DOI 10.1007/s11746-012-2148-1 ORIGINAL PAPER Chrcteriztion of Oxidtive Stility of Fish Oil- nd Plnt Oil-Enriched Skimmed Milk Lind C. Sg Ver Kristinov Bente Kirkhus Chrlotte Jcosen Josefine Skret Kristin Hovde Lilnd Elling-Olv Rukke Received: 28 Mrch 2012 / Revised: 12 Septemer 2012 / Accepted: 17 Septemer 2012 Ó AOCS 2012 Astrct The ojective of this reserch ws to determine the oxidtive stility of fish oil lended with crude plnt oils rich in nturlly occurring ntioxidnts, cmelin oil nd ot oil, respectively, in ulk nd fter supplementtion of 1 wt% of oil lends to skimmed milk emulsions. Aility of crude ot oil nd cmelin oil to protect fish oil in ulk nd s fish oil-enriched skimmed milk emulsions ws evluted. Results of oxidtive stility of ulk oils nd lends ssessed y the Schl oven weight gin test nd y the rncimt method showed significnt increse in oxidtive stility when ot oil ws dded to fish oil in only 5 nd 10 %, wheres no protective effect of cmelin oil ws oserved when evluted y these methods. Moreover, fish oil lended with ot oil conferred the lowest PV nd lower mounts of voltile compounds during the storge period L. C. Sg (&) K. H. Lilnd E.-O. Rukke Deprtment of Chemistry, Biotechnology nd Food Science, Norwegin University of Life Sciences, PO Box 5003, 1432 As, Norwy e-mil: lind.sg@1life63.com V. Kristinov Deprtment of Biotechnology, Norwegin University of Science nd Technology, 7491 Trondheim, Norwy V. Kristinov SINTEF Fisheries nd Aquculture Limited, PO Box 4762, Sluppen, 7465 Trondheim, Norwy B. Kirkhus J. Skret Nofim AS, Norwegin Institute of Food, Fisheries nd Aquculture Reserch, Osloveien 1, 1430 As, Norwy C. Jcosen Division of Industril Food Reserch, Ntionl Food Institute, Technicl University of Denmrk, Building 221, Søltofts Plds, 2800 Kongens Lyngy, Denmrk of 14 dys t 4 C. Surprisingly, skimmed milk supplemented with fish-ot oil lend gve the highest scores for off-flvors in the sensory evlution, demonstrting tht severl methods, including sensory nlysis, should e comined to illustrte the complete picture of lipid oxidtion in emulsions. Keywords Antioxidnts Emulsion Fish oil Oxidtion PUFA Plnt oils Introduction Mrine n-3 polyunsturted ftty cids (PUFA) hve received incresed ttention during the lst decde due to potentil helth enefits in humn nutrition [1, 2]. Fish oil is the min dietry source of the long-chin (LC) n-3 PUFA, especilly eicospentenoic cid (EPA C20:5) nd docoshexenoic cid (DHA C22:6). However, intke of mrine foods is elow the recommended level in mny countries [3]. Enrichment of commonly consumed foods with LC n-3 PUFA is wy of incresing consumption of these ftty cids in the diet. Oil-in-wter (O/W) emulsions re the sis of mny frequently consumed food products, such s myonnise nd sld dressings. A gret del of reserch hs een focused on oil-in-wter emulsions, mong other ecuse of its interesting properties s medium for the enrichment of oil with eneficil ftty cid content. Due to the high degree of unsturtion of the frequently dded LC n-3 PUFA EPA nd DHA, triglycerides rich in these ftty cids re prone to oxidtion. Lipid oxidtion cn dversely ffect the nutritionl vlue, shelf-life nd sensory qulity of foods. Oxidtive deteriortion of lipids results in the formtion of primry oxidtion products, lipid hydroperoxides, which re
tsteless nd odorless. When these primry oxidtion products re decomposed they form mixtures of voltile nd non-voltile secondry oxidtion products [4]. In order to get complete picture of the oxidtion process, the degree of oxidtion should e mesured y more thn one method, including methods detecting oth the primry nd secondry oxidtion products [4]. In complex systems such s lipid contining emulsions, series of fctors cn ffect the initition nd propgtion of oxidtion [5, 6]. Processing conditions nd physicl nd chemicl properties of the dded ingredients re mong these fctors [7]. Such ingredients my include mrine oils, ntioxidnts, wter, emulsifiers, proteins nd so forth. It hs een suggested tht specil emphsis should e on the use of nturl emulsifying nd stilizing compositions with regrds to food emulsion technologies [8]. Plnt oils contin nturlly occurring ntioxidnt compounds, where the most undnt ones re tocopherols [9]. Cmelin stiv lso known s flse flx is n oilseed crop with high levels (30 40 %) of the essentil -linolenic cid (C18:3 n-3), mking it vulnerle to oxidtion. However, cmelin oil hs een found to e very resistnt to oxidtion nd rncidity prtly due to high content of c-tocopherol [10]. Ot oil is nother plnt oil with interesting properties. Among which re its wide rnge of compounds with ntioxidtive qulities, including nturlly occurring tocopherols nd tocotrienols [11]. Blending plnt oils with more unsturted fish oils hs een climed to exert protective effect of fish oil in ulk [12]. Eidhin nd O Beirne 2010 [13] showed tht odor scores of spreds produced with lends of fish oil nd cmelin oil improved compred to spreds produced with only fish oil. In ddition, such lends would provide high levels of the nutritionlly importnt n-3 ftty cids ALA, EPA nd DHA. Industril clims tht ot rn oil lended with PUFA contining oil, such s fish oil, cn exert protective effect when dded to cow milk hve een mde (ptent no Norge 325446, Lipid composition nd use hereof ). In ddition to the content of nturlly occurring emulsifiers in the form of protein mteril, cow s milk is widely consumed oil-in-wter emulsion, thus n interesting medium for enrichment of fish oil rich in LC n-3 PUFA. The ojective of this study ws to investigte the oxidtive stility of fish oil lended with crude plnt oils high in PUFA nd nturl ntioxidnts, cmelin nd ot oil, respectively, efore nd fter ddition to n oil wter emulsion in the form of skimmed milk. Mterils nd Methods Mterils Refined food grde fish oil (lend of cod liver oil nd slmon oil) (FO) with dded ntioxidnts (totl mount less thn 2 % w/w stted y the supplier) ws provided y Borregrd Industries Ltd., division Denomeg Pure Helth, Norwy. Crude cold pressed cmelin oil (CO) ws provided y Bioforsk Øst (Apelsvoll, Norwy). After hrvest, seeds were stored in cold-storge chmer t 5 C. Crude oil ws otined y using pilot press for smll smples (BT Bio Presse Type 50, BT iopresser ps, Dyvd, Denmrk). Oil frctions were frozen t -40 C immeditely fter pressing. Crude food grde Ot Oil (OO), extrcted with ethnol ws otined from CreNutrition (Swedish Ot Fier AB, Sweden). Skimmed milk powder ws otined from TINE BA (Oslo, Norwy) with ft content of \1.0 %. Chrcteriztion of Oils Initil peroxide vlue of the three oils ws mesured y the AOCS Officil method Cd8-90 [14]. The ftty cid composition of FO ws provided y the mnufcturer, wheres the ftty cid compositions of CO nd OO were provided y Nofim (As, Norwy). The contents of ftty cids were mesured s ftty cid methyl esters [15] using gs chromtogrphy (GC) [16] with flme ioniztion detection (FID). Peks were identified y mens of externl stndrds. The concentrtion of the individul ftty cids ws expressed in % of totl ftty cids. The tocopherol nd tocotrienol (only OO) profile of the oils ws nlyzed y Eurofins Scientific (Moss, Norwy), n ccredited lortory, nd Nofim, y using norml phse high-performnce liquid chromtogrphy (HPLC) sed on method descried y Pnfili et l. [17]. Determintion of Oxidtive Stility of Oils Blends of FO with CO or OO, s well s pure oils, were tested for their oxidtive stility y mesuring the oil stility index (OSI) ccording to AOCS officil method Cd 12 92 [18]. The inry lend rtios were 90:10 nd 95:5 for oth FO:CO nd FO:OO. Ech inry rtio ws prepred s well-mixed tch; the minor oil component (i.e., plnt oil) ws weighed first, nd the remining ws filled up with fish oil to otin the desired rtio. Smples (5.00 ± 0.04 g) were plced in glss tues, seled with two-hole ruer stopper equipped with ertion nd effluent tues, nd instlled in the oxidtive stility instrument (Omnion Inc., Rocklnd, MA, USA). The proe mesuring the conductivity signl ws connected to computer which processed the dt nd generted OSI curves nd OSI times utomticlly. All the smples were run t 70.0 ± 0.1 C, ir pressure ws set t 4.0 4.25 psi. A reltively low temperture of 70 C compred to the temperture of 110 C descried in the AOCS officil method ws chosen
due to the high susceptiility of FO to oxidtion. The ir pressure ws reduced from the one prescried in the AOCS officil method (5.5 psi), due to extensive foming of OO under the flow of oxygen, to prevent contmintion of the conductivity mesurement tue contining deionized wter nd proe y the oily fom. The determintions were crried out in six replictes. The sme lends s for the OSI test were used for the Schl oven weight gin test. Oil smples (5.00 ± 0.01 g) were weighed into open glss petri-dishes (inner dimeter 7.0 cm, height 1.2 cm) nd plced in lortory drying oven (TS 8136, Termks AS, Bergen, Norwy) t 70 ± 1 C in the drk with no ir circultion. The dishes were tken out of the oven for weighing every 8 h during the first 21 dys nd then twice or once dy, cooled to mient temperture, reweighed nd returned to the oven. The time required to rech 0.5 % weight gin ws clculted nd tken s n index of stility. All of the smple tches were nlyzed in triplicte. Preprtion of Emulsions Skimmed milk powder (100 g/l) ws mixed with wter (20 C) using n Ultr Turrx Super Dispx SD 45/2 (IKA- Werke GmH nd Co. KG, Stufen, Germny). The resulting milk (3 L) ws psteurized y heting to 72 C within 3 min, holding for 15 s nd then cooled to room temperture. Three different tches of emulsions were then prepred s descried in Tle 1; FO:CO nd FO:OO were mixed together in rtio 90:10 nd the oil lend (1 wt%) were then dded to the skimmed milk. For pure