Avaiiabte online at www.sciencedirect.com
`__ f do
`"3? ScienceDirect
`
`Food Control xxx [200'l'} xxx—xxx
`
`FOOD
`CONTROL
`
`www.elsevier.oomllocatei'f'oodcont
`
`\Dtm‘-JC7~
`
`ll
`l2
`l3
`
`I5
`
`1'?
`
`l9
`20
`
`2|
`22
`23
`
`24
`
`Chemical changes in omega—3—enhanced farmed rainbow trout
`(0nc0rhynchus mykiss) fillets during
`abusiVe—tempe-rature storage
`
`Yi—Chen Chen 5‘, Jason Nguyen *3, Kenneth Semmens b, Sarah Beamer b, Jacek Jaczynski b’*
`
`'1 School 0fNtrrrr'l'r'orr, Chung Slrwi Merlical Um't:t?rst'.'_t’. No. H0. SECHON I, Jrcmgt.-0 N. Roma’, Trticlttairg, Tttfttwt
`b /lmmro’ and Ntm't'.'irm(Jl' 3t‘t'e'tr(.‘t’s. West Vfrgirrirr Univerfity. R0. Box M03. M0rg:'mn‘oti')t, WV. USA
`
`Received I6 October 2006; received in revised form 20June 2007: accepted 26 June 200'?
`
`EXHIBIT
`
`%
`E
`
`Abstract
`
`Chemical changes of omega~3—enhanced Farmed rainbow trout fillets developed by dietary modification with flaxseecl oil {F0} and
`alpha—tocopheryl acetate [or-TA) were determined during 10 “C storage for 6 or 8 days. Regardless of storage period and packing meth-
`ods, higher [P < 0.05) thiobarbituric acid—reactive substances (TBARSJ and lower (P 4 0.05) alpha~tocophero| content were measured in
`stored fillets from FO—supplemented groups. Packaging method {vacuum vs. non-vacuum] did not (P > 0.05) aliect fatty acid profile
`(PAP), but vacuum packaging suppressed lipid oxidation (TBARS) (P < 0.05) during storage. However, alpha-tocopherol unlike vac-
`uum packaging protected omega-3 FA in the tested fillets. Regardless of storage period, stored fillets obtained from trout supplemented
`with 15% of F0 and 900 ppm of at-TA had the highest (P 4 0.05) proportions of unsaturated FA, total t-u—3 FA, and alpha—linolenic acid
`(ALA) as well as the lowest (P < 0.05) proportions of saturated FA and docosahexaenoic acid (DHA] when compared with those from
`other treatment groups.
`© 200? Published by Elsevier Ltd.
`
`Kc-,1-ii'ord.-.-.' Flaxseed oil; AIpha—tocop|teryl acetate: Vacuum packing; Trout fillet: Abusive storage; TEARS values; Saturated Fatty acids: Unsaturated
`fatty acids; Omega—3 fatty acids: Omega-6 fatty acids
`
`
`I. Introduction
`
`According to the American Heart Association statistics
`in 2006,
`the cardiovascular disease (CVD) has been the
`number one leading cause of human death in the United
`States since I990 (Thom et al., 2006). This report also
`showed that 34.2% of American adults had one or more
`
`types of CVD in 2003 and over 0.9 million people died
`due to CVD in 2003. Omega-3 fatty acids (co~3 FA) in fish
`and fish-derived food products can reduce the risk of CVD.
`Hence, consumption of at least two fish servings per week
`is
`recommended by the American Heart Association
`(Krauss et al., 1996}. Institute of Medicine (2002) also sug-
`
`' Corresponding author. Tel..' +1 304 293 2406; Fax: +1 304 293 2232.
`E-rnail address.‘ jacek.jaczynski@mai|.wv1|.edu (J. Jaczyn.-;ki)_
`
`0956-7] 351$ — see front matter © 200? Published by Elsevier Ltd.
