Degradation products formed from long‐chain PUFA during deodorization of fish oil

Omega‐3 long‐chain polyunsaturated fatty acids (LC‐PUFA) are sensitive to heat and may be destroyed by thermal processes such as deodorization. For example, deodorization of fish oil may induce polymerization, geometrical isomerization and cyclization of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. In this paper, we review our main findings on the effects of deodorization at three different temperatures on semi‐refined fish oil LC‐PUFA. Cyclic structures have been elucidated and mechanisms responsible for ring formation have been discussed. Polymers were found to be the most abundant degradation products formed during fish oil deodorization. A method for quantitative measurement of geometrical isomers of EPA and DHA by gas‐liquid chromatography (GLC) has been developed and validated. Overall assessment of the results obtained with this method suggests that deodorization at temperatures above 180°C affects the quality and the content of LC‐PUFA in fish oil.     

Geometrical isomerization of polyunsaturated fatty acids in physically refined rapeseed oil during plant‐scale deodorization

The effect of the operating temperature (between 220 and 270 °C) on the formation of trans isomers of linoleic and linolenic acids in physically refined rapeseed oil during deodorization in a plant‐scale semicontinuous tray‐type deodorizer (capacity 10 t/h) was investigated. The industrial procedures of physical refining consisted of a two‐step bleaching and deodorization process. The degree of isomerization of linoleic acid ranged from 0.33 to 4.77% and that of linolenic acid from 4.43 to 45.22% between 220 and 270 °C, respectively. A relation between the logarithm of the degree of isomerization and the deodorization temperature can be approximated by statistically highly significant linear functions for both linoleic and linolenic acids. Oleic acid was resistant to the heat‐induced geometrical isomerization. The values found for the ratio between the degrees of isomerization of linolenic and linoleic acids, slightly decreasing with increasing temperature, were equal to 13.6 and 12.9 at 230 and 240 °C, respectively. Two trans isomers of linoleic acid, exclusively with one double bond isomerized into trans configuration, and four trans isomers of linolenic acid, mostly with one double bond isomerized into trans configuration, were determined in deodorized rapeseed oils. Linolenic acid was observed to be the main source responsible for the formation of nearly all trans fatty acids in physically refined rapeseed oil. At 235 °C, a deodorization temperature considered as a reasonable technological compromise, the content of trans fatty acids in plant‐scale physically refined rapeseed oil was less than 1% of total fatty acids, which would be acceptable for further application.     

Heat treatment of vegetable oils III. GC-MS characterization of cyclic fatty acid monomers in heated sunflower and linseed oils after total hydrogenation

Fractions of cyclic fatty acid monomers (CFAM) were isolated from linseed oil heated at 275°C for 12 hr under nitrogen, at 240°C for 10 hr under nitrogen and at 240°C for 10 hr under air. Cyclic fatty acid monomers fractions were also isolated from a sunflower oil heated at 275°C for 12 hr under nitrogen and at 200°C for 48 hr in a commercial fryer. The CFAM fractions were hydrogenated and their composition studied by gas liquid chromatography coupled with mass spectrometry (GC-MS). The CFAM in the fraction isolated from heated linseed oil samples were a mixture (1:1) ofcis andtrans cyclopentyl and cyclohexyl isomers, while the CFAM in the fractions isolated from heated sunflower oils were mostly cyclopentyl isomers. The major cyclopentyl isomers weretrans andcis methyl 7-(2′-hexylcyclopentyl) -heptanoate, methyl 9-(2′-butyl-cyclopentyl)-nonanoate and methyl 10-(2′-propylcyclo-pentyl)-decanoate. The major cyclohexyl isomers were thetrans andcis methyl 9-(2′-propylcyclohexyl)-nonanoate which represented about 50% of the CFAM isomers isolated from heated linseed oil samples. For part II in this series see Ref. 1.     

