Monoglyceride and Diglyceride Production Through Lipase-Catalyzed Glycerolysis and Molecular Distillation

Distilled glycerides are obtained through distillation of the system mono-diglycerides which is produced from the esterification reaction between a triglyceride with glycerol. In this work, monoglycerides (MG) and diglycerides (DG) are produced through lipase-catalyzed glycerolysis of soybean oil using Candida antarctica B in a solvent-free system. To separate the products of the reaction in order to obtain essentially MG and an oil of DG, it is necessary to use a suitable process in order to preserve the stability of the components and to keep the products free of inappropriate solvents. So, after 24 h of enzymatic reaction, the mixture of acylglycerols and fatty acids was distilled into a centrifugal molecular distiller, since it provides a free solvent and lower temperature environment to increase the desired product concentration. Starting from a material with 25.06% of triglycerides (TG), 46.63% of DG, 21.72% of MG, 5.38% of free fatty acids (FFA), and 1.21% of glycerol, the MG purity in the distillate stream was 80% at evaporator temperature (T _E) equal to 250 °C and feed flow rate (Q) equal to 10.0 mL/min. At these conditions, the MG recovery was 35%. The material collected in the residue stream presented DG-enriched oil with TG unhydrolyzed, residual MG, and low acidity (29.83% of TG, 53.20% of DG, 15.64% of MG, and 1.33% of FFA), which is suitable to replace TG oil in the human diet.     

Monoglycerides and Diglycerides Synthesis in a Solvent-Free System by Lipase-Catalyzed Glycerolysis

Five lipases were screened (Thermomyces lanuginosus free and immobilized forms, Candida antarctica B, Candida rugosa, Aspergillus niger, and Rhizomucor miehei) to study their ability to produce monoglycerides (MG) and diglycerides (DG) through enzymatic glycerolysis of soybean oil. Lipase from C. antarctica was further studied to verify the enzyme load (wt% of oil mass), the molar ratio glycerol/oil, and the water content (wt% of glycerol) on the glycerolysis reaction. The best DG and MG productions were in the range 45–48% and 28–30% (w/w, based on the total oil), respectively. Using immobilized lipases, the amount of free fatty acids (FFA) produced was about 5%. However, the amount of FFA produced when using free lipases, with 3.5% extra water in the system, is equivalent to the MG yield, about 23%. The extra water content provides a competition between hydrolysis and glycerolysis reactions, increasing the FFA production.     

Lipase-Catalyzed Preparation of Diacylglycerol-Enriched Oil from High-Acid Rice Bran Oil in Solvent-Free System

The ability of immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) to catalyze the reaction of high-acid rice bran oil (RBO) and monoglyceride (MG) for diacylglycerol-enriched rice bran oil (RBO-DG) preparation was investigated. The effects of substrate ratio, reaction temperature, time, and enzyme load on the respective content of free fatty acid (FFA) and DG in the final RBO-DG products was investigated. Enzyme screening on the reaction was also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (50?C70?°C), the enzyme load (2?C6?%; relative to the weight of total substrates), and the reaction time (4?C8?h) on the respective content of FFA and DG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values. The optimum preparation conditions were as follows: MG/RBO, 0.25; temperature, 56?°C; enzyme load, 4.77?%; and reaction time, 5.75?h. Under the suggested conditions, the respective content of FFA and DG was 0.28 and 27.98?%, respectively. Repeated reaction tests indicated that Lipozyme RM IM could be used nine times under the optimum conditions with 90?% of its original catalytic activity still retained.     

Preparation of a Crosslinked Bioimprinted Lipase for Enrichment of Polyunsaturated Fatty Acids from Fish Processing Waste

Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator (benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase. Crude fish oil was treated at 45 °C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA. The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according to the described crosslinking–bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield.     

