Thermal degradation of long-chain polyunsaturated fatty acids during deodorization of fish oil

Véronique Fourniera

Frédéric Destaillatsb

Pierre Juanédaa

Fabiola Dionisib

Pierre Lambeletb

Jean-Louis Sébédioc

Olivier Berdeauxa

a INRA, UMR FLAVIC,Dijon, France

b Nestlé Research Center,Vers-chez-les-Blancs,Switzerland

c INRA,Clermont-Ferrand, France

Thermal degradation of long-chain polyunsaturatedfatty acids during deodorization of fish oil

Long-chain polyunsaturated fatty acids (LC-PUFA) of the n-3 series, particularly eico-sapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities espe-cially in the functionality of the central nervous system. Due to the occurrence ofnumerous methylene-interrupted ethylenic double bonds, these fatty acids are verysensitive to air (oxygen) and temperature. Non-volatile degradation products, whichinclude polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPAand DHA, were evaluated in fish oil samples obtained by deodorization under vacuumof semi-refined fish oil at 180, 220 and 250 7C. Polymers are the major degradationproducts generated at high deodorization temperatures, with 19.5% oligomers beingformed in oil deodorized at 250 7C. A significant amount of CFAM was produced duringdeodorization at temperatures above or equal to 220 7C. In fact, 23.9 and 66.3 mg/g ofC20 and C22 CFAM were found in samples deodorized at 220 and 250 7C, respec-tively. Only minor changes were observed in the EPA and DHA trans isomer contentand composition after deodorization at 180 7C. At this temperature, the formation ofpolar compounds and CFAM was also low. However, the oil deodorized at 220 and250 7C contained 4.2% and 7.6% geometrical isomers, respectively. Even after adeodorization at 250 7C, the majority of geometrical isomers were mono- and di-trans.These results indicate that deodorization of fish oils should be conducted at a maximaltemperature of 180 7C. This temperature seems to be lower than the activation energyrequired for polymerization (intra and inter) and geometrical isomerization.

Keywords: Deodorization, fish oil, geometrical isomerization, long-chain poly-unsaturated fatty acids, thermal degradation.

1 Introduction

The nutritional importance of long-chain polyunsaturatedfatty acids (LC-PUFA) has been well established. PUFA ofthe n-3 series, and especially eicosapentaenoic (EPA,20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), havespecific roles particularly in blood clotting [1], in the inflam-matory systems [2], in the functionality of the retina [3] and inthe central nervous system [4]. Fish oils and marine prod-ucts are the major food sources of n-3 LC-PUFA.

Fish oils have to undergo refining steps before their con-sumption or their utilization as food supplements. Refin-ing is usually divided into four steps: degumming, neu-tralization, bleaching and deodorization. The degummingstep is usually applied only to vegetable oils. The last stepis critical as it involves high temperatures (180–270 7C)that could give rise to side reactions [5]. Deodorizationprimarily removes undesirable volatile substances andconverts the oil into a bland-tasting, odorless and color-less liquid. Therefore, this process improves the oil’s

quality and stability. Less unsaturated fats and oils weredeodorized successfully, but then this method wasapplied to polyunsaturated oil [6]. Nowadays, steamrefining is the only large-scale practicable method used inthe industry [6] and has been used for fish oil deodoriza-tion. However, its repercussion on the production of deg-radation products from LC-PUFA still needs to be eval-uated.

Due to the occurrence of numerous methylene-inter-rupted ethylenic double bonds, LC-PUFA are unstableand heat treatment induces a number of chemical trans-formations (oxidation, polymerization, cyclization, geo-metrical isomerization and/or double bond migration) [7].Although deodorization aims to remove undesirablecompounds that affect the taste and the smell of fish oil, asimultaneous loss of valuable components could occur.For this reason, it is essential to be able to quantify deg-radation products in refined fish oils and to find deodor-ization conditions to prevent their formation during pro-cessing. Among the degradation products formed duringheat treatment in the absence of air, polar compounds(mostly oligomers), cyclic fatty acid monomers (CFAM)and geometrical isomers (trans fatty acid isomers) aremore likely to be produced.

