Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

Enzymatic transesterification of palm olein with nonspecific and 1,3-specific lipases

The enzymatic transesterification of palm olein was conducted in a low-moisture medium with nonspecific and 1,3-specific lipases from microbial sources. The enzymes were first immobilized on Celite, lyophilized for 4 h and then added to a reaction medium that consisted of 10% (wt/vol) palm olein in water-saturated hexane. The catalytic performance of the enzymes was evaluated by determining the changes in triglyceride (TG) composition and concentrations by reverse-phase high-performance liquid chromatography (HPLC) and the formation of free fatty acids by titration. Studies with lipase fromCandida rugosa showed that the degree of hydrolysis was reduced by drying the immobilized preparation and that the best drying time was 4 h. In all cases, the transesterification process resulted in the formation of PPP, a TG initially undetected in the oil, and increases in the concentrations of OOO (1.3–2.1-fold), OOL (1.7–4.5-fold), and OLL (1.7–4.3-fold), where P, O, and L are palmitic, oleic, and linoleic acids, respectively. SOS (where S is stearic acid), another TG not detected in the oil, was synthesized byRhizomucor miehei andPseudomonas lipases, with the latter producing more of this TG. There was a corresponding decrease in the concentrations of POP, PLP, POO, and POL. PPP concentration ranged from 1.9% (w/w) forMucor javanicus lipase to 6.2% (w/w) forPseudomonas lipase after 24 h. The greatest degree and fastest rate of change were caused byPseudomonas lipase, followed by the enzymes fromR. miehei andAspergillus niger. The effects of transesterification and hydrolysis of palm olein by the various lipases resulted in changes in the overall degree of saturation of the triglyceride components. There seems to be no clear correlation between the enzyme positional specificity and the products formed. Possible mechanisms for the formation of PPP, OOL, OLL, OOO, and SOS are discussed.     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

Frying Quality Characteristics of French Fries Prepared in Refined Olive Oil and Palm Olein

The objective of this study was to compare two oils with different polyunsaturated/saturated (P/S) fatty acid ratios, refined olive oil (P/S 0.75) and palm olein (P/S 0.25), in frying French fries. The chemical qualities of the oil residues extracted from the French fries were assayed for five consecutive batches fried at 1-h intervals. The levels of total polar compounds, free fatty acids, p-anisidine value and phytosterol oxidation products (POPs) were elevated in French fries fried in both oils. The level of total polar compounds increased from 4.6 in fresh refined olive oil to 7.3% in final batches of French fries. The corresponding figures for palm olein were 9.8–13.8%. The level of free fatty acid in fresh refined olive oil increased from 0.06 to 0.11% in final products. These figures for palm olein were 0.04–0.13%. The p-anisidine value increased from 3.7 to 32.8 and 2.5 to 53.4 in fresh oils and in final batches of French fries in refined olive oil and palm olein, respectively. The total amount of POPs in fresh refined olive oil increased from 5.1 to 9.6 μg/g oil in final products. These figures were 1.9 to 5.3 μg/g oil for palm olein.     

Partial purification and properties of lipase from germinating seeds of Jatropha curcas L.

Lipase present in the seeds of Jatropha curcas L. was isolated and some of its properties studied. Lipase activity was detected in both dormant and germinating seeds. The lipase was partially purified using a combination of ammonium sulfate precipitation and ultrafiltration, which increased the relative activity of the lipase by 28- and 80-fold, respectively. The lipase hydrolyzed palm kernel, coconut, and olive oils at comparable rates (approximately 5 μg FFA/μg protein/min); palm—Raphia hookeri and Jatropha curcas L.—oils at about twice the rate of the first group of oils; and palm and fish oils at a higher rate than all other oils. The lipase, however, had the highest activity with monoolein. Optimal pH and temperature for maximal lipase activity were 7.5 and 37°C, respectively. The addition of ferric ion (15 mM) to the lipase assay medium caused 90% inhibition of lipase activity, whereas calcium and magnesium ions enhanced lipase activity by 130 and 30%, respectively.     