Isolation and Purification of Lipids by Centrifugal Partition Chromatography

Abstract

Methodologies for the separation of various lipid classes including free fatty acids (FA) and fatty acid ethyl esters (FAEE), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phosphoglycerides (PG), and glycosphingolipids (GSL) by Centrifugal Partition Chromatography are presented. The fatty acid ethyl esters, Hexadecanoate, Octadecanoate, cis-9-Octadecenoate; and cis,cis-9,12-Octadecadienoate, and all-cis-9,12,15-Octadecatrienoate, were separated at 800 rpm; flow rate of 2.0 ml/min; using n-hexane/acetonitrile (1:1, v/v) as the solvent system and normal ascending and reversed descending elution modes respectively. The fatty acid ethyl esters, all-cis-5,8,11,14,17-Icosapentaenoate and all-cis-4,7,10,13,16,19-Docosahexaenoate, were separated at 600 rpm; flow rate of 3.3 ml/min; using n-hexane/methanol/water (1/0.9/0.1, v/v/v) as the solvent system and normal ascending mode. FA and MG; and DG and TG were separated at 600 rpm; 10     

Lipid content and fatty acid composition in lemon wax

Lipids were extracted from lemon wax and fractionated into four classes on a silicic acid glass packed column by thin-layer chromatography (TLC). The free fatty acids, the fatty acid composition and the amount of each separated lipids were determined by capillary column gas chromatography (GC). Total lipids (TL) were 60 mg per 100 g raw weight and the ratio of nonpolar lipids (NPLs): glycolipids (GLs): phospholipids (PLs) was about 47:2:2. The main free fatty acids in lemon wax were hexadecanoic acid, cis-9-octadecenoic acid and cis,cis-9,12-octadecadienoic acid, while in the lipid fractions the main fatty acids were hexadecanoic acid in all the fractions, cis-cis-9,12-octadecadienoic and decanoic acids in triglyceride (TG) fraction, dodecanoic and cis-9-octadecenoic acids in diglyceride (DG) fraction and tetradecanoic, octadecanoic and cis-9-octadecenoic acids in GL and PL fractions. The ratio of unsaturated to saturated fatty acids showed a remarkable difference among these four lipid fractions. In PL and GL fractions this ratio was similar, 47.7% and 47.1% respectively, and in TG fraction it was 42.4% while in DG fraction this value was 23.5%.     

Isolation of 6,9,12,15-Hexadecatetraenoic Fatty Acid (16:4n-1) Methyl Ester from Transesterified Fish Oil by HSCCC

Fish oil is considered a healthy food due to the presence of large amounts of polyunsaturated fatty acids (PUFAs), especially in the form of n-3 fatty acids 5,8,11,14,17-eicosapentaenoic acid (20:5n-3; EPA) and 4,7,10,13,16,19-docosahexaenoic acid (22:6n-3; DHA). However, fish oil is known to contain many other PUFAs, some of which are uncommon and whose bioactivity is scarcely investigated. In this study, we isolated the rare PUFA 6,9,12,15-hexadecatetraenoic fatty acid (16:4n-1) which bears a double bond on the terminal carbon from fish oil in form of its methyl ester. We used high-speed counter-current chromatography (HSCCC) for the fractionation of 500 mg-portions of fatty acid methyl esters prepared from a fish oil capsule and investigated the fractions by GC/MS. Twenty-eight 13-mL fractions were collected and fatty acid methyl esters were detected in fractions 11–23. The elution was carried out in normal phase mode, providing the long-chained saturated and monoenoic fatty acids first. More than 100 fatty acids ranging from 10:0 to 26:0 could be identified in the HSCCC fractions, and most of them were polyunsaturated. The reproducibility of the HSCCC method was shown by repeated injection of the fish oil and the fractions containing 6,9,12,15-hexadecatetraenoic fatty acid (16:4n-1). The late eluting 16:4n-1 methyl ester was isolated in pure form and its structure was verified.