Correspondence: Olivier Berdeaux, UMR FLAVIC, INRA, 17 rueSully BP 86510, 21065 Dijon Cedex, France. Phone: 133 380693540, Fax: 133 3 80693223, e-mail: [email protected]

Eur. J. Lipid Sci. Technol. 108 (2006) 33–42 DOI 10.1002/ejlt.200500290 33

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© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

34 V. Fournier et al. Eur. J. Lipid Sci. Technol. 108 (2006) 33–42

In 1974, Ackman et al. found that ordinary “non-hydro-genated” vegetable oils contained small amounts of PUFA(linoleic and a-linolenic) with trans bonds instead of ordinarycis bonds, and the author showed that these trans fattyacids were produced during deodorization of oils at ele-vated temperatures [8]. Investigations on heat treatmenteffects on lipids have already been done for mono-, di- andtri-unsaturated fatty acids [5, 6, 8–19]. Only few papersevaluate the effect of deodorization on LC-PUFA [7, 20–22].

In the present study, effects of deodorization temperaturewere evaluated based on three degradation productsofLC-PUFA: polar compounds, CFAM and geometrical isomers.

2 Material and methods

2.1 Samples and reagents

The semi-refined fish oil (NissuiFine ChemicalDept., NipponSuisan Kaisha, Ltd., Tokyo, Japan) was deodorized with alab deodorizer. The semi-refined fish oil had a free fatty acid(FFA) content of 0.01 (expressed as oleic acid) and a per-oxide value of ,0.20 meq O2/kg oil. Briefly, fish oil washeatedateither180, 220or250 7Cfor3 hundera pressure of1.5 mbar and with 2%/h (based on oil) direct steam injection.The control sample was not submitted to any deodorization.Hexane, petroleum ether (b.p. 40–65 7C), chloroform,dichloromethane, methanol, acetone, tetrahydrofuran, andacetonitrile were purchased from SDS (Peypin, France).Platinum oxide was purchased from Merck (Munich, Ger-many). Standards of fatty acid ethyl and methyl estersand 2-amino-2-methyl-1-propanol were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

2.2 Quantification and composition of polarcompounds

During oil refining or frying processes, a complex mixtureof degradation products, e.g. polar compounds, can beformed [9]. These degradation products not only differ bytheir polarity, but also by their molecular weight. They areprincipally due to the action of atmospheric oxygen andthe water content of foodstuffs in the case of frying, and thehigh temperatures in the case of the degradations thatoccur during refining. Thus, polar compound determina-tion stands out as the most commonly used methodologyto evaluate oil degradation and has been included toestablish the limits of alteration acceptable for the oilsintended for human consumption [9]. Fish oils were frac-tionated by column chromatography according to astandard method [23] using a column filled with a homog-enized blend of 20 g silica with water at a ratio of 95 : 5.Briefly, the apolar fraction was eluted by 150 mL petroleum

ether/diethyl ether 87 : 13 (vol/vol). Then, the polar fractionwas recovered by elution with 150 mL diethyl ether. Thetwo fractions (polar and apolar) were further submitted tofractionation by high-performance size-exclusion chro-matography (HPSEC) for the separation of oligomers bymolecular size [9]. The separation was achieved with aSpectraSystem P1000XR pump (ThermoElectron, Cour-taboeuf, France) using two columns (Waters, Milford,USA): an Ultrastyragel column (500 Å) and a GPC KF-8025column having 8 mm internal diameter and 300 mm length,and a Shimadzu RID-10A differential refractometricdetector (Kyoto, Japan). An isocratic program using tetra-hydrofuran at 1 mL/min was employed for elution.

