Enzymatic production of human milk fat substitutes containing palmitic and docosahexaenoic acids at sn-2 position and oleic acid at sn-1,3 positions

This paper studies the synthesis of structured triacylglycerols (STAGs) rich in palmitic and docosahexaenoic acids (PA and DHA) at sn-2 position and oleic acid (OA) at sn-1,3 positions by a four step process. First, triacylglycerols (TAGs) were obtained with 63–66 mol PA/100 mol total fatty acids and 10 mol DHA/100 mol by acidolysis of tuna oil and commercial PA, catalyzed by the non-positionally specific lipase Novozym 435. Then these TAGs were purified neutralizing the free fatty acids (FFAs) by KOH hydroethanolic solutions and extracting TAGs with hexane; these TAGs were completely recovered as pure TAGs (without FFAs). The third step involved the displacement of fatty acids located at sn-1,3 positions by acidolysis of PA and DHA enriched TAGs with OA rich FFAs, catalyzed by the sn-1,3 specific lipase DF from Rhizopus oryzae, immobilized on Accurel MP-1000; TAGs with 67 mol OA/100 mol at sn-1,3 positions and 52.1 and 15.4 mol PA and DHA, respectively, per 100 mol at sn-2 position were obtained. Both acidolysis reactions were carried out in stirred tank reactors (STRs) with lipase both dispersed in the reaction medium and contained in a cartridge filter attached to the stirrer rod. Finally STAGs were purified and obtained with yields of over 80 mol STAGs/100 mol STAGs in the reaction product (no FFAs were detected).     

Stereospecific analysis of triacylglycerols in camel (Camelus dromedarius) milk fat

In the lipid fraction of camel (Camelus dromedarius) milk the percentage of saturated fatty acids (SFAs) was 62.8% with a content of palmitic acid of 28.5%. The unsaturated fatty acid (UFA) fraction was dominated by oleic and palmitoleic acids. Enzyme digestion and chemical degradation methods were used to determine the intramolecular fatty acid (FA) composition and then the intermolecular FA distribution in the three sn-positions of the triacylglycerols of the camel milk. FAs showed a specific preference for a particular position: in all samples studied, SFAs were prevalently esterified in the sn-2 position, while UFAs occupied mainly the sn-1 and sn-3 position. As the carbon chain lengthened from 8 to 16 the percentages of SFAs decreased in the sn-2 position and increased in the outer positions. Such data indicated that the length of the carbon chain could be a discriminating factor in the acylation process of SFAs.     

Roasting effects on fatty acid distributions of triacylglycerols and phospholipids in sesame (Sesamum indicum) seeds

Sesame seeds were roasted at different temperatures (180–220 °C) using a domestic electric oven. The positional distribution of fatty acids in triacylglycerols (TAGs) and phosphatidylcholine (PC) isolated from total lipids in these seeds was investigated as well as the naturally occurring antioxidants that are present. Major lipid components were TAGs and phospholipids (PLs), while steryl esters (SEs), free fatty acids (FFAs) and sn‐1,3‐ and sn‐1,2‐diacylglycerols (DAGs) were minor ones. Following roasting, a significant increase (P < 0.05) was observed in FFAs and in both forms of DAG (primarily sn‐1,3‐DAG). The greatest PL losses (P < 0.05) were observed in phosphatidylethanolamine (PE), followed by PC and phosphatidylinositol (PI). On the other hand, the amounts of γ‐tocopherol and sesamin remained at over 80 and 90% respectively of the original levels after roasting at 220 °C. The principal characteristics of the positional distribution of fatty acids were still retained after 25 min of roasting: unsaturated fatty acids, especially linoleic and/or oleic, were predominantly concentrated in the sn‐2‐position, and saturated fatty acids, especially stearic and/or palmitic, primarily occupied the sn‐1‐ or sn‐3‐position. The results suggest that unsaturated fatty acids located in the sn‐2‐position are significantly protected from oxidation during roasting at elevated temperatures. © 2001 Society of Chemical Industry     

Distribution of fatty acids in triacylglycerols and phospholipids from peas (Pisum sativum L.)

