TMAH-Catalyzed Transesterification of EPA and DHA in Encapsulated Fish Oil Products

Tetramethylammonium hydroxide (TMAH)-catalyzed transesterification was developed as a rapid and reliable method using gas chromatography (GC) to determine the total fatty acid profile and to quantify the ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in 20 brands of encapsulated fish oil products. The AOAC method with boron trifluoride (BF₃) as a catalyst was used as a reference. After the respective transesterifications of BF₃ and TMAH, seven brands of encapsulated fish oil showed a single peak of EPA or DHA in the chromatograms, while 13 brands showed a single peak in the chromatograms after BF₃ esterification, but doublet peaks of EPA or DHA after TMAH esterification. By comparing with the GC/MS NIST library and authentic standard fatty acids of ethyl esters, the two pairs of doublet peaks were confirmed the ethyl and methyl esters of EPA and DHA, while the sum of the peak areas of the doublet represented the content of EPA or DHA. The reaction time course concluded that optimal TMAH transesterification was obtained at 25 °C for 10 min and using GC columns of low to medium polarity including Rtx-wax and Rtx-2330 were able to differentiate and quantify the ethyl- and methyl-esterified EPA and DHA, while RT-2560 column with higher polarity than the two other columns was unable to resolve the ethyl ester from the methyl ester of EPA or DHA. An EPA/DHA ratio of ≥1.10 may serve as an indicator of fish oil fortified with the ethyl ester of EPA.     

Kinetic Study of the Prooxidant Effect of α-Tocopherol. Hydrogen Abstraction from Lipids by α-Tocopheroxyl Radical

A kinetic study of the prooxidant effect of α-tocopherol was performed. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) by α-tocopheroxyl radical in toluene were determined, using a double-mixing stopped-flow spectrophotometer. The second-order rate constants (k _p) obtained are <1 × 10⁻² M⁻¹ s⁻¹ for 1, 1.90 × 10⁻² M⁻¹ s⁻¹ for 2, 8.33 × 10⁻² M⁻¹ s⁻¹ for 3, 1.92 × 10⁻¹ M⁻¹ s⁻¹ for 4, and 2.43 × 10⁻¹ M⁻¹ s⁻¹ for 5 at 25.0 °C. Fatty acid esters 3, 4, and 5 contain two, four, and six –CH₂– hydrogen atoms activated by two π-electron systems (–C=C–CH₂–C=C–). On the other hand, fatty acid ester 2 has four –CH₂– hydrogen atoms activated by a single π-electron system (–CH₂–C=C–CH₂–). Thus, the rate constants, k _abstr/H, given on an available hydrogen basis are k _p/4 = 4.75 × 10⁻³ M⁻¹ s⁻¹ for 2, k _p/2 = 4.16 × 10⁻² M⁻¹ s⁻¹ for 3, k _p/4 = 4.79 × 10⁻² M⁻¹ s⁻¹ for 4, and k _p/6 = 4.05 × 10⁻² M⁻¹ s⁻¹ for 5. The k _abstr/H values obtained for 3, 4, and 5 are similar to each other, and are by about one order of magnitude higher than that for 2. From these results, it is suggested that the prooxidant effect of α-tocopherol in edible oils, fats, and low-density lipoproteins may be induced by the above hydrogen abstraction reaction.     

Lipase-catalyzed incorporation of n−3 PUFA into palm oil

Two immobilized lipases, IM60 from Rhizomucor miehei and QLM from Alcaligenes sp., were used as biocatalysts for the modification of the FA composition of palm oil by incorporating n−3 PUFA. Acidolysis and interesterification reactions were conducted with hexane as organic solvent, and the products were analyzed by using GLC. After a 24-h incubation in hexane, there was an average incorporation of 20.8% EPA and 15.6% DHA into palm oil, respectively, while the percentages of palmitic and oleic acids in palm oil decreased by 28.8 and 11.8%, respectively. Higher EPA and DHA incorporation was obtained when EPAX (fish oil concentrate high in n−3 PUFA) was used in the ethyl ester form (interesterification reaction) than in the free acid form (acidolysis) in the presence of Lipozyme (IM60 lipase. Lipase QLM was found to discriminate against EPA, and it showed slightly better catalytic activity for DHA in the free acid form than in the ethyl ester form. Generally, as the mole ratio of the acyl donor to TAG increased, the percentage incorporation of EPA and DHA increased; however, reactions catalyzed by Lipozyme IM60 did not show increases in the incorporation beyond a TAG/EPAX mole ratio of 3. When limitations due to mass transfer were not a factor, an increase in the reactant amount also gave an increase in the percentage incorporation of the n−3 PUFA. Palm oil containing EPA and DHA was successfully produced and may be beneficial in certain food and nutritional applications.     

