Characterization and Oxidative Stability of Human Milk Fat Substitutes Enzymatically Produced from Palm Stearin

Production of human milk fat substitutes (HMFSs) from three types of palm stearin with palmitic acid (PA) of 91.3, 70.3 and 62.6 %, respectively, was scaled up to a kilogram scale. The physiochemical properties of these products including fatty acid profiles, triacylglycerol compositions, tocopherol contents, oxidative stability and melting and crystallization profiles were compared with those of HMFSs from lard, butterfat and tripalmitin and fats from infant formulas. Based on their chemical compositions, HMFSs from palm stearin with PA contents of 70.3 and 62.6 % produced by enzymatic acidolysis were found to have the highest degree of similarity to human milk fat, which indicated that these HMFSs were the most suitable for use in infant formulas. However, HMFSs from palm stearin with PA content of 91.3 % had the highest tocopherol contents. By investigation of the primary and secondary oxidation products during accelerated oxidation, the oxidative stability of HMFSs was found to be positively correlated to the contents of tocopherols, and the volatile oxidation compounds with the highest relative contents in HMFSs were aldehydes analyzed by solid-phase microextraction-GC–MS. All HMFSs had final melting points lower than body temperature.     

Characterization and Oxidative Stability of Structured Lipids: Infant Milk Fat Analog

Structured lipids (SLs) for infant milk formulation were produced by enzymatic interesterification of tripalmitin with vegetable oil blends and fish oil. The SLs were characterized by fatty acid content and structure, melting profiles, oxidative stability index, free fatty acid (FFA) concentration, and tocopherol content. Oxidative stability was studied using accelerated methods by quantifying FFA, peroxides (peroxide value) and aldehydes (p-anisidine value) production. Total oxidation (TOTOX value) was calculated as 2 × (peroxide value) + (p-anisidine value). The structured lipids after purification by distillation and addition of antioxidants had melting profiles, oxidative stability index, and initial FFA concentration that were similar to that of the starting oil blends, while the fatty acid composition and structure of the SLs were similar to that of human milk fat. Oxidative stability of the SLs was improved with tocopherol addition as antioxidants and was comparable to that of the vegetable oils and oil blends.     

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 Interesterification of Lauric Fat Blends Formulated by Grouping Triacylglycerol Melting Points

Lauric fat blends (appreciable amount of lauric fat with liquid oil and hard fat) initially formulated for shortening production by grouping triacylglycerol (TAG) melting points were further modified by enzymatic interesterification (EIE) to improve their key functionalities as plastic fats. At a similar fat blend formulation, only the high melting fat and medium melting fat were interesterified in binary‐EIE. Meanwhile, both fats and the liquid oil were interesterified in ternary‐EIE. The solid fat content (SFC) of all binary‐EIE blends was generally retained as similar in the temperature range between 0 and 20 °C when the amount of unsaturated TAGs was limited by excluding the liquid oil during EIE. However, the SFC was significantly reduced at temperatures above 20 °C compared to that of the initial blends. Furthermore, the melting point of binary‐EIE blends at BH50H15 formulation prepared with palm stearin and fully hydrogenated rapeseed oil as the hard fat was found to be drastically reduced from 54.6 to 35.3 °C and from 62.8 to 39.2 °C, respectively. In contrast, the SFC of ternary‐EIE blends was generally reduced when more unsaturated TAGs were available for EIE by including the liquid oil. However, higher SFC was noticed at temperatures around 10 °C in ternary‐EIE blends, as the amount of high‐melting fractions in their initial blends was increased from BH50H5 to BH50H15. Eventually, both binary and ternary‐EIE were also found to significantly alter the crystal microstructure of lauric fat blends, in terms of crystal morphology, size and network density.     

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel^® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).     

