Synthesis and Structural Analysis of Structured Triacylglycerols with CLA Isomers in the <Emphasis Type="Italic">sn</Emphasis>-2- Position

The present research deals with the chemical esterification of the sn-2- position of sn-1,3-diacylglycerol (sn-1,3-DAG) with 9cis,11trans (c9,t11) and 10trans,12cis (t10,c12) conjugated linoleic acid (CLA) isomers to obtain structured triacylglycerols (TAG); the sn-1,3-DAG substrates were produced from extra virgin olive oil by means of enzymatic reactions while CLA isomers were obtained using a three-step procedure based on alkaline hydrolysis of sunflower oil, urea purification of linoleic acid (LA) and alkaline isomerization of LA. The results showed good levels of CLA incorporation in structured TAG at the tested temperatures: 37.5% at 4 °C and 39.1% at 14 °C. To evaluate the incorporation of CLA isomers in sn-2- position of sn-1,3-DAG structural analysis of the newly synthesized TAG was carried out using an enzymatic and a chemical method. The results of the structural analysis also showed up the occurrence of acyl migration. The pancreatic lipase method allowed the direct determination of the fatty acid composition of TAG sn-2- position but this enzymatic method showed different results (p < 0.05) in respect to the chemical one; this occurrence could be due to an acylic specificity of the lipase. High incorporation of CLA isomers in sn-2- position of TAG was observed, 77.0% at 4 °C and 81.5% at 14 °C, considering the results of the chemical procedure.     

Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

Stereospecific analysis of TAG from sunflower seed oil

Stereospecific analysis of TAG from a sunflower seed oil of Tunisian origin was performed. The TAG were first fractionated according to chain length and degree of unsaturation by RP-HPLC. The four major diacid- and triacid-TAG fractions were palmitoyldilinoleoyl-glycerol, dioleoyllinoleoylglycerol, oleoyldilinoleoylglycerol, and palmitoyloleoyl-linoleoyl-glycerol, amounting to 7.2, 16.6, 29.5, and 12 mol%, respectively. The TAG of the four fractions were individually submitted to stereospecific analysis, using a Grignard-based partial deacylation, separation of sn-1,2(2,3)-DAG from sn-1,3-DAG by boric acid-impregnated silica gel TLC plates, conversion of the sn-1,2(2,3)-DAG to their 3,5-dinitrophenylurethane (DNPU) derivatives, fractionation of DNPU derivatives by RP-HPLC, resolution of the DNPU-DAG by HPLC on a chiral column, transmethylation of each sn-DNPU-DAG fraction, and analysis of the resulting FAME by GC. The data obtained were used to determine the triacyl-sn-glycerol composition of the main TAG of the oil. Fifteen triacyl-sn-glycerols were identified and quantified, representing, along with the monoacid-TAG, trilinoleoylglycerol and trioleoylglycerol, more than 90% of the total oil TAG. The two major triacyl-sn-glycerols were trilinoleoyl-glycerol and 1-linoleoyl-2-linoleoyl-3-oleoyl-glycerol (18.6 and 18.5% of the total, respectively). Results clearly identified linoleic acid as the major FA at the sn-2 position, whereas oleic and palmitic acids were the major FA at the sn-3 position. The sn-1 position was occupied to nearly the same extent by linoleic and oleic acids, and to a greater extent by palmitic acid, which was practically absent at the sn-2 position.     

Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

Lipolysis of different oils using crude enzyme isolate from the intestinal tract of rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Crude enzyme isolate was prepared from the intestine of rainbow trout. Positional specificity of the crude enzyme isolate was determined from both 1(3)- and 2-MAG products after in vitro lipolysis of radioactive-labeled triolein. The ratio of 2-MAG/1(3)-MAG was 2∶1, suggesting that the overall lipase specificity of the enzyme isolate from rainbow trout tended to be 1,3-specific; however, activity against the sn-2 position also was shown. In vitro lipolysis of four different unlabeled oils was performed with the crude enzyme isolate. The oils were: structured lipid [SL; containing the medium-chain FA (MCFA) 8∶0 in the sn-1,3 positions and long-chain FA (LCFA) in the sn-2 position], DAG oil (mainly 1,3-DAG), fish oil (FO), and triolein (TO). MCFA were rapidly hydrolyzed from the SL oil. LCFA including n−3 PUFA were, however, preserved in the sn-2 position and therefore found in higher amounts in 2-MAG of SL compared with 2-MAG of FO, DAG, and TO. Lipolysis of the DAG oil produced higher amounts of MAG than the TAG oils, and 1(3)-MAG mainly was observed after lipolysis of the DAG oil. The positional specificity determined and the results from the hydrolysis of the different oils suggest that n−3 very long-chain PUFA from structured oils may be used better by aquacultured fish than that from fish oils.     