Synthesis of star polymethyl acrylate by SET-LRP at ambient temperature

The star-shaped polymethyl acrylate (PMA) was synthesized by single electron transfer living radical polymerization (SET-LRP) at 30 °C in dimethyl sulfoxide, using 2,2-dibromomethyl-1,3-dibromopropane as the multifunctional initiator, Cu⁰ powder and tris-(2-dimethylamino ethyl)amine (Me₆-TREN) as catalyst. The structure of polymer was analyzed by ¹H NMR, and the results showed that the star-shaped PMA had perfect chain ends (–Br) retention. In addition, the polymerization proceeded smoothly and the time dependence of ln([M]₀/[M]) was linear, which could indicate a first order propagation rate with respect to both radicals and monomer concentration, the polymerization was the living polymerization. The M _n and M _w/M _n of polymer were being measured by Gel Permeation Chromatography. The k _p^app = 0.0367 h⁻¹ and the conversion was 36.3% at 16 h, meanwhile the M _n^GPC of the polymer was 13,300 and the M _w/M _n was 1.40.     

Modeling the Primary Oxidation in Commercial Fish Oil Preparations

The quality of commercial fish oil products can be difficult to maintain because of the rapid lipid oxidation attributable to the high number of polyunsaturated fatty acids (PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). While it is known that oxidation in fish oil is generally the result of a direct interaction with oxygen and fatty acid radicals, there are very few studies that investigate the oxidation kinetics of fish oil supplements. This study uses hydroperoxides, a primary oxidation product, to model the oxidation kinetics of two commercially available fish oil supplements with different EPA and DHA contents. Pseudo first order kinetics were assumed, and rate constants were determined for temperatures between 4 and 60 °C. This data was fit to the Arrhenius model, and activation energies (E _a) were determined for each sample. Both E _a agreed with values found in the literature, with the lower PUFA sample having a lower E _a. The oil with a lower PUFA content fit the first-order kinetics model at temperatures ≥20 °C and ≤40 °C, while the higher PUFA oil demonstrated first-order kinetics at temperatures ≥4 °C and ≤40 °C. When the temperature was raised to 60 °C, the model no longer applied. This indicates that accelerated testing of fish oil should be conducted at temperatures ≤40 °C.     

Analysis of EPA and DHA and distinction between fish oils and concentrates

Fish oil is the major dietary source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Adequate dietary intake of these n‐3 PUFA is beneficial to reduce the risks of cardiovascular mortality, prostate cancer, and neurological disorders in children and adults. There is a surge in demand for fish oil in the functional food market. Microencapsulation of fish oil is the trend to improve its stability and sensory quality. The EPA and DHA content of the fish oil products may vary markedly from the label due to their susceptibility to oxidation. Quick and reliable methods other than the AOAC BF₃ method have been exploited to quantify EPA and DHA in the encapsulated fish oil and the microencapsulated powdered products such as infant formula. This article describes a method to differentiate the ethyl‐ester form from the TAG form of EPA and DHA in encapsulated fish oil which may be a mixture of natural triacylglycerol enriched with ethyl esterified EPA and DHA. A method is recommended due to the difference in apparent potency of these two esterified forms, which may be a concern in infant formula and elsewhere.     