2.3 Fatty acid methyl ester preparation

Prior to gas chromatography (GC) analysis, the acylglycer-ols were transesterified using a basic catalyst (0.5 Msodium methanolate in methanol). About 20 mg of fish oilwas weighed accurately and diluted in 1 mL toluene. Of thesodium methanolate solution, 2 mLwas added and the tubeheld at 50 7C for 5 min. The transesterification was stoppedby adding 0.1 mL acetic acid. Ethyl arachidate (1.8 mg) wasadded as internal standard and the esters were washedwith 5 mL distilled water, then extracted successively with 5and 3 mL hexane. The solvents were evaporated and theesters diluted in hexane to a concentration of 0.1 mg/mL.

2.4 GC analysis

Fatty acid methyl esters (FAME) were analyzed on a Hew-lett Packard Model 4890 capillary gas chromatograph(Palo Alto, CA, USA). Most GC analyses were performedusing a CP-Sil88 capillary column (100 m60.25 mm ID,0.2 mm film; Varian, Courtaboeuf, France) except for GC-mass spectrometry (GC-MS) analysis of CFAM which wasdone using a BPX70 (120 m60.25 mm ID capillary col-umn, 0.25 mm film; SGE, Melbourne, Australia). Theinstrument was equipped with a split/splitless injector(splitless for 0.5 min). Linear velocity of hydrogen was37.0 cm/s at 60 7C. The temperature was held at 60 7C for5 min, programmed to 165 7C at 15 7C/min and held for1 min, and then to 225 7C at 2 7C/min and finally held at225 7C for 17 min [24]. The injection port was held at 250 7Cand a flame ionization detector (FID) was used at 250 7C(hydrogen at 35 mL/min and air at 350 mL/min).

2.5 Quantification of C20 and C22 CFAM

CFAM were quantified according to literature procedure[18]. About 100 mg of the oil samples were weighed andthe FAME prepared as described previously. The result-


Eur. J. Lipid Sci. Technol. 108 (2006) 33–42 Degradation of LC-PUFA during deodorization 35

ing FAME were weighed and a known amount of inter-nal standard was added [0.125% of two standards,stearic and eicosanoic fatty acid ethyl esters (FAEE)].Catalytic hydrogenation using platinum oxide under astream of hydrogen was carried out on FAME of deo-dorized fish oils. Platinum oxide, around 5 mg, wasadded along with a magnetic stirrer before hermeticallyclosing the tube and opening the hydrogen supply. After3 h, the reduction reaction was completed. The hydro-genated FAME were dried under a stream of nitrogenand then solubilized in 700 mL acetone. The totallyhydrogenated FAME were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) (Ultrabase C18 column 250610 mm, particlesize 5 mm; Shandon HPLC, Cheshire) as described bySebedio et al. [25]. The analysis was run in isocraticmode with a flow rate of 4 mL/min using a mixture ofacetonitrile and acetone (90 : 10, vol/vol). A differentialrefractometric detector was used for detection. Solvent,column and detector were maintained at 58 7C to pre-vent crystallization of the sample. Fractions were furtheranalyzed by GC-FID and GC-MS.

2.6 Separation of geometrical isomers byargentation thin-layer chromatography

FAME fractions containing isomers of EPA and DHA wereseparated according to their number of trans doublebonds by argentation thin-layer chromatography (Ag-TLC) [26]. Briefly, TLC plates (Silica gel, 20620 cm;Merck, Darmstadt, Germany) were impregnated byimmersion in a silver nitrate solution (10% wt/vol in ace-tonitrile) for 30 min. Plates were eluted with toluene/methanol (85 : 15, vol/vol). Bands containing mono-, di-,tri-, tetra-, penta-trans isomers for EPA and DHA andhexa-trans isomer for DHA were scraped off the plate,and the FAME were recovered by adding 5 mL of a 1%NaCl methanol/water 90 : 10 (vol/vol) blend, then extract-ed twice with 2 mL hexane.

2.7 Purification of 20:5 and 22:6 FAME byRP-HPLC

FAME prepared from deodorized oil were separated usinga Kromasil-C18 25 cm610 mm ID 5 mm (Thermo Quest,Courtaboeuf, France) column. FAME were dissolved in100 mL acetone and eluted with an isocratic programusing distilled and filtrated acetonitrile at a flow rate of4 mL/min, as described by Juaneda and Sebedio [27].Fractions containing EPA, DHA and their geometrical iso-mers were collected to recover enough material (seventimes, at around 3 mg per run) for further separation byAg-TLC.

