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.     

Effect of phospholipids on enzyme-catalyzed transesterification of oils

The effect of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the activity of an immobilized lipase, Lipozyme, during transesterification of oils has been studied. PC concentrations less than 0.05% did not affect the initial rate of transesterification. Higher concentrations of PC as well as PE and PI-rich phospholipids at the 0.5% level caused a reduction in the initial reaction rate, but after ten hours transesterification had progressed to the same extent as the control sample. Reuse of Lipozyme for ten batch reactions showed that a PC content of less than 0.05% did not cause significant inactivation of the enzyme, but PC contents above this level caused progressive inactivation of the enzyme and the inactivation increased with PC content. The ability of phospholipids to inactivate the Lipozyme was in the order PE>PC>PI. Variation in the acyl groups of PC did not significantly affect inactivation of the enzyme. It is concluded that the phospholipid content of edible oils should be reduced below 200 ppm by degumming if unacceptable inactivation of Lipozyme is to be avoided.     

酶法酯交换过程中不饱和脂肪酸对其酰基位移的影响

酶法制备结构酯的过程中,由于酰基位移的发生而生成了副产品．并影响了产品质量。研究了Lipozyme IM和猪胰脂酶催化橄榄油与硬脂酸甲酯反应,油酸、亚油酸、亚麻酸的相对酰基位移率的比较。结果显示：双键越多酰基位移越小,猪胰脂酶催化时脂肪酸酰基位移程度大于Lipozyme IM。     

Enrichment of DHA from Tuna Oil in a Packed Bed Reactor via Lipase-Catalyzed Esterification

A packed-bed reactor (length 6.5 cm; id 4.65 mm) has been used to enrich docosahexaenoic acid (DHA) via the lipase-catalyzed esterification of the fatty acid from tuna oil with ethanol. Lipozyme RM IM (from Rhizomucor miehei) was used for the esterification reaction because of its ability to discriminate between different fatty acids, and several reaction parameters, including the temperature, molar ratio of substrates, and water content were explored as a function of residence time. In this way, the optimum conditions for the enrichment process were determined to be a temperature of 20 °C, a molar ratio of 1:5 (i.e., fatty acid to ethanol), and a water content of 1.0 % (based on the total substrate weight). Under these conditions, a residence time of 90 min gave a DHA concentration of 70 wt% and a DHA recovery yield of 87 wt% in the residual fatty acid fraction.     

Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosa lipase in a large-scale study

Interesterification of a blend of palm stearin and coconut oil (75∶25, w/w), catalyzed by an immobilized Thermomyces lanuginosa lipase by silica granulation, Lipozyme TL IM, was studied for production of margarine fats in a 1- or 300-kg pilot-scale batch-stirred tank reactor. Parameters and reusability were investigated. The comparison was carried out between enzymatic and chemical interesterified products. Experimentally, Lipozyme TL IM had similar activity to Lipozyme IM for the interesterification of the blend. Within the range of 55–80°C, temperature had little influence on the degree of interesterification for 6-h reaction, but it had slight impact on the content of free fatty acids (FFA). Drying of Lipozyme TL IM from water content 6 to 3% did not affect its activity, whereas it greatly reduced FFA and diacylglycerol contents in the products. Lipozyme TL IM was stable in the 1-kg scale reactor at least for 11 batches and the 300-kg pilot-scale reactor at least for nine batches. Due to regiospecificity of the lipase (sn-1,3 specific), enzymatically interesterified products had different fatty acid distribution at sn-2 position from the chemically randomized products, implying the potential nutritional benefits of the new technology. Presented at the 91st American Oil Chemists' Society Annual Meeting in San Diego, April 28, 2000.     

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.     