Preparation of core–shell latex particles by emulsion co-polymerization of styrene and butyl acrylate,and evaluation of their pigment properties in emulsion paints

A series of core–shell polymeric particles with poly(n-butyl acrylate-co-methacrylic acid-co-ethylene glycol dimethylacrylate) as core and poly(styrene-co-methyl methacrylate) as shell were prepared by seeded emulsion polymerization. The role of ethylene glycol dimethylacrylate (EGDMA) is to crosslink the core so as to avoid any probability of gel formation and to bind both the core and the shell phase together. The spherical morphology of the core–shell structure was achieved at 60:40 core to shell ratio. The core–shell morphology was confirmed by SEM and TEM analyses. GPC analysis of the particles reveals that the polymer shows a bimodal mode. The first peak has M _w = 382700 and M _n = 245200 with polydispersity index of 1.6, and the second peak has M _w = 21200 and M _n = 14800 with polydispersity index of 1.4. These core–shell latexes were applied as a pigment/binder in emulsion paint and the paint properties like gloss, rock hardness, washability, opacity, etc. were compared with the standard. The results show that these core–shell latexes can provide similar hiding power with 17% reduction of TiO₂ in the paint formulation.     

Effects of Seal Oil and Tuna-Fish Oil on Platelet Parameters and Plasma Lipid Levels in Healthy Subjects

Fish are a rich source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two long-chain polyunsaturated n-3 fatty acids (LC n-3 PUFA) with cardiovascular benefits. A related but less-investigated LC n-3 PUFA, docosapentaenoic acid (DPA), is more common in seal oil and pasture-fed red meats. This study compared indicators of platelet function and plasma lipids in healthy volunteers given supplements containing these different fatty acids (FA) for 14 days. Subjects, randomised into three groups of ten, consumed capsules of tuna oil (210 mg EPA, 30 mg DPA, 810 mg DHA), seal oil (340 mg EPA, 230 mg DPA, 450 mg DHA) or placebo (sunola) oil. Supplementary LC n-3 PUFA levels were approximately 1 g/day in both fish and seal oil groups. Baseline dietary FA and other nutrient intakes were similar in all groups. Both fish and seal oil elevated platelet DHA levels (P < 0.01). Seal oil also raised platelet DPA and EPA levels (P < 0.01), and decreased p-selectin (P = 0.01), a platelet activation marker negatively associated with DPA (P = 0.03) and EPA (P < 0.01) but not DHA. Plasma triacylglycerol decreased (P = 0.03) and HDL-cholesterol levels increased (P = 0.01) with seal oil only. Hence, seal oil may be more efficient than fish oil at promoting healthy plasma lipid profiles and lowering thrombotic risk, possibly due to its high DPA as well as EPA content.     

Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans

The aim of this study was to determine whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), or both, were responsible for the triglyceride (TG)-lowering effects of fish oil. EPA (91% pure) and DHA (83% pure), a fish oil concentrate (FOC; 41% EPA and 23% DHA) and an olive oil (OO) placebo (all ethyl esters) were tested. A total of 49 normolipidemic subjects participated. Each subject was given placebo for 2–3 wk and one of the n-3 supplements for 3 wk in randomized, blinded trials. The target n-3 fatty acid (FA) intake was 3 g/day in all studies. Blood samples were drawn twice at the end of each supplementation phase and analyzed for lipids, lipoproteins, and phospholipid FA composition. In all groups, the phospholipid FA composition changed to reflect the n-3 FA given. On DHA supplementation, EPA levels increased to a small but significant extent, suggesting that some retroconversion may have occurred. EPA supplementation did not raise DHA levels, however, FOC and EPA produced significant decreases in both TG and very low density lipoprotein (VLDL) cholesterol (C) levels (P<0.01) and increases in low density lipoprotein (LDL) cholesterol levels (P<0.05). DHA supplementation did not affect cholesterol, triglyceride, VLDL, LDL, or high density lipoprotein (HDL) levels, but it did cause a significant increase in the HDL₂/HDL₃ cholesterol ratio. We conclude that EPA appears to be primarily responsible for TG-lowering (and LDL-C raising) effects of fish oil.     