     

Phenylmethanesulfonyl fluoride pretreatment stabilizes plasma lipidome in lipidomic and metabolomic analysis

Though it is standard practice to test the stability of analytes in the matrix for routine bioanalytical method, stability evaluation is always impractical and skipped in untargeted lipidomic and metabolomic analysis because analytes in these studies are enormous, diverse and sometimes unknown. Lipidome represents a major class of plasma metabolome and shows great potential to be diagnostic and prognostic biomarkers. However, lipidome also faces stability problems because plasma contains kinds of lipid degradation enzyme. Here, using liquid chromatography time of flight mass spectrometry based lipidomic methodology, plasma levels of various lipids including triglyceride (TG), diglyceride (DG), free fatty acid (FFA), phosphatidylethanolamine (PE) phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), and sphingomyelin (SM) were dynamically determined within 4 h at ambient temperature. In mouse and rat plasma, the levels of most TG, DG, PC and PE species significantly decreased with respect to time, whereas those of LPC, LPE and FFA significantly increased with respect to time. However, such changes did not occur in human plasma, thus indicating hepatic lipase and esterase might involve in the species-specified degradation of lipid classes in plasma. Phenylmethanesulfonyl fluoride (PMSF) pretreatment prevented such lipidome instability in mouse plasma. The results suggested the instability of plasma lipidome should be highly concerned, and the enhancement of ex vivo stability of plasma lipidome could enable more reliable clinical translation of lipidomic data for biomarker discovery.     

Kinetic properties of soluble and immobilizedCandida rugosa lipase

Immobilized lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) fromCandida rugosa has been immobilized on commercially available microporous polypropylene and used for the batch hydrolysis of different animal fats. The effect of the reaction products at concentrations similar to those obtained at 90% hydrolysis, both on soluble and immobilized lipase, was studied. Glycerol showed low inhibitory effect but oleic acid caused 50% inhibition. A mixture of free fatty acids present in the complete hydrolysis of beef tallow inhibited lipase activity more than 70%. The stability of the enzyme (both soluble and immobilized) was highest in the presence of 20% isooctane. The apparent Michaelis constant for each substrate for the soluble enzyme did not change on immobilization.     

Composition of the seed oils ofOriganum tyttanthum andMentha asiatica

The fatty oils of the seeds ofOriganum tyttanthum andMentha asiatica (family Labiatae) have been investigated. The glyceride and fatty-acid compositions of the two oils have been determined. From the oil ofM. asiatica have been isolated the new acid 9-hydroxyoctadeca-12-cis,15-cis-dienoic acid and a mixture of six unsaturated oxo acids.     

Oxidative stabilities of enzymatically interesterified goose fat and rapeseed oil blend by rancimat and PDSC

Goose fat (GF) and rapeseed oil (RSO) 2:3 m m^?1 blend was enzymatically interesterified at 60 °C with and without microwaves assistance. As the catalyst, a commercial preparation of the immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) containing 2 % of water was used, and the catalyst load was 8 % in each case. The starting mixture and the interesterified products were separated by column chromatography into pure triacylglycerols fraction (TAG) and a non-triacylglycerol fraction, which contained free fatty acids (FFA), mono- and diacylglycerols (MAG and DAG). The oxidative stabilities of fats studied and TAG derived from them were assessed by Rancimat at 100 °C and by pressure differential scanning calorimetry (PDSC) under oxygen at 110–140 °C. Interesterification reduced the oxidative stability of GF and RSO blend. The main factors influenced on the oxidative stabilities of fats studied were concentrations of tocopherols and the presence of FFA, MAG and DAG. The structures of TAGs were of minor importance. From the resulting PDSC exotherms, their times to reach the onset (τ _on) and peak maximum (τ _max) were measured and used for calculations of parameters of the Arrhenius type kinetics for thermaloxidative decomposition of fats studied.     

Preparation of mono- and diacylglycerols by enzymatic esterification of glycerol with conjugated linoleic acid in hexane

Esterification of glycerol with conjugated linoleic acid (CLA) was carried out in hexane. Lipase from Rhizomucor miehei provided a high degree of esterification (80%) in 8 h at 50°C when used at 15% (w/w) in a system containing a 1∶2 molar ratio of glycerol to free fatty acids. Esterification levels >80% were obtained in 8 h at 40°C with 15% (w/w) lipase from Candida antarctica at the same molar ratio of reactants. The extent of esterification of CLA was >90% after 4h of reaction at 50°C with a 5% (w/w) loading of either R. miehei or C. antarctica lipase, together with a 1∶1 molar ratio of substrates. Both enzymes incorporated the original CLA as acylglycerol residues in primarily 1,3-diacylglycerol and 1-monoacylglycerol. The CLA-rich acylglycerols can be employed as emulsifiers or as substitutes for natural fats and oils.     