FO, 1 wt% ws dded to the skimmed milk. The smples were susequently homogenized (18 MP) in two-vlve Rnnie homogenizer (Model LAB 4580/71, Copenhgen, Denmrk) under cooling conditions (7 9 C). The ph of the emulsions ws 6.7. The three seprte tches of smples were stored in closed Pyrex ottles (50 ml for PV nd HS-GC/MS nlysis, 1 L for sensory nlysis) nd sujected to storge t 4 C in the drk, nd following nlysis. Smples for PV nd voltile nlysis were tken t dy 0, 5, 8 nd 14, immeditely flushed with nitrogen Tle 1 Experimentl design over the ddition of oils for preprtion of enriched emulsions Smple nme Addition of oil to milk (wt%) FO CO OO FO 1.0 FO? CO 0.9 0.1 FO? OO 0.9 0.1 FO fish oil, FO? CO fish oil nd cmelin oil mixture, FO? OO Fish oil nd ot oil mixture (quntity 99.9, AGA AS, Oslo, Norwy), nd stored t -25 C. Smples were thwed just efore nlysis. Sensory evlution of the emulsions ws crried out fter 0, 5 nd 8 dys of storge. Anlysis of Primry Oxidtion Products Lipids were extrcted from the emulsions y chloroform:methnol (1:1 w/w) [19], using reduced mount of solvent [20]. Extrcts were evported to dryness under N 2. PV were mesured directly in the oil extrcted from the milk emulsion y colorimetric ferric-thiocynte method [21]. Ech of the three smple tches were nlyzed in duplicte. Anlysis of Voltile Secondry Oxidtion Products Voltiles were trpped in Tenx tues (Perkin Elmer, Norwlk, CN, USA) y purging 4 g of the milk emulsions with N 2 (150 ml/min) for 30 min t 45 C. 4-methyl-1- pentnol in rpeseed oil ws used s internl stndrd. An utomtic therml desorer (ATD-400, Perkin Elmer, Norwlk, CN) ws used to desor (200 C) the voltiles, nd susequently they were cryofocused on Tenx GR cold trp. Seprtion of the voltile compounds ws chieved y gs chromtogrphy (HP 5890 IIA, Hewlett Pckrd, Plo Alto, CA, USA) s descried y Timm- Heinrich et. l. (2003) [22]. The voltiles were nlyzed y mss spectrometry (HP 5972 mss-selective detector) nd identified y MS lirry serches (Wiley138 K, John Wiley nd Sons, Hewlett Pckrd) nd y uthentic externl stndrds. The individul compounds were quntified through clirtion curves. The formtion of nine voltiles 1-penten-3-one, 1-penten-3-ol, (E)-2-pentenl, 2-penten-1-ol, hexnl, (E)-2-hexenl, 2,4-heptdienl, (E,E)-2,4-heptdienl nd (E,Z)-2,6-nondienl, responsile for off-flvors [23, 24], ws followed for 14 dys of storge t 4 C. All of the three smple tches were nlyzed in triplicte. Sensory Evlution To descrie the ojective perception of the vrious emulsions, trined pnel performed qulity descriptive nlysis ISO 6564:19865(E) nd ISO 13299:2003(E) of the smples. The pnel consisted of 12 sujects employed exclusively to work s sensory ssessors t Nofim (Ås, Norwy). Ech of the 12 memers tke prt in the sensory nlyses conducted t Nofim 12 h per week nd hs etween 3 nd 25 yers of experience using descriptive nlysis on vrious kinds of food nd everges including milk. The pnelists hve een selected nd trined ccording to recommendtions in ISO 8586-1:1993(E).
Prior to the ssessments, the pnel went through trining session to select relevnt sensory ttriutes. The session lsted for 1 h nd included some of the ctul smples from the project. The ssessors developed list of ttriutes for the milk smples nd greed on consensus list of ttriutes for the profiling nd on the definition of ech ttriute. Two smples were ssessed in trining session for the purpose to gree on the vrition in ttriute intensity nd this session lsted for nother hour. The descriptors used for odor nd flvor ssessment were fishy, metllic, sterin/prffin nd pint. The coded smples (50 ml) were served in lind trils t 0, 5 nd 8 dys of storge nd rndomized ccording to smple, ssessor nd replicte. The pnelists evluted the smples in duplicte, during two sessions. Emulsions were evluted on continuous intensity scle rnging from 1 to 9, where 9 is the mximum intensity. The sensory lortory hs een designed ccording to guidelines in ISO 8589:1988(E) with seprte ooths. Dt were collected using Eye Question, v. 3.8.6 (Logic 8, Nederlnd). Sttisticl Anlysis Dt were evluted y one-wy nlysis of vrince nd Tukey s test using Minit Sttisticl softwre (Addison- Wesley, Reding, MS, USA). Differences were considered to e significnt t p \ 0.05. Sensory descriptions ws plotted in R version 2.14.1, which is free softwre environment mintined y the R Development Core Tem (http://www.r-project.org/). Results nd Discussion Properties of Oils FO, CO nd OO used in the present work were chrcterized in terms of ftty cid profile, initil PV nd tocopherol profile (Tle 2). Levels of ftty cids nd tocopherols in CO nd OO were in good greement with previous reports [10, 11, 25]. The FO used in this study contined 10 nd 12 % of the n-3 LC PUFA EPA nd DHA, respectively (Tle 2). In CO nd OO the mjority of the PUFA consisted of the n-3 PUFA -linolenic cid (ALA) nd n-6 linoleic cid (LA), respectively. The high ALA content (37.9 %) in CO might e nutritionl dvntge, ut it cn lso e driving fctor for oxidtion. PUFA re susceptile to lipid oxidtion, in the order DHA [ EPA [ ALA [ LA. The order reflects the mount of rective methylene groups ville for peroxidtion processes. Compred to FO nd CO, the ftty cid profile of OO showed lower degree of unsturtion. In ddition to triglycerides, the reltively high content of Tle 2 Ftty cid composition, initil peroxide vlue nd tocopherol content of the fish oil (FO), cmelin oil (CO) nd ot oil (OO), nd lends of FO:CO nd FO:OO (90 % nd 10 %, respectively) Ftty Acids % FO CO OO 90:10 FO:CO 90:10 FO:OO SFA C14:0 3.8 0.1 0.2 3.4 3.4 C16:0 13.5 5.4 15.8 12.7 13.7 C18:0 4.0 2.4 1.3 3.8 3.7 C20:0 1.3 0.1 0.1 0.0 Sum SFA 21.3 9.2 17.4 20.0 20.8 MUFA C16:1(n-7) 5.5 0.2 5.0 5.0 C18:1(n-7) 4.0 0.7 0.7 3.7 3.7 C18:1(n-9) 19.0 12.5 37.7 18.4 20.9 C20:1(n-9) 14.7 0.7 1.5 0.1 C20:1(n-11) 1.3 C22:1(n-9) 3.0 0.1 0.3 0.0 C22:1(n-11) 4.6 4.1 4.1 Sum MUFA 34.4 30.9 39.4 32.9 33.8 PUFA C18:2(n-6) 4.4 15.6 41.5 5.5 8.1 C18:3(n-3) 1.2 37.9 1.4 4.9 1.2 C18:4(n-3) 1.9 1.7 1.7 C20:2(n-6) 2.2 0.2 0.0 C20:4(n-3) 0.9 1.9 1.0 0.8 C20:5(n-3) 10.0 9.0 9.0 C21:5(n-3) 0.6 0.5 0.5 C22:5(n-3) 2.2 2.0 2.0 C22:6(n-3) 11.9 10.7 10.7 Sum PUFA 33.1 57.6 42.9 35.6 34.1 Other 11.2 2.4 0.3 Degree of unsturtion 193.4 187.8 126.6 191.7 185.6 Initil PV (mequiv/kg) \0.1 1.5 2.2 Tocopherols (ppm) - 740 26 90 c- 1310 784 13 d- 600 13 6.5 phospholipids (C 12 % w/w) in crude ot [26] my influence oxidtive stility. Given tht the quntities of CO nd OO to FO were reltively low (10 90 %), less significnt chnges in ftty cid profile of FO? CO nd FO? OO re expected, hence no considerle dilution effect of FO. Fish oil nd CO hd the highest c-tocopherol levels (1310 nd 784 ppm, respectively), wheres OO hd the highest levels of -tocopherol (90 ppm). FO hd the highest totl content of tocopherols, ut the predominnt prt is dded tocopherol, wheres CO nd OO were crude oils with only nturlly occurring tocopherols. In ddition to the tocopherol content, OO contined tocotrienols
(193 ppm -tocotrienol nd 11 ppm -tocotrienol). The oils my lso contin other ntioxidnts not nlyzed in this study. In prticulr, phenolic compounds in CO nd OO my contriute to protection ginst oxidtion [10]. Tocopherols nd tocotrienols ct y donting their phenolic hydrogens to lipid free rdicls, nd hve donting power in the order [ [ c [ d [27]. Reltive ntioxidnt ctivity of tocopherols depends on fctors such s the concentrtion, lipid composition, physicl stte (ulk or emulsion) nd temperture. All the oils hd low initil peroxide vlues. Peroxides, primry oxidtion products in CO were higher y only 0.7 mequiv/kg thn in OO, indicting tht the two oils hd similr levels of oxidtion. The initil PV in the FO ws very low (\0.1 mequiv/kg). In fish oil enriched emulsion, low initil peroxide vlue ws shown to fcilitte the control of oxidtive deteriortion [24]. The three pure oils showed no significnt increse (p [ 0.05) in the peroxide vlue during storge t 4 C for 21 dys (dt not shown). Results of Stility Tests of Selected Oil Blends The oxidtive stility of oils nd their lends ws evluted sed on the mesurement of the induction period (OSI) nd the Schl oven test t 70 C (Tle 3). The stility tests gve consistent results, indicting tht ddition of OO to the lends gve the est protection ginst oxidtion (Tle 3). Blends of FO? OO contining 5 nd 10 % OO were roughly two-times s oxidtive stle s lends of FO? CO with the sme proportions of CO. Incresing the proportions of OO in the FO? OO lend incresed the stility significntly, while incresing the proportions of CO in the FO? CO lends hd only minor effects on the oxidtive stility. The ftty cid composition nd unsturtion indices of the oils used in this study suggest tht FO would e the lest stle, followed y CO, nd then OO. Despite the high ALA content (*40 %) in CO, the induction period for CO ws doule nd significntly different to tht oserved for FO s seen in Tle 3, which cn e explined y differences in presence of minor compounds such s phenolic compounds nd tocopherols s well s ftty cid profile [13]. The present results indicte tht OO is very resistnt to oxidtion. However, the OSI test conditions did not llow determintion of OSI vlues for pure OO. This ws due to extensive foming of the oil in the glss tues under the ir flow which inevitly contminted the mesurement proe. The vlue is therefore denoted s Nd (not determined). Content of polr lipids nd free ftty cids my hve cused the foming [28]. Pure OO showed no increse in weight fter more thn 50 dys in the oven t 70 C, so the weight oservtion ws terminted. Addition of only 5 % OO gve incresed protection, indicting tht the Tle 3 Oxidtive Stility Index (OSI) nd weight gin vlues of oils nd inry