Effect of Palm Oil Enzymatic Interesterification on Physicochemical and Structural Properties of Mixed Fat Blends

The physicochemical properties of binary and ternary fat systems made of commercial samples of palm oil (PO) blended with anhydrous milk fat (AMF) and/or rapeseed oil (RO) were studied. Physical properties such as solid fat content by pulsed‐Nuclear Magnetic Resonance (p‐NMR), melting profile by differential scanning calorimetry (DSC), and polymorphism of the blends were investigated. Palm oil was then batch enzymatically interesterified for 27 h, using Lipozyme^® TL IM as biocatalyst, and further blended with AMF and/or RO in the same way. The objective of the present work was to evaluate the effect of batch enzymatic interesterification (B‐EIE) of palm oil on physical characteristics of the investigated fat blends. For that purpose, iso‐solid diagrams have been constructed from p‐NMR data. It was shown that B‐EIE of palm oil modifies its melting behaviour, but also its polymorphic stability and miscibility with other fats. Under dynamic conditions, after B‐EIE, the non‐ideal behaviour (eutectic) detected at low temperatures in the ternary PO/AMF/RO system disappears in the corresponding EIE‐PO/AMF/RO. After static crystallization followed by a tempering, the hardness of palm oil is increased after B‐EIE, as well as the hardnesses of the blends containing this fat compared to the native one. Polymorphism stability of the binary and ternary fat systems is also modified after B‐EIE compared to the corresponding native systems.     

Enzymatic Interesterification of Palm Oil and Fractions: Monitoring the Degree of Interesterification using Different Methods

Interesterification is an important modification technique for fats and oils resulting in the redistribution of the fatty acids among the glycerol backbone and thus changing the physico-chemical properties of the modified fat. In this study palm oil, palm olein and soft palm mid fraction (PMF) were subjected to both enzymatic (batchwise) and chemical interesterification. The reaction products were characterized before, during and after interesterification by HPLC, pulsed NMR (p-NMR) and differential scanning calorimetry (DSC). Interesterification led to more uniform triacylglycerol (TAG) compositions with smaller differences in final physico-chemical properties between the studied substrates. The degree of interesterification was evaluated on the basis of TAG composition and solid fat content (SFC). Significant differences between both methods were observed. The degree of interesterification based on SFC is therefore a better tool to evaluate the rate constant of the reaction as the TAG composition method does not take into consideration the formation of positional isomers at the end of the enzymatic process.     

Enzymatic Interesterification of Extra Virgin Olive Oil with a Fully Hydrogenated Fat: Characterization of the Reaction and Its Products

The lipase-catalyzed interesterification of extra virgin olive oil (EVOO) and fully hydrogenated palm oil (FHPO) was studied in a batch reactor operating at 75 °C. The compositions of the semi-solid fat products depend on the reaction conditions and the initial ratio of EVOO to FHPO. The dependence of the quasi-equilibrium product TAG profile on the reaction time was determined for initial weight ratios of EVOO to FHPO from 80:20 to 20:80. Lipozyme TL IM, Lipozyme RM IM and Novozym 435 were employed as biocatalysts. The interesterification reaction was optimized with respect to the type and loading of biocatalyst. Equilibrium was approached in the shortest time with Novozym 435 (80% conversion in 4 h). The chemical, physical, and functional properties of the products were characterized. Appropriate choices of the reaction conditions and the initial ratio of EVOO to FHPO lead to TAG with melting profiles and solid fat contents similar to those of commercial products. Differences were observed in the solid fat contents, melting profiles, and oxidative stabilities of the various interesterified products and also between the indicated properties of each category of product and the corresponding physical blend of the precursor reagents.     

Studies in Interesterification — Part I Production of Edible Fats from Vegetable Oils by Directed Interesterification

A modification of the Eckey's directed interesterification process of individual fats and their mixtures has been studied. The fat after directed interesterification with NaOMe catalyst (0.4% w/w) was first chilled at 15° C for variable lengths of time (30 mts. — 180 mts.) and then subjected to further reaction at ca. 23° C from 12 hrs. — 168 hrs. depending on the type of fat and products desired to produce fats of different melting ranges. It was established that the above pretreatments influenced the end products but a minimum content of ca. 20% saturated fatty acids over C₁₆ was necessary to produce a fat of melting range 36°–38° C.     

Microstructure and Thermal Profile of Structured Lipids Produced by Continuous Enzymatic Interesterification

Enzymatic interesterification has been shown to be an alternative for the production of structured lipids resembling human milk fat. The knowledge of the physical properties of fat is an important tool for the implementation of this fat in a food matrix. The enzymatic interesterification reaction modifies the composition of triacylglycerols changing the crystallization properties and polymorphic form of fats. Blends containing different proportions of lard and soybean oil (80:20, 70:30, 40:60, 30:70 and 20:80) were enzymatically interesterified in a continuous flow tubular reactor and analyzed for crystalline structure by polarized light microscopy, the polymorphic form using X-ray diffraction and thermal properties by differential scanning calorimetry. The structural modifications resulting from continuous enzymatic interesterification changed the crystallization behavior and thermal profile of the samples, reducing enthalpy values. Structural changes were also evident on polarized light microscopy images, disclosing an increase in the crystallization rate among the samples after the continuous enzymatic reaction.     