Improving Stability and Activity of Cross-linked Enzyme Aggregates Based on Polyethylenimine in Hydrolysis of Fish Oil for Enrichment of Polyunsaturated Fatty Acids

Cross-linking of enzyme aggregates from recombinant Geotrichum sp. lipase based on polyethylenimine (PEI) was applied to hydrolyze fish oil for enrichment of polyunsaturated fatty acids successfully. Through acetone precipitation and cross-linking of physical aggregates using glutaraldehyde in the presence of PEI, firmly cross-linked enzyme aggregates (PEI-CLEAs) were prepared. They could maintain more than 65% of relative hydrolysis degree after incubation in the range of 50–55 °C for 4 h and maintain more than 85% of relative hydrolysis degree after being treated by acetone, tert-butyl alcohol and octane for 4 h. PEI-CLEAs increased hydrolysis degree to 42% from 12% by free lipase. After five batch reactions, PEI-CLEAs still maintained 72% of relative hydrolysis degree. Hydrolysis of fish oil by PEI-CLEAs produced glycerides containing concentrated EPA and DHA in good yield. PEI-CLEAs had advantages over general CLEAs and free lipase in initial reaction rate, hydrolysis degree, thermostability, organic solvent tolerance and reusability.     

Characterization of <Emphasis Type="Italic">Opuntia ficus indica</Emphasis> seed oil from Tunisia

The lipid fraction of Opuntia ficus indica seeds was extracted and analyzed for its chemical and physical properties such as acid value, free fatty acid percentage (% FFA), iodine index, peroxide value, and saponification value as well as refractive index and density. The yield of seed oil was calculated as 11.75%. The acid and free fatty acid values indicated that the oil has a fairly low acidity. The triacylglycerols, fatty acids, sterols, and tocopherols were identified and their concentrations determined. The main TAGs were LLL (25.60%), OLL (21.53%), PLL (15.53%), and POL + SLL (12.73%). Linoleic acid (60.69%) was the dominant fatty acid, followed by oleic (21.42%) and palmitic (12.76%) acids, respectively. A high level of sterols making up 16.06 g/kg seed oil was present. The sterol marker, β-sitosterol, accounted for 71.60% of the total sterol content in the seed oil. Among the tocopherols present in the oil, γ-tocopherol (421.08 mg/kg) was the main constituent.     

A kinetic study on lipase-catalyzed interesterification of soybean oil with oleic acid in a continuous packed-bed reactor

To provide a mathematical basis for the design and operation of a continuous, packed-bed reactor for the interesterification of soybean oil, soybean oil that contains 22.7% oleoyl and 54.3% linoleoyl moieties as molar acyl moiety composition was interesterified in hexane with oleic acid, using an immobilized sn-1,3-specific lipase (Lipozyme IM) from Mucor miehei. The reaction was carried out in a U-shaped Pyrex glass-made packed-bed reactor at 37°C in the following system: concentration of soybean oil in the feed stream=12.5 wt%, molar ratio of fatty acid to soybean oil=3.0, and water content in the feed stream=1340–2340 ppm. At these water contents, Lipozyme IM gave practically the same catalytic activity, and the content of triacylglycerols in the product oil was 91–94 wt%. Rate equations for the change in oleoyl and linoleoyl moiety compositions in soybean oil were derived and their validity was confirmed experimentally. On the other hand, the catalytic activity of Lipozyme IM decayed in the first-order fashion. Based on these deactivation kinetics, the flow rate of the feed stream is simulated for the operation of a continuous, packed-bed reactor at 37°C that produces an oil of a fixed composition of oleoyl moiety.     