Preparation of 1,3‐Diolein by Irreversible Acylation

Earlier findings on the nutritional benefits of diacylglycerols (DAGs) have attracted much attention on the synthesis of DAGs. In this study, we reported an improved method for the lipase‐catalyzed synthesis of 1,3‐diolein by the irreversible glycerolysis of vinyl oleate with glycerol. The effects of reaction system, lipase loading, molar ratio of vinyl oleate to glycerol, reaction temperature and time on 1,3‐diolein content in crude reaction mixture were investigated. When the reaction was conducted in a solvent‐free system at 30 °C for 8 h by reacting 2 mmol vinyl oleate with 1 mmol glycerol with 8 % (w/w, relative to total reactants) Lipozyme RM IM (Novozymes, Beijing, China) as catalyst, there were 90.5 ± 2.9 % (area/area) 1,3‐diolein and (3.3 ± 0.3) % 1,2‐diolein produced. After purification, 1,3‐diolein was obtained at 81.4 % yield with 98.2 % purity. The lipase‐catalyzed synthesis of 1,3‐diolein using vinyl oleate as acyl donor by glycerolysis was also conducted using a medium with 50 mmol of glycerol and 100 mmol vinyl oleate. Compared to enzymatic esterification in a solvent, enzymatic glycerolysis for the synthesis 1,3‐diolein is more effective due to the irreversible reaction, mild due to the low reaction temperature, and environmentally benign due to the use of solvent‐free reaction system.     

Lipase-catalyzed preparation of palmitic and stearic acid-rich phosphatidylcholine

Saturated FA enhance the oxidative stability of phospholipids. In the present study phosphatidylcholine (PC) rich in palmitic and stearic acids was prepared using lipase-catalyzed transesterification from PC isolated from egg and soybean lecithins. Two different lipases, namely, Novozym 435 and Lipozyme TL IM, were used for the transesterification. The reaction conditions were optimized by varying the lipase dosage, molar ratio of PC to FA, and reaction period. Palmitic acid could be incorporated up to 58.6 and 57.1% using Lipozyme TL IM and 56 and 61% using Novozym 435 in egg and soybean PC from an initial content of 37.4 and 16.8%, respectively. Similarly, stearic acid incorporation was up to 44.7 and 46.3% using Lipozyme TL IM and 37.2 and 55.8% using Novozym 435 in egg and soybean PC from an initial content of 8.6 and 2.1%, respectively.     

Modification of menhaden oil by enzymatic acidolysis to produce structured lipids: Optimization by response surface design in a packed bed reactor

Structured lipids from menhaden oil were produced by enzymatic acidolysis in a packed bed reactor. Response surface methodology was applied to optimize the reaction. Lipozyme IM from Rhizomucor miehei lipase was the biocatalyst, and caprylic acid was the acyl donor. Parameters such as residence time, substrate molar ratio, and reaction temperature were included for the optimization. High incorporation of acyl donor and retention of high levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in the original menhaden oil were obtained. Good quadratic models were obtained for the incorporation of caprylic acid and for the content of EPA plus DHA retained, by multiple regression with backward elimination. The coefficients of determination (R ²) for the two models were 0.91 and 0.87, respectively. The regression probabilities (P) were below 0.003 for both models. Also, the predicted values from the two models had linear relationships with the observed responses. All parameters studied had positive effects on the incorporation of caprylic acid, but only residence time and substrate molar ratio had negative effects on the content of EPA plus DHA retained. The optimal conditions generated from models were temperature =65°C, substrate molar ratio=4–5, and residence time=180–220 min. Incorporated caprylic acid did not replace DHA, but the content of EPA decreased somewhat with an increase in caprylic acid incorporation.     

Enzymatic incorporation of stearic acid into a blend of palm olein and palm kernel oil: Optimization by response surface methodology

Response surface methodology was used to model the incorporation of stearic acid into a blend of palm olein and palm kernel oil in hexane using the sn-1,3-regiospecific lipase Lipozyme RM IM. The factors investigated were incubation time, temperature, and substrate molar ratio. A second-order model with interaction was used to fit the experimental data. The coefficients of determination, R ² and Q ², were 0.96 and 0.90, respectively. The adjusted R ² was 0.95. The regression probability was less than 0.001, and the model showed no lack of fit. Also, a linear relationship was observed between the predicted and observed values. All parameters studied had positive effects on incorporation of stearic acid, with substrate molar ratio having the greatest effect. The interaction terms of substrate molar ratio with temperature and time also had positive effects on incorporation, whereas the effect of the squared term of substrate molar ratio was negative. The quadratic terms of temperature and time, as well as their interaction term, had no significant effect on incorporation at α_0.05. Model verification was done by performing a chi-square test, which showed that there was no significant difference between predicted values and a new set of observed responses.     

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.     

