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 fat hydrolysis and synthesis

The hydrolysis of tallow, coconut oil and olive oil, by lipase fromCandida rugosa, was studied. The reaction approximates a firstorder kinetics model. Its rate is unaffected by temperature in the range of 26–46 C. Olive oil is more rapidly hydrolyzed compared to tallow and coconut oil. Hydrolysis is adversely affected by hydrocarbon solvents and a nonionic surfactant. Since amounts of fatty acids produced are almost directly proportional to the logarithms of reaction time and enzyme concentration, this relationship provides a simple means of determining these parameters for a desired extent of hydrolysis. All three substrates can be hydrolyzed, almost quantitatively, within 72 hr. Lipase fromAspergillus niger performs similarly. The lipase fromRhizopus arrhizus gives a slow hydrolysis rate because of its specificity for the acyl groups attached to the α-hydroxyl groups of glycerol. Esterification of glycerol with fatty acid was studied with the lipase fromC. rugosa andA. niger. All expected five glycerides are formed at an early stage of the reaction. Removal of water and use of excess fatty acid reverse the reaction towards esterification. However, esterification beyond a 70% triglyceride content is slow.     

Hydrolysis of edible oils by lipases immobilized on hydrophobic supports: Effects of internal support structure

The hydrolysis of edible oil by immobilized lipases on novel support materials was investigated. Six hydrophobic polymers were studied with the following techniques: (i) determination of the surface area of each support by BET (Brunauer-Emmett-Teller) analysis of nitrogen adsorption isotherms; (ii) electron photomicrography; and (iii) measuring lipase activity by hydrolysis of olive oil with lipase fromCandida cylindracea immobilized on each support. A detailed structural analysis on one support also was carried out by mercury porosimetry. The composition and porosity of a support are more important than the surface area in determining activity for immobilized lipases. Furthermore, having selected the “most efficient” support, five lipases fromC. cylindracea, Rhizomucor miehei, andPseudomonas cepacia, were immobilized, and their hydrolytic activities were determined at several temperatures and pH values with olive oil and beef tallow as substrates in solvent-free systems. For each parameter, twelve successive 2.5-h hydrolysis reactions were conducted on a laboratory-scale under batch conditions. Lipase AY fromC. cylindracea had the highest hydrolytic activity, in the range of 30–50°C at pH 5.5 with olive oil as the substrate. For beef tallow, lipase PS, fromP. cepacia, displayed the highest activity at 50°C and pH 7.     

In vivo determination of triglyceride secretion using radioactive glycerol in rats fed different dietary saturated fats

Our objective was to determine the relative rates ofin vivo triglyceride (TG) secretion and the composition of very low density lipoproteins (VLDL) in rats fed different dietary saturated fats. Male Sprague-Dawley rats (150–200 g) were fed diets containing 16% corn oil, or 14% butterfat, 14% beef tallow, 14% olive oil, or 14% coconut oil plus 2% corn oil for 5 wk. Changes in plasma TG specific radioactivity were determined in individual, unanesthetized fasted rats after injection of 100 μCi [2-³H]glycerol. Nonlinear regression analysis using a 2-compartment model was used to determine the fractional rate constant for TG turnover in plasma. The plasma TG pool was 33–40% larger with beef tallow than with corn, olive or coconut oil feeding (p<0.05), and 20% larger with beef tallow than with butterfat feeding. The rate of TG secretion into plasma (mg/min/100 g body weight) was 60% higher in animals fed beef tallow than corn or coconut oil (p<0.05) and 26–33% higher in animals fed beef tallow than olive oil or butterfat. Differences in VLDL composition (% wt) were also noted. Our data suggest that greater TG secretion is the primary factor contributing to the larger TG pool with ingestion of beef tallow relative to butterfat, corn or coconut oil. These results suggest that different dietary saturated fats have unique effects on TG metabolism in rats. Presented in part at the 1990 meeting of the Federation of American Societies for Experimental Biology in Washington, D.C. (see ref. 1).     