mixtures of oils % of dded oil OSI vlues (h) t 70 C presence of tocopherols, tocotrienols, phenolics nd other compounds in OO proly contriuted to the drmticlly prolonged induction period, given tht no dilution effect cn e expected. Nturlly, FO with its high unsturtion conferred the lowest OSI time (*57 h) nd lso reched 0.5 % weight gin t the erliest time point. The mesured induction periods of FO using the two methods were reltively high compred with rncimt mesurements of nchovy, hke liver nd srdine oils under the sme temperture conditions [29]. The reltively high stility of FO cn e ttriuted to the high totl tocopherol content of 2660 ppm, nd the presence of scoryl plmitte nd other ntioxidnts (not shown), which my ehve synergisticlly in reinforcing the ntioxidnt ctivity of tocopherols. Oxidtive Stility of Enriched Emulsions 0.5 % weight increse (h) t 70 C FO 100 56.9 ± 2.2 51.1 ± 0.4 FO ± CO 0? 100 139.5 ± 2.5 x.6 ± 2.5 x 90? 10 58.1 ± 2.5 y 51.8 ± 1.8 y 95? 5 54.1 ± 3.3 y 53.1 ± 0.1 y FO ± OO 0? 100 Nd Nd 90? 10 159.4 ± 1.7 x 162.6 ± 1.1 x 95? 5 113.3 ± 2.3 y 117.9 ± 0.9 y The vlues re expressed in hours ± stndrd devition (n = 6 for OSI nd n = 3 for the weight gin method) x y indictes significnt differences (p \ 0.05) within oil types in the column. Nd not determined Oil-in-wter emulsions, enriched with LC n-3 PUFA, were prepred with 1 wt% FO, nd 90:10 FO:CO nd FO:OO lends (Tle 1). Stility tests indicted tht rtio of 95:5 ws sufficient to increse oxidtive stility in lends with OO in ulk, wheres rtios higher thn 90:10 were needed for lends with CO. However, preliminry experiments showed tht inclusion of higher levels of the plnt oils (more thn 10 %) resulted in more chrcteristic tste nd smell nd lso poorer physicl emulsion stility ws oserved, hence 90:10 rtio of fish oil to plnt oil ws chosen for oxidtive stility tests in ulk nd in the skimmed milk emulsions. Skimmed milk ws chosen in this study due to its low ft content (\0.1 %) which cuses less interference with the mesurements nd lso due to the presence of nturlly occurring emulsifier s form of protein mteril.
Formtion of Primry Oxidtion Products Peroxide vlues re shown in Tle 4. Peroxide vlues of the oil extrcted from the emulsions t dy 0 were higher thn the initil PV of the oils, proly due to the fct tht vlues otined from the ferric thiocynte method re generlly higher thn vlues otined with the iodometric method [4]. In ddition, it my lso e presumed tht the oxidtion process ws initited s erly s during the processing of the emulsions. Since het is known to increse the oxidtion rte of lipids, the temperture should in generl e kept s low s possile during processing nd storge [4]. With this in mind, the emulsions in this study were homogenized t low temperture to void the effect of temperture on the oxidtion rte. Nevertheless, the emulsions reched high peroxide vlues during storge. The FO emulsion incresed more thn 40-fold in peroxide vlue fter 5 dys of storge. During further storge, the PV in FO enriched emulsion incresed significntly. In contrst, the FO? OO emulsion showed no significnt difference in PV during the 14-dy storge. The protective effect of fish oil y ddition of 10 % crude ot oil my e scried to its content of tocopherols nd tocotrienols nd other minor compounds with ntioxidnt effects [11]. It hs een suggested tht low initil PV (\0.1 mequiv/ kg) is more criticl for oxidtion rtes thn PUFA or content of tocopherol in oils [24, 30]. In this study FO oxidized very rpidly when emulsified into skimmed milk despite low initil PV. A significnt increse in peroxide vlue ws lso shown in the FO? CO emulsion, with n initil vlue of 5.8 to 42.7 mequiv/kg fter 5 dys of storge. This indictes tht ddition of crude CO to FO did not hve protective effect ginst oxidtion in the emulsion when considering the primry oxidtion products, which ws lso oserved in ulk. A decrese in PV ws shown in FO? CO from dy 5 to 8, nd from dy 8 to 14, which could indicte decomposition of hydroperoxides to secondry oxidtion products. When relting the PV with results from the induction time mesurements (Tle 3), the sme trend ws oserved concerning the oxidtion rte of the oils nd lends, which ws found in the order; FO [ FO? CO [ FO? OO. Previous reserchers hve reported tht milk emulsion prepred with liquid skim milk with less thn 0.01 % nd lso with 1 % milk ft content, enriched with 1.5 wt% fish oil nd 0.5 wt% lend of fish oil nd rpeseed oil, respectively, resulted in reltively low peroxide vlues during storge t 2 C for 14 dys [24, 30]. The fish-rpeseed oil lend resulted in PV \ 1 mequiv/kq, while skimmed milk enriched with cod liver oil (without ntioxidnts) gve peroxide vlues up to 5.3 mequiv/kq. Milk is complex medium with severl fctors tht cn either inhiit or promote oxidtion of the dded oils. The present results indicted