Novel triazole-based aluminum complex for ring-opening polymerization of lactones

Novel triazole-based aluminum complex {O,O′-[4,5-P(O)Ph₂tz]-AlMe₂ was studied as the catalyst for the ring-opening polymerization of caprolactone (ε-CL) in chlorobenzene. In the presence of methanol, isopropanol, and bifunctional poly(ethylene glycol), the catalytic system produced polymers with high conversion (81–85 %) but broader distribution (M _w/M _n = 1.5–1.8). The system of catalyst and benzyl alcohol produced relative monodisperse PCLs (M _w/M _n ~ 1.2) with defined molecular weight at 1/1ratio, 60 °C and an initial concentration of ε-CL equal to 0.5 mol/L.     

Supercritical fluid fractionation of fish oil esters using incremental pressure programming and a temperature gradient

Incremental pressure programming was demonstrated to be an effective technique for increasing the yield of 90% pure allcis-5,8,11,14,17-ethyl eicosapentaenoate (EPA) in the fractionation of urea-crystallized fish oil ethyl esters using supercritical fluid CO₂. The fractionations, which also produced high purity allcis-4,7,10,13,16,19-ethyl docosahexaenoate (DHA), were performed using a column temperature gradient. In initial experiments, the maximum temperature of the superimposed gradient was 80°C, and processing pressures ranged from 1900–2200 psi. By reduction of processing pressures, comparable yields of EPA were obtained from fractionations in which the maximum temperatures in the gradient were 70°C and 60°C.     

Lipase selectivity toward fatty acids commonly found in fish oil

The main objective of this study was to compare the fatty acid selectivity of numerous commercially available lipases toward the most ubiquitous fatty acids present in fish oils in form of their corresponding ethyl esters. Special interest was taken in their ability to separate the n‐3 long‐chain polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from the more saturated fatty acids as well as exploiting the putative discrimination between these highly valuable n‐3 PUFA. Hydrolysis of sardine oil ethyl esters in a Tris buffer solution by 12 microbial lipases is described. The results reveal that all of the lipases strongly discriminate against the n‐3 PUFA and prefer the more saturated fatty acids as substrates. Most of the lipases discriminate between EPA and DHA in favor of EPA, however, 2 bacterial lipases from Pseudomonas were observed to prefer DHA to EPA. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed fatty acid selectivity when their fish oil triacylglycerol hydrolysis was studied. Thus, the n‐3 PUFA including EPA and DHA were observed to be hydrolyzed at a considerably higher rate than the more saturated fatty acids.     

Separation of Sunflower Oil from Hexane by Use of Composite Polymeric Membranes

Vegetable oil extraction, as performed today by the oilseed-crushing industry, usually involves solvent extraction with commercial hexane. After this step, the vegetable oil–hexane mixture (miscella) must be treated to separate its components by distillation. If solvent-resistant membranes with good permeation properties can be obtained, membrane separation may replace, or be used in combination with, conventional evaporation. Two tailor-made flat composite membranes, poly(vinylidene fluoride) (PVDF–Si and PVDF–CA) and a commercially available composite membrane (MPF-50), were used to separate a crude sunflower oil–hexane mixture. The effects of temperature, cross-flow velocity (v), transmembrane pressure (Δp), and feed oil concentration (C _f) on membrane selectivity and permeation flux were determined. The PVDF–Si membrane achieved the best results, being stable in commercial hexane and having promising permselectivity properties for separation of vegetable oil–hexane miscella. Improved separation performance was obtained at C _f = 25%, Δp = 7.8 bar, T = 30 °C, and v = 0.8 m s⁻¹; a limiting permeate flux of 12 Lm⁻² h⁻¹ and 46.2% oil retention were achieved. Low membrane fouling was observed under all the experimental conditions studied.     

Metabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil but at Lower Dose of EPA and DHA,in Healthy Volunteers