`doi:10.l0l6lj.foodcont.200?'.06.0l I
`
`gcsted intake levels of omega-3 PUFA that were set for
`wlinolenic acid and based on median intakes in the US
`
`as 1.6 and 1.1 g/d for men and women, respectively.
`Although docosahexaenoic (DHA, 22:6n3} and eicosa-
`pentaenoic (EPA, 20:5n3} acids are considered more
`important than or-iinolenic acid (ALA,
`lS:3n3) for human
`nutrition, Zhao et al. (2004) investigated the effects of aver-
`age American diet, a linoleic acid (LA, l8:2n6) diet, and an
`ALA diet on heart disease in humans. These authors found
`
`that the level of C-reactive protein, a marker of inflamma-
`tion strongly associated with heart disease, declined for
`both the LA and ALA diets, but much more significantly
`for the ALA diet. Therefore, Zhao et al. (2004) concluded
`that ALA seems to lower the CVD risk by inhibiting vascu-
`Iar inflammation beyond its lipid-lowering ctfects. Harper,
`Edwards, DeFilipis, and Jacobson (2006) reported similar
`
`36
`3?
`
`45439490—C-'\-D00
`
`42
`
`éfififi
`
`4?
`48
`
`§J1\J'|-I5—D\D
`
`
`
`AKBM 1096
`
`1
`
`

`
`
`
`2
`
`}'.—C. Chen et at‘.
`
`4' Food Control xxx (200.7) xxx-xxx
`
`S3
`54
`55
`56
`S‘?
`58
`59
`60
`6 I
`62
`63
`
`65
`66
`6?
`68
`69
`T0
`7 I
`T2
`T3
`T4
`T5
`76
`7'?
`T8
`T9
`80
`8 I
`32
`83
`
`85
`So
`8?
`88
`
`89 Q1
`90
`9!
`92
`93
`94
`95
`96
`9?
`98
`99
`1013
`WI
`I02
`I03
`I04
`I05
`I06
`107
`I08
`
`results that ALA increases cardioprotective (co-3} fatty
`acids in humans. In addition, ALA can be converted to
`EPA and DHA in mammals (Voss, Reinhart, Sankarappa,
`& Sprecher, 1991]. Therefore, it is likely that regardless of
`the chain length of the w—3 FA, increasing overall concen-
`tration ofthese FA as well as the omega-3 to omega-6 fatty
`acid ratio (co—3,t'cu-6 FA ratio) in the human diet will be ben-
`eficial to human health. This is probably why ‘several orga-
`nizations recommend that higher co-3;’w-6 FA ratio in the
`human diet contributes to human health.
`
`While worldwide fish consumption has been steadily
`increasing, the capture of marine fish has been fairly stable
`(Williams, 1998). This is likely why the aquaculture pro-
`duction had quadrupled and currently makes up for that
`shortfall {Sargent &. Tacon, 1999). However, the content
`of the (B-3 FA in farmed rainbow trout fillets is typically
`lower than in the wild counterparts (Hardy, I990]. Greene
`and Selivonchick (1990) suggested that addition of ALA in
`the diet can maintain concentrations ofthe EPA and DHA
`
`at a physiologically optimum level in cultured trout. Buzzi,
`Henderson, and Sargent (1996) also showed that the ALA
`serves as a precursor for formation of EPA and DHA in
`hepatocytes of rainbow trout. However, during desatura-
`tion of fatty acids, the ALA and LA both compete for 6-
`desaturase, and therefore, the conversion of ALA to EPA
`and subsequently to DHA is relatively inefficient (Emken,
`Adlof, Duval, & Nelson, 1999). A potential way to reduce
`cost and provide great flexibility in tailoring lipids in fish
`fillets to tneet
`the health concern of consumers can be
`
`achieved via lipid modification of the fish diet. Therefore,
`an opportunity exists to fortify tr_out fillets with co-3 PUFA
`by dietary modification. Flaxseed oil (F0) contains the
`highest concentration of ALA compared to other plant-
`derived lipid sources used in fish feeds (National Research
`Council, 1993]. Our research has shown that it is possible
`to increase Lt)-3 FA content
`in cultured, rainbow trout
`(0ncorhyncl'm.r mykiss] by supplementing trout diets with
`F0 (Chen, Nguyen, Semmens, Beamer, & Jaezynski, 2006].
