Biocatalysed synthesis of sn-1,3-diacylglycerol oil from extra virgin olive oil

Enzyme and Microbial Technology 41 (2007) 727–732

Biocatalysed synthesis of sn-1,3-diacylglycerol oil from extra virgin olive oil F. Blasi, L. Cossignani, M.S. Simonetti, P. Damiani ∗ Section of Food Chemistry and Analysis, Department of Economic Science and Food Sciences, University of Perugia, Via S. Costanzo, 06126 Perugia, Italy Received 19 December 2006; received in revised form 14 June 2007; accepted 15 June 2007

Abstract Enzymatic synthesis of sn-1,3-diacylglycerols (sn-1,3-DAG) in two steps without isolation of the intermediates was investigated. Firstly ethanolysis of extra virgin olive oil (EVO) using immobilized non-regiospecific lipase from Candida antarctica (Novozym 435) was carried out to obtain glycerol (Gly) and fatty acid ethyl esters (FAEE). In the second step the ethanolysis products have been re-esterificated testing different sn-1,3regiospecific lipases, both immobilized and non-immobilized, in different reaction media, that is in the presence of solvents or in a solvent-free system, for different times, at different temperatures (12, 25 and 40 ◦ C). The lipase from Rhizomucor miehei (Lipozyme IM) has been the most effective among the sn-1,3-specific lipases screened. © 2007 Elsevier Inc. All rights reserved. Keywords: sn-1,3-Diacylglycerols; Extra virgin olive oil; Novozym 435; Lipozyme IM

1. Introduction Diacylglycerols (DAG), naturally present as minor components in various edible oils and fats, are known to be used as additives or carriers in the food, medicine and cosmetic industry, sometimes in association with monoacylglycerols (MAG) [1]. DAG have been approved by United States Food and Drug Administration (USA FDA) for use as component of emulsifiers and accepted as “Generally Recognized As Safe” (GRAS) in 2000 and approved for many uses in foods [2]. Recently, lipid matrices containing more than 80% of DAG have been reported to be used as cooking oils with health benefits. In Japan, DAG oil has been approved for sale as a “Food for Specified Health Use” (FOSHU) in 1999 [3]. It has been shown recently that DAG oil has beneficial effects on the prevention and management of obesity compared with triacylglycerols (TAG), the main components of edible oils [4]. Clinical studies have shown that DAG oil reduces the postprandial increase in serum chylomicron TAG levels [5–7] prevents accumulation of body fat, especially visceral fat [6,8].

∗

Corresponding author. Tel.: +39 075 5857921; fax: +39 075 5857921. E-mail address: [email protected] (P. Damiani).

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.06.005

The taste and physicochemical properties of DAG oil are comparable to those of traditional vegetable edible oil [2]. The energy value of DAG oil and TAG oil measured by bomb calorimeter was virtually identical, that is 9.30 and 9.46 kcal/g, respectively; moreover no significant differences in absorption rates, 96.3% in both cases, were observed [5]. So the characteristic of DAG oil to reduce body fat accumulation is not caused by a difference in energy value or absorption rate, but it is attributable to the different metabolic mechanism of TAG and DAG [9]. It has been reported that heated or unheated DAG oil did not show any genotoxic effect [4]. Dietary intake of DAG at concentrations up to 6.0% for 24 months produced no signs of systemic toxicity and had no effect on the incidence of neoplastic findings in mice [10]. DAG at dietary concentrations up to 9.5% for 1 year had no effect on normal canine growth and development, in comparison to TAG [11]. Several chemoenzymatic [12] and biotechnological methods are available for the preparation of DAG [13]. Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are a group of ubiquitous enzymes, that can catalyse not only hydrolysis of fat and oils, but also various reverse reactions, such as esterification, acydolysis, inter-esterification and many others. Such biocatalysts present many advantages over chemical catalysts: their specificity, regioselectivity and enantioselectivity allow them to catalyze reactions under mild conditions of

