Isolation and structural analysis of the cyclic fatty acid monomers formed from eicosapentaenoic and docosahexaenoic acids during fish oil deodorization

Long-chain polyunsaturated fatty acids (LC-PUFAs) present in fish oils are thermolabile molecules. Among the degradation reactions encountered, thermal cyclization occurs during refining or other heat treatments. Numerous studies have been carried out in the past to quantify and determine the structures of cyclic fatty acid monomers (CFAMs) formed from oleic, linoleic and linolenic acids in heated vegetable oils. Recently, much attention have been given to LC-PUFAs due to their potential health benefits. However, data on quantification of CFAMs formed from these fatty acids, such as eicosapentaenoic acid (EPA, cis-5, cis-8, cis-11, cis-14, cis-17 20:5) and docosahexaenoic acid (DHA, cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 22:6), the two main LC-PUFAs in fish oils, are scarce. In the present study, structural analyses of CFAMs formed from EPA and DHA during the deodorization of fish oil are presented. Fish oil sample was deodorized at 250 degrees C for 3 h under a pressure of 1.5 mbar in a laboratory deodorizer. The CFAMs formed during heat treatment of fish oil were isolated by a combination of saponification, esterification, urea fractionations and column chromatography. Structural analyses of C20- and C22-CFAMs were achieved by gas-chromatography electronic-ionization mass-spectrometry (GC-EI-MS) of their 4,4-dimethyloxazoline (DMOX) derivatives. We identified seven out of 13 possible structures of hydrogenated CFAMs formed from EPA, and nine out of 16 possible structures of CFAM formed from DHA. Major CFAMs from both EPA and DHA were cyclohexyl isomers. All possible cyclohexyl isomers were found but only nine out of 18 of the cyclopentyl isomers were present in concentration sufficient for identification. Chemical mechanisms involved in the formation of polyunsaturated LC-PUFAs have been investigated. The results have shown that general principle involved in the cyclization of LC-PUFAs is same as that for the thermal cyclization of oleic, linoleic and alpha-linolenic acids.     

深海鱼油中脂肪酸的分析

用氢氧化四甲胺／甲醇酯化法使深海鱼油内的三酸甘油酯中的脂肪酸和游离脂肪酸转化成脂肪酸甲酯,用气相色谱和气相色谱／质谱进行分析,鉴定出４１个组分,并对其中的主要组分顺式－５,８,１１,１４,１７－十二碳五稀酸（ＥＰＡ）和顺式－４,７,１０,１３,１６,１９－二十二碳六烯酸（ＤＨＡ）建立了定量分析方法。方法的相对标准偏差分别为４．３％（ＥＰＡ）和４．７％（ＤＨＡ）。ＥＰＡ和ＤＨＡ的回收率分别为１００．     

In Vitro and In Vivo Digestibility of Soybean,Fish, and Microalgal Oils,and Their Influences on Fatty Acid Distribution in Tissue Lipid of Mice

The digestion rates of microalgal (docosahexaenoic acid, DHA, 56.8%; palmitic acid, 22.4%), fish (DHA, 10.8%; eicosapentaenoic acid, EPA, 16.2%), and soybean oils (oleic, 21.7%; linoleic acid, 54.6%) were compared by coupling the in vitro multi-step and in vivo apparent digestion models using mice. The in vitro digestion rate estimated based on the released free fatty acids content was remarkably higher in soybean and fish oils than in microalgal oil in 30 min; however, microalgal and fish oils had similar digestion rates at longer digestion. The in vivo digestibility of microalgal oil (91.49%) was lower than those of soybean (96.50%) and fish oils (96.99%). Among the constituent fatty acids of the diet oils, docosapentaenoic acid (DPA) exhibited the highest digestibility, followed by EPA, DHA, palmitoleic, oleic, palmitic, and stearic acid, demonstrating increased digestibility with reduced chain length and increased unsaturation degree of fatty acid. The diet oils affected the deposition of fatty acids in mouse tissues, and DHA concentrations were high in epididymal fat, liver, and brain of mice fed microalgal oil. In the present study, microalgal oil showed lower in vitro and in vivo digestibility, despite adequate DHA incorporation into major mouse organs, such as the brain and liver.     