Geometrical isomerisation of eicosapentaenoic and docosahexaenoic acid at high temperatures

Concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were heated at 140–240 °C for 2–8 h under nitrogen. The trans isomers were analysed by gas chromatography‐mass spectrometry on a BPX‐70 cyanopropyl column. All geometrical isomers of EPA and DHA with one trans double bond were observed. The rate constants (k) for the isomerisation of the all‐cis isomers were calculated and found to be higher than previously reported for linoleic acid and α‐linolenic acid. Arrhenius plots showed a linear relationship between ln k and the reciprocal absolute temperature above 180 °C. The distribution patterns of isomers with one trans double bond are approximately constant up to a degree of isomerisation of 25%. The degree of isomerisation can therefore be estimated from selected trans peaks.     

Combined Urea Complexation and Argentated Silica Gel Column Chromatography for Concentration and Separation of PUFAs from Tuna Oil: Based on Improved DPA Level

Eicosapentaenoic acid (EPA, 20:5n‐3), docosapentaenoic acid (DPA) isomers (22:5n‐6 and 22:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) derived from tuna oil were concentrated by three stages of urea fractionation at various crystallization temperatures and different fatty acid/urea ratios. Thereafter, polyunsaturated fatty acids concentrate containing comparatively enriched DPA levels was purified by argentated silica gel column chromatography. A product containing 22.2 ± 0.6 % EPA, 4.6 ± 0.0 % DPAn‐6, 5.9 ± 0.1 % DPAn‐3 and 42.3 ± 1.2 % DHA was obtained at 1:1.6 fatty acid/urea ratio (w/w) by crystallization at ?8 °C for 16 h, ?20 °C for 8 h, and ?8 °C for 16 h. A DPA isomer concentrate containing 26.1 ± 0.5 % DPAn‐6 and 22.3 ± 0.4 % DPAn‐3 was achieved by argentated silica gel chromatography in the 6 % acetone/n‐hexane solvent fraction (v/v), and the recovery of both fatty acids was 66.1 ± 3.2 and 70.7 ± 2.2 %, respectively. Furthermore, 91.9 ± 2.5 % EPA and 99.5 ± 2.1 % DHA with recoveries of 47.8 ± 2.0 and 56.7 ± 3.3 %, respectively, were obtained in various fractions.     

Deodorization of lipase‐interesterified butterfat and rapeseed oil blends in a pilot deodorizer

A mixture of butterfat and rapeseed oil (7 : 3, wt/wt) was interesterified using immobilized lipase from Thermomyces lanuginosus at 50 °C. The interesterified mixture was then deodorized at five temperatures (60–180 °C) and three samples were withdrawn at 1, 2, and 3 h. The operation was monitored by free fatty acid (FFA) content, peroxide value (PV), volatiles, and the sensory evaluation of the samples with respect to flavor and odor (most importantly the butter flavor and odor and the off‐flavor and odor from butyric acid). ANOVA partial least squares regression analysis showed that deodorization time, and especially deodorization temperature, significantly affected the sensory properties and levels of volatiles, FFA and peroxides in the samples. The best compromise between removing undesirable off‐flavors while maintaining the desirable butter flavor seemed to be obtained by using a deodorization temperature of 120 °C for 2 h. Response surface methodology analysis showed a significant effect of deodorization temperature and, to a lesser extent, deodorization time. The butter flavor and odor had an optimum at a deodorization temperature of approximately 100–120 °C for 1–2 h. These conditions are therefore recommended in order to remove the off‐flavor and odor, while maintaining as much as possible of the attractive butter flavor and odor.     