BACKGROUND: The fatty acid distribution of triacylglycerols (TAG) and major phospholipids (PL) obtained from four varieties of peas (Pisum sativum) was investigated. The total lipids extracted from the peas were separated by thin layer chromatography into seven fractions. RESULTS: The major lipid components were PL (52.2–61.3%) and TAG (31.2–40.3%), while hydrocarbons, steryl esters, free fatty acids and diacylglycerols (sn‐1,3 and sn‐1,2) were also present in minor proportions (5.6–9.2%). The main PL components isolated from the four varieties were phosphatidylcholine (42.3–49.2%), phosphatidylinositol (23.3–25.2%) and phosphatidylethanolamine (17.7–20.5%). Significant differences (P < 0.05) in fatty acid distribution were found for different pea varieties. Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. However, the principal characteristics of the fatty acid distribution in the TAG and three PL were evident among the four varieties: unsaturated fatty acids were predominantly located in the sn‐2 position while saturated fatty acids primarily occupied the sn‐1 or sn‐3 position in the oils of the peas. CONCLUSION: These results should be useful to both producers and consumers for the manufacture of pea foods in Japan. Copyright © 2007 Society of Chemical Industry     

Production and structural analysis of triacylglycerols containing capric acid and conjugated linoleic acid isomers obtained by enzymatic acidolysis

BACKGROUND: Structured lipids containing medium‐chain fatty acids have interesting applications as reduced‐calorie fats; moreover, conjugated linoleic acid (CLA) isomers have shown interesting biological properties. The aim of this study was to synthesize triacylglycerols (TAGs) containing capric acid in the sn‐1‐ and sn‐3‐ positions and CLA isomers in the sn‐2‐ position, using different commercial available lipases. RESULTS: The homogeneous CLA‐TAGs (Tri‐CLA) were chemically synthesized starting from glycerol and CLA isomers, 9‐cis,11‐trans and 10‐trans,12‐cis CLA. The acidolysis reactions of Tri‐CLA with capric acid were carried out at 55 °C for different times in hexane; after 96 h the acidolysis average yield was 65%. The best capric acid incorporation in total TAGs was obtained after 96 h with Lipozyme IM (56.6%). The results of structural analysis carried out on the obtained TAGs showed that both Novozyme 435 and anhydrous Lipozyme IM gave the best incorporation of capric acid in sn‐1(3)‐ positions (61.8%). However, anhydrous Lipozyme IM gave also the highest CLA percent content in sn‐2‐ position (73.2%). CONCLUSION: Anhydrous Lipozyme IM appears to be the more effective enzyme in acidolysis reactions to obtain structured TAGs containing CLA isomers in the central position and capric acid at external positions. Copyright © 2009 Society of Chemical Industry     

The composition and structure of the lipids of sheep lymph

Phosphatidylcholine was the principal phospholipid component in the intestinal lymph of sheep and was accompanied by small amounts of phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine and sphingomyelin. Stereo-specific analysis of the triacylglycerols confirmed that palmitic acid was concentrated in position sn-2 and showed that there was a higher proportion of the remaining saturated fatty acids in position sn-1 than in position sn-3. In the phosphatidylcholine, saturated fatty acids were concentrated in position sn-1 and unsaturated in position sn-2, contrary to an earlier report.     

The effect of feed supplementation with dietary sources of n‐3 polyunsaturated fatty acids,flaxseed and algae Schizochytrium sp., on their incorporation into lipid fractions of Japanese quail eggs

The influence of feed supplemented with dietary sources of n‐3 polyunsaturated fatty acids (PUFAs), namely flaxseed and algae Schizochytrium sp., on the fatty acid composition of lipids from Japanese quail eggs was studied. Two groups of quails were fed for 19 weeks, starting from 6th week of life. Lipid fractions from dried egg mass of total of 4300 eggs of control and experimental group were analysed. The fatty acid profile was affected in both triacylglycerols and phospholipids. The significant (P < 0.05) increase in n‐3 PUFAs up to 7.33% in the total lipids, α‐linolenic acid (ALA, 18:3) up to 4.57% in triacylglycerols, docosahexaenoic acid (22:6, DHA) up to 23.91% in phosphatidylethanolamine and up to 4.35% in phosphatidylcholine as well as the significant (P < 0.05) reduction in n‐6/n‐3 ratio in the lipid fractions to the range of 0.9–3.7 was observed in experimental group. The incorporation of different n‐3 into lipids was selective: ALA was found in the triacylglycerol fraction, whereas EPA, DPA and DHA were detected in the phospholipid fractions, exclusively at sn‐2 position. The studies showed that the feeding quails with modified diets is effective way of biofortifying the eggs with nutritionally desired n‐3 PUFAs.     

Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil

BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn‐2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn‐3 position. RESULTS: Two types of lipase‐catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn‐2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn‐2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase‐catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase‐catalysed re‐esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn‐2 position was synthesised from pine nut oil via lipase‐catalysed redistribution and re‐esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts. Copyright © 2011 Society of Chemical Industry     

Optimisation of tripalmitin‐rich fractionation from palm stearin by response surface methodology

BACKGROUND: Solvent fractionation is effective in improving separation at low temperature, resulting in higher yield and purity of the final product. Tripalmitin (PPP) is an important substrate for the synthesis of human milk fat substitute (HMFS). In this study a fraction rich in PPP was separated from palm stearin by solvent fractionation. RESULTS: The PPP‐rich fraction was concentrated from palm stearin by acetone fractionation. Response surface methodology (RSM) was employed to optimise PPP purity (Y₁, %) and PPP content (Y₂, g kg^?1 palm stearin) with the independent variables fractionation temperature (X₁, 25, 30 and 35 °C) and weight ratio of palm stearin to acetone (X₂, 1:3, 1:6 and 1:9). The predictive models for PPP purity and PPP content of the solid fraction were adequate and reproducible, with no significant lack of fit and satisfactory levels of R². PPP purity showed a positive correlation with temperature and acetone ratio, whereas PPP content exhibited a negative correlation. The optimised fractionation condition for a targeted PPP‐rich fraction with > 92% PPP purity and > 225 g kg^?1 PPP content from palm stearin was predicted. CONCLUSION: The RSM model for optimising PPP purity and PPP content in the PPP‐rich fraction from palm stearin by acetone fractionation was valid. The scaled‐up PPP‐rich fraction obtained can be used as a substrate for the synthesis of 1,3‐dioleoyl‐2‐palmitoylglycerol, which is a main component of HMFS in infant formulas. Copyright © 2010 Society of Chemical Industry     

Enzymatic interesterification of tripalmitin with vegetable oil blends for formulation of caprine milk infant formula analogs

The structure of triacylglycerols in vegetable oil blends was enzymatically modified, and the blends were incorporated into skim caprine milk to produce goat milk-based infant formula analogs, homologous to human milk. A modified lipid containing palmitic, oleic, and linoleic acids, resembling the composition of human milk fat, was synthesized by enzymatic interesterification reactions between tripalmitin and a vegetable oil blend containing a 2.5:1.1:0.8 ratio of coconut, safflower, and soybean oils. A commercial sn-1,3-specific lipase obtained from Rhyzomucor miehei, Lipozyme RM IM, was used as the biocatalyst. The effects of substrate molar ratio and reaction time on the incorporation of palmitic, oleic, and linoleic acids at the sn-2 position of the triacylglycerols were investigated. The fatty acid composition and sn-2 position of the experimental formulas were analyzed using gas chromatography. Results showed that the highest incorporation of palmitic acid was obtained at 12 h of incubation at 55°C with a substrate molar ratio of 1:0.4 of tripalmitin to vegetable oil blend. However, the modified milk interesterified for 12 h at a 1:1 molar ratio had a greater resemblance to human milk compared with the other formulas. The level of oleic acid incorporation at the sn-2 position increased with the molar ratio of tripalmitin to vegetable oil blend. It was concluded that, unlike the original goat milk and other formulas, the formulated caprine milk with a molar ratio of 1:1 and a 12-h incubation was similar to the fatty acid composition of human milk.     

Seasonal variation in the positional distribution of fatty acids in bovine milk fat

The aim of this study was to determine the seasonal variation in the positional distribution of fatty acids (FA) in bovine milk fat. Bovine milk samples were collected from May 2017 to April 2018 in the Netherlands, and the FA composition in the sn-2 position was determined by using sn-1(3)-selective transesterification of Candida antarctica lipase B. The majority of the FA showed significant variation at sn-2 and sn-1(3) positions between different seasons. The seasonal variation in sn-2 position was higher than the sn-1(3) positions. Parallel to the changes in the diet of the cows throughout a year, we observed an increase in blood-derived FA (i.e. C18:0, C18:1 cis-9) concentrations and a decrease in de novo–synthesized FA during summer. In winter, more saturated FA were esterified in sn-2 position of milk fat. Highest concentrations of palmitic acid, C16:0, was observed in sn-2 position in winter, whereas the amount of unsaturated FA at this position was highest in summer. These results showed that the FA compositions in different regiospecific positions changed due to season; however, the proportions of a specific FA within the 3 positions of the triacylglycerols in milk fat did not change upon seasonal variation.     