Optimization of Fatty Acid Determination in Selected Fish and Microalgal Oils

The use of ten fatty acid methyl ester reference standards coupled with a detailed quantification method was shown to significantly optimize the fatty acid determination of selected fish and microalgal oils when compared to methods that use only one reference standard (C19:0 or C23:0) as a relative response factor. When using the mixture of ten reference standards after transesterifying oils with NaOH/BF₃, determination of total fatty acids, eicosapentaenoic acid and docosahexaenoic acid improved by an average of 7.3, 11.5 and 8.4%, respectively. Furthermore, improvements of 13.9, 18.9 and 6.8% of total fatty acids, EPA and DHA, respectively, were obtained when using the mixture of reference standards for fatty acid determination after directly extracting and transesterifying oil contained in microalgal cells with a mixture of methanol, HCl and chloroform. Fatty acid methyl ester standards dissolved in isooctane showed <5% variability throughout 130 days of stability testing when stored at ?20 °C. The optimized method can be used for improving the quantification of fatty acids in both oils (fish and microalgal oils) and dry microalgal cells.     

毛细管气相色谱法测定深海鱼油中的EPA和DHA含量

用氢氧化钾－甲醇酯化法将深海鱼油中的脂肪酸快速、有效地转化成脂肪酸甲酯,然后进行毛细管气相色谱法测定,以二十二碳饱和脂肪酸甲酯为内标物,建立了5,8,11,14,17－二十碳五烯酸（EPA）和4,7,10,13,16,19－二十二碳六烯酸（DHA）的定量分析方法,并用于实际样品分析。该方法对EPA和DHA定量分析的相对标准偏差分别为4．6％和5．0％,回收率分别为99．7％和99．4％。     

Determination of docosahexaenoic acid and n-3 fatty acids in refined fish oils by 1H-NMR spectroscopy: IUPAC interlaboratory study

A high-resolution proton nuclear magnetic resonance (NMR) method for determining the concentration (mg/g) of docosahexaenoic acid (DHA), the molar proportion (mol%) of DHA, and the molar proportion of total n-3 fatty acids in fish oils was validated by an IUPAC interlaboratory study (the Commission VI-6 on Oils, Fats, and Derivatives WG 3/98). Thirteen laboratories from 5 countries tested 6 pairs of blind duplicate fish oils: a refined tuna oil, 2 extracted tuna oils, an extracted bonito oil, an extracted salmon oil, and an extracted sardine oil ranging from 9 to 30 mol% DHA and from 20 to 35 mol% n-3 fatty acids. Before 1 D-proton NMR measurements with 300-500 MHz instruments, oil samples were weighed and diluted with deuterochloroform solution containing ethylene glycol dimethyl ether as internal standard. To achieve precise performance, a detailed procedure for signal area measurement was described in the protocol, and all participants were instructed about the critical importance of following the protocol. Statistical performances with invalid and outlier data removed were as follows: repeatability relative standard deviations (RSDr) ranged from 0.91 to 2.62% and reproducibility relative standard deviation (RSDR) ranged from 1.73 to 4.27% for DHA concentration (mg/g); RSDr ranged from 0.39 to 2.06%, and RSDR ranged from 0.59 to 3.46% for mol% DHA; RSDr ranged from 0.23 to 0.90% and RSDR ranged from 0.85 to 2.01 % for mol% total n-3 fatty acids. The method is expected to be recommended by IUPAC.     