Lipase-catalyzed triglyceride hydrolysis in organic solvent

An investigation of lipases fromRhizomucor miehei,Candida rug osa and porcine pancreas revealed that these enzymes hydrolyzed triglycerides in an organic solvent system. The presence of secondary amines,i.e., diethylamine,N-methylbutylamine, or the tertiary amine, Methylamine, greatly increased the extent of hydrolysis. The lipolysis of tallow took place under mild conditions,e.g., room temperature, moderate shaking and within 20 hr. At 45°C, complete hydrolysis of tallow was obtained in 6 hr. Vegetable oils and a fish oil (cod liver oil) were also hydrolyzed at 20°C byR. miehei lipase in the presence of iV-methylbutylamine for 20 hr. The lipases were recovered for reuse with some loss of activity. Optimum yields of free fatty acids were obtained by usingR. miehei lipase as catalyst. Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

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.     

Enzymatic interesterification of tallow-sunflower oil mixtures

In an effort to improve the physical and/or thermal characteristics of solid fats, the enzymatic interesterification of tallow and butterfat with high-oleic sunflower oil and soybean oil was investigated. The two simultaneously occurring reactions, interesterification and hydrolysis, were followed by high-performance liquid chromatography of altered glycerides and by gas-liquid chromatography of liberated free fatty acids. The enzymes used in these studies were immobilized lipases that included either a 1,3-acyl-selective lipase or acis-9-C₁₈-selective lipase. The degree of hydrolysis of the fat/oil mixtures was dependent upon the initial water content of the reaction medium. The extent of the interesterification reaction was dependent on the amount of enzyme employed but not on the reaction temperature over the range of 50–70°C. Changes in melting characteristics of the interesterified glyceride mixtures were followed by differential scanning calorimetry of the residual mixed glycerides after removal of free fatty acids. Interesterification of the glyceride mixes with the two types of enzymes allowed for either a decrease or increase in the solid fat content of the initial glyceride mix.     

Further improvements in the yield of monoglycerides during enzymatic glycerolysis of fats and oils

Three approaches were used in an effort to increase the yield of monoglycerides (MG) during the lipase catalyzed reaction of glycerol with triglyceride fats and oils: i) various commercially available lipases were screened for ability to catalyze MG synthesis; ii) mixtures of lipases were compared with single lipases; and iii) two-step temperature programming was applied during the reaction. Of these, temperature programming was found to be the most effective. With an initial temperature of 42°C for 8–16 hr followed by incubation at 5°C for up to 4 days, a yield of approximately 90 wt% MG was obtained from beef tallow, palm oil and palm stearin. When the second incubation temperature was greater than 5°C, the yield of MG was progressively lower with increasing temperature. In the case of screening of newly available commercial lipase preparations, lipases fromPseudomonas sp. were found to be most effective, giving a yield of approximately 70 wt% MG at 42°C from tallow. Lipases fromGeotrichum candidum, Penicillium camembertii (lipase G) andCandida rugosa were inactive. A mixture of lipases fromPenicillium camembertii andHumicola lanuginosa was found to be more effective than either enzyme alone, giving a yield of approximately 70 wt% MG using beef tallow or palm oil. A mixture ofPenicillium camembertii lipase with eitherPseudomonas fluorescens lipase orMucor miehei lipase was not more effective thanPseudomonas fluorescens orMucor miehei lipase alone.     

Catalytic Behaviour of Lipase Free and Immobilized in Biphasic Membrane Reactor with Different Low Water-Soluble Substrates