The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol

The objective of this study was to investigate the use of lipases as catalysts for producing concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil as an alternative to conventional chemical procedures. Transesterification of fish oil with ethanol was conducted under anhydrous solvent-free conditions with a stoichiometric amount of ethanol. Among the 17 lipases tested, the results showed that Pseudomonas lipases had the highest activity toward the saturated and monounsaturated fatty acids in the fish oil, much lower activity toward EPA and DHA and, at the same time, good tolerance toward the anhydrous alcoholic conditions. With 10 wt% of lipase, based on weight of the fish oil triacylglycerol substrate (15% EPA and 9% DHA initial content), a 50% conversion into ethyl esters was obtained in 24 h at 20°C, in which time the bulk of the saturated and monounsaturated fatty acids reacted, leaving the long-chain n-3 polyunsaturated fatty acids unreacted in the residual mixture as mono-, di-, and triacylglycerols. This mixture comprised approximately 50% EPA+DHA. Total recovery of DHA and EPA was high, over 80% for DHA and more than 90% for EPA. The observed fatty acid selectivity, favoring DHA as a substrate, was most unusual because most lipases favor EPA.     

Influence of heat and refining on formation of CLA isomers in sunflower oil

The aims of this study were to determine whether CLA are formed during refining of vegetable oils and to study the level and composition of CLA during heating. The effects of three refining steps (neutralization, bleaching, and deodorization) were analyzed with respect to their effect on CLA content. Two temperatures (180 and 220°C) were used for heating; CLA appeared only after deodorization. The level of CLA was positively influenced by temperature. More CLA were present after treatment at 220°C than at 180°C (1.3 and 0.2% of total FA, respectively). The high temperature modified the relative proportions of the CLA isomers. The main CLA isomers in fresh or heated oils were the trans,trans ones (mainly 9,11 and 10,12 isomers).     

Deodorization of vegetable oils: Prediction of trans polyunsaturated fatty acid content

Kinetics of the formation of trans linoleic acid and trans linolenic acid were compared. Pilot plant-scale tests on canola oils were carried out to validate the laboratory-scale kinetic model of geometrical isomerization of polyunsaturated fatty acids described in our earlier publication. The reliability of the model was confirmed by statistical calculations. Formation of the individual trans linoleic and linolenic acids was studied, as well as the effect of the degree of isomerization on the distribution of the trans fatty acid isomers. Oil samples were deodorized at temperatures from 204 to 230°C from 2 to 86 h. Results showed an increase in the relative percentage of isomerized linolenic and linoleic acid with an increase in either the deodorization time or the temperature. The percentage of trans linoleic acid (compared to the total) after deodorization ranged from <1 to nearly 6%, whereas the percentage of trans linolenic acid ranged from <1 to >65%. Applying this model, the researchers determined the conditions required to produce a specially isomerized oil for a nutritional study. The practical applications of these trials are as follows: (i) the trans fatty acid level of refined oils can be predicted for given deodorization conditions, (ii) the conditions to meet increasingly strict consumer demands concerning the trans isomer content can be calculated, and (iii) the deodorizer design can be characterized by the deviation from the theoretical trans fatty acid content of the deodorized oil.     

Application of Silver Ion High-Performance Liquid Chromatography for Quantitative Analysis of Selected n-3 and n-6 PUFA in Oil Supplements

The aim of this study was to develop a simple method for simultaneous determination of selected cis/cis PUFA–LNA (18:2), ALA (18:3), GLA (18:3), EPA (20:5), and DHA (22:6) by silver ion high‐performance liquid chromatography coupled to a diode array detector (Ag‐HPLC‐DAD). The separation was performed on three Luna SCX Silver Loaded columns connected in series maintained at 10 °C with isocratic elution by 1 % acetonitrile in n‐hexane. The applied chromatographic system allowed a baseline separation of standard mixture of n‐3 and n‐6 fatty acid methyl esters containing LNA, DHA, and EPA and partial separation of ALA and GLA positional isomers. The method was validated by means of linearity, precision, stability, and recovery. Limits of detection (LOD) for considered PUFA standard solutions ranged from 0.27 to 0.43 mg L^?1. The developed method was used to evaluate of n‐3 and n‐6 fatty acids contents in plant and fish softgel oil capsules, results were compared with reference GC‐FID based method.     