`It is well known that lipid oxidation is one of the major
`problems in fish-derived food products due to flavor
`deterioration and loss of nutritional value. Lipid oxidation
`typically results in a formation of aldehydes (acids, hydro-
`carbons, and epoxides}, alkyl
`radicals (hydrocarbons,
`alcohols} and semialdehydes (or oxo-esters) (Ladilcos &
`Lougovois, 1990; Nawar, 1996). These oxidation products
`are generated depending on the oxidative conditions. Poly-
`unsaturated fatty acids (PUFA) are more easily oxidized
`than saturated fatty acids, and therefore, food products
`enhanced with the cu-3 PUFA are more prone to lipid oxi-
`dation and rancidity development. There are potential
`human health risks associated with increased consumption
`of food products containing oxidized cu-3 PUFA (Fritsche
`& Johnston, 1990; Kubow, 1993]. Another important fac-
`tor to limit a more common use of (.0-3 PUFA-enhanced
`
`food products is the development of oft“-flavors due to the
`lipid oxidation that may be offensive
`to consumers
`[Waagbo, Sandnes, Torrissen, Sandvin, & Lie, 1993). The
`
`composition of fish fillets typically correlates with the com-
`position of the fish diet. Therefore, an antioxidant supple-
`mentation of trout diet may yield higher concentration of
`that antioxidant in the fillets, resulting in decreased lipid
`oxidation of the fillets during storage and distribution.
`In order to prevent quality loss, fish-derived food prod-
`ucts require an effective antioxidant system due to high
`unsaturation offish muscle lipids (Jia et al., 1996}. Fat-sol-
`uble antioxidants, such as vitamin E, play an important
`role in preventing the oxidation of unsaturated lipids in fish
`muscle (Pope, Burtin, Clayton, Madge, & Muller, 2002].
`The ct-tocopherol shows the greatest antioxidant activity
`among four homologue pairs (or—, B-, 7-, 5-tocopherols,
`and tocotrienols} [Burton & Ingold, 1981). When compar-
`ing the antioxidant effectiveness among at-, 7-, and 5-toc-
`opherols, or-tocopherol typically shows the highest rate of
`scavenging of lipid peroxyl and alkoxyl radicals (Kulas &
`Ackman, 2001}. Hence, ct—tocopheryl acetate (ct-TA), a
`vitamin E derivative, is usually used as an antioxidant to
`reduce lipid oxidation in food. However, the loss of antiox-
`idant efficacy in postmortem muscle is due to the depletion
`of the antioxidant in the muscle {Petillo, Hultin, I-{rzyno—
`wek, & Autio, I998}. It has also been speculated that lipid
`oxidation of Lt)-3 PUFA is initiated due to the depletion of
`tissue vitamin E in meat systems (Ajuyah, Ahn, Hardin, &
`Sim, 1993). The addition of antioxidants in animal diets
`distributes the antioxidant effects at cellular levels, which
`promotes post-slaughter stability (Buckley, Morrissey, &
`Gray, I995]. Dietary Ct‘.-TA showed antioxidant effects on
`w—3 enriched pork (Rey et al., 2001), dry-cured ham (Isabel
`et al., 2003], broilers (Ahn, Wolfe, & Sim, 1995), eggs
`{Galobart, Barroeta, Baucells, Codony, & Ternes, 2001),
`salmon {Scaife, Onibi, Murray, Fletcher, & Houlihan,
`2001}, and trout (Frigg, Probucki, & Ruhdel, 1990). In
`addition, exclusion of oxygen decreases lipid oxidation of
`meat (Higgins, Kerry, Buckley, & Morrissey, I998; Nam
`& Ahn, 2003}.