tht interctions etween the milk medium nd the dded PUFA seemed to promote oxidtion rther thn inhiit it. As opposed to ulk oils where oxidtion minly tkes plce t the interfce of oil nd ir, oxidtion of oil-in-wter emulsions is likely to occur t the interfce etween the oil nd wter phse, which results in differences in fctors tht ffect the lipid oxidtion process. Protein mteril intrinsic in cow s milk functioned s emulsifiers t the interfce of the oil droplets in the present study, thus no externl emulsifier ws dded. Due to the ph vlue (6.7) of the milk emulsions, the surfce chrge of the emulsion droplets is expected to e negtive nd my therefore hve potentilly ttrcted positively chrged trce metls which re highly pro-oxidtive, therey enhncing lipid oxidtion in the studied emulsions, especilly FO nd FO? CO which gve the highest peroxide vlues [6]. The emulsions were exposed to oxygen for short period of time during the homogeniztion process, which is lso fctor for the initition of oxidtion. The high levels of n-3 LC-PUFA in FO nd the high ALA levels in the CO re nother fctor tht cn ccelerte oxidtion, due to their polrity nd orienttion towrds the interfce [31], which my e reson for PV vlues [40 mequiv/kg fter only 5 dys of storge. The significnt increse in oxidtion in the emulsions fter just 5 dys of storge t 4 C isin contrst to the stility in ulk oils which further confirms Tle 4 Peroxide vlue of enriched skimmed milk emulsions during 14 dys storge t 4 C Peroxide vlue (mequiv O 2 /kg oil) Smple Dys of storge 0 5 8 14 FO 1.4 ± 0.7,x 47.0 ± 0.4,x 62.4 ± 1.0 c,x 90.6 ± 1.5 d,x FO? CO 5.8 ± 3.1,x 42.6 ± 0.5,x 32.3 ± 3.6 c,y 17.0 ± 1.0 d,y FO? OO 7.1 ± 1.2,x 5.8 ± 0.0,y 10.6 ± 0.2,z 9.0 ± 0.7,z PV expressed s mequiv O 2 /kg ± stndrd devition (n = 2) -d x z in the row indicte significnt difference (p \ 0.05) etween dys of storge within smple in the columns indicte significnt difference (p \ 0.05) etween smples within dys
tht oxidtive stility of PUFA is highly dependent on the food mtrix, lipid composition nd form [5]. Formtion of Secondry Voltile Compounds Nine voltile secondry oxidtion products derived from the degrdtion of n-3 nd n-6 ftty cids [23, 24] were selected s mrkers of oxidtion during storge of the emulsions, including 1-penten-3-one, 1-penten-3-ol, (E)-2- pentenl, 2-penten-1-ol, hexnl, (E)-2-hexenl, 2,4-heptdienl, (E,E)-2,4-heptdienl nd (E,Z)-2,6-nondienl. Except for 2-penten-1-ol nd hexnl, the formtion of the selected secondry voltile compounds incresed even during the first storge dys (Tle 5). 2-penten-1-ol ws elow the detection limit in ll the emulsions during the storge period. Hexnl ws lredy present in high vlues in the emulsions from dy 0. Significntly higher initil vlues were found in the emulsions contining the crude plnt oils, OO (out 56 9 10 4 ng/g) nd CO (out 20 9 10 4 ng/g) compred with the FO emulsion (out 12 9 10 4 ng/g). Hexnl is common degrdtion product from the utoxidtion of linoleic cid hydroperoxides, nd hs very low threshold vlue for flvor nd odor [4]. In ot nd in cmelin oil, hexnl is one of the most undnt voltile compounds [32, 33]. Both the OO nd CO contin high levels of linoleic cid, 41.5 nd 15.6 %, respectively (Tle 2). During the storge period, degrdtion of hexnl ws detected in OO nd CO emulsions, while hexnl incresed in the FO emulsion (Tle 5). This ws somewht surprising, ut could indicte tht hexnl prticipted in other rections in the OO nd CO emulsions to higher extent thn in the FO emulsion. In previous studies on the oxidtive stility of fish oilenriched milk [24, 30], the development of typicl voltile secondry oxidtion products were elow 12 ng/g emulsions fter 11 dys of storge t 2 C, even without the ddition of ntioxidnts. In our study only 1-penten-3-one nd 2-pentenl were elow this vlue for the FO enriched milk t dy 14. Peroxide vlues showed significnt nd high increse in the FO nd FO? CO emulsion from dy 0 to 5 (Tle 4). As result of the increse in hydroperoxides, corresponding formtion of secondry voltile compounds especilly in FO nd FO? CO ws seen in this storge period, indicting high decomposition of peroxides (Tle 5). The numer of rective methylene groups is higher, nd the ctivtion energy for strcting proton from methylene group in conjugtion in FO nd FO? CO is lower thn for FO? OO with lower degree of unsturtion in the ftty cid profile. At 8 nd 14 dys of storge, the FO? OO emulsion showed significntly lower vlues of 1-penten-3-ol, 2,4-heptdienl nd (E,E)- 2,4-heptdienl thn in the FO nd FO? CO emulsions (Tle 5). (E,Z)-2,6-nondienl ws not detected in the FO? OO emulsion during the storge period, wheres significnt increse from 10.8 to 41.7 ng/g, during 5 to 14 dys storge ws found for (E,Z)-2,6-nondienl in the FO emulsion. Development of the vinyl ketone 1-penten-3- one ws higher in FO emulsion t dy 8 nd 14 compred to FO? CO nd FO? OO emulsions. 