Modeling the Primary Oxidation in Commercial Fish Oil Preparations

The quality of commercial fish oil products can be difficult to maintain because of the rapid lipid oxidation attributable to the high number of polyunsaturated fatty acids (PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). While it is known that oxidation in fish oil is generally the result of a direct interaction with oxygen and fatty acid radicals, there are very few studies that investigate the oxidation kinetics of fish oil supplements. This study uses hydroperoxides, a primary oxidation product, to model the oxidation kinetics of two commercially available fish oil supplements with different EPA and DHA contents. Pseudo first order kinetics were assumed, and rate constants were determined for temperatures between 4 and 60 °C. This data was fit to the Arrhenius model, and activation energies (E _a) were determined for each sample. Both E _a agreed with values found in the literature, with the lower PUFA sample having a lower E _a. The oil with a lower PUFA content fit the first-order kinetics model at temperatures ≥20 °C and ≤40 °C, while the higher PUFA oil demonstrated first-order kinetics at temperatures ≥4 °C and ≤40 °C. When the temperature was raised to 60 °C, the model no longer applied. This indicates that accelerated testing of fish oil should be conducted at temperatures ≤40 °C.     

Formation Kinetics of Monomeric Cyclic Fatty Acid Methyl Esters of Alpha-Linolenic Acid: Effects of Mono cis/trans Isomers

Cyclic fatty acid monomers (CFAM) are formed at low levels in edible oils during thermal processing operations such as frying or refining, and inevitably become part of the diet. These proatherogenic agents may increase the levels of oxidative stress markers, and induce hepatomegaly and steatosis. However, the kinetics involved in their formation is not well known. The objective of the present study was to evaluate the effects of cis and trans isomers on cyclization reactions involved in the thermal transformation of alpha-linolenic acid (ALA). Geometrical isomers of ALA were obtained from all-cis ALA by nitric acid treatment. Mono-trans isomers were concentrated using silver nitrate-silica gel chromatography. All-cis ALA, isomerized ALA, and a fraction at 85% mono-trans isomers were heat treated at 275 °C in hexadecane for periods up to 24 hours, and the formation of geometrical isomers and CFAM was monitored by GC. The results show that mono-trans isomers at carbon 9 and carbon 15 form CFAM at an accelerated rate, compared to the corresponding cis isomers, resulting in the formation of higher levels of CFAM over shorter time periods. The validation of the kinetic model was performed by solving simultaneously and nonlinearly fitting the system of coupled differential equations with experimental data. Good agreement was found between the experimental data and the predicted values. This work suggests that the use of polyunsaturated vegetable oils over extended periods for thermal processing of food may result in the formation of CFAM, in particular, if mono-trans isomers are present in the oil.     

EPA and DHA Levels in Whole Blood Decrease More Rapidly when Stored at −20 °C as Compared with Room Temperature, 4 and −75 °C

High‐throughput n‐3 fatty acid profiling is enabled by collection techniques such as venous whole blood and fingertip prick (FTP) sampling, but the resulting increased sample numbers increases storage demand. Highly unsaturated fatty acids (HUFA) in erythrocytes are susceptible to oxidation, but this tendency is poorly characterized in venous and FTP whole blood. Presently, whole blood samples with low and high n‐3 content collected with ethylenediaminetetraacetic acid were stored on chromatography paper with and without BHT pre‐treatment for up to 180 days at different temperatures (room, 4, ?20, ?75 °C). Whole blood prepared with heparin and BHT and stored in cryovials was also examined. Eicosapentaenoic acid (EPA, 20:5n‐3) + docosahexaenoic acid (DHA, 22:6n‐3) is relatively stable when stored at ?75 °C under various conditions but rapidly decreases in whole blood when stored at ?20 °C. At ?20 °C, BHT + heparin prepared whole blood can prevent decreases in cryovials up to 180 days but BHT only slows the decreases on chromatography paper. Surprisingly, whole blood stored at 4 °C and room temperature was less susceptible to decreases in EPA + DHA as compared with ?20 °C storage. Assessments of n‐3 blood biomarkers indicate the % n‐3 HUFA in total HUFA was more stable as compared with the sum of the relative % of EPA + DHA. In conclusion, FTP and venous whole blood for fatty acid analysis should be stored at ?75 °C whenever possible. In the absence of ?75 °C storage conditions, BHT should be added and 4 °C or room temperature appear to be better alternatives to ?20 °C.     