Silk-Fiber Immobilized Lipase-Catalyzed Hydrolysis of Emulsified Sunflower Oil

Lipase was immobilized in silk fibers through glutaraldehyde cross-linking to a maximum loading of 59 U/g silk-fiber and the immobilized lipase was utilized for the hydrolysis of sunflower oil (Helianthus annuus). The hydrolytic activity of the lipase, which was poor in biphasic oil in water system, was increased significantly when the sunflower oil was emulsified in aqueous medium. The hydrolytic activities of the immobilized lipase were 48.73 ± 1.26 U, 36.11 ± 0.96 U, and nil when the substrate sunflower oil was used as emulsion created by a rhamnolipid biosurfactant, Triton X100, and ultrasonication, respectively. Although the efficiency of the immobilized lipase was less than 12% than the corresponding free lipase, the immobilized lipase could be reused for the biosurfactant-mediated hydrolysis of sunflower oil up to third cycle of the reaction. The yield of the fatty acids in the second, third, and fourth cycles were 49.45%, 22.91%, and 5.09%, respectively, of the yield obtained in the first cycle.     

Improvement of a solid phase extraction method for analysis of lipid fractions in muscle foods

The most used method for muscle lipid fractionation into major lipid classes was modified for improving its separation efficiency. Extracted lipids from a masseter muscle of one Iberian pig were separated into neutral lipids (NL), free fatty acids (FFA) and polar lipids (PL) using aminopropyl minicolumns, following the extensively used method of Kaluzny et al. [1] (old method-OM-) and a method based on that, developed by Pinkart et al. [2] with some (modifications modified method–MM). Obtained lipid classes were further analysed by TLC and lipid fractions were identified. TLC evidenced the presence of a certain amount of PL in the NL fraction obtained with the OM. On the other hand, using the MM only an almost undetectable presence of PL was evidenced in the NL fraction. Fatty acid composition of NL, PL and FFA obtained with each method was studied by gas chromatography. Fatty acid profile of NL was strongly influenced by the separation method used. Thus, NL obtained using the OM showed higher amounts of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and lower of monounsaturated fatty acids (MUFA) than those obtained using the MM. Moreover, NL obtained using the OM showed the presence of fatty alcohols, constituents of phospholipids (PhL) absent or present only in trace amounts in acylglycerols. This profile reflects the coelution of PL in the NL fraction. Fatty acid profile of FFA and PL fractions was also influenced by the solid phase extraction (SPE) method used, but to a lesser extent.     

Lipase-catalyzed interesterification of soybean oil with an omega-3 polyunsaturated fatty acid concentrate prepared from sardine oil

To reduce the content of linoleoyl moiety in soybean oil, soybean oil that contains 53.0% linoleoyl moiety as molar acyl moiety composition was interesterified with an omega-3 polyunsaturated fatty acid (PUFA) concentrate (24.0 mol% eicosapentaenoic acid [EPA], 40.4 mol% docosahexaenoic acid [DHA]) prepared from sardine oil, using an immobilized sn-1,3-specific lipase from Rhizomucor miehei (Lipozyme IM). The reaction was carried out in a batch reactor at 37 degrees C under the following conditions: 500 micromol of soybean oil, molar ratio of omega-3 PUFA concentrate to soybean oil = 1.0-6.0,5.0 mL of heptane, and 30 batch interesterification units of enzyme. After the reaction time of 72 h, modified soybean oil, which contains 34.9% linoleoyl, 10.1% eicosapentaenoyl, and 14.2% docosahexaenoyl moieties, was produced at the molar reactant ratio of 6.0. In this oil, the total omega-3 acyl moiety composition reached 34.1%; the molar ratio of omega-3 to omega-6 acyl moieties was enhanced by five times compared with soybean oil. Compared with palmitic acid, DHA was kinetically six times less reactive, although the EPA was by 16% more reactive.     

Composition of the seed oils ofOriganum tyttanthum andMentha asiatica

Summary The fatty oils of the seeds ofOriganum tyttanthum andMentha asiatica (family Labiatae) have been investigated. The glyceride and fatty-acid compositions of the two oils have been determined. From the oil ofM. asiatica have been isolated the new acid 9-hydroxyoctadeca-12-cis,15-cis-dienoic acid and a mixture of six unsaturated oxo acids.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 149–155, March–April, 1976.     

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, α-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.