Enzymatic synthesis of geranyl acetate by transesterification with acetic anhydride as acyl donor

Pseudomonas sp. lipase (PS) was immobilized by adsorption technique onto glass beads and tested for its ability to synthesize geranyl acetate by transesterification with acetic anhydride as the acyl donor. Reactions were carried out inn-hexane containing 0.1 M geraniol, 0.1 M acetic anhydride, and 200 units of lipase PS. Enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse were studied. Yields of up to 96% were obtained with 200 units (approximately 11% w/w of reactants) of enzyme. Increasing amounts of geraniol inhibited lipase activity, while excess acyl donor concentration enhanced ester production. Yields as high as 97% were obtained at 50°C, 24 h incubation, with no added water. Solvents with logP values ≥3.0 showed the highest conversion yields. Solvent-free samples also performed well. The pH range of 4–9 gave good yields (92–98.4%). Enzyme reuse studies showed the lipase remained active after 15 runs.     

Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

Lipase-catalyzed hydrolysis of palm oil

The hydrolysis of palm oil, palm olein and palm stearin, soybean oil, corn oil and peanut oil by the commercial lipase fromCandida rugosa (formerly known asC. cylindracea) was studied. The optimal conditions for the hydrolysis of palm oil by the lipase were established. The lipase fromC. rugosa exhibits an optimal activity at 37 C and at pH 7.5. The optimal oil to hexane ratio is 1 g of oil to 0.5 ml hexane. The rate of hydrolysis of palm oil by the lipase is linear on a logarithmic scale. Under the same conditions, palm oil and palm olein were hydrolyzed at the same rate, whereas palm stearin was hydrolyzed much more slowly.     

Thermal Stability of Immobilized Lipase from Candida antarctica in Glycerols with Various Water Contents at Elevated Temperatures

The immobilized lipase from Candida antarctica (fraction B, CALB) was incubated in glycerols with various water contents at 80–100 °C to measure the residual activity as a function of time. The glycerol-containing water stabilized the immobilized CALB, especially at 30–60 wt% water contents. The thermal inactivation behaviors of the immobilized CALB were expressed by a model in which the free energy of activation for the inactivation of the immobilized lipase molecules obeyed a Gaussian distribution.     

Lipase immobilized on a hydrophobic,microporous support for the hydrolysis of fats

Enzymatic hydrolysis of triglycerides has been studied as a low energy-consuming alternative to the present steam (Colgate-Emery) process. Cost analysis of the enzymatic process indicates that use of immobilized lipase compares favorably with the present steam process. This paper discusses the search for an adsorbent to use as the support material for the lipase. Hydrophobic microporous powders, membranes and fibers were found to give the best performance, as little of the lipase’s activity was lost upon immobilization. Lipase immobilized on Accurel powder has been studied in various reactor configurations for the hydrolysis of triglycerides. Reactor designs studied include cocurrent and counter-current fixed beds, continuous stirred tank reactors, and the diaphragm reactor. Productivities of the latter two reactor types were 1100 and 1700 kg fatty acid per kg immobilized lipase. Presented at the symposium “The Biology, Biochemistry and Technology of Lipases” at the 78th annual meeting of the American Oil Chemists’ Society held May 17–21, 1987, in New Orleans, Louisiana.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

Enzymatic synthesis of fatty hydrazides from palm oils

Fatty hydrazides (FH) have been successfully synthesized from palm oils by a one-step lipase catalyzed reaction. The synthesis was carried out by treating the oils with hydrazine hydrate at neutral pH using an immobilized lipase, Lipozyme as the catalyst. The percentages of conversion of RBD (refined, bleached and deodorized) palm oil (PO), RBD palm olein (POn), RBD palm stearin (PS) and RBD palm kernel olein (PKOn) into their fatty hydrazides are 95, 97, 97 and 99, respectively.     

