis> lipase

Lipase-catalyzed interesterification between fish oil and medium-chain TAG has been investigated in a packedbed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), has recently been developed for fat modification. This study focuses on the new characteristics of the lipase in a packed-bed reactor when applied to interesterification of TAG. The degree of reaction was strongly related to the flow rate (residence time) and temperature, whereas formation of hydrolysis by-products (DAG and FFA) were only slightly affected by reaction conditions. The degree of reaction reached equilibrium at 30–40 min residence time, and the most suitable temperature was 60°C or higher with respect to the maximal degree of reaction. The lipase was stable in a 2-wk continuous operation without adjustment of water content or activity of the column and the substrate mixture.     

Production of structured lipids by lipase-catalyzed acidolysis in supercritical carbon dioxide: Effect on acyl migration

Structured lipids were synthesized by the acidolysis of corn oil by caprylic acid in supercritical carbon dioxide (SCCO₂) with Lipozyme RM IM from Rhizomucor miehei. The effects of pressure and temperature on the reaction were studied. To compare the degrees of acyl migration in the SCCO₂ and solvent-free reaction systems, the effects of reaction time on the degree of acyl migration were also studied. The highest mole percentage incorporation of caprylic acid (62.2 mol%) occurred at 24.13 MPa in SCCO₂. The overall incorporation of caprylic acid in the SCCO₂ system remained higher than that in the solvent-free system at every temperature tested. This trend was observed more clearly at lower temperatures (35–55°C) than at higher temperatures (65–75°C). Acyl migration with both reaction systems was low, with a negligible difference between them up to 12 h, after which the degree of acyl migration in the solvent-free system increased rapidly with time up to 24 h compared with the SCCO₂ system.