2.8 Preparation of 4,4-dimethyloxazolinederivatives

4,4-Dimethyloxazoline (DMOX) derivatives were preparedas described by Dobson and Christie [28]. Briefly, 250 mLof 2-amino-2-methyl-1-propanol was added to 1 mgFAME. The mixture was flushed with nitrogen, then heldfor 8 h at 170 7C. The DMOX derivatives were extractedwith 3 mL dichloromethane and washed with distilledwater until neutrality. The solvent was evaporated and theDMOX derivatives were diluted in hexane to a final con-centration of 0.1 mg/mL.

2.9 GC-MS analysis

GC-MS analysis was performed using an Agilent tech-nologies gas chromatograph 6890A Network coupled to aselective mass detector 5973 (Palo Alto, CA, USA) usingthe same temperature program as described before,except that the final temperature was kept for a longerperiod of time to allow heavier derivatives to elute. Thecolumn outlet was directly connected to the ion source ofthe mass spectrometer operated at 230 7C and using anionization energy of 70 eV. Spectral data was acquiredover a mass range of 50–450 amu.

3 Results and discussion

The deodorization process was found to affect the con-centration of LC-PUFA in fish oil. Deodorization of fish oilat 220 and 250 7C led to critical losses of LC-PUFA.Actually more than 60% of LC-PUFA was lost after deo-dorization at 250 7C (Fig. 1). It could be noticed (Fig. 1)that at 220 and 250 7C, n-6 LC-PUFA, as arachidonic(20:4 n-6) and 22:5 n-6, are less prone to thermal degra-dation than all other n-3 LC-PUFA reported. Degradationof fish oil during deodorization was investigated by de-termining the total amount of the non-volatile com-pounds. Three classes of degradation products are pre-dominantly formed during heat treatment in the absenceof air: polar compounds (mostly polymers), CFAM andtrans LC-PUFA isomers. This study was done to evaluatethe effect of the deodorization temperature on the deg-radation of a fish oil containing 5.8% EPA and 19.5%DHA, and to find the optimal experimental conditionsthat could be used to prevent the degradation of LC-PUFA during deodorization. Fatty acid compositions ofcontrol and heated oils are reported in Tab. 1. Threedeodorization temperatures were selected: 180 7C;220 7C, which appears to be the mean temperaturerecommended for industrial deodorization of vegetableoils in France [19]; and 250 7C, which is a positive controlfor degradation.



Tab. 1. The fatty acid composition (g/100 g of oil, n = 3) of deodorized fish oil as a function of deo-dorization temperature.

Fatty acids Control Deodorization temperature [ 7C]

180 220 250

MV SD MV SD MV SD MV SD

14:0 2.77 0.03 2.88 0.08 2.81 0.04 2.85 0.0616:0 15.79 0.26 16.02 0.24 15.67 0.30 16.02 0.1616:1 4.57 0.07 4.65 0.04 4.54 0.11 4.61 0.0418:0 3.96 0.06 3.96 0.03 3.90 0.07 3.98 0.0418:1 16.63 0.26 16.65 0.16 16.41 0.30 16.60 0.1618:2 1.30 0.02 1.29 0.00 1.24 0.02 1.15 0.0120:1 2.55 0.02 2.53 0.01 2.54 0.04 2.62 0.0222:1 1.04 0.01 1.03 0.01 1.03 0.01 1.05 0.01all-cis 20:4 n-6 1.76 0.01 1.74 0.01 1.52 0.01 0.62 0.02all-cis 20:5 n-3 5.81 0.07 5.64 0.03 4.18 0.07 0.90 0.06all-cis 22:4 n-6 0.23 0.00 0.22 0.00 0.19 0.00 0.06 0.02all-cis 22:5 n-6 1.11 0.01 1.06 0.01 0.85 0.01 0.24 0.01all-cis 22:5 n-3 1.25 0.01 1.20 0.00 0.92 0.01 0.22 0.00all-cis 22:6 n-3 19.54 0.06 18.70 0.14 12.68 0.10 2.15 0.01Other 21.68 0.88 22.42 0.73 31.53 1.06 46.92 0.54