Diacylglycerol acyltransferases from Vernonia and Stokesia prefer substrates with vernolic acid

Genetic engineering of common oil crops for industrially valuable epoxy FA production by expressing epoxygenase genes alone had limited success. Identifying other key genes responsible for the selective incorporation of epoxy FA into seed oil in natural high accumulators appears to be an important next step. We investigated the substrate preferences of acyl CoA: diacylglycerol acyltransferases (DGAT) of two natural high accumulators of vernolic acid, Vernonia galamensis and Stokesia laevis, as compared with a common oilseed crop soybean. Developing seed microsomes were fed with either [¹⁴C]oleoyl CoA or [¹⁴C]vernoloyl CoA in combinations with no exogenous DAG or with 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-vernoloyl-sn-glycerol, 1,2-divernoloyl-sn-glycerol, 1,2-dioleoyl-rac-glycerol, or 1,2-divernoloyl-rac-glycerol to determine their relative incorporation into TAG. The results showed that in using sn-1,2-DAG, the highest DGAT activity was from the substrate combination of vernoloyl CoA with 1,2-divernoloyl-sn-glycerol, and the lowest was from vernoloyl CoA or oleoyl CoA with 1,2-dioleoyl-sn-glycerol in both V. galamensis and S. laevis. Soybean DGAT was more active with oleoyl CoA than vernoloyl CoA, and more active with 1,2-dioleoyl-sn-glycerol when oleoyl CoA was fed. DGAT assays without exogenous DAG, or with exogenous sn-1,2-DAG fed individually or simultaneously showed consistent results. In combinations with either oleoyl CoA or vernoloyl CoA, DGAT had much higher activity with rac-1,2-DAG than with their corresponding sn-1,2-DAG, and the substrate selectivity was diminished when rac-1,2-DAG were used instead of sn-1,2-DAG. These studies suggest that DGAT action might be an important step for selective incorporation of vernolic acid into TAG in V. galamensis and S. laevis.     

Positional distribution of Δ5-olefinic acids in triacylglycerols from conifer seed oils: General and specific enrichment in the sn-3 position

Triacylglycerols (TAG) were purified from the storage lipids extracted from the seeds of several conifer species (Taxus baccata, Larix decidua, Sciadopytis verticillata, and Juniperus communis), each species belonging to one of the four families Taxaceae, Pinaceae, Taxodiaceae, and Cupressaceae, respectively. Each species was characterized by a high content of 5,9-18:2, 5,9,12-18:3, 5,11,14-20:3, or 5,11,14,17-20:4 acids, respectively. TAG were partially deacylated with ethylmagnesium bromide, and the resulting 1,2-, 2,3-diacylglycerols (DAG), and 2-monoacylglycerols (MAG) were purified by thin-layer chromatography. 1,2- and 2,3-DAG were further fractionated by chiral column high-performance liquid chromatography of the 3,5-dinitrophenylurethane derivatives. Alternately, TAG were subjected to porcine pancreatic lipase, and the resulting 2-MAG were purified for further analysis. Gas-liquid chromatography of fatty acid methyl esters prepared from the separated DAG and MAG, coupled with appropriate calculations, indicated that the Δ5-olefinic acids, irrespective of the species, chainlengths and number of ethylenic bonds, were considerably enriched in the sn-3 position of TAG where they accounted for ca. 35 to 74 mole% of fatty acids esterified to this position (depending on the initial level of total Δ5-olefinic acids in TAG), which corresponded to 79–94% of Δ5-olefinic acids esterified to the three positions. On the other hand, Δ5-olefinic acids were less than 10% in the sn-2 position and less than 6% in the sn-1 position of TAG. This specific enrichment of Δ5-olefinic acids in the sn-3 position thus appears to be a general characteristic of conifer seed TAG. These results were extended to TAG from the seeds of two pine species (Pinus koraiensis and P. pinaster) that are rich in Δ5-olefinic acids and available commercially on a ton-scale.     