The purpose of the present study is to investigate the effects of krill oil and fish oil on serum lipids and markers of oxidative stress and inflammation and to evaluate if different molecular forms, triacylglycerol and phospholipids, of omega-3 polyunsaturated fatty acids (PUFAs) influence the plasma level of EPA and DHA differently. One hundred thirteen subjects with normal or slightly elevated total blood cholesterol and/or triglyceride levels were randomized into three groups and given either six capsules of krill oil (N = 36; 3.0 g/day, EPA + DHA = 543 mg) or three capsules of fish oil (N = 40; 1.8 g/day, EPA + DHA = 864 mg) daily for 7 weeks. A third group did not receive any supplementation and served as controls (N = 37). A significant increase in plasma EPA, DHA, and DPA was observed in the subjects supplemented with n-3 PUFAs as compared with the controls, but there were no significant differences in the changes in any of the n-3 PUFAs between the fish oil and the krill oil groups. No statistically significant differences in changes in any of the serum lipids or the markers of oxidative stress and inflammation between the study groups were observed. Krill oil and fish oil thus represent comparable dietary sources of n-3 PUFAs, even if the EPA + DHA dose in the krill oil was 62.8% of that in the fish oil.     

Improvement of Oil Quality in Soybean [<Emphasis Type="Italic">Glycine max </Emphasis>(L.) Merrill] by Mutation Breeding

Low oxidative stability, off-flavor and rancidity are the major drawbacks of soybean oil. Modification of the fatty acid composition of soybean [Glycine max (L.) Merrill] oil can improve its quality and value for processors and acceptability among consumers. Mutation breeding of soybean was therefore initiated with the objective of identifying stable soybean mutants with altered fatty acid composition for improved oxidative stability and nutritional quality. Seeds of soybean cultivar ‘MACS 450’ were treated with γ-radiation and/or ethyl methane sulfonate (EMS). The harvest of M₁ plants was evaluated for fatty acid composition by gas chromatography. Highly significant variation in all the fatty acids except palmitic acid was observed. Treatment of EMS in higher concentrations as well as combined treatment of both the mutagens, i.e., γ-radiation and EMS were effective in increasing the variability for the fatty acid content in soybean oil. The variability was skewed towards high levels of oleic (35–42%) and low levels of linolenic acid (3.77–5.00%). M₃ and M₄ generations of desirable variants were analyzed for the stability of the mutated trait_. Only high oleic variants were stable in M₃ and M₄ generations. Based on fatty acid values, oxidative stability index (OSI), nutritional quality index (NQI) and ratio of essential fatty acids (ω₆/ω₃) were calculated for the control and M₂, M₃ and M₄ generations. The ω₆/ω₃ ratio in all the high oleic mutants was within the World Health Organization (WHO) recommended value (5–10%). A significant positive correlation between OSI and oleic acid content (P < 0.001) indicated improved oxidative stability of the oil while retaining nutritional quality. These high oleic lines could be utilized further in breeding programs for improvement of soybean oil quality.     

Enhanced incorporation of n−3 fatty acids from fish compared with fish oils

This work was undertaken to study the impact of the source of n−3 FA on their incorporation in serum, on blood lipid composition, and on cellular activation. A clinical trial comprising 71 volunteers, divided into five groups, was performed. Three groups were given 400 g smoked salmon (n=14), cooked salmon (n=15), or cooked cod (n=13) per week for 8 wk. A fourth group was given 15 mL/d of cod liver oil (CLO) (n=15), and a fifth group served as control (n=14) without supplementation. The serum content of EPA and DHA before and after intervention revealed a higher rise in EPA and DHA in the cooked salmon group (129% rise in EPA and 45% rise in DHA) as compared with CLO (106 and 25%, respectively) despite an intake of EPA and DHA in the CLO group of 3.0 g/d compared with 1.2 g/d in the cooked salmon group. No significant changes were observed in blood lipids, fibrinogen, fibrinolysis, or lipopolysaccharide (LPS)-induced tissue factor (TF) activity, tumor necrosis factor-α (TNFα), interleukin-8 (IL-8), leukotriene B₄ (LTB₄), and thromboxane B₂ (TxB₂) in whole blood. EPA and DHA were negatively correlated with LPS-induced TNFα, IL-8, LTB₄, TxB₂, and TF in whole blood. In conclusion, fish consumption is more effective in increasing serum EPA and DHA than supplementing the diet with fish oil. Since the n−3 FA are predominantly in TAG in fish as well as CLO, it is suggested that the larger uptake from fish than CLO is due to differences in physiochemical structure of the lipids.     