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temperature and pressure, with lower side products and waste treatments costs [13]. Enzymatic synthesis of sn-1,3-DAG can be obtained by esterification of glycerol (Gly) with free fatty acid (FA) [14] or a variety of acyl donators (fatty acids alkyl and vinyl esters) [15], by glycerolysis of a fat (beef tallow, soybean oil, rapeseed oil) and Gly [3,16,17] or by selective hydrolysis [18]. A Japanese company patented method [19] is a preparation process of DAG which comprises reacting FA or lower alkyl ester thereof with Gly in the presence of immobilized lipase. The objective of this research is the biocatalysed synthesis of sn-1,3-DAG in two steps, without intermediate isolation in order to obtain a DAG oil for nutritional purposes using as starting material the extra virgin olive oil (EVO). The first step is represented by ethanolysis reaction of EVO with immobilized nonspecific lipase from Candida antarctica (Novozym 435), to obtain Gly and fatty acid ethyl esters (FAEE); these products have been re-esterified in the second step using a sn-1,3-specific lipase, to produce sn-1,3-DAG. The effects of different lipases, reaction time, medium and temperature were studied. FA compositions of EVO and of sn-1,3-DAG, produced from EVO, were determined too. Concerning the novelty of the presented paper, to the best of our knowledge this is the first work on exclusively enzymatic synthesis of DAG oil, starting from a vegetable oil without isolation of intermediates. 2. Materials and methods Immobilized lipases: lipase B from C. antarctica (Novozym 435), immobilized on acrilic macroporous resin, minimum 10,000 U/g, Sigma; lipase from Rhizomucor miehei (Lipozyme IM), immobilized on an anionic exchange resin, 42 U/g, Fluka; hog pancreas lipase, immobilized in Sol–Gel-AK, 43 U/g, Fluka; lipase from Pseudomonas cepacia, immobilized on ceramic elements, 15,156 U/g, Fluka. Non-immobilized lipases: lipase from Mucor javanicus, 400 U/mg, Sigma; hog pancreas lipase, 21 U/mg, Fluka; lipase from Pseudomonas sp. (Lipase PS), 30,000 U/g, Amano Pharmaceutical Co. Ltd. All chemicals and solvents were of analytical grade or of high-performance liquid chromatography (HPLC) grade. EVO was produced in Umbria (Italy).

2.1. Ethanolysis reaction EVO (100 mg) was mixed with ethanol 96% (0.5 mL) for emulsification of the reaction mixture, after which Novozym 435 (50 mg) was added. The reaction was carried out in an amber screw-capped vial at 45 ◦ C overnight, under magnetic stirring at 100 rpm. Initially, to set up the experimental conditions, an aliquot (2 ␮L) of the reaction mixture was withdrawn, added with 0.2 mL of the mixture chloroform/methanol (1:1, v/v) and filtered; 10 ␮L were analyzed by HPLC equipped with light scattering detector (HPLC-LSD), as reported in Section 2.3. The reaction was stopped by catalyst filtration (0.2 ␮m nylon membrane filter, Corning Incorporated, Corning, Germany); then the filtered enzyme was washed three times with the mixture chloroform/methanol (1:1, v/v), finally the solvents were evaporated under nitrogen stream. The reaction products, dissolved in the mixture chloroform/methanol (1:1, v/v), were analyzed by HPLC-LSD, as reported in Section 2.3.

viously washed three times with fresh dry ethanol, filtered under vacuum, oven-dried at 50 ◦ C and then stored in anhydrous ambient) at 12, 25 and 40 ◦ C under magnetic stirring in an amber opened vial, in a solvent-free system under vacuum (20 hPa, measured at room temperature). The reactions were also carried out with 1 mL of solvent (hexane or hexane:t-butanol mixture, 1:1), at the same temperatures. All the cited enzymatic reactions were carried out for 96 h; the reaction time was extended to 144 h, when the temperature was 12 ◦ C. Every 24 h an aliquot (2 ␮L) of the reaction mixture was withdrawn, then 0.2 mL of the mixture chloroform/methanol (1:1, v/v) was added and the solution was filtered; 10 ␮L were analyzed by HPLC-LSD, as reported in Section 2.3. Finally, the reactions were stopped by catalyst filtration and solvent evaporation. All reactions were carried out in triplicate. The data were calculated using the relative response factors (RRFs), obtained by analyzing in triplicate standard compound solutions at three different dilutions (the oleic acid concentration was double compared to that of the other compounds).