Na2SiO3-Catalyzed Glycerolysis of Sacha Inchi (Plukenetia volubilis L.) Oil into Di- and Monoacylglycerols

Direct glycerolysis of novel edible Sacha Inchi (Plukenetia volubilis L.) seed oil (PvLO) into diacylglycerols (DAG) and monoacylglycerols (MAG) was studied over solid Na₂SiO₃ with or without microwave assistance. The glycerolysis yield was calculated by qualitative and semiquantitative analyses of ¹H NMR, ¹³C NMR, and FT-IR spectra. The yields of ~33% 1, 3-DAG, ~16% 1, 2-DAG, ~40% 1-MAG, and ~2.3% 2-MAG were achieved after 16 hours at 120 °C in three consecutive cycles using acetone, with an interesterification rate of 92%. The modified oil showed enhanced gelation ability at low temperatures. The yield of 1, 2-DAG can be increased by adding acetone as solvent. The fatty acid compositions and unsaturated structure of lipids were less destroyed after alkaline glycerolysis. However, more α-linolenic and linoleic acids were transferred to the sn-2 position of glyceryl skeleton. The oxidative stability of the modified oil was still controllable. In summary, this work provides a feasible method to convert polyunsaturated plant oils into oils rich in DAG and MAG with less destructive impact on the olefinic structure of oil. Also, it provides a useful example of how to quickly evaluate the influence of chemical modification on the chemical structure of plant oils by using various spectral technologies.     

Triacylglycerols of Apiaceae seed oils: Composition and regiodistribution of fatty acids

Oils from the seeds of caraway (Carum carvi), carrot (Daucus carota), celery (Apium graveolens) and parsley (Petroselinum crispum), all from the Apiaceae family, were analyzed by gas chromatography for their triacylglycerol (TAG) composition and fatty acid (FA) distribution between the sn‐1(3) and sn‐2 positions of TAG. Twenty‐two TAG species were quantified. Glyceryl tripetroselinate was the major TAG species in seed oils of carrot, celery and parsley, with levels ranging from 38.7 to 55.3%. In caraway seed oil, dipetroselinoyllinoleoylglycerol was the major TAG species at 21.2%, while the glyceryl tripetroselinate content was 11.4%. Other TAG species were linoleoyloleoylpetroselinoylglycerol and dipetroselinoyloleoylglycerol. Predominantly, TAG were triunsaturated (72.2–84.0%) with diunsaturates at 14.4–25.9%, and small amounts of monounsaturated TAG. Results for regiospecific analysis showed a non‐random FA distribution in Apiaceae for palmitic, petroselinic, linoleic and oleic acids. Petroselinic acid was predominantly located at the sn‐1(3) position in carrot, celery and parsley seed oils, while it was mainly at the sn‐2 position in caraway seed oil. The distribution of linoleic acid was opposite to that of petroselinic acid. Oleic acid was mostly located at the sn‐2 position, except for caraway, where it was evenly distributed between the sn‐1(3) and sn‐2 positions. Both the saturated FA, palmitic and stearic acid, were located mainly at the sn‐1(3) position. The presence of a high level of tripetroselinin in parsley seed oil (55.3%) makes it a potential source for the production of petroselinic acid.     