Roasting effects on fatty acid distribution of triacylglycerols and phospholipids in the kernels of pumpkin (Cucurbita spp) seeds

In this study, changes in fatty acid distributions of pumpkin seeds (Cucurbita spp) in the course of the roasting process were investigated. Whole pumpkin seeds were exposed to microwaves for 6, 12, 20 or 30 min at a frequency of 2450 MHz. The kernels were separated from the seeds, and the lipid components and the fatty acid distributions of triacylglycerols (TAGs) and phospholipids (PLs) were analysed by a combination of thin layer chromatography and gas chromatography. Major lipid components were TAGs, free fatty acids (FFAs) and PLs, while steryl esters and diacylglycerols were also present in minor proportions. The greatest PL losses (p < 0.05) were observed in phosphatidyl ethanolamine, followed by phosphatidyl choline or phosphatidyl inositol. With a few exceptions, significant differences (p < 0.05) in fatty acid distributions occurred when the seeds were microwaved for 20 min or more. Nevertheless, the positional characteristics of fatty acid distributions in the TAGs were still retained after 20 min of roasting: unsaturated fatty acids were predominantly concentrated in the sn‐2‐position, and saturated fatty acids primarly occupied the sn‐1‐ or sn‐3‐position. These results suggest that no significant changes in fatty acid distribution of TAGs and PLs would occur within 12 min of microwave roasting, thus ensuring that a good‐quality product would be obtained. Copyright © 2005 Society of Chemical Industry     

Fatty acid distribution in triacylglycerols and phospholipids of broad beans (Vicia faba)

Fatty acid distribution of triacylglycerols (TAG) and phospholipids (PL) obtained from the four cultivars of broad beans (Vicia faba) was investigated. Total lipids extracted from the beans were separated by thin-layer chromatography into eight fractions. The major lipid components were TAG (47.7–50.1%) and PL (47.5–50.5%), while hydrocarbons, steryl esters, free fatty acids, diacylglycerols (1,3-DAG and 1,2-DAG) and monoacylglycerols were present in minor proportions (1.8–2.4%). The major PL components were phosphatidylcholine (56.4–58.4%), phosphatidylethanolamine (20.3–21.7%) and phosphatidylinositol (16.6–18.6%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. No significant differences (P > 0.05) in fatty acid distribution existed when the different cultivars were compared. The fatty acid distributions in the TAG were evident among the four cultivars: unsaturated fatty acids were predominantly concentrated in the sn−2 position, and saturated fatty acids primarily occupied the sn−1 or sn−3 position in the oils. These results could be useful to both consumers and producers for manufacture of traditional foods in Japan and elsewhere.     

Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn-1 and sn-2 positions

Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [¹⁴C]palmitic acid (90%) and [³H]oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A₂-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.     

Intramuscular Lipids and Triglyceride Structures in Range and Feedlot Steers

Longissimus muscle of feedlot steers contained less moisture and more triglycerides (TG) but similar amounts of cholesterol and lipid phosphorus when compared to muscle of range steers. A 100-g portion of the muscle from feedlot steers provided more of all fatty acids analyzed except iso- and anteiso-tridecanoic and pentadecanoic acids (br 13:0, br 15:0) and octadecatrienoic acid (18:3). Structures of the intramuscular TG were not influenced by dietary treatment. Medium chain saturated acids (10:0 to 15:0) were distributed throughout all 3 positions, 16:0 predominated in the sn-1 position, 18:0 and 20:0 in the sn-1 and sn-3 positions. Br 13:0 and br 15:0 predominated in the sn-2 position and were essentially absent from the sn-3 position. Monounsaturated acids, with the exception of trans octadecenoic acid (trans 18:1), were found mainly in the sn-2 and sn-3 positions. Trans 18:1 predominated in the sn-1 and sn-3 positions thereby resembling a long chain saturated acid, Octadecadienoic acid (18:2) and 18:3 were about equally divided between positions sn-2 and sn-3. Tram 18:1 comprised 1.9 and 1.3% of total intramuscular fatty acids from range and feedlot steers, respectively. The structures of intramuscular TG, with respect to 18:2 and saturated acids, did not resemble those of native peanut oil which has been reported to be more atherogenic than randomized peanut oil.     