2.3. HPLC-LSD analysis 2.3.1. Apparatus The HPLC analyses were carried out using a gradient pump, Models 305 and 307 (Gilson, Middletown, WI, USA), a Lichrosorb Si-60 (5 ␮m, 250 mm × 4.0 mm i.d., Merck, Darmstad, Germany) column and a LSD (Sedex 55, S.E.D.E.RE., France), operating at 40 ◦ C and nitrogen pressure of 240 kPa. The chromatograms were acquired and the data handled using the Class-VP software (Shimadzu, Kyto, Japan). 2.3.2. Chromatographic conditions The samples were analyzed by gradient elution: the mixture hexane:isopropyl alcohol (95:5, v/v) was maintained for the first 6 min at flow rate of 0.7 mL/min, then it was changed to the mixture hexane:isopropyl alcohol (80:20, v/v) at flow rate of 1.0 mL/min, that was held for 20 min; then the column was reconditioned with hexane:isopropyl alcohol (95:5, v/v), flow rate 0.7 mL/min.

2.4. Analysis of fatty acid composition (mol%) The mol% acidic compositions of EVO and sn-1,3-DAG samples, analysed as fatty acid methyl esters (FAME), were determined by high resolution gas chromatography (HRGC). 2.4.1. Isolation of sn-1,3-DAG sn-1,3-DAG were isolated by thin layer chromatography (TLC) on silica gel plates (SIL G-25, 20 cm × 20 cm, 0.25 mm, Macherey-Nagel, Germany), treated with 5% boric solution (methanol/water 80:20, v/v), using hexane:diethyl ether (1:1, v/v) as developing mixture. The sn-1,3-DAG fraction (RF ∼ = 0.35) was scraped off and extracted with diethyl ether (2 mL × 3), then derivatized as reported in Section 2.4.2. 2.4.2. Preparation of FAME The EVO and sn-1,3-DAG FAME were prepared by direct transesterification: about 10 mg were dissolved with 3 mL of hexane and then 0.5 mL of 2N methanolic KOH was added; after 3 min, water (3 mL) was added and the upper organic phase was dried over anhydrous Na2 SO4 and then concentrated under nitrogen stream for HRGC analysis. 2.4.3. Apparatus A DANI 1000DPC gas-chromatograph (Norwalk, CT, USA), equipped with a split-splitless injector and with a flame ionization detector (FID) was used. The HRGC capillary column was a 100% polyethylene glycol, Zebron ZBWAX (30 m × 0.25 mm i.d., 0.25 ␮m f.t.) from Phenomenex, Torrance, CA. The chromatograms were acquired and processed using Clarity integration software (DataApex Ltd., Prague, Czech Republic).

2.2. Synthesis of sn-1,3-DAG The products of ethanolysis reaction, obtained without isolation, were reacted with Lipozyme IM or anhydrous Lipozyme IM (the enzyme was pre-

2.4.4. Chromatographic conditions The injector and detector temperature was 250 ◦ C. The oven temperature increased from 130 ◦ C to 250 ◦ C, at 3 ◦ C/min; the final temperature was

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Fig. 1. LSD-HPLC profile of TAG, FAEE, sn-1,3-DAG, sn-1,2(2,3)-DAG, FA and sn-1(3)-MAG standard compounds. held for 10 min. Carrier gas (He) flow rate was 1 mL/min; split ratio was 1:70.

2.5. HPLC-UV analysis 2.5.1. Derivatizing procedure About 10 mg of the reaction products (Section 2.2) were derivatized with a chiral agent (S)-(+)-1-(1-naphthyl)ethyl-isocyanate; the urethane derivatives, isolated by solid-phase extraction columns (SPE-ODS octadecylsilane (C18 ) ˚ 500 mg, Baker Analyzed) as described bonded to silica gel (40 ␮m ADP, 60 A); in a previous paper [20], were added with 1 mL of mobile phase, 0.4% (v/v) n-propanol (containing 2% water) in hexane, and filtered; 20 ␮L of the obtained solution were analyzed by HPLC equipped with UV detector. 2.5.2. Apparatus The HPLC analyses were carried out using a gradient pump Model LC-10AD VP (Shimadzu, Kyto, Japan), equipped with two silica gel columns in series (3 ␮m, 250 mm × 4.6 mm i.d., Hypersil, HiChrom Ltd.) and an UV detector, operating at 280 nm (Model 140 M, Portlab). The chromatograms were acquired and the data handled using the Class-VP software (Shimadzu, Kyto, Japan). 2.5.3. Chromatographic conditions The samples were analyzed by isocratic elution with the mixture 0.4% (v/v) n-propanol (containing 2% water) in hexane. The mobile phase flow was 1 mL/min.