Effects of dietary triacylglycerol structure on triacylglycerols of resultant chylomicrons from fish oil- and seal oil-fed rats

We investigated the influence of the intramolecular fatty acid distribution of dietary triacyl-sn-glycerols (TAG) rich in n-3 polyunsaturated fatty acids (PUFA) on the structure of chylomicron TAG. Fish oil and seal oil, comparable in fatty acid compositions but with different contents of major n-3 PUFA esterified at thesn-2 position (20:5n-3, 46.6%, and 5.3%; 22:6n-3, 75.5%, and 3.8%, respectively), were fed to rats. Mesenteric lymph was collected and the chylomicrons were isolated by ultracentrifugation. The fatty acid composition of chylomicrons largely reflected the fatty acid composition of the oils administered. The intramolecular fatty acid distributions of the TAG fed were reflected in the chylomicron TAG as the fraction of the total contents observed in thesn-2 position of 20:5n-3 were 23.6 and 13.3%, and of 22:6n-3 were 30.6 and 5.4% for resultant chylomicrons following fish oil and seal oil administration, respectively. Thus, after seal oil administration, significant higher load of n-3 PUFA was esterified in thesn-1,3 positions of chylomicron TAG compared with fish oil administration (P<0.05).     

Energy value and digestibility of dietary oil containing mainly 1,3-diacylglycerol are similar to those of triacylglycerol

Diacylglycerol (DAG) is a component of various vegetable oils. Approximately 70% of the DAG in edible oils are in the configuration of 1,3-DAG. We recently showed that long-term ingestion of dietary oil containing mainly 1,3-DAG reduces body fat accumulation in humans as compared to triacylglycerol (TAG) oil with a similar fatty acid composition. As the first step to elucidate the mechanism for this result, we examined the difference in the bioavailabilities of both oils by measuring food energy values and digestibilities in rats. Energy values of the DAG oil and the TAG oil, measured by bomb calorimeter, were 38.9 and 39.6 kJ/g, respectively. Apparent digestibility expressed according to the formula: (absorbed) x (ingested)⁻¹x100=(ingested—excreted in feces)x(ingested)⁻¹x100 for the DAG oil and the TAG oil were 96.3±0.4 and 96.3±0.3% (mean±SEM), respectively. The similarity in the bioavailabilities of both oils supports the hypothesis that the reduced fat accumulation by dietary DAG is caused by the different metabolic fates after the absorption into the gastrointestinal epithelial cells.     

Positional distribution of fatty acids in triacylglycerols and phospholipids from adzuki beans (Vigna angularis)

The fatty acid distributions of triacylglycerols (TAG) and major phospholipids (PL) obtained from adzuki beans (Vigna angularis) were investigated. The total lipids extracted from the beans were separated by thin‐layer chromatography (TLC) into eight fractions. The major lipid components were PL (63.5 wt‐%), TAG (21.2 wt‐%), steryl esters (7.5 wt‐%) and hydrocarbons (5.1 wt‐%), while free fatty acids, diacylglycerols (1,3‐DAG and 1,2‐DAG) and monoacylglycerols were also present in minor proportions (0.2–1.1 wt‐%). The major PL components isolated from the beans were phosphatidylcholine (45.3 wt‐%), phosphatidylethanolamine (25.8 wt‐%) and phosphatidylinositol (21.5 wt‐%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. With a few exceptions, however, the principal characteristics of the fatty acid distribution in the TAG and three PL were evident in the beans: Unsaturated fatty acids were predominantly concentrated in the sn‐2 position while saturated fatty acids primary occupied the sn‐1 or sn‐3 position in the oils of the adzuki beans. In general, these results could be useful to both consumers and producers for the manufacture of traditional adzuki foods in Japan.     

Changes in olive oil composition due to microwave heating

The effects of microwave heating on some components of extra-virgin olive oil were studied. Traditional parameters, including free acidity, peroxide value and ultraviolet absorbance values at 232 and 268 nm, were determined in six extra-virgin olive oil samples before and after the microwave treatment. Significant differences (P<0.01) were detected for free acidity, peroxide, and ultraviolet absorbance at 268 nm; also, the absorbances at 232 nm showed significant differences (P<0.05) between treated and untreated samples. The glycerolic fractions, triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG), were isolated by thin-layer chromatography. The respective percentage fatty acid (FA) composition and percentage amount were obtained by high-resolution gas chromatography with an internal standard. For the most abundant TAG fraction, the stereospecific analysis was carried out to obtain the FA percentage compositions of the three sn-positions. Small but significant modifications were observed regarding the decrease in the TAG percentage and increases in the DAG and MAG percentage amounts. No significant changes were observed for the FA compositions of TAG, DAG, and MAG fractions before and after the treatment. Nevertheless, the results of TAG stereospecific analysis showed losses of unsaturated FA in all sn-positions. Higher percentage changes in the sn-1- than in sn-2-position of TAG were observed. Regarding the volatile fraction, different profiles were obtained after the treatment.     