Fig. 1. Degradation of LC-PUFA relative to deodorizationtemperature. ARA, arachidonic acid; EPA, eicosapentae-noic acid; DPA, docosapentaenoic acid; DHA, doc-osahexaenoic acid.

3.1 Polymerization of LC-PUFA duringdeodorization of fish oil

As a first step to assess the effect of the deodorizationtemperature on polar compound appearance, HPSECwas directly used for oil fractionation. Fig. 2 illustrates thesize-exclusion chromatogram of undeodorized and deo-dorized fish oils giving triacylglycerol (TAG) polymers anddimers as a function of the deodorization temperature.

We note an accumulation of polar compounds already inoil deodorized at 180 7C, but more significantly at 220 and250 7C.

The application of size-exclusion chromatography (SEC)to the direct separation of polar compounds producedduring heating of fats has been explored by Marquez-Ruizet al. [29]. With this method, it is only necessary to dilutethe fat in the appropriate solvent before the chromato-graphic determination, but the resolution and detection ofminor compounds are very poor owing to the presence ofunaltered TAG as major components having molecularweights similar to oxidized compounds. For this reason,in a second step, we separated the samples into totalpolar and nonpolar fractions using adsorption chroma-tography. Therefore, a IUPAC column chromatographymethod was used prior to HPSEC to investigate thecomposition of the fractions.

Then, the two fractions were separated by HPSEC. Anapolar fraction always gave one peak in HPSEC, corre-sponding to TAG. Tab. 2 shows the evolution of thepolar compounds separated by HPSEC, including totallevels and their composition, during deodorization.Control oil contains 2.6% of polar compounds. A part ofthese polar compounds in control oil are partial acyl-glycerols which naturally occur in crude oil. Mono-acylglycerols are removed by deodorization at a tem-perature of 220 and 250 7C. An explanation for thepresence of other polar compounds is that the controloil has already been semi-refined. The polar fraction



Fig. 2. Size-exclusion chromatogram of fish oilshowing formation of TAG polymers and dimerapparition as a function of deodorization tempera-ture. 1: Triacylglycerol polymers (TAGP), 2: tria-cylglycerol dimers (TAGD), 3: triacylglycerols (TAG)and oxidized triacylglycerol monomers (oxTAGM),4: diacylglycerols (DAG), 5: monoacylglycerols(MAG), and 6: free fatty acids (FFA).

Tab. 2. Total polar compounds (mg/g of oil, n = 2) and polar compounds distribution in deodorizedfish oil as a function of deodorization temperature.

Polar compound Control Deodorization temperature [ 7C]

180 220 250


Total 26.4 1.1 34.1 3.7 51.0 1.3 194.9 1.1Triacylglycerol polymers 1.3 0.3 1.5 0.2 8.1 0.2 104.4 2.9Triacylglycerol dimers 4.6 0.3 5.4 0.6 16.4 0.0 67.7 1.0Oxidized triacylglycerols 5.6 0.5 12.7 2.1 11.1 0.7 6.6 0.4Diacylglycerols 6.5 0.4 7.5 0.8 10.0 0.3 13.2 2.3Monoacylglycerols 7.0 0.5 6.0 0.5 4.5 0.1 1.7 0.3Free fatty acids 1.0 0.1 0.9 0.0 0.9 0.1 1.3 0.9

increased slightly with temperature for the 180 and 220 7Csamples and greatly for the oil deodorized at 250 7C, toreach 19.5 wt-%.