3. Results and discussion The ethanolysis and re-esterification reactions were monitorated by a LSD-HPLC procedure. In Fig. 1 the HPLC separation of reference compounds was shown; the RRFs resulted in the following: 1.0 for triolein, 1.4 for ethyloleate, 0.8 for sn-1,3-dioleilglycerol, 1.1 for sn-1,2(2,3)-dioleilglycerol, 4.0 for oleic acid, 3.2 for sn-1(3)-monoleilglycerol. The first step in the synthesis of DAG oil was the enzymatic ethanolysis of EVO using a positionally nonspecific lipase, the Novozym 435. EVO was chosen as starting vegetable oil not only because it contains a valid antioxidant fraction (tocoferols, carotenoids and polyphenols) important for its beneficial influence on human health, but also because it is very easily available in Italy. At 45 ◦ C Novozym 435 catalysed the deacylation of EVO acylglycerols with very high yields; in fact the crude product (110.8 ± 1.5 mg, n = 6) contained FAEE (98.0%) and minor % contents of TAG (0.9%), sn-1(3)-MAG (0.3%), FA (0.8%) and only traces of DAG, as shown by HPLC profile reported in Fig. 2. For the successive step, the re-esterification reaction, seven commercial sn-1,3-specific lipases, immobilized and

Fig. 2. LSD-HPLC profile of EVO ethanolysis products.

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Fig. 3. Products of the re-esterification reaction in a solvent-free system catalysed at 40 ◦ C by Lipozyme IM (a) and anhydrous Lipozyme IM (b). The presented data are the average of triplicate experiments and error bars indicate standard deviation around the mean.

Fig. 4. Products of the re-esterification in a solvent-free system catalysed at 25 ◦ C by Lipozyme IM (a) and anhydrous Lipozyme IM (b). See legend of Fig. 3 for an explanation of error bars.

non-immobilized, from different sources were screened. The immobilized ones have the advantages of easy stopping of the reaction by filtration, the easy recover of the enzyme and the opportunity of reuse it. Lipozyme IM was found to give the best yield of esterification products in respect to the other six screened lipases (data not shown) and, therefore, was selected for use in the subsequent studies. Lipozyme IM exhibited both high regiospecificity and activity toward the FA, derived from EVO, in fact sn-1,2(2,3)-DAG in every reactions, at all tested temperatures, were maximum 3% (Figs. 3–5). Since Gly is immiscible with hydrophobic organic solvents, all attempts for its enzymatic esterification with FAEE both in hexane and in hexane:t-butanol (1:1) gave not good results (sn-1,3-DAG < 10%); for this reason the successive reactions were carried out in a solvent-free medium, at different temperatures. In synthetic reactions, the equilibrium composition at constant temperature can be modified in two ways: the first requires the removal of by-products and the other involves the use of excess reagent. When FAEE were used as acyl donors, ethanol produced during the esterification was removed carrying out the reaction in opened vials and applying vacuum (solvent-free system), to avoid acyl migration and an unfavourable equilibrium. Moreover, another important parameter in determining the type of products formed in a reversible reaction as esterification is the molar ratio of the substrates. In this study no excess reagent was used; in fact the molar ratio between FAEE and Gly, derived from ethanolysis of EVO, was not modified, because the aim of the work was not to change the qualitative composition of the starting oil.

Fig. 5. Products of the re-esterification reaction in a solvent-free system catalysed at 12 ◦ C by Lipozyme IM (a) and anhydrous Lipozyme IM (b). See legend of Fig. 3 for an explanation of error bars.

F. Blasi et al. / Enzyme and Microbial Technology 41 (2007) 727–732

As regards the reaction parameters, time and temperature, the results have been reported in Figs. 3–5. The MAG% composition was not represented because ever less than 1.0%. At 40 ◦ C both the reactions carried out with Lypozime IM (Fig. 3a) and with anhydrous Lypozime IM (Fig. 3b) after 24 h produced about 40% of sn-1,3-DAG and about 30% of TAG. The formation of sn-1,3-DAG decreased with the increase of the reaction time; in fact after 72 h any DAG were not detected, while TAG gradually increased up to 100% after 96 h. At 25 ◦ C the reactions carried out with Lypozime IM produced about 45% of sn-1,3-DAG and about 15% of TAG after 24 h (Fig. 4a), but in this case the percentage of DAG was constant up to 72 h, while TAG gradually increased up to 100% after 96 h. At the same temperature the reactions carried out with anhydrous Lypozime IM (Fig. 4b), already after 24 h produced about 55% of sn-1,3-DAG, with a percentage of TAG less then 5%; in fact at this temperature TAG was 30% after 96 h. At 12 ◦ C the reactions carried out with Lypozime IM (Fig. 5a) produced about 60% of sn-1,3-DAG after 72 h, but this value changed when the reactions were carried out for longer times; in fact after 144 h there were about 38% of TAG and about