1-penten-3-one, the diunsturted ldehyde (E,E)-2,4-heptdienl nd (E,Z)- 2,6-nondienl re compounds derived from degrdtion of n-3 PUFA, nd hve een chrcterized s very potent odornts, contriuting to unplesnt rncid nd fishy offflvors in fish oil enriched milk nd myonnise [23, 24]. In generl the FO emulsion developed higher levels of 1-penten-3-one, 1-penten-3-ol, (E)-2-pentenl, (E)-2-hexenl, 2,4-heptdienl, nd (E,Z)-2,6-nondienl during the storge period, closely followed y the FO? CO emulsion. A degrdtion of 1-penten-3-one, (E)-2-pentenl, 2,4-heptdienl nd (E,E)-2,4-heptdienl ws shown fter 5 dys of storge for the FO? CO emulsion, which indicte further oxidtion or rections with proteins to tertiry products. Overll the evlution of voltile compounds showed the lowest vlues for the FO? OO emulsion for ll compounds except for hexnl, followed y the FO? CO emulsion, s lso oserved when PV of the sme emulsions were mesured. Sensory Evlution of Enriched Emulsions The verge sensory scores for the off-odors nd off-flvors in milk emulsions stored for 8 dys in the drk t 4 C re shown in Fig. 1. Only smll chnges were detected in sterin/prffin odors nd flvors during the storge period, with FO? OO t dy 8 hving the highest score on 2.0 for odor nd 2.3 for flvor (not shown). Fish nd pint odor nd flvor resulted in the overll highest vlues nd incresed from dy 0 to 8, in prticulr for the FO nd FO? OO emulsions. A significnt increse in fishy nd pint off flvors from dy 0 to 5 were shown for FO, wheres the FO? OO emulsion incresed significntly from dy 0 to 8. The highest scores were found for FO? OO emulsion t dy 8. Pint odor nd flvor showed significntly higher intensity in the FO? OO emulsion t dy 8 (odor score 6.8 nd flvor score 7.0), compred with the FO nd FO? CO emulsions (odor score 4.5 nd 1.7, respectively, flvor score 4.8 nd 2.2). These results contrdicts with results from the nlysis of secondry voltile compounds, where the FO? OO emulsion showed etter oxidtive stility compred with FO nd FO? CO emulsions. Voltile compounds resulting in prticulr fish nd pint odor nd flvor in the FO? OO emulsion compred with FO nd FO? CO were not found (Tle 5). Whether this cn e explined y compounds not mesured y HS GC/MS is unknown. This clerly demonstrtes tht oth sensory nlysis nd instrumentl
Tle 5 Development of selected voltile oxidtion products in the enriched skimmed milk emulsions during 14 dys of storge t 4 C Storge time (dys) 0 5 8 14 1-Penten-3-one FO Nd 4.7 ± 1.1,x 6.4 ± 1.1,x 6.2 ± 0.4,x FO? CO Nd 4.7 ± 1.1,x 3.6 ± 0.8,y Nd FO? OO Nd 3.88 ± 0.81,x 4.1 ± 0.8,y 1.3 ± 0.2,y 1-Penten-3-ol FO 0.5 ± 0.0,x 7.4 ± 1.1,x 18.8 ± 1.9 c,x 39.1 ± 2.8 d,x FO? CO 1.1 ± 0.1,x 7.4 ± 0.4,x 16.4 ± 1.6 c,x 24.2 ± 1.5 d,y FO? OO 1.9 ± 0.1,x 4.02 ± 0.13,x 6.3 ± 0.7,y 18.7 ± 2.0 c,z (E)-2-Pentenl FO Nd 4.7 ± 0.9,x 8.1 ± 1.5,x 10.3 ± 1.0,x FO? CO Nd 4.5 ± 1.0,x 4.8 ± 1.1,y 2.7 ± 0.2,y FO? OO 1.6 ± 0.0,x 3.49 ± 0.48,x 3.9 ± 0.5,y 2.6 ± 0.1,y 2-Penten-1-ol FO Nd Nd Nd Nd FO? CO Nd Nd Nd Nd FO? OO Nd Nd Nd Nd Hexnl FO 125850 ± 2335,x 137256 ± 8043,x 219224 ± 10914,x 272187 ± 11629 c,x FO? CO 203317 ± 20938,y 144327 ± 10376,x 109798 ± 6468,y 31997 ± 3591 c,y FO? OO 557172 ± 40888,z 314844 ± 11756,y 332771 ± 15715,z 57733 ± 1617 c,y (E)-2-Hexnl FO Nd 6.8 ± 0.2,x 11.8 ± 0.6,x 19.6 ± 1.0 c,x FO? CO Nd 6.3 ± 0.6,x 8.4 ± 1.0,y 8.0 ± 0.5,y FO? OO Nd 4.4 ± 0.2,y 5.1 ± 0.5,,z 6.6 ± 0.5,y 2,4-Heptdienl FO Nd 208 ± 31,x 525.5 ± 61.1,x 385.6 ± 73.5,,x FO? CO 10.1 ± 1.6,x 324.2 ± 121.0,x 460.0 ± 140.0,xy 365.5 ± 131.5,x FO? OO 14.4 ± 8.4,x 165.8 ± 28.0,x 262.5 ± 23.04,y 355.0 ± 70.5,x (E,E)-2,4-Heptdienl FO Nd 17.8 ± 0.7,x 27.2 ± 2.1,x 28.8 ± 6.4,x FO? CO 0.7 ± 0.1,x 20.1 ± 3.1,x 37.0 ± 3.2 c,y 15.0 ± 4.4,y FO? OO 1.8 ± 1.4,x 9.0 ± 2.0,y 11.3 ± 1.0,z 22.8 ± 4.9 c,x,y (E,Z)-2,6-Nondienl FO Nd 10.9 ± 0.9,x 25.2 ± 3.9,x 41.7 ± 3.5 c,x FO? CO Nd 9.0 ± 2.1,x 14.0 ± 4.6,y 14.2 ± 5.2,y FO? OO Nd Nd Nd Nd Expressed s ng/g of milk emulsion ± stndrd devition (n = 3) -d x z in the row indicte significnt difference (p \ 0.05) etween dys of storge within smple in the columns indicte significnt difference (p \ 0.05) etween smples within dys (comintions shring letter re not significntly different) methods re needed for more complete evlution when monitoring lipid oxidtion in lipid enriched emulsions. One possile explntion for the high scores for off-flvors in the FO? OO emulsion cn e relted to the content of minor components including free ftty cids in ot oil [32]. The free ftty cids re formed during lipid extrction y hydrolysis of triglycerides either y lipses or y high temperture in the presence of wter [34]. In emulsions, the polrity of free ftty cids nd hydroperoxides cn drive them to the surfce of n emulsion droplet nd interctions with queous-phse oxidtion ctlysts cn occur [6].