Changes in Tissue Lipid and Fatty Acid Composition of Farmed Rainbow Trout in Response to Dietary Camelina Oil as a Replacement of Fish Oil

Camelina oil (CO) replaced 50 and 100 % of fish oil (FO) in diets for farmed rainbow trout (initial weight 44 ± 3 g fish^?1). The oilseed is particularly unique due to its high lipid content (40 %) and high amount of 18:3n‐3 (α‐linolenic acid, ALA) (30 %). Replacing 100 % of fish oil with camelina oil did not negatively affect growth of rainbow trout after a 12‐week feeding trial (FO = 168 ± 32 g fish^?1; CO = 184 ± 35 g fish^?1). Lipid and fatty acid profiles of muscle, viscera and skin were significantly affected by the addition of CO after 12 weeks of feeding. However, final 22:6n‐3 [docosahexaenoic acid (DHA)] and 20:5n‐3 [eicosapentaenoic acid (EPA)] amounts (563 mg) in a 75 g fillet (1 serving) were enough to satisfy daily DHA and EPA requirements (250 mg) set by the World Health Organization. Other health benefits include lower SFA and higher MUFA in filets fed CO versus FO. Compound‐specific stable isotope analysis (CSIA) confirmed that the δ¹³C isotopic signature of DHA in CO fed trout shifted significantly compared to DHA in FO fed trout. The shift in DHA δ¹³C indicates mixing of a terrestrial isotopic signature compared to the isotopic signature of DHA in fish oil‐fed tissue. These results suggest that ~27 % of DHA was synthesized from the terrestrial and isotopically lighter ALA in the CO diet rather than incorporation of DHA from fish meal in the CO diet. This was the first study to use CSIA in a feeding experiment to demonstrate synthesis of DHA in fish.     

Lipase selectivity toward fatty acids commonly found in fish oil

The main objective of this study was to compare the fatty acid selectivity of numerous commercially available lipases toward the most ubiquitous fatty acids present in fish oils in form of their corresponding ethyl esters. Special interest was taken in their ability to separate the n‐3 long‐chain polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from the more saturated fatty acids as well as exploiting the putative discrimination between these highly valuable n‐3 PUFA. Hydrolysis of sardine oil ethyl esters in a Tris buffer solution by 12 microbial lipases is described. The results reveal that all of the lipases strongly discriminate against the n‐3 PUFA and prefer the more saturated fatty acids as substrates. Most of the lipases discriminate between EPA and DHA in favor of EPA, however, 2 bacterial lipases from Pseudomonas were observed to prefer DHA to EPA. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed fatty acid selectivity when their fish oil triacylglycerol hydrolysis was studied. Thus, the n‐3 PUFA including EPA and DHA were observed to be hydrolyzed at a considerably higher rate than the more saturated fatty acids.     

Retention behavior of trans isomers of eicosapentaenoic and docosahexaenoic acid methyl esters on a polyethylene glycol stationary phase

A polyethylene glycol (PEG) stationary phase was evaluated for the separation of mono‐trans isomers of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) methyl esters. The resolution patterns were compared to patterns achieved with previously applied conditions on a cyanopropyl phase. There were no overlaps between all‐cis EPA/DHA and their mono‐trans isomers on the PEG phase. Because of overlap between 22:0 and 22:1 isomers, the PEG column is not a good choice for analyses of EPA trans isomers in crude fish oils. However, if the saturated and monounsaturated fatty acids are not present in significant amounts, PEG can be a better choice than cyanopropyl columns.     

Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol

The objective of this study was to investigate the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their FA selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40°C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the FA, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. As an example, when FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained after 48 h. The residual FFA contained 78% DHA and only 3% EPA, in 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration.     