`Based on the results from our previous research, supple-
`menting rainbow trout with 900 ppm of ct-TA for I20 days
`of feeding increased or-tocopherol content
`in the fillets
`obtained from to-3-enhanced rainbow trout (Chen et al.,
`2006}. Therefore, the addition of antioxidants to the fish
`diet may also promote post-slaughter oxidative stability
`during storage and distribution of fish-derived food p1'od—
`ucts. However, fish-derived food products are sometimes
`exposed to temperature abuse during storage and distribu-
`tion, particularly if they are transported in remote areas.
`The temperature abuse poses accelerated risks of lipid oxi-
`dation and subsequent rancidity development. The acceler-
`ated lipid oxidation could be particularly applicable to the
`to-3-enhanced trout fillets due to high unsaturation level of
`their lipids. However, there are no reports concerning phys-
`icochemical changes and strategies to prevent the quality
`degradation during post-slaughter abusive-temperature
`refrigerated storage of trout enriched with to-3 FA via die-
`tary modification with fiaxseed oil and et—tocopheryl acc-
`tate. Therefore, our objectives were to (1) determine
`
`109
`III]
`Ill
`I12
`II3
`I14
`I15
`I16
`IIT
`I18
`[I9
`120
`I2}
`I22
`I23
`I24
`125
`I26
`I27
`I28
`I29
`I30
`13}
`I32
`I33
`I34
`135
`I36
`I37’
`133
`I39
`I40
`14}
`I42
`I43
`I44
`I45
`I46
`14'!
`I48
`149
`150
`I5]
`I52
`I53
`I54
`I55
`I56
`I52
`I58
`I59
`I60
`I6|
`I62
`I63
`164
`I65
`
`
`
`2
`
`

`
`
`
`
`
`Y.~C. Chen er of. I Food Control xxx (200?) xx.r—XxX
`
`3
`
`166
`I 6'?
`-I 68
`l 69
`I 70
`
`l?|
`
`122
`
`I23
`I24
`175
`N6
`I7?
`178
`I79
`[30
`ISI
`I82
`I83
`I84
`185
`I86
`13?
`I88
`I89
`I90
`I91
`I92
`I93
`l94
`195
`196
`I9?
`
`I98
`
`I99
`200
`
`changes in lipid oxidation (TBARSL fatty acid profile, and
`content of ct-tocopherol in to-3-enhanced trout fillets sub-
`jected to abusive-temperature refrigerated storage; and
`(2) devise strategies to prevent lipid oxidation {i.e., rancid-
`ity} and degradation of co-3 fatty acids in the fillets.
`
`2. Materials and methods
`
`2.1. Feeding trio! and dit?t.r
`
`A gravity-fed flow-through raceway system composed of
`four levels was used for this study. Each level had two par-
`allel
`lanes and each lane had two tanks. Tanks were
`
`stocked with T5 rainbow trout (0. mflciss) fingerlings per
`tank (91 x l22><9l cm). Rainbow trout was fed dry pel-
`leted diets formulated with 0 (basal diet) or l5.0% (wfw)
`of fiaxseed oil (F0) supplementation (Ziegler Brothers
`Inc:., Gardners, PA]. Each level ofthe F0 supplementation
`was also enhanced with 0 or 900 ppm of ot—tocopheryl ace-
`tate (ot-TA). Hence,
`there were four dietary treatments.
`Major ingredients of the trout basal diet are shown in
`Table 1. The fatty acid profiles of the experimental feeds
`at'e shown in Table 2. The dietary treatments were ran-
`domly assigned to the tanks in each level of the raceway
`system. The fat
`in the basal diet was supplemented with
`the F0 in the experimental diets. Feed was stored at 4 °C.
`Approximately l512L,/min of spring water
`flowed
`through the raceway system. It was aerated entering the
`system and half way through the system to maintain a dis-
`solved oxygen concentration above ?0"/» of saturation.
`Water temperature was approximately 12°C during the
`feeding trial. Fish were fed 6 days each week and main-
`tained on a natural photoperiod. Fish were hand fed to
`satiation twice a day for 120 days. The animal experiments
`were approved by app1'0pt‘i::tte institutional authorities.