Physical properties of Pseudomonas and Rhizomucor miehei lipase-catalyzed transesterified blends of palm stearin:palm kernel olein

The physical properties of Pseudomonas and Rhizomucor miehei lipase-catalyzed transesterified blends of palm stearin:palm kernel olein (PS:PKO), ranging from 40% palm stearin to 80% palm stearin in 10% increments, were analyzed for their slip melting points (SMP), solid fat content (SFC), melting thermograms, and polymorphic forms. The Pseudomonas lipase caused a greater decrease in SMP (15°C) in the PS:PKO (40:60) blend than the R. miehei lipase (10.5°C). Generally, all transesterified blends had lower SMP than their unreacted blends. Pseudomonas lipase-catalyzed blends at 40:60 and 50:50 ratio also showed complete melting at 37°C and 40°C, respectively, whereas for the R. miehei lipase-catalyzed 40:60 blend, a residual SFC of 3.9% was observed at 40°C. Randomization of fatty acids by Pseudomonas lipase also led to a greater decrease in SFC than the rearrangement of fatty acids by R. miehei lipase. Differential scanning calorimetry results confirmed this observation. Pseudomonas lipase also successfully changed the polymorphic forms of the unreacted blends from a predominantly β form to that of an exclusively β′ form. Both β and β′ forms existed in the R. miehei lipase-catalyzed reaction blends, with β′ being the dominant form.     

Preparation of diacylglycerol‐enriched palm olein by phospholipase A1‐catalyzed partial hydrolysis

Partial hydrolysis of palm olein catalyzed by phospholipase A₁ (Lecitase Ultra) in a solvent‐free system was carried out to produce diacylglycerol (DAG)‐enriched palm olein (DEPO). Four reaction parameters, namely, reaction time (2–10 h), water content (20–60 wt‐% of the oil mass), enzyme load (10–50 U/g of the oil mass), and reaction temperature (30–60 °C), were investigated. The optimal conditions for partial hydrolysis of palm olein catalyzed by Lecitase Ultra were obtained by an orthogonal experiment as follows: 45 °C reaction temperature, 44 wt‐% water content, 8 h reaction time, and an enzyme load of 34 U/g. The upper oil layer of the reaction mixture with an acid value of 54.26 ± 0.86 mg KOH/g was first molecularly distilled at 150 °C to yield a DEPO with 35.51 wt‐% of DAG. The DEPO was distilled again at 250 °C to obtain a DAG oil with 74.52 wt‐% of DAG. The composition of the acylglycerols of palm olein and the DEPO were analyzed and identified by high‐performance liquid chromatography (HPLC) and HPLC/electrospray ionization/mass spectrometry. The released fatty acids from the partial hydrolysis of palm olein catalyzed by phospholipase A₁ showed a higher saturated fatty acid content than that of the raw material.     

Invert emulsion as a medium for fungal lipase activity

An extracellular lipase from the fungusPythium ultimum was active in an invert [water-in-oil] emulsion consisting of 4% water emulsified into edible oils with taurocholic acid as the surfactant. The pH range for optimum lipolytic activity was 7.5–8.5, and the optimum temperature for activity was 45°C. Specific activity of the purified lipase was 919.5 μmol/min/mg protein in the invert emulsion. Water content of the invert emulsion influenced activity of the lipase differently, depending on the substrate. The rate of olive oil hydrolysis with thePythium lipase decreased with time, possibly due to inactivation of the enzyme and inhibition by free fatty acid products of the reaction. Total hydrolysis of olive oil by thePythium lipase was compared with that by lipases fromCandida rugosa andRhizopus arrhizus in the invert emulsion. Hydrolysis essentially ceased within 24 h or less for the lipases from each source. However, the addition of aqueous solution at 8 h from the beginning of incubation stimulated hydrolysis byC. rugosa andR. arrhizus lipases by 1.8-fold and 2.5-fold, respectively, but not by theP. ultimum lipase, over corresponding controls after 48 h.     