In the polar fraction, six groups of compounds could beidentified by HPSEC, i.e. TAG polymers (TAGP), TAGdimers (TAGD), oxidized TAG monomers (oxTAGM), dia-cylglycerols (DAG), monoacylglycerols (MAG) and FFA.While TAGP, TAGD and oxTAGM are compounds formedthrough oxidation and polymerization reactions, DAG,MAG and FFA are components arising from hydrolysis [9].oxTAGM is the major polar compound formed at 180 7C.Polymers are the major degradation products generatedat high deodorization temperatures; 6.8% and 10.4% ofdimers and polymers, respectively, are formed in oil deo-dorized at 250 7C.

Also, quantification by GC-FID of FAME prepared fromfish oil with an internal standard showed a loss in mattervisible in GC (non-volatile matter), which correlated with

results obtained with SEC. This is in agreement with theresults of Grandgirard and Julliard [13] who demonstratedthat the non-volatile fraction was well correlated withresults obtained by HPSEC. Our results are in agreementwith those of Marquez-Ruiz et al. [29] who showed thatpolymers and dimers occurred at the highest levels in themost unsaturated oils. Results provided in Tab. 1 suggestthat LC-PUFA are more prone to thermal degradationcompared to C18 PUFA. It could be hypothesized thatdue to the higher number of ethylenic double bonds, LC-PUFA are the prevalent substrate of polymerization reac-tions. On the contrary, opposite results were observed byCmolik and Pokorny [6]. It was observed for a-linolenicacid-rich vegetable oils that the overall content of mostpolyunsaturated TAG changed slightly compared to largevariations due to isomerization of linolenic acid. Thisresult concerned oil deodorized at temperatures from 265to 269 7C; these temperatures favored isomerization overpolymerization reactions.



3.2 Cyclization of LC-PUFA duringdeodorization of fish oil

First evidence for the presence of CFAM in heated fats wasreported in 1953 [30]. Toxicity experiments 3 years later onfractions isolated from heated fats had shown a possibletoxic effect of some of the CFAM. Then, a great deal ofwork has been done on the identification and quantifica-tion of linoleic and linolenic cyclic monomers. Sebedio etal. [18] demonstrated that ten times less CFAM are formedin an oil rich in C18:2 than in a C18:3-rich oil. This suggeststhat PUFA are more prone to intramolecular cyclizationthan other, less unsaturated fatty acids. Moreover, Sebe-dio and De Rasilly [21] demonstrated the occurrence ofcyclic fatty acids in refined fish oil concentrate.

Three independent quantifications of CFAM were per-formed for each sample (control oil and oils deodorized at180, 220 and 250 7C) using a previously described meth-od [25]. FAEE of stearic and eicosanoic acids were usedas internal standards instead of odd fatty acids foundnaturally in fish oil. Fully hydrogenated fish oil was ana-lyzed by HPLC and two fractions were collected (seeFig. 3). The fraction collected between C18:0 FAME andC20:0 FAME contained a mixture of C19:0 FAME, the firstinternal standard C18:0 FAEE and C20 CFAM. The frac-tion collected between C20:0 FAME and C22:0 FAMEcontained a mixture of C21:0 FAME, the second internal

Fig. 3. HPLC chromatogram of the hydrogenated FAMEof fish oil deodorized at 250 7C.

standard C20:0 FAEE and a mixture of C22 CFAM. Thetwo fractions were converted into DMOX derivatives priorto GC-MS analysis.

GC-MS analyses of the two isolated fractions confirmedthe presence of CFAM of C20 and C22 with the corre-sponding molecular weights and fragmentation patternalready observed by Sebedio and De Rasilly [21]. Frag-mentation of CFAM DMOX derivatives gives character-istic spectra (see Fig. 4). Electron impact mass spectrawith molecular ion at m/z 363 for C20 species and 391 forC22 species were obtained for each peak, confirming thatthe peaks are DMOX derivatives of C20 and C22 hydro-genated CFAM. Fig. 5A shows the single ion monitoring

Fig. 4. Example of mass spectra of DMOXderivatives of hydrogenated CFAM; (A) cor-responds to a C20 CFAM with a six-mem-bered carbon ring located at position C9–C14 (R1, C12NOH22; R2, C6H13), and (B) cor-responds to a C22 CFAM with a five-mem-bered carbon ring located at position C9–C13 (R1, C12NOH22; R2, C9H19).