`
`2.2. Srtmptle pt‘epom.'t'on
`
`Trout from the four dietary treatments were harvested
`on day I20 and then killed by a blow to the head. All
`
`Table I
`Major ingredients of the trout basal diet {g.t'kg)
`
`Ingredients
`Wheat middlings
`Fish meal
`Hydrolyaed feather meal
`Dehulled soybean meal
`Blood rneal
`Ground extruded whole soybean
`Corn gluten meal
`Minerals
`Vitamins
`Soy lecithin
`Yeast culture
`Crude protein [g/kg}
`Metabolizable energy {MJt'l<g]
`
`tgfkg}
`230
`250
`100
`100
`100
`60
`50
`25
`IS
`I0
`I0
`42
`l2.6
`
`Table 2
`Total fat and fatty acid composition“ of experimental diets
`
`Parameter
`
`|8:3n3
`205113
`22:61:}
`182116
`20:49i6
`Total unsaturates
`Total saturates
`Total cm}
`Total rt:-6
`to-3l'nJ-6
`Total fat (“/n, dry basis]
`
`'34: F0 supplementation
`0%
`
`15%
`
`st Fatty acid in total fatty acids”
`3.4? :l: l.l4
`46.2 :l: 139'
`I l.0 :: 0.64'
`[.22 i 0.32
`l2.'.-’ :l: 0.7?
`L62 2.: 015'
`20.9 :: L03
`21.6 :I: 0.90
`0.2? :: 0.1?
`0.33 i 0.03
`61.0 :: l.0|
`82.5 :: 1.39‘
`39.0 :: |.0|
`I15 i 1.39’
`22.5 $1.44
`49.] :: 2.l3'
`22.2 :: 1.08
`22.6 :I: 0.86
`1.27 i 0. I 0
`2. I 7 :l: 0.04‘
`l4.4 :: 2.32
`24.9 :: 0.65’
`
`" * indicates significant differences [P < 0.05) between mean va‘ues of the
`same type of fatty acid.
`b Data are given as mean :: SEM (n = 6).
`
`harvested fish were stored at 4 “C before filleting. The har-
`vested fish were filleted to obtain boneless and skinless but-
`
`terfly fillets on the same day when the fish were harvested.
`Following filleting, the fillets were either placed in nylon
`vacuum pouches (3 mil standard barrier, Koch, Kansas
`City, MO), labelled and vacuum packed or the fillets were
`placed on plastic trays and aerobically over-wrapped {non-
`vaccum) with a typical household plastic wrapping mate-
`rial (Aldi® clear plastic wrap, Batavia, IL). The vaccum-
`and non-vacuum packed fillets were subjected to storage
`at 10 “C for 6 or 8 days. Following the storage period, fil-
`lets were homogenized in a laboratory blender [Model
`5lBL3l, Waring Commercial, Torrington, CT), placed in
`nylon vacuum pouches (3 mil standard barrier, Koch, Kan-
`sas City, MO},
`labelled, vacuum packed and stored at
`-80 °C until analyzed. The storage periods were selected
`based on correlation between TEARS values in fish and
`
`rancidity development reported by Kc, Cervantes, and
`Robles-Martinez H984). In addition, an un-trained panel
`monitored overall appearance and smell
`(non-vacuum
`only) of the fillets during storage in order to assess when
`the storage should be stopped and the fillets analyzed.
`The objective was to analyze the fillets when the rancidity
`development was slightly in progress (6 days) and more
`advanced (8 days}.
`
`2.3. Moismre {“/3) and toraifat (96)
`
`Two grams of homogenized fillets were placed onto an
`aluminum dish (Fisher Scientific Co., Fairlawn, NJ} and
`dispersed evenly across the dish. The moisture content of
`fillets was determined by using the oven-drying method
`(105 “C for 24 h] (AOAC, 1995].