Cholestane as a digestibility marker in the absorption of polyunsaturated fatty acid ethyl esters in Atlantic salmon

Salmonid fish require long-chain n−3 fatty acids in their diet. The digestibility of different chemical forms of fish oil fatty acids, fed as triacylglycerols, free fatty acids or ethyl esters, was examined in 300 g farmed Atlantic salmon (Salmo salar) using cholestane as an indicator of fat absorptionin lieu of the chromium oxide (Cr₂O₃) which is commonly used as a marker in digestibility studies. It was established that the two digestibility markers gave similar results. Conveniently, cholestane does not require a separate analysis if fatty acids are to be determined by appropriate gas-liquid chromatography. The long-chain polyunsaturated fatty acids were particularly well absorbed, the apparent digestibility being 90–98% when feeding triacylglycerols or free fatty acids. However, the digestibility of monounsaturated fatty acids (75–94%) was lower, and lower still for saturated fatty acids (50–80%). Ethyl esters of fatty acids were significantly less well absorbed (P<0.05) than were the corresponding fatty acids in free acid or triacylglycerol form. Irrespective of dietary fat type, only free fatty acids were identified in feces, indicating total hydrolysis of triacylglycerols and ethyl esters. Presented in part at the World Aquaculture Society meeting, June 10–14, 1990, Halifax, Canada.     

Thermally induced polymerization and copolymerization with styrene of diazoketones in the presence of benzoquinone

Thermally induced polymerization of diazoketones, (E)-1-diazo-3-nonen-2-one 1 and (E)-1-diazo-4-phenyl-3-buten-2-one 2, is described. Heating 1 and 2 in a solvent at 60–100 °C afforded polymers, where all the main chain carbons bear acyl groups derived from the monomers and the main chain contains ca. 25–35 mol% of azo group. Molecular weight of the resulting polymers increased up to M _n = 8,400 by the addition of benzoquinone to the reaction mixtures. The polymerization was supposed to proceed via radical propagating chain end and copolymerization of 1 with styrene gave copolymers (M _n = 11,000–15,000) having acylmethylene, azo group, and repeating unit from styrene in their main chains.     

New Binuclear Complexes with Mixed Ligands and μ<Subscript>2</Subscript>-Phenol Oxygen Bridges: Synthesis Crystal Structures and Magnetism

Two new complexes, [M₂(L)₂(bpy)₂](ClO₄)₂ [L = methyl salicylate, bpy = 2,2′-bipyridine, M = Cu (1) and Zn (2)] have been synthesized and structurally characterized by X-ray structure analyses. The centrosymmetric dimeric structure consists of a binuclear unit, in which M (M = Cu or Zn) atoms are bridged by two phenol oxygen atoms as a μ ₂-bridged. The neighboring binuclear units interact with each other by two kinds of weak contacts: one kind is π–π stacking by π–σ attraction with an edge-to-face C–H···π interaction, and the second type is a H-bonding interaction, which extends the binuclear unit into a 3D network. Magnetic measurements confirm that 1 presents a very strong intradinuclear ferromagnetic coupling between the copper(II) ions.     

Transesterification of Fish Oil to Produce Fatty Acid Ethyl Esters Using Ultrasonic Energy

This study evaluated the production of fatty acid ethyl esters from fish oil using ultrasonic energy and alkaline catalysts dissolved in ethanol. The feasibility of fatty acid ethyl ester production was determined using an ultrasonic bath and probe, and between 0.5 and 1% KOH (added to the fish oil). Furthermore, factors such as ultrasonic device (bath and probe), catalyst (KOH and C₂H₅ONa), temperature (20 and 60 °C), and duration of exposure (10–90 min) were assessed. Sodium ethoxide was found to be a more efficient catalyst than KOH when transesterifying fish oil. Ultrasonic energy applied for greater than 30 min at 60 °C using 0.8% of C₂H₅ONa as a catalyst transesterified over 98% fish oil triglycerides to fatty acid ethyl esters. It is reasonable to conclude that the yield of fatty acid ethyl esters produced by applying ultrasonic energy to fish oil is related to the sonication time. Due to increases in the surface area contact between the reactants and the catalyst, ultrasonic energy has the potential to reduce the production time required by a conventional large-scale commercial transesterification method that uses agitation as a way of mixing.