Parameters affecting diacylglycerol formation during the production of specific-structured lipids by lipase-catalyzed interesterification

Diacylglycerols (DAG) are important intermediates in lipase-catalyzed interesterification, but a high DAG concentration in the reaction mixture results in a high DAG content in the final product. We have previously shown that a high DAG concentration in the reaction mixture increases the degree of acyl migration, thus adding to the formation of by-products. In the present study we examined the influence of water content, reaction temperature, enzyme load, substrate molar ratio (oil/capric acid), and reaction time on the formation of DAG in batch reactors. We used response surface methodology (RSM) to minimize the numbers of experiments. The DAG content of the product was dependent on all parameters examined except reaction time. DAG formation increased with increasing water content, enzyme load, reaction temperature, and substrate ratio. The content of sn-1,3-DAG was higher than that of sn-1,2-DAG under all conditions tested, and the ratio between the contents of the former compounds and the latter increased with increasing temperature and reaction time. The water content, enzyme load, and substrate ratio had no significant effect on this ratio. The DAG content was positively correlated with both the incorporation of acyl donors and the degree of acyl migration.     

Rapid and direct quantitative analysis of positional fatty acids in triacylglycerols using 13C NMR

There is increasing evidence that the positional fatty acid composition (FAC) of TAG is more important than total FAC with regard to nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of positional fatty acids in TAG using ¹³C NMR was developed to overcome the tedious conventional methods which involve enzymatic hydrolysis, Grignard chemical degradation, and chromatography analysis. A set of NMR data acquisition parameters and processing methods had proven their excellent versatility and applicability on various types of oils and fats, with systematic error of 1.0 mol%. It was found that there are discrepancies between the regiospecific analysis results obtained by the current method and by conventional methods. Probable acyl migration occurring in the hydrolysis process in conventional methods is a noted problem. As the current ¹³C NMR method is a direct measurement and no hydrolysis of the sample is needed, acyl migration during the analysis is eliminated. As a result, the saturation level is always higher at sn‐1, 3 positions and lower at sn‐2 position in TAG structure of oils in the regiospecific data obtained from the current ¹³C NMR than that from conventional methods. In the absence of laborious chemical derivatization, this method is simple, accurate, and user‐friendly for researchers, especially for nutritionists to support their nutritional studies from the perspective of positional FAC of edible oils and fats.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.     

Regiospecific Analysis of Shark Liver Triacylglycerols

The liver oils of six shallow-water shark species, silky (Carcharhinus falciformis), thresher (Alopias superciliosus), oceanic whitetip (Carcharhinus longimanus), blue (Prionace glauca), hammerhead (Sphyrna lewini) and salmon (Lamna ditropis) were analyzed with particular attention to the regioisomeric composition of triacylglycerols (TAG). The TAG compositions were analyzed by using an HPLC-evaporative light scattering detector and each molecular species identified by HPLC-atmospheric pressure chemical ionization/mass spectrometry. Major lipid components of all sharks’ oils were TAG (~80 %) made up of omega-3 polyunsaturated fatty acids at 26–40 % and 20–25 % docosahexaenoic acid (DHA). Forty different molecular species were detected in the TAG fractions. TAG consisting of one palmitic acid, one DHA and one oleic acid (12.5–19.9 %) and TAG consisting of two palmitic acids and one DHA (8.4–15.4 %) were the predominant form while 30–50 % TAG molecular species were bound to one or more DHA. Distribution of fatty acids in the primary (sn-1 and sn-3) and secondary (sn-2) position of the glycerol backbones was examined by regiospecific analysis by using pancreatic lipase and it was found that DHA was preferentially positioned at sn-2. These findings greatly extend the utilization of shark liver oils in food productions and may have a significant impact on the future development of the fish oil industry.     