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor. In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.     

Characterization of Oxidative Stability of Fish Oil- and Plant Oil-Enriched Skimmed Milk

The objective of this research was to determine the oxidative stability of fish oil blended with crude plant oils rich in naturally occurring antioxidants, camelina oil and oat oil, respectively, in bulk and after supplementation of 1 wt% of oil blends to skimmed milk emulsions. Ability of crude oat oil and camelina oil to protect fish oil in bulk and as fish oil-enriched skimmed milk emulsions was evaluated. Results of oxidative stability of bulk oils and blends assessed by the Schaal oven weight gain test and by the rancimat method showed significant increase in oxidative stability when oat oil was added to fish oil in only 5 and 10 %, whereas no protective effect of camelina oil was observed when evaluated by these methods. Moreover, fish oil blended with oat oil conferred the lowest PV and lower amounts of volatile compounds during the storage period of 14 days at 4 °C. Surprisingly, skimmed milk supplemented with fish-oat oil blend gave the highest scores for off-flavors in the sensory evaluation, demonstrating that several methods, including sensory analysis, should be combined to illustrate the complete picture of lipid oxidation in emulsions.     

Utilization of Olive-Pomace Oil for Enzymatic Production of Cocoa Butter-like Fat

Refined olive-pomace oil (ROPO) was utilized as a source for the production of a cocoa butter (CB)-like fat. Immobilized sn-1,3 specific lipase-catalyzed acidolysis of ROPO with palmitic (PA) and stearic (SA) acids was performed at various substrate mole ratios (ROPO:PA:SA) to produce major triacylglycerols (TAGs) of CB. Products obtained for various substrate mole ratios were compared to a commercial CB in terms of TAG content, melting profile, solid fat content (SFC) and microstructure. The fat produced at a substrate mole ratio of 1:2:6 was the most similar to CB. The product contained 11% POP, 21.8% POS, 15.7% SOS while commercial CB contained 18.9% POP, 33.1% POS and 24.7% SOS. The product had a melting peak of 29.9 °C while CB had one of 28.5 °C. Polarized light microscope (PLM) images showed that fat crystal network microstructures of this product and CB were very similar.     

Preparation and Characterization of Human Milk Fat Substitutes Based on Triacylglycerol Profiles

Human milk fat substitutes (HMFS) having similarity in (TAG) composition to human milk fat (HMF) were prepared by Lipozyme RM IM‐catalyzed interesterification of lard blending with selected oils in a packed bed reactor. Four oil blends with high similarity in fatty acid profiles to HMF were first obtained based on the blending model and then the blending ratios were screened based on TAG composition similarity by enzymatic interesterification in a batch reactor. The optimal ratio was determined as 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. This blending ratio was used for a packed bed reactor and the conditions were then optimized as residence time, 1.5 h; reaction temperature, 50 °C. Under these conditions, the obtained product showed high degrees of similarity in fatty acid profile with 39.2 % palmitic acid at the sn‐2 position, 0.5 % arachidonic acid (n‐6) and 0.3 % docosahexaenoic acid (n‐3) and the scores for the degree of similarity in TAG composition was increased from 58.4 (the oil blend) to 72.3 (the final product). The packed bed reactor could be operated for 7 days without significant decrease in activity. The final product presented similar melting and crystallization profiles to those of HMF. However, due to the loss of tocopherols during deacidification process, the oxidative stability was lower than that of the oil blend. This process for the preparation of HMFS from lard with high similarity in TAG composition by physical blending and enzymatic interesterification, as optimized by mathematical models in a packed bed reactor, has a great potential for industrialization.     