`Fat content in fillets was determined according to Soxh-
`let extraction method (AOAC, 1995}. Sample size was 5 g
`and extraction time was 16h at a drip rate of approxi-
`
`201
`202
`203
`204
`205
`206
`20'?
`208
`209
`210
`2! 1
`212
`2 l 3
`2 l 4
`21 5
`2 I -S
`21'?
`2 I 8
`219
`220
`221
`222
`223
`224
`225
`
`226
`
`22?
`223
`229
`230
`231
`232
`233
`234
`
`
`
`3
`
`

`
`
`
`4
`
`Y.-C. Chair at at.
`
`J Food Control xxx (200?) .rxx—x,rx
`
`235
`236
`237
`
`238
`239
`
`240
`24 I
`242
`243
`244
`245
`246
`24'}
`248
`249
`250
`25]
`252
`253
`254
`255
`
`256
`
`25'?
`258
`259
`260
`261
`262
`263
`264
`265
`266
`26'?
`268
`269
`220
`271
`272
`2'33
`274
`225
`226
`2??
`223
`279
`280
`28 I
`282
`283
`284
`285
`286
`28'?
`
`mately 10 mLt'min. Extractions were performed with petro-
`leum ether. Fat content was determined on a gravimetric
`basis and expressed as percent by weight on dry basis.
`
`2.4. Measarerttent of tt'tiobarbitttrt'c (tct'd-reactive substances
`{ TBA RS)
`
`Oxidative rancidity of fillets was measured by a 2~thio—
`barbituric acid—re-active substances (TBARS) assay of ma]-
`ondialdehyde (MBA) as described by Jaczynski and Park
`(2003). Three drops of antioxidant (Tenox 6, Eastman
`Chemical Div., Kingsport, TN, USA) and 3mL of TBA
`were added to 0.2g of homogenized fillet sample. Then,
`17 ml. trichloroacetic acid—HCl reagent was added. The
`solution was flushed with nitrogen and closed. A blank
`was prepared in the same manne1', but without sample.
`The tubes were boiied for 30 min, and then cooled. The col»
`ored solution (15 mL} was centrifuged at 5000g for IS min.
`A clear and colored supernatant was transferred to a cuv-
`ette, and the absorbance was measured at 535 nm using a
`UV,(Vis
`spectrophotometer
`(model DU530, Beckman
`Instruments, Fullerton, CA). The results were reported as
`mg MDAt'kg of sample.
`
`2.5. Measareinent of alp!'ia—tocopherof content
`
`The content of ct-tocopherol in trout fillets was mea-
`sured by a modified saponification method using high pres-
`sure liquid chromatography (HPLC) described by Liu and
`Lee (1998). An aliquot of 0.25 g of L-ascorbic acid (Fisher
`Scientific, Fair Lawn, NJ) and 0.01 g of TBHQ (tert-butyi-
`hydroquinone, Sigma-Aldrich, St. Louis, M0) were added
`to approximately lg ofa homogenized fillet sample in a
`test tube. Exact weight of the fillet sample was recorded
`and used in calculations. A freshly prepared 7.3 mL diges-
`tion solution (ll% (w,t'v] potassium hydroxide, 555% (vt'v)
`ethanol, 45% (vfv) distilled and deionized water} was added
`to the fillet sample, followed by vortexing [Vortex Genie 2,
`Scientific Industries, Bohemia, NY) for 30 s to dissolve the
`ascorbic acid. The tubes were incubated at 80 °C for 20 min
`
`with continuous and gentle shaking in order to digest the
`fillet sample. Immediately following the incubation,
`the
`tubes were cooled in ice slush for 10 min. An aliquot of
`4 mL of isooctane {Fisher Scientific, Fair Lawn, NJ] was
`added to the cooled solution and the tubes were vortexed
`for 2 min. Then,
`the tubes were allowed to stand in the
`ice slush for 5 min. An aliquot of 1 mL of the top clear iso-
`octane layer was transferred to a small tube and stored at
`-80 °C until the solution was injected into the HPLC (Per-
`kin—Elmer series 4, Perkin—Elmer, Norwalk, CT) using a
`Rheodyne injector (Rheodyne, Rohnert Park, CA). The
`HPLC was equipped with a Waters Resolve C-18 spherical
`silica column (5 pm, 3.9x 150 mm} {Water Guard-Pak,
`Waters Corp., Milford, MA) and a spectrophotometric
`(UVt'Vis) detector (Perkin—Elmer LC-75, Perkin—Elmer,
`Norwalk, CT). A mixture of isooctaneftetrahydrofuran
`(96t'4, v/v) was prepared and filtered (0.45 pm} daily and
`
`the mixture was used as a mobile phase. The fiow rate
`was 1 mlfmin and the injection volume was 30 1.1L. The er-
`tocopherol was detected at excitation wavelength of
`296 nm and emission wavelength of 325 nm. The blank
`was run as described above, but without the fillet sample.