Fig. 5. [M]1 and [M–15]1 SIM chromatograms ofDMOX derivatives of CFAM. (A) C20 CFAM(m/z = 348 and 363), and (B) C22 CFAM (m/z =376 and 391), in fish oils deodorized at 250 7C.

(SIM) chromatogram of C20 CFAM (m/z = 363 [M]1 andm/z = 348 [M–15]1). Fig. 5B shows the SIM chroma-togram of C22 CFAM (m/z = 391 [M]1 and m/z = 376[M–15]1). These results confirm the location of CFAMpeaks on the GC-FID chromatograms used for the quan-tification step. Analysis by GC-MS confirmed the absenceof CFAM in vicinal HPLC fractions. Minor C20 and C22cyclic fatty acids from arachidonic and 22:5 acids cannotbe distinguished from cyclic fatty acids from EPA andDHA, respectively, obtained after hydrogenation of oil.Consequently, cyclized PUFA having 20 or 22 carbons areall confounded with dominant EPA and DHA CFAM, givinga total quantification for C20 and C22.

A good reproducibility was obtained for quantification byGC-FID of both C20and C22species. A significant amountof CFAM was produced during deodorization at tempera-tures above or equal to 220 7C (Tab. 3). In fact, 23.9 and

66.3 mg/g of C20 and C22 CFAM were found in samplesdeodorized at 220 and 250 7C, respectively. As for polarcompounds, the control (semi-refined) oil already con-tained CFAM at 1.7 mg/g. The amount of CFAM formed inoil deodorized at 180 7C (3.8 mg/g) was higher than thatreported by Sebedio and De Rasilly [21]. They measuredonly 0.4–0.6 mg/g of CFAM in encapsulated fish oils, evenif the EPA and DHA concentrations were higher in the sup-plement as compared to the oil used in this study.

3.3 Geometrical isomerization of EPA (20:5 n-3)and DHA (22:6 n-3) during deodorization of fishoil

Geometrical isomerization occurs during deodorization,which is generally conducted at temperatures in the rangeof 180–270 7C under vacuum in the presence of steam for



Tab. 3. CFAM derived from C20 and C22 unsaturated fatty acids formed during deodorization of fishoil (mg/g, n = 3)

CFAM Control Deodorization temperature [ 7C]

180 220 250


C20 0.2 0.0 0.6 0.0 4.7 0.7 17.0 1.8C22 1.5 0.1 3.2 0.2 19.2 1.0 49.3 2.3

a few minutes to several hours [5]. These isomers havebeen shown to be components in edible vegetable oilsthat have been subjected to heat treatment, provided thetemperature was higher than 190 7C [15]. They have beendetected in infant food formulas, either liquid or pow-dered, from France [5], Canada [31] and the USA [32].

By geometrical isomerization of EPA and DHA, 32 and64 isomers, respectively, can theoretically be formed.Geometrical isomerization of PUFA is not a negligiblephenomenon, even if other reactions (polymerization,oxidation) are quantitatively more important in edible fatsand oils. Fatty acid compositions of the fish oils (Fig. 6)show that deodorization has an important impact on thegeometrical isomerization of EPA and DHA. Tab. 4 pre-sents the evolution in trans isomers of EPA and DHA as afunction of deodorization temperature. Due to possibleoverlapping of trans fatty acids with other degradationproducts, e.g. CFAM, the reported values for trans fattyacids might be slightly overestimated. Only minoramounts of EPA and DHA trans isomers were formedduring deodorization at 180 7C. However, the oil deodor-ized at 220 7C contained about 7.7 and 34.1 mg/g of EPAand DHA geometrical isomers, respectively. Deodoriza-tion at 250 7C was so detrimental to PUFA that only 0.9%EPA and 2.2% DHA remained in the fish oil after such atreatment (see Tab. 1). Furthermore, 20.0 and 55.6 mg/gof geometrical isomers of EPA and DHA, respectively,were detected in the oil deodorized at 250 7C. Our resultsindicate that geometrical isomerization of fish oil is mini-mized when the deodorization temperature does notexceed 180 7C.