Enzymatic Interesterification of High Oleic Sunflower Oil and Tripalmitin or Tristearin

The objective of this study was to produce low saturated, zero‐trans, interesterified fats with 20 or 30 % saturated fatty acids (SFA) such as C16:0 or C18:0. Tripalmitin (TP) or tristearin (TS) was blended with high oleic sunflower oil (HOSO) at different ratios (0.1:1, 0.3:1, and 0.5:1 [w/w]). Total C16:0 and C18:0 compositions of the resulting TP/HOSO and TS/HOSO blends, respectively, were plotted against blending ratios. Linear interpolation was used to estimate blending ratios that would yield physical blends (PB) with 20 or 30 % SFA. Interesterified blends (IB) were then synthesized from the customized PB using Lipozyme TL IM as the biocatalyst. Total and sn‐2 fatty acid compositions, triacylglycerol (TAG) molecular species, thermal behavior, and oxidative stability of PB and IB were compared. The total fatty acid compositions of PB and IB were similar but fatty acid positional distributions and TAG molecular species composition differed. IB contained 5–10 % more SFA at the sn‐2 position than corresponding PB. Furthermore, interesterification generated mono‐ and disaturated TAG species which resulted in broader melting profiles for IB. However, IB had lower oxidative stability than PB. The reformulation of food products with zero‐trans interesterified fats may be advantageous to the reduction of cardiovascular disease burden in the population.     

Synthesis of a Cocoa Butter Equivalent by Enzymatic Interesterification of Illipe Butter and Palm Midfraction

This study aims to synthesize a cocoa butter equivalent (CBE)‐structured lipid from a blend of illipe butter (IB) and palm midfraction (PMF) by means of enzymatic interesterification using Rhizomucor miehei sn‐1,3 specific lipase, Lipozyme^® RM IM (Novozymes North America, Inc., Franklinton, NC, USA) as the biocatalyst. Physical and chemical attributes of the CBE and cocoa butter (CB) were analyzed. The synthesized CBE matched the triacylglycerol (TAG) profile range of a commercial CB and is therefore hypothesized to show similar physical and chemical characteristics to CB. The TAG profile, fatty‐acid constituents, melting and cooling behavior, polymorphism, and crystal morphology were determined using high‐performance liquid chromatography, gas chromatography, differential scanning calorimetry, X‐ray diffraction (XRD), and polarized light microscopy, respectively. Four enzymatically interesterified blends of IB:PMF at different weight ratios were analyzed for their TAG profiles, and a ratio of IB:PMF 10:3 (%, w/w) at 5% enzyme load and a reaction time of 30 min gave similar TAG results to CB. The TAG values of the IB:PMF 10:3 interesterified product (IP) were 1,3‐dipalmitoyl‐2‐oleoylglycerol at 19.1 ± 1.0%, 1‐palmitoyl‐2‐oleoyl‐3‐stearoylglycerol at 42.7 ± 1.0%, and 1,3‐distearoyl‐2‐oleoylglycerol at 29.9 ± 0.3%. The melting and the cooling profile of IP and CB showed no significant difference. XRD of IP and CB displayed similar dominant peaks at 4.6 Å, representing a β polymorph. Both CB and IP have similar granular spherulitic crystals.     

Oxidative Alkene Cleavage by Chemical and Enzymatic Methods

The cleavage of alkenes to the corresponding carbonyl products is a widely employed method in organic synthesis, especially to introduce oxygen functionalities into molecules, remove protecting groups and tailor large molecules. Chemical methods available for alkene cleavage include, for instance, ozonolysis, several metal‐based variants (KMnO₄, OsO₄, RuO₄, etc.), electrochemical alternatives, singlet oxygen, hypervalent iodine and organic molecules in combination with oxygen. Furthermore, several enzymatic methods for alkene cleavage have been described to establish safe, mild and selective oxidation methods. Various heme and non‐heme iron‐dependent enzymes catalyse the alkene cleavage at ambient temperature and atmospheric pressure in an aqueous buffer, showing good chemo‐ and regioselectivities in selected cases. Quite recently some Cu‐, Mn‐ and Ni‐dependent enzymes have been identified for this reaction. This review gives an overview of the different chemical and enzymatic methods available for the cleavage of alkenes.

     

Dairy Lecithin from Cheese Whey Fat Globule Membrane: Its Extraction, Composition, Oxidative Stability, and Emulsifying Properties