Preparation of Human Milk Fat Substitutes from Lard by Lipase‐Catalyzed Interesterification Based on Triacylglycerol profiles

Human milk fat substitutes (HMFSs) with triacylglycerol profiles highly similar to those of human milk fat (HMF) were prepared from lard by physical blending followed by enzymatic interesterification. Based on the fatty acid profiles of HMF, different vegetable and single‐cell oils were selected and added to the lard. Blend ratios were calculated based on established physical blending models. The blended oils were then enzymatically interesterified using a 1,3‐regiospecific lipase, Lipozyme RM IM (RML from Rhizomucor miehei immobilized on Duolite ES562; Novozymes A/S, Bagsværd, Denmark), to approximate HMF triacylglycerol (TAG) profiles, particularly with respect to the distribution of palmitic acid in the sn?2 position. The optimized blending ratios were determined to be: lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. The optimized reaction conditions were determined to be: enzyme load of 11 wt%, temperature of 60 °C, water content of 3.5 wt%, and reaction time of 3 hours. The resulting product was evaluated for total and sn?2 fatty acids, polyunsaturated fatty acids, and TAG composition. A high degree of similarity was obtained, indicating the great potential of the product as a fat alternative for use in infant formulas.     

Enzymatic acidolysis of tristearin with lauric and oleic acids to produce coating lipids

Triacylglycerols with potential for coating application were prepared by acidolysis of tristearin with lauric and oleic acids using Lipozyme IM60 lipase in n-hexane. The effects of reaction parameters such as time, temperature, substrate mole ratio, water content, enzyme load, and enzyme reuse were studied. Five-gram scale synthesis was carried out to obtain the melting profile of products by differential scanning calorimetry (DSC). An acceptable melting profile was obtained for the product obtained with a 1∶4∶1 (tristearin/lauric acid/oleic acid) mole ratio of reactants. The DSC melting peak for this product was 31.4°C. Synthesis of 1200 g of this product was carried out at a 1∶4∶1 substrate ratio in a stirred tank batch reactor under optimal conditions. The reaction product, purified by short-path distillation, was coated onto crackers and studied for its moisture inhibition ability, under water vapor-saturated atmosphere, in a desiccator over different time intervals. The effectiveness of the synthesized lipid as a coating material was compared against uncoated crackers as a control and with cocoa butter-coated crackers. The synthesized lipid was better in preventing moisture absorption than cocoa butter. This work was presented at the Biocatalysis Symposium in April 2000, held at the 91 st Annual Meeting and Expo of the American Oil Chemists’ Society, San Diego, CA.     

Feed batch addition of saccharide during saccharide-fatty acid esterification catalyzed by immobilized lipase: Time course,water activity,and kinetic model

A conversion of 80–93% was achieved for esterification of oleic acid and fructose (or sucrose) catalyzed by immobilized Rhizomucor miehei lipase (Lipozyme IM; Novozymes, Franklinton, NC) at 65°C using near-stoichiometric amounts of substrates. The product consisted of mono- and diester at a ratio of 9∶1 gg⁻¹. The main obstacle for achieving a high rate of reaction, the poor miscibility of the substrates, was overcome by taking advantage of the greatly increased solubility of fructose as the proportion of ester increased. A phase diagram demonstrated that the solubility of fructose increased linearly from 0.002 to 0.07 to 0.13 gg⁻¹ as the ester mass fraction increased from 0.00 to 0.47 to 0.80, respectively. Solvent (tert-butanol) was present only during the first phase of the time course of the reaction to enhance fructose solubility and was allowed to evaporate away completely on reaching 25% conversion. A conversion higher than 80–93% could not be achieved by reducing the bioreactor's water content through use of vacuum pressure or water activity control. Water adsorption isotherms demonstrate the significant increase of equilibrium liquid phase water content as the reaction progressed, which was due to higher water adsorption by the monoester relative to oleic acid. Increased removal of liquid phase water may result in the loss of water from the lipase, resulting in a reduction of its biocatalytic activity. Initial rate experiments were used to derive a Ping-Pong Bi Bi kinetic model that strongly agreed with measured data for the time course of the reaction. Lipozyme IM did not lose activity when employed for three successive fructose-oleate esterification batch reactions or, equivalently, for a 24-d reaction period.