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64% of sn-1,3-DAG. Better results were obtained at 12 ◦ C using anhydrous Lypozime IM (Fig. 5b); after 24 h about 40% of sn1,3-DAG was produced. This percentage gradually increased up to 70 after 144 h, while at this time TAG was 20%. To confirm the isomeric purity of the synthetized sn-1,3DAG, the reaction mixtures were derivatized with the chiral reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate and analyzed by normal phase (NP) HPLC. Using standard compounds, the order of elution was determined (Fig. 6a): sn-1,3-DAG (tr = 25 min), sn-1,2-DAG (tr = 45 min) and sn-2,3-DAG (tr = 55 min). In Fig. 6b the NP-HPLC chromatogram relative to a sample of the re-synthesis reaction (anhydrous Lypozime IM, 12 ◦ C, 144 h) was shown; the exclusive presence of sn-1,3-DAG confirms the absolute specificity of Lipozyme IM during the esterification of Gly and FAEE from EVO. The total % FA composition of synthesized DAG is reported in Table 1 together with that of the starting EVO; the % compositions showed little differences in palmitic and oleic acids. The experimental conditions to obtain high yields of sn-1,3DAG have been reported; the use of the obtained product as edible oil should be possible after the removal of residual FAEE

Fig. 6. UV-HPLC profile of naphthylurethane derivatives of DAG standard mixture (a) and of a re-esterification sample-anhydrous Lipozyme IM, 12 ◦ C, 144 h (b).

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Table 1 Fatty acid composition (mean value mol% ± S.D.) of EVO and synthetized sn1,3-DAG Fatty acids

EVO

Palmitic acid, C16:0 Palmitoleic acid, C16:1n − 7 Stearic acid, C18:0 Oleic acid, C18:1n − 9 Linoleic acid, C18:2n − 6 ␣-Linolenic acid, C18:3n − 3 Arachic acid, C20:0

12.0 0.7 1.7 78.4 5.7 0.8 0.4

[6]

sn-1,3-DAG ± ± ± ± ± ± ±

0.36 0.09 0.08 0.29 0.08 0.12 0.05

14.8 0.9 1.7 75.9 5.2 0.7 0.5

± ± ± ± ± ± ±

0.25 0.12 0.16 0.31 0.24 0.17 0.14

[7]

[8]

[9]

without influencing the residual antioxidant compounds and other minor components, for example by molecular distillation [21].

[10]

[11]

4. Conclusion The enzymatic synthesis of sn-1,3-DAG, in two steps, without intermediate isolation, represents a simple and effective way to obtain a DAG oil, starting from a high quality vegetable oil, as EVO. The Lipozyme IM was the most effective among the sn-1,3-specific lipases screened, in particular the best results were obtained at 12 ◦ C using anhydrous Lipozyme IM. The sn-1,3-DAG, besides the use in foods for their healthy characteristics, could be used as precursors for the synthesis of other lipid classes, such as phospholipids, glycolipids and lipoproteins or as drug carriers too. References [1] Fureby AM, Tian L, Adlercreutz P, Mattiasson B. Lipase-catalysed preparation of diglycerides. Enzyme Microb Technol 1997;20:198–206. [2] Soni MG, Kimura H, Burddock GA. Chronic study of diacylglycerol oil in rats. Food Chem Toxicol 2001;39:317–29. [3] Lo SK, Baharin SB, Tan CP, Lai OM. Lipase-catalysed production and chemical composition of diacylglycerols from soybean oil deodoriser distillate. Eur J Lipid Sci Technol 2004;106:218–24. [4] Kasamatsu T, Ogura R, Ikeda N, Morita O, Saigo K, Watabe H, et al. Genotoxicity studies on dietary diacylglycerol (DAG) oil. Food Chem Toxicol 2005;43:253–60. [5] Taguchi H, Nagao T, Watanabe H, Onizawa K, Matsuo N, Tokimitsu I, et al. Energy value and digestibility of dietary oil containing

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