Antioxidant Effect of Nanoemulsions Based on Citrus Peel Essential Oils: Prevention of Lipid Oxidation in Trout

The antioxidant effects of oil‐in‐water nanoemulsion based on edible citrus peel essential oils on the fatty acid composition of rainbow trout fillets stored at 4 ± 2 °C are investigated. Fish fillets are treated with nanoemulsion and stored for 16 days. Lipid samples are converted into fatty acid methyl esters which are then detected by gas chromatagrophy (GC). The results show that palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), vaccenic acid (C18:1?‐7), oleic acid (C18:1?9), eicosenoic acid (C20:1?9), linoleic acid (C18:2?6), linolenic acid (C18:3?3), eicosapentaenoic acid (EPA) (C20:5?3), and docosahexaenoic acid (DHA) (C22:6?3) are the most important fatty acids in fish meat. While polyene index and hypocholesterolemic:hypercholesterolaemic fatty acid ratios decrease in trout fillets during cold storage, thrombogenicity index and atherogenicity index generally increase (especially in control and Tween 80 groups). The concentrations of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) are higher in the treatment groups and the saturated fatty acids (SFAs) are lower in all groups compared to those of the control group. Application of nanoemulsion based on citrus essential oils prevents oxidation of PUFA especially EPA and DHA, thus has potential as a preservative for fish oil. Practical Applications: In recent years, nanotechnological applications have been increasingly applied to the protection of food. Similarly, natural essential oils are used to increase the shelf life of foods. This study demonstrates the combined effect of a new method of nanoemulsions and essential oils on the safety of foods.     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: III—Cyclic fatty acid monomers study

The influence of isomeric triacylglycerols (TG) containing 18:3n-3 and 18:2n-6 on the formation of cyclic fatty acid monomers (CFAM) after heat treatment was assessed. Diacid TG, containing linoleic acid (L) or linolenic acid (α-Ln) along with palmitic acid (P), and positioned either in the central position (PLP and PLnP, respectively), or in one of the two outer positions (PPL and PPLn, respectively) were synthesized. Monoacid TG of trilinolein and trilinolenin mixed with tripalmitin were also prepared. The CFAM formed after heating were analyzed after total hydrogenation. The results obtained with the model TG were compared to another model consisting of a canola oil and its randomized counterpart. In diacid TG, the location of α-Ln in the central position of the TG molecule (PLnP) greatly enhanced the formation of the CFAM upon heating at temperatures below 240°C. On the other hand, 18:3n-3 in monoacid TG (LnLnLn) was highly resistant to CFAM formation within the same range of temperatures (180–220°C). The TG structure, more than the TG composition, seemed to explain the differences in the CFAM formation between the original canola oil and its interesterified counterpart. Like α-Ln, 18:2n-6 was more prone to cyclization when attached at the central position of the model TG. Conversely, the influence of the TG composition on the cyclization rate was less important for L than for α-Ln. It was concluded that positioning the C18 polyunsaturated fatty acid in the central position of TG rendered the oils much more sensitive to the cyclization reaction upon heat treatment.     

One-Step Extraction and Concentration of Polyunsaturated Fatty Acids from Fish Liver

The fatty acids (FA) eicosapentaenoic acid (20:5ω-3; EPA) and docosahexaenoic acid (22:6ω-3; DHA), which have several health benefits, have been concentrated from mako shark liver (Isurus oxyrinchus). The process was carried out in one single step, in which fish liver oil was simultaneously extracted, saponified and concentrated. Additionally, the polyunsaturated fatty acids (PUFA) concentrate was winterized to crystallize the remaining saturated FA, resulting in a further increase in the concentration of DHA and EPA. Two variables, temperature and water concentration in the saponification mixture, were optimized to increase the concentration of ω-3 PUFA. Best results were obtained at 12 °C and 0% water content in the mixture, obtaining 17.8% purity and 77.6% yield of EPA; DHA purity and yield were 33.3 and 82.2%, respectively.