In order to gain information on the nature of these geo-metrical isomers of LC-PUFA, i.e. the number of transethylenic double bonds, the polyunsaturated FAME puri-fied by RP-HPLC were fractionated according to theirnumber of trans double bonds by Ag-TLC. The first bandin the Ag-TLC analysis contained all-cis DHA (Rf = 0.2),the second band mono-trans of DHA and all-cis EPA(Rf = 0.4), and the third band mono-trans of EPA and di-trans of DHA (Rf = 0.6), and so on.

Tab. 4. Evolution of geometrical isomers from the PUFAEPA and DHA during deodorization of fish oil (mg/g).

Fatty acid Deodorization temperature [ 7C]

Control 180 220 250

Total trans-EPA 1.4 1.5 7.7 20.0Relative distribution

[%]Mono-trans 100.0 100.0 100.0 62.1Di-trans –§ – tr# 37.9Other – – – trTotal trans-DHA 0.7 1.8 34.1 55.6Relative distribution

[%]Mono-trans 100.0 100.0 96.4 50.2Di-trans – – 3.6 39Other – – tr 10.8

§ Under the limit of detection.# Trace amount, under the limit of quantification.

Methyl tricosanoate (23:0 FAME) was used as internalstandard after recovery of FAME collected by Ag-TLC, toperform the relative quantification of geometrical isomerclasses by GC.

Results reported in Tab. 4 show that the composition oftrans isomers changes with the deodorization tempera-ture, with di-trans being detected at 220 7C and tri-transat 250 7C. Results clearly indicate that DHA is more sen-sitive than EPA to geometrical isomerization and thatmore di-trans geometrical isomers are formed at 250 7C.Mono-trans fatty acids constitute the majority of the geo-metrical isomers of EPA and DHA found in the deodorizedfish oil sample analyzed.

4 Conclusion

LC-PUFA degradation products were monitored by de-termining the amount of the new compounds formedduring heating: polymers, CFAM and LC-PUFA geomet-



Fig. 6. GC-FID chromatograms of theEPA/DHA methyl ester fish oil fractionsobtained by RP-HPLC of (A) non-deo-dorized and (B) deodorized oil at180 7C, (C) 220 7C and (D) 250 7C.(E, F) Enlargement of chromatogramzones of EPA and DHA methyl esterspurified from fish oil deodorized at250 7C.

rical isomers. We showed that polymers are the majordegradation products generated at high deodorizationtemperatures, with 19.5% oligomers being formed inthe oil deodorized at 250 7C. A significant amount ofCFAM was produced during deodorization at tempera-tures above or equal to 220 7C. Only minor changeswere observed in the EPA and DHA trans isomer con-tent and distribution after deodorization at 180 7C. Atthis temperature, the formation of polar compounds andCFAM was also low. However, the oil deodorized at 220and 250 7C contained about 4.2% and 7.6% geomet-rical isomers, respectively. Even after a deodorization at250 7C, the majority of geometrical isomers were mono-and di-trans. All together, these results indicate that

deodorization of fish oils should be conducted at amaximal temperature of 180 7C. This temperatureseems to be lower than the activation energy requiredfor polymerization (intra and inter) and geometrical iso-merization. Further studies are in progress in order toidentify geometrical isomers and CFAM from EPA andDHA.

Acknowledgments

The authors kindly acknowledge Mrs. Bole-Richard forher technical support and Mr. Semon for GC-MS analysis(Analytical platform, PPM, UMR-FLAVIC, Dijon, France).



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[Received: October 18, 2005; accepted: November 24, 2005]


Thermal degradation of long-chain polyunsaturated fatty acids during deodorization of fish oil

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