Effect of Lipase Activity and Specificity on the DAG Content of Olive Oil from the Shodoshima-Produced Olive Fruits

Olive oils have a higher relative diacylglycerol (DAG) content than other plant oils. The lipase in olive fruits is involved in DAG production and is directly related to the acidity of the olive oil. However, the lipase activity and positional selectivity have not been clarified. To investigate the properties of olive fruit lipase, olive fruits of the Mission variety harvested during mid-December of 2005 on Shodoshima Island (Japan) were stored at 20, 30 or 40 °C for 4 weeks. Changes in the acidity and acylglycerol content of the oils extracted from the stored fruits were analyzed. The acidity and DAG content of the olive oils increased due to triacylglycerol (TAG) hydrolysis during storage. sn-1,2-DAGs preferentially increased during the early stages of storage, indicating that the olive fruit lipase is enantioselective for the sn-3 position, while non-enzymatic isomerization of sn-1,2-DAGs was observed throughout the entire duration of storage. Kinetic analysis revealed that the enantioselectivity of olive fruit lipase for the sn-3 position was approximately four times greater than for the sn-1 position. The lipase was gradually inactivated at temperatures of 30 °C or higher, and the ratios of the rate constant for inactivation to TAG hydrolysis at the sn-3 position was 0.2, 13, and 23 at 20, 30, and 40 °C, respectively.     

Metabolities of dietary triacylglycerol and diacylglycerol during the digestion process in rats

The present study investigated the metabolic fate of dietary TAG and DAG and also their digestion products in the stomach and small intestine. A diet containing 10% TAG or DAG oil, enriched in 1,3-DAG, was fed to Wistar rats ad libitum for 9 d. After 18 h of fasting, each diet was re-fed ad libitum for 1 h. The weights of the contents of the stomach and small intestine were measured, and the acylglycerol and FFA levels were analyzed by GC at 0, 1, and 4 h after the 1-h re-feeding. The amounts of re-fed diet ingested and the gastric and small intestinal content were not different between the two diet groups. In the TAG diet group, the main products were TAG and DAG, especially 1(3),2-DAG. In addition, 1,3-DAG and 1(3)-MAG were present in the stomach, and the 1,3-DAG levels increased over time after the re-feeding period. In the DAG diet group, the main products in the stomach were DAG, MAG, FFA, and TAG. There were significantly greater amounts of 1,3-DAG, 1(3)-MAG, and FFA in the DAG diet group in the stomach compared with the TAG diet group. The amount of FFA in the stomach relative to the amount of ingested TAG plus DAG in the DAG diet group was higher than that in the TAG diet group. Acylglycerol and FFA levels were considerably lower in the small intestine than in the stomach. These results indicate that, in the stomach, where acyl migration might occur, the digestion products were already different between TAG and DAG oil ingestion, and that DAG might be more readily digested by lingual lipase compared with TAG. Furthermore, almost all of the dietary lipid was absorbed, irrespective of the structure of the acylglycerol present in the small intestine.     

Study of Some Experimental Parameters in the Synthesis of Triacylglycerols with CLA Isomers and Structural Analysis

The present research deals with the synthesis of structured triacylglycerols (TAG) by enzymatic treatment of sn-1,3-diacylglycerol (sn-1,3-DAG) with conjugated linoleic acid (CLA) isomers using the immobilized lipase from Rhizomucor miehei (Lipozyme^® IM) under different experimental conditions. In particular, the influence of reaction parameters, such as temperature, enzymatic load, reaction time and DAG/CLA ratio has been evaluated using an experimental design software with a screening objective. Two responses have been selected, they are the percentage of CLA isomers in total TAG and in the sn-2- position and a three-level-4-factor fractional factorial experimental design was used to screen the variables. The results showed that the selected experimental variables have an influence on the enzymatic reaction, in particular, the DAG/CLA substrate ratio and the temperature, both of which inversely correlated with CLA incorporation, but also the enzymatic load and the reaction time, both directly correlated with CLA incorporation. The best results for CLA isomer % content both in total TAG (46.3%) and in the sn-2- position (52.2%) were obtained at 40 °C for 96 h, with 20% enzymatic load and a 0.5 reactive ratio.