Simultaneous quantification of total eicosapentaenoic acid,docosahexaenoic acid and arachidonic acid in plasma by high‐performance liquid chromatography–tandem mass spectrometry

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC–tandem mass spectrometry (HPLC‐MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid–liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r² > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30–90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega‐3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre‐treatment concentrations varied between subjects for EPA (17–68 mg/L), DHA (36–63 mg/L) and AA (121–248 mg/L). During the dosing period EPA increased 460–480% from the baseline concentration, while DHA increased 150–160%. The EPA–AA ratio increased from 0.07–0.56 to 0.3–3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of polyunsaturated fatty acids in blood serum after fish oil administration.

Free and total fatty acids in the blood serum of patients with hyperlipoproteinemia have been analysed as their methyl esters by capillary gas chromatography using an FFAP column. In one-step reactions the free fatty acids in serum react with methanol-acetyl chloride (50:1, v/v) at 25 degrees C, the total fatty acids (free plus esterified) are transesterified with methanol-toluene-acetyl chloride (8:2:1, v/v) at 100 degrees C. The quantification of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is based on an internal standard (13,16,19-docosatrienoic acid) and on calibration standards. Under normal diet the concentrations of EPA and DHA are as follows (mean +/- S.D., n = 27): free EPA, 0.2 +/- 0.1 mg/dl; free DHA, 0.6 +/- 0.2 mg/dl; total EPA, 3.6 +/- 2.1 mg/dl; total DHA 11.4 +/- 3.1 mg/dl. Under a fish oil intake of 9 g per day, free and total EPA concentrations rise by ca. five- to six-fold, and free and total DHA concentrations by ca. two-fold.     

An Analysis of Oxidative Changes and the Fatty Acid Profile in Stored Poultry Sausages with Liquid and Microencapsulated Fish Oil Additives

This study deals with the fatty acid profile and oxidative changes (TBARS) in vacuum-packed (VP) or modified-atmosphere-packed (MAP) finely-comminuted poultry sausages with liquid fish oil and microencapsulated fish oil (MC) additives. An analysis of omega-3 fatty acids (EPA and DHA) showed that their content in the samples with the fish oil additive decreased from the initial value of 0.22 g∙100 g⁻¹ of the product to 0.18 g∙100 g⁻¹ (MAP) and 0.17 g∙100 g⁻¹ (VP), respectively. After in vitro digestion, the total EPA and DHA content in the sample with microencapsulated oil amounted to 0.17 g∙100 g⁻¹ of the product. The TBARS values showed the VP samples with both forms of the fish oil additive had the lowest values on the first day of storage. Storage of the samples for 21 days caused a slight increase in the degree of lipid oxidation. The research indicated that the forms of the oil additive did not have a negative influence on the sensory features or the physicochemical properties of the sausages. The EPA and DHA levels in samples with liquid fish oil and those with oil microcapsules were sufficient for the sausage producer to declare high content of these fatty acids in accordance with the current EC regulation.     

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.

     

Syntheses of some polyunsaturated sulfur- and oxygen-containing fatty acids related to eicosapentaenoic and docosahexaenoic acids.

With the aim of enhancing selectively the beneficial biological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) a number of polyunsaturated fatty acids containing sulfur or oxygen atoms in the chain has been synthesized starting from EPA and DHA, respectively. Oxidative degradation of these acids led to the corresponding aldehydes all-(Z)-3,6,9,12-pentadecatetraenal and all-(Z)-3,6,9,12,15-octadecapentaenal. Reactions with DBU converted these aldehydes quantitatively into the conjugated isomers (2E,6Z,9Z,12Z)-pentadecatetraenal and (2E,6Z,9Z,12Z,15Z)-octadecapentaenal, respectively. The four aldehydes were transformed by a sequence of reactions comprising reduction to the alcohols, halogenation and substitution with mercapto esters into the corresponding sulfur containing polyunsaturated fatty acid esters. The oxygen containing esters were prepared from the respective alcohol by boron trifluoride catalysed reaction with ethyl diazoacetate.     

Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid method was developed for the simultaneous profiling of 29 free fatty acids in plasma using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). Barium acetate was used as the cationization agent in the positive ion mode for sensitive multiple reaction monitoring (MRM) experiments. The cis- and trans-C18:1 and -C18:2 isomers were baseline-separated using two tandem reversed-phase C18 UPLC columns, while identification of two pairs of positional isomers of C18:3 and C20:3 required isomer-specific product ions, as the analytes were not chromatographically resolved. The assay linearity was greater than three orders of magnitude and correlation coefficients were >0.99; the limits of detections were typically less than 0.2 microM. The method was successfully applied to plasma free fatty acid profiling of samples from volunteers who participated in a randomized crossover study involving the administration of either placebo or fish oil capsules. The results clearly indicate the ability to measure the time profiles of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma for the volunteers given fish oil capsules while the concentrations of the other free fatty acids and the total free fatty acid concentration in plasma remained virtually constant.     

Synthesis of EPA- and DHA-Enriched Structured Acylglycerols at the sn-2 Position Starting from Commercial Salmon Oil by Enzymatic Lipase Catalysis under Supercritical Conditions

There is consistent evidence that long-chain polyunsaturated fatty acids (LCPUFA) belonging to the n-3 series, i.e., eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids, decrease the risk of heart, circulatory and inflammatory diseases. Furthermore, the bioavailability of such fatty acids has been shown to depend on their location in triacylglycerol (TG) molecules at the sn-2 position. Consequently, great attention has been accorded to the synthesis of structured acylglycerols (sAG), which include EPA or DHA at the sn-2 position. The aim of this work was to synthesize sAG starting from deodorized refined commercial salmon oil. For this, immobilized lipase B from Candida antarctica (nonspecific) was used as a catalyst for the intra–interesterification process under CO₂ supercritical conditions (CO₂SC). According to the CO₂SC reaction time, three different fractions including sAG compounds were obtained. The location of EPA and DHA at the sn-2 position in the resulting glycerol backbone was identified by mass spectrometry (MALDI-TOF) analysis. In all fractions obtained, a marked decrease in the starting TG content was observed, while an increase in the DHA content at the sn-2 position was detected. The fraction obtained after the longest reaction time period (2 h) led to the highest yield of sn-2 position DHA in the resulting sAG molecule.     

气相色谱全分析海水和淡水养殖南美白对虾组织中脂肪酸组成与含量

采用超声提取-毛细管气相色谱法分析测定了海水养殖和淡水养殖的南美白对虾的肌肉和肝胰腺(中肠腺)中脂肪酸的组成与含量,以用于对虾繁殖与营养的研究。采用Folch 液超声提取对虾组织中的脂质,脂质经氢氧化钾-三氟化硼法甲酯化后上机分析。采用BPX70(60 m×0.25 mm i.d.)石英毛细管柱分离,火焰离子化检测器检测。以线性程序升温等当碳长度(ECLT)值结合标准品对照的方法对17种脂肪酸定性,用面积归一化法定量。结果表明,两种对虾的脂肪酸组成均以不饱和脂肪酸为主,而且不饱和多烯酸的含量明显高于不饱和单烯酸。两种对虾的肌肉脂肪酸中高度不饱和脂肪酸,如二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的总含量均较高,达20%以上;肝胰腺中EPA和DHA的总含量较低,为11%～17%。淡水虾中n-6族不饱和脂肪酸含量比海水虾高,海水虾中n-3族与n-6族脂肪酸含量之比值比淡水虾高。     

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.     