Determination of partially hydrogenated terphenyls-based thermal heating fluid in vegetable oils by HPLC with fluorescence detection

An HPLC method for the determination of partially hydrogenated terphenyls-based thermal heating fluid, Therminol 66^™, in various vegetable oils is described. Direct analysis of palm olein showed that the 3- and 4-cyclohexylbiphenyl peaks of the Therminol 66^™ used in quantitative analysis co-eluted with other fluorescent peaks present naturally in the oil. However, those interfering peaks were readily removed after saponification of palm olein. The concentrations of the 3- and 4-cyclohexylbiphenyls of Therminol 66^™ were monitored by fluorescence detection at 257 (excitation) and 320 nm (emission). The calibration graph obtained by using the peak areas of the 3- and 4-cyclohexylbiphenyls against the concentrations of Therminol 66^™ was linear, with a correlation coefficient of 0.994. The limit of quantitation, using spiked palm olein, was as low as 0.2 μg/g. The coefficients of variation obtained from the intra- and interday studies obtained by using three spiked concentrations (0.2, 0.5, and 1.0 μg/g) were 1.76–6.43 and 3.77–10.4%, respectively. The mean recovery value obtained from sunflower, soybean, and canola oils was more than 88.7%.     

Lipase-catalyzed randomization of fats and oils in flowing supercritical carbon dioxide

Enzymes can frequently impart more selectivity to a reaction than chemical catalysts. In addition, the use of enzymes can reduce side reactions and simplify post-reaction separation problems. In combination with an environmentally benign and safe medium, such as supercritical carbon dioxide (SC-CO₂), enzymatic catalysis makes supercritical fluids extremely attractive to the food industry. In this study, randomization of fats and oils was accomplished with an immobilized lipase in flowing SC-CO₂. Triglycerides, adsorbed onto Celite, are solubilized in CO₂ and carried over 1–10 g immobilized lipase derived from Candida antarctica. The degree of randomization and rate of triglyceride throughput could be controlled by CO₂ pressure and flow rate and quantity of enzyme used. The dropping points and solid fat indices of the resulting randomized oils were compared to oils that were randomized by conventional methods with sodium methoxide. Reversed-phase high-performance chromatography with flame-ionization detection was used to quantitate changes in triglyceride composition of various substrates, such as palm olein and high-stearate soybean oil. The resultant randomized oil mixtures have properties, e.g., solid fat index, that make them potential candidates for incorporation into traditional margarine formulations.     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

Enzyme hydrolysis of babassu oil in a membrane bioreactor

This work deals with the enzymatic hydrolysis of babassu oil by immobilized lipase in a membrane bioreactor using unmixed aqueous and lipid streams. The experimental work was carried out in a flat plate membrane module with two different membranes: hydrophobic (nylon) and hydrophilic [mixed cellulose esters (MCE)], with different nominal pore sizes ranging from 0.10 to 0.65 μm. Candida cylindracea lipase was adsorbed on the membrane surface area, and the reactor was operated in batch mode. The initial enzymatic rate increased from 80 to 150 μmol H⁺/min when the organic phase velocity increased from 1.0×10⁻³ to 3.0×10⁻³ m/s, indicating that mass transfer in that phase was the process-limiting step. Calcium ions had a marked effect on immobilized lipase activity, increasing around twofold the lipolytic activity. Long-term experimental runs showed that the immobilized lipase remained stable for at least 8 d. The values for immobilized protein and maximal productivities observed for 0.45 μm membranes were: 1.01 g/m² and 193 μmol H⁺/m²·s for MCE membrane and 0.78 g/m² and 220 μmol H⁺/m²·s for nylon membrane. The productivities obtained are among the highest values reported in the technical literature.     

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.