The catalytic activity and reaction rate of lipase have been studied using the biocatalyst free in an organic/aqueous emulsion and immobilized in a biphasic organic/aqueous membrane reactor. The first reaction system was realized in a stirred tank reactor. The other was obtained by immobilizing the enzyme in the sponge layer of an asymmetric capillary membrane and recirculating the two phases along the two separate circuits of the membrane module. The performance of the reactors has been studied using two different low water-soluble substrates: triglycerides present in commercial olive oil and (R,S)-cyanomethyl-[2-(4-isobutylphenyl)propionate] (CNE). The effects of substrate viscosity and flow dynamics conditions, such as organic phase flow rate, on the biphasic membrane reactor performance have been evaluated on the basis of observed reaction rate and catalytic activity of free and immobilized lipase for both substrates. It has been observed that free lipase showed higher catalytic activity with olive oil, while immobilized lipase showed higher catalytic activity with CNE which has a lower viscosity than olive oil. The increase of organic phase flow rate negatively affected the reactor performance, with a minor effect when using CNE rather than olive oil. The influence of temperature on the biocatalyst performance with the two substrates has also been investigated. The optimal temperature value of lipase was different for the two substrates: 28°C with CNE and 40°C with olive oil. © 1997 SCI.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Interactions Between Oil Substrates and Glucose on Pure Cultures of Ruminal Lipase-Producing Bacteria

The hydrolysis of free fatty acids from lipids is a prerequisite for biohydrogenation, a process that effectively saturates free fatty acids. Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens have long been thought to be the major contributors to ruminal lipolysis; however, Propionibacterium avidum and acnes recently have been identified as contributing lipase activity in the rumen. In order to further characterize the lipase activity of these bacterial populations, each was grown with three different lipid substrates, olive oil, corn oil, and flaxseed oil (3 %). Because different finishing rations contain varying levels of glycogen (a source of free glucose) this study also documented the effects of glucose on lipolysis. P. avidum and A. lipolyticus 5s demonstrated the most rapid rates (P < 0.05) of lipolysis for cultures grown with olive oil and flaxseed oil, respectively. A. lipolyticus, B. fibrisolvens, and P. avidum more effectively hydrolyzed flaxseed oil than olive oil or corn oil, especially in the presence of 0.02 % glucose. Conversely, P. acnes hydrolyzed corn oil more readily than olive oil or flaxseed oil and glucose had no effect on lipolytic rate. Thus, these bacterial species demonstrated different specificities for oil substrates and different sensitivities to glucose.     

Processing for industrial fatty acids - II

Fatty acids are produced industrially from tallow, palm oil, palm stearin, palm kernel oil and coconut oil. The current and future supply situations of these raw materials and market economics favor palm stearin and palm kernel oil as major raw materials for fatty acids. The Malaysian oleochemical industry has adopted high-temperature and high-pressure “splitting” of triglycerides. Variations in product yields occurring in the processing of tallow and palm stearin and of coconut oil and palm kernel oil are indicated. Developments on the enzymic hydrolysis of triglycerides to fatty acids have been made, particularly in Japan. Enzymic hydrolysis at low temperature has the advantage of energy conservation compared to the high-temperature and pressure-splitting process. But enzymic hydrolysis is only applicable to triglycerides of low titre, such as palm kernel oil.     

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.     

Solid phase enzymatic glycerolysis of beef tallow resulting in a high yield of monoglyceride

A mixture of mono-, di- and triglycerides was obtained when beef tallow was reacted with glycerol using lipase enzyme as a catalyst. The reaction was carried out batchwise in a small vessel with agitation by magnetic stirring. The yield of monoglyceride (MG) was greatly influenced by the reaction temperature—at higher temperatures (48–50°C) a yield of approximately 30% MG was obtained, while at lower temperatures (38–46°C) a yield of approximately 70% MG was obtained. A sharp transition was observed between the high and low yield equilibrium states. The temperature at which this transition occurred is called the critical temperature (T_c) and was found to be 46°C in the case of tallow. During the course of the reaction, when approximately 40% MG had been synthesized, the reaction mixture became solid but the reaction continued until approximately 70% MG had been synthesized. A yield of 70% MG also was obtained with tallow at 42°C when a glycerol/tallow mole ratio ranging from 1.5 to 2.5 was used. The free fatty acid content at equilibrium depended on the water concentration in the glycerol phase and varied from 0.5% to 11.0% when the water content ranged from 0.6% to 12.5%. Above 8% water content, the yield of MG was reduced. Of the commercially available lipases that were investigated, lipase fromPseudomonas fluorescens orChromobacterium viscosum resulted in the highest yield of MG.     

Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by ¹³C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.     

Recovery of hydroxy fatty acids from lesquerella oil with lipases

Two hydroxy acids, lesquerolic (53 wt%) and auricolic (4%), are present at significant quantities inLesquerella fendleri seed oil. Results reported here indicate the selective release of hydroxy fatty acids during hydrolysis of this oil catalyzed byRhizopus arrhizus lipase. For example, hydroxy acids composed 85–90 wt% of the free fatty acids released during lipolysis, as compared to 54% present overall in the oil. In addition, over 80% of the lesquerolic acid is released from the triglycerides. The reason for this lipase’s success was determined to be its 1,3-positional specificity. The vast majority of lesquerella oil’s hydroxy acids is at the 1- and 3-positions of its triglycerides, as confirmed by the compositional analysis of partial glycerides formed during lipolysis.     

Spoilage of coconut oil purification and properties of a fungal lipase that attacks coconut oil

A lipase was isolated from a strain ofAspergillus flavus which attacked coconut kernel and oil with the liberation of free fatty acids. The enzyme was purified 109-fold by ammonium sulphate precipitation, diethyl aminoethyl-cellulose and Sephadex G-200 chromatography. The optimum pH of the enzyme reaction was 6.2. The action of the enzyme on pure triglycerides was studied. The triglycerides of the shorter chain fatty acids were more rapidly hydrolyzed, while hydrolysis of tristearin was not detected under the conditions of assay. K _m for trilaurin and trimyristin were 9.09×10⁻⁴ and 1.42×10⁻³ M, respectively. Para-chloromercuricbenzoate was an inhibitor. Thin layer chromatography and gas liquid chromatography of the esterified products of enzymatic hydrolysis of coconut oil showed the presence of oleate, palmitate, myristate, laurate, caprate, caproate and caprylate but not stearate, although stearate was present in coconut oil.     

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1∶2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40°C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7–36.5°C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5°C; JAOCS 80, 341–346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.     

Enzymatic conversion of steryl esters to free sterols

Conversion of oleic acid phytosteryl esters (OASE) to free phytosterols (referred to as sterols) by an enzymatic process was attempted. Enzymatic hydrolysis of OASE reached a steady state at 55–60% hydrolysis, but addition of methanol (MeOH) significantly accelerated the conversion of OASE to sterols. Screening of commercially available enzymes indicated that Pseudomonas aeruginosa lipase was most effective for the conversion. Based on the study of several factors affecting the reaction, the optimal conditions were determined as follows: ratio of OASE to MeOH, 1∶2 (mol/mol); water content, 10 wt%; lipase amount, 20 U/g by weight of reaction mixture; temperature, 30°C. When the reaction was conducted for 48 h with stirring, the conversion reached 98%. FAME accumulated in the reaction mixture, but FFA did not, indicating that the FAME was poorly recognized as a substrate in the reverse conversion of sterols to OASE but the FFA was easily recognized as a substrate. The high conversion of OASE to sterols was therefore due to elimination of FFA from the reaction system. After the enzymatic reaction, the oil layer was fractionated at −20°C with 5 vol parts of n-hexane. Sterols were efficiently purified in the resulting precipitate (92% recovery, 99% purity).     

Enzymatic activity of Chromobacterium viscosum lipase in an AOT/Tween 85 mixed reverse micellar system

To enhance the Chromobacterium viscosum lipase (glycerol‐ester hydrolase; EC 3.1.1.3) activity for the reaction of water‐insoluble substrates, the AOT/isooctane reverse micellar interface was modified by co‐adsorption of a non‐ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non‐ionic surfactant and olive oil as a water‐insoluble substrate. An appreciable increase of lipase activity was observed and at higher W_o values (where W_o = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell‐shaped profile. The kinetic model for the lipase‐catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems. © 1999 Society of Chemical Industry