气相色谱-质谱联用鉴定基于水酶法制备的虹鳟鱼骨油组分

采用水酶法制备虹鳟鱼骨油,单因素分析法优化虹鳟鱼骨酶解的工艺条件,考察了料液比、pH值、酶解时间、酶解温度、加酶量5个因素对鱼油提取率的影响,采用气相色谱-质谱联用 (GC-MS) 技术对鱼骨油的脂肪酸组成和含量进行了分析鉴定.结果表明,在55℃、pH 7.5、酶解时间为3 h、料液比为1∶1、加酶量为2000 U/g的条件下,利用碱性蛋白酶提取的虹鳟鱼骨油中的油脂含量最高.GC-MS分析结果表明,虹鳟鱼骨油中主要成分是不饱和脂肪酸,含量为脂肪酸总量的80.4% (w/w),其中单不饱和脂肪酸和多不饱和脂肪酸分别约占不饱和脂肪酸的76.9%和23.1% (w/w), DHA和EPA的总量为3.4% (w/w).本研究优化了虹鳟鱼油的提取技术,对虹鳟鱼油的主要挥发性物质进行了分析鉴定,初步确定了其中对鱼油风味起主要贡献的物质,对鱼油产品的分析与鉴别具有参考价值.     

蛇油的挥发性成分分析和脱腥

采用水蒸汽蒸馏、黏土吸附、乙醇-乙酸乙酯共沸的方法除去蛇油的腥味,运用顶空固相微萃取(SPME)-气相色谱(GC)-质谱(MS)联用技术分析脱腥前后蛇油中挥发性成分的变化,并运用GC-MS联用技术分析脱腥后的蛇油中的脂肪酸.结果表明水蒸汽蒸馏是效果最佳的脱腥方法,它可使蛇油挥发性成分的量降至脱腥前的38%,使腥味的主要成分减少60%～100%,同时较大程度地保留了多不饱和脂肪酸.     

碱催化法衍生化气相色谱/质谱法分析青海湖裸鲤鱼油中的脂肪酸

用碱催化法将青海湖裸鲤鱼油甲酯化,以气相色谱/质谱法分析鱼油中的脂肪酸。青海湖裸鲤可食用部分中鱼油含量为25.13%。从鱼油中共鉴定出47种脂肪酸,包括直链、单支链、多支链饱和脂肪酸,单不饱和、多不饱和脂肪酸,环丙烷基、呋喃基脂肪酸等。不饱和脂肪酸含量为73.6%,其中多不饱和脂肪酸含量为25.4%,以C18∶2(4.9%),C18∶3(3.1%),C20∶4(1.3%),C20∶5(二十碳五烯酸（EPA）, 9.4%)和C22∶6(二十二碳六烯酸（DHA）, 6.7%)为主。单不饱和脂肪酸含量为48.2%,主要由C16∶1(20.3%),C18∶1(25.9%)构成。饱和脂肪酸含量为25.7%,主要有C14∶0(3.4%),C16∶0 (19.4%)和C18∶0(1.1%)。青海湖裸鲤鱼油中还存在不常见的环丙烷基和呋喃基脂肪酸及多种奇数碳链和支链脂肪酸。因此,青海湖裸鲤是功能性脂肪酸的重要膳食来源。     

Use of Isopropanol as a Modifier in a Hexane-Acetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids

A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).     

Preparation of Triacylglycerols Rich in Omega-3 Fatty Acids from Sardine Oil Using a Rhizomucor miehei Lipase: Focus in the EPA/DHA Ratio

The increasing evidence on the differential biochemical effects of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acid concentrates with different amounts of these fatty acids. In the present work, physicochemical and enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols (TAG), rich in n-3 fatty acids. Sardine oil was obtained by washing sardine (Sardina pilchardus) mince with a NaHCO₃ solution, hydrolyzed in a KOH–ethanol solution, and concentrated with urea. The esterification reaction was performed in the stoichiometric proportion of substrates for re-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight of substrates, without any solvent, during 48 h. This procedure led to approximately 88 % of acylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids was higher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in the unesterifed fraction (free fatty acids) increased from 20 to 54 %, while the EPA level in the same fraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (density functional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explain some of the experimental results.