Characterization of Low Saturation Palm Oil Products after Continuous Enzymatic Interesterification and Dry Fractionation

ABSTRACT:  The lipase-catalyzed interesterification of refined, bleached, deodorized palm olein with iodine value (IV) of 62 was studied in a pilot continuous packed-bed reactor operating at 65 °C. Sn- 1,3 specific immobilized enzyme; Lipozyme® TL IM ( Thermomyces Lanuginosa ) from Novozyme A/S was used in this study. The interesterification reaction produced fully solidified fats at ambient temperature due to the production of trisaturated triacylglycerols (TAG) (PPP and PPS, where P = palmitic acid, S = stearic acid). The reaction also increased the percentage of triunsaturated TAG (OLL, OLO, and OOO, where O = oleic acid, L = linoleic acid). The interesterified product was then dry fractionated at temperatures of 9, 12, 15, 18, and 21 °C to separate the saturated fats from the unsaturated. The results show that IV of olein increased when the fractionation temperature (T_FN) decreased. The highest IV of olein was 72, obtained from T_FN at 9 °C. After interesterification and laboratory-scale fractionation, the olein fractions contained higher unsaturation content ranging from 64.7% to 67.7% compared to the starting material (58.3%), while the saturation content was reduced from 41.7% to the range of 32.3% to 35.3%. The yields of these oleins were low with the range of 24.8% to 51.8% due to the limitation of the vacuum filtration. Ten kilograms of pilot-scale fractionation with membrane press filter was used to determine the exact olein yield. At T_FN of 12 °C, 67.1% of olein with saturation content of 33.9% was obtained.     

Stereospecific Positional Distribution of Fatty Acids of Camellia (Camellia japonica L.) Seed Oil

Abstract: The stereospecific positional distribution of fatty acids of camellia seed oil (also called camellia oil) was determined. The camellia oil was mainly composed of neutral lipids (88.2%), and the oleic acid (86.3%) was found to be a major fatty acid of neutral lipids. In the glycolipids and phospholipids, the oleic acid was also found to be a major fatty acid at 62.5% and 54.2%, respectively. The oleic acid was distributed abundantly in all sn‐1, 2, and 3 positions. It was found that the oleic acid was present more at sn‐2 (93.6%) and 3 positions (94.7%), than at sn‐1 position (66.0%). Practical Application: The information of stereospecific positional distribution of fatty acids in the camellia oil can be used for the development of the structured lipids for food, pharmaceutical, and medical purposes.     

Preparation of palm olein enriched with medium chain fatty acids by lipase acidolysis

Medium chain (MC) fatty acids, caprylic (C8:0) and capric (C10:0) were incorporated into palm olein by 1,3-specific lipase acidolysis, up to 36% and 43%, respectively, when added as mixtures or individually after 24 h. It was found that these acids were incorporated into palm olein at the expense of palmitic and oleic acids, the former being larger in quantity and reduction of 18:2 was negligible. The modified palm olein products showed reduction in higher molecular weight triacylglycerols (TGs) and increase in concentration of lower molecular weight TGs compared to those of palm olein. Fatty acids at sn-2 position in modified products were: C10:0, 4%; C16:0, 13%; C18:1, 66%; and C18:2, 15.4%. DSC results showed that the onset of melting and solids fat content were considerably reduced in modified palm olein products and no solids were found even at and below 10 °C and also the onset of crystallisation was considerably lowered. The cloud point was reduced and iodine value dropped from 55.4 to 38 in modified palm olein. Thus, nutritionally superior palm olein was prepared by introducing MC fatty acids with reduced palmitic acid through lipase acidolysis.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Cholesterol-lowering food additives: lipase-catalysed preparation of phytosterol and phytostanol esters

We have shown recently that plant steryl and stanyl esters, used as plasma cholesterol-lowering food supplements, can be efficiently prepared from the sterols and stanols via lipase-catalysed esterification with fatty acids and transesterification with fatty acid methyl esters or triacylglycerols [Weber, N., Weitkamp, P., & Mukherjee, K. D. (2001). Journal of Agricultural and Food Chemistry, 49, 67–71]. In continuation of this work, sterols contained in three different steam distillates obtained from rapeseed oil by conventional deodorization or from rapeseed oil or a mixture of soybean and rapeseed oils by physical refining have been converted to a high degree in situ to the corresponding long-chain acyl esters via esterification and/or transesterification with fatty acids and/or triacylglycerols using lipase from Candida rugosa as biocatalyst in vacuo (20–40 mbar) at 40 °C. The steryl esters formed were purified to ⩾90% by deacidification, flash chromatography on silica gel and solvent fractionation using acetone-water (9:1,v/v).