An ethanol extraction method was studied for the production of dairy lecithin from cheese whey-derived milk fat globule membrane (MFGM). A two-step ethanol extraction of MFGM involving first extraction at pH 6.5, followed by second extraction at pH 4.5 yielded 17.2 % lipids. The extracted material contained about 90 % lipids, 4.5 % ash, and 1.2 % moisture. The phospholipid content of the ethanol extract was 31 % and the remainder was mostly neutral lipids. The phospholipid fraction contained 34 % sphingomyelin, 31 % phosphatidylcholine, 27 % phosphatidylethanolamine, 4.6 % phosphatidylserine, and 3.1 % phosphatidylinositol. Since the ethanol extract contained 31 % phospholipids, it can be technically termed as dairy lecithin. The major fatty acid components were linoleic acid (5.1 %), myristic acid (8.3 %), palmitic acid (29 %), stearic acid (14 %), oleic acid (25 %), and the remainder was minor fatty acids with chain length ranging from C4:0 to C22:5. The dairy lecithin was semi-solid at room temperature and exhibited a major phase transition at about 35 °C. Owing to its low polyunsaturated fatty acid content, the dairy lecithin was reasonably stable to oxidation as measured by the rate and extent of hexanal production during 35 days of storage at 45 °C. Oil-in-water emulsions made with less than 2 % dairy lecithin (relative to the total emulsion weight) were unstable; however, emulsions made with greater than 4 % dairy lecithin were very stable for more than 60 days at room temperature. The results of this study indicated that a highly functional dairy lecithin can be commercially produced using cheese whey-derived MFGM as the starting material.     

Lipase-Catalyzed Synthesis of Human Milk Fat Substitutes from Palm Stearin in a Continuous Packed Bed Reactor

Structured lipids (SL) with similar fatty acid (FA) composition and distribution to human milk fat (HMF) were synthesized by lipase-catalyzed acidolysis of chemically interesterified palm stearin (IV = 35.6) with mixed FA of stearic acid and myristic acid and FA from rapeseed oil, sunflower oil, and palm kernel oil in a continuous packed bed reactor. Response surface methodology (RSM) was used to optimize the reaction system with three selected parameters, namely residence time, temperature, and substrate molar ratio. The best-fitting quadratic models were obtained for the contents of palmitic acid (PA) and PA at the sn-2 position (sn-2 PA) by multiple regressions and the determination coefficient (R ²) values for the models were 0.9886 and 0.9799, respectively. The optimal conditions generated from the models were as follows: residence time, 2.7 h; temperature, 58 °C; substrate molar ratio, 9.5 mol/mol. Under these conditions, the contents of PA and sn-2 PA were 28.8 and 53.2%, respectively, and other FA observed in the experiments were all within the range of corresponding FA of HMF. The similarity of the product obtained to HMF was evaluated by the cited model. The scores for total and sn-2 FA of the product were 45.2 and 38.4, respectively, and the total score for the product was 83.6, which indicated a high degree of similarity of the product to HMF.     

Enzymatic Interesterification Between Pine Seed Oil and a Hydrogenated Fat to Prepare Semi-Solid Fats Rich in Pinolenic Acid and Other Polyunsaturated Fatty Acids

Enzyme catalyzed interesterification (EIE) of pine seed oil (PSO) and a fully hydrogenated soybean oil (FHSBO) were studied in batch reactors in solvent-free media to prepare different semisolid fats rich in polyunsaturated fatty acids (PUFA). Optimal operation conditions found were: 10 % (w/w) enzyme loading, 75 °C and magnetic agitation at 300 rpm. Quasi-equilibrium conditions were reached after 2, 3 and 6 h, when immobilized lipases from Thermomyces lanuginosus (Lipozyme^® TL IM), Candida antarctica B. (Novozym^® 435) and Rhizomucor miehei (Lipozyme^® RM IM) from Novozymes A/S (Bagsvaerd, Denmark) were employed, respectively. Similar distributions of unsaturated to saturated fatty acid (UFA/SFA) residues along the glycerol backbone of the fat products were obtained with both non-selective and sn-1(3) regioselective lipases due to significant spontaneous acyl migration during the reaction. The products had higher UFA/SFA ratios at the sn-2 position (2.4–2.5, 1.4–1.7, and 0.5–0.8 for the trials involving 20, 40 and 70 % FHSBO, w/w, respectively) than the corresponding physical blends (0.8, 0.4 and 0.5, respectively). Fat products containing 3.1–11.6 % (w/w) pinolenic acid (Pn) and 16.1–35.7 % (w/w) linoleic acid (L) at the sn-2 position were prepared. The free acid contents of EIE products prepared with Lipozyme^® TL IM and Novozym^® 435 were 6.1–6.4 and 2.5–2.6, respectively. Residual activities of Lipozyme^® TL IM and Novozym^® 435 diminish by ca. 20 % after 9 reaction cycles.