Intensity [1-9] FO FO+CO FO+OO Metl odor 0 5 8 Metl flvor 0 5 8 compounds during storge t 4 C for 14 dys storge. However, sensory nlysis of the sme emulsion gve the highest scores for undesirle off-flvors, indicting tht severl methods, including sensory nlysis, should e comined to illustrte the complete picture of lipid oxidtion in emulsions. Intensity [1-9] Intensity [1-9] The intensity of fish nd pint off-flvors in the FO? CO emulsions hd low intensity in the rnge 1.1 2.2 during the entire storge period nd showed no significntly increse in ny of the off-flvors, in contrst to FO nd FO? OO emulsions which gve higher sensory scores nd lso incresed significntly during storge. Crude oils my hve strong chrcteristic product-relted flvor, thus, crude CO hd very distinct odor nd flvor even when mixed with FO nd dded to milk, which my hve cused msking effect of the off-flvors relted to lipid oxidtion in the FO? CO emulsion, resulting in low sensory scores for these ttriutes. This finding is in ccordnce with recent study y Eidhin nd O Beirne (2010) [13] showing tht cmelin oil hd msking effect on fish odors when lended with fish oil. Conclusion Fishy odor c Fishy flvor 0 5 8 0 5 8 Pint odor c This study demonstrted tht inclusion of only 5 nd 10 % crude ot oil significntly incresed the oxidtive stility of fish oil in ulk, demonstrting interesting ntioxidtive properties. The oxidtive stility of skimmed milk emulsion enriched with lend of 90 % fish oil nd 10 % ot oil lso reveled the lowest peroxide vlues nd voltile 0 5 8 0 5 8 Dys Dys Pint flvor Fig. 1 Averge intensity [1 9] of sensory descriptions in skimmed milk enriched with fish oil (FO), inry lend of fish oil nd cmelin oil (FO? CO) nd inry lend of fish oil nd ot oil (FO? OO) during 8 dys of storge t 4 C. Error rs indicte stndrd devitions. Letters c indicte significnt difference etween dys of storge for ech emulsion type c c Acknowledgments The uthors thnk Denomeg A/S, Bioforsk nd Swedish ot fier AB for providing the oils used in this study. References 1. Simopoulos AP (1999) Essentil ftty cids in helth nd chronic disese. Am J Clin Nutr 70:560 569 2. Ruxton CHS, Reed SC, Simpson MJA, Millington KJ (2007) The helth enefits of omeg-3 polyunsturted ftty cids: review of the evidence. J Nutr Diet 20:275 285 3. Trutwein EA (2001) n-3 Ftty cids physiologicl nd technicl spects for their use in food. Eur J Lipid Sci Technol 103:45 55 4. Frnkel EN (2005) Lipid oxidtion, 2nd edn. The oily press, Englnd 5. Couplnd JN, McClements DJ (1996) Lipid oxidtion in food emulsions. Trends Food Sci Technol 7:83 91 6. Mei L, McClements DJ, Decker EA (1999) Lipid oxidtion in emulsions s ffected y chrge sttus of ntioxidnts nd emulsion droplets. J Agric Food Chem 47:2267 2273 7. Nielsen NS, Klein A, Jcosen C (2009) Effect of ingredients on oxidtive stility of fish oil-enriched drinking yoghurt. Eur J Lipid Sci Technol 111:337 345 8. Nicovsk K (2010) Oxidtive stility nd rheologicl properties of oil-in-wter emulsions with wlnut oil. Adv J Food Sci Technol 2:172 177 9. Duqn EMA, Adullh A, Sni AH (2011) Nturl ntioxidnts, lipid profile, lipid peroxidtion, ntioxidnt enzymes of different vegetle oils. Adv J Food Sci Technol 3:308 316 10. Armovic H, Butinr B, Nikolič V (2007) Chnges occurring in phenolic content, tocopherol composition nd oxidtive stility of cmelin stiv oil during storge. Food Chem 104:903 909 11. Peterson DM (2001) Ot ntioxidnts. J Cer Sci 33:115 129 12. Pdely FB, Freemn, IP, Polmn, RG, vnlookern GJ (1989) Europen ptent 304 115 13. Eidhin D, O Beirne D (2010) Oxidtive stility nd cceptility of cmelin oil lended with selected fish oils. Eur J Lipid Sci Technol 112:878 886 14. Americn Oil Chemists Society (AOCS) (2009) Officil methods nd recommended prctices of the Americn oil chemists society. Method Cd 8 90. AOCS Press, Chmpign 15. Americn Oil Chemists Society (AOCS) (1997) Officil methods nd recommended prctices of the Americn oil chemists society. Method Ch 1 91. AOCS Press, Chmpign 16. Americn Oil Chemists Society (AOCS) (1992) Officil methods nd recommended prctices of the Americn oil chemists society. Method Ce 1 89. AOCS Press, Chmpign 17. Pnfili G, Frtinni A, Irno M (2003) Norml phse high-performnce liquid chromtogrphy method for the determintion of tocopherols nd tocotrienols in cerels. J Agric Food Chem 51:3940 3944 18. Americn Oil Chemists Society (AOCS) (1993) Officil methods nd recommended prctices of the Americn oil chemists society. Method Cd 12 92. AOCS Press, Chmpign
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