`Various isomeric forms of tocopherol (Sigma—Aldrich, St.
`Louis, M0) were also run on the HPLC as standards as
`well as the extraction efficiency of cstocopherol was deter-
`mined and used in calculations. An experimental standard
`curve was used to calculate or-tocopherol content in the
`trout fillets, which is reported as mg of ct-tocopherol per
`kilogram of fillets.
`
`2.6. Lipid extraction andfatty acid analysis
`
`Lipids were extracted using methodology described by
`Foleh, Lees, and Sloane (1957) and used for analysis of
`fatty acid profile. According to the procedure of Fritsche
`and Johnston (1990), fatty acids were transmethylated by
`the addition of 4 mL of 4% (w,fv} methanolic H2304 and
`heated in a 90 °C water bath for 60 min. The mixture was
`
`saponified by transferring through a Na2SO4 filled glass
`Pasteur pipette and subsequently dried under N2 in a
`60 °C water bath for 60 min. The fatty acid methyl esters
`(FAME) were re—suspended in filtered isooctane. The
`FAME and standards were analyzed by using a gas chro-
`matograph (Va1'ian CP-3800 gas chromatograph, Varian
`Analytical Instruments, Walnut Creek, CA] and a flame
`ionization detector fitted with a wall—coated open tubular
`(WCOT} fused silica capillary column (CP-S088, 50m
`length, 0.25 mm inside diameter) [Varian Analytical Instru-
`ments, Walnut Creek, CA}. Injection and detection temper-
`ature was maintained at 220 °C and column temperature
`was 190 °C. Nitrogen was the carrier gas, and a split ratio
`of l—t0 was used. The fatty acids were identified by com-
`paring their retention times with known standards (Sigma,
`St. Louis, MO) and references (Ackman, 1998). Peak area
`and the amount of each fatty acid were computed by an
`integrator using the Star GC workstation version 6 soft-
`ware (Varian Analytical Instruments, Walnut Creek, CA).
`
`2.7. Statistical analysis
`
`The experiment was conducted using a 2x2 factorial
`arrangement of treatment in a randomized block design.
`The interaction effect (FO><a-TA}, main effect (F0 and
`ct-TA}, and blocking effect (packaging method] were ana-
`lyzed. All significant differences in the interaction effect,
`main effect, and blocking effect were tested using an
`ANOVA test at 0.05 probability level. When a significant
`difference in the interaction effect was determined, the least
`significant difierence {LSD} test at 0.05 probability level
`was used to test differences between combination treat-
`
`ments. At least six trout (n= 6) from each experimental
`treatment and six feeds (n = 6) from each experimental diet
`were randomly obtained and analyzed. All statistical anal-
`yses of data were performed using SAS (2002).
`
`288
`289
`290
`291
`292
`293
`294
`295
`296
`297
`298
`299
`
`300
`
`30]
`302
`303
`304
`305
`306
`307
`308
`309
`3|0
`31 l
`312
`313
`314
`315
`316
`31 2
`3| 8
`3E9
`320
`32]
`322
`323
`324
`325
`
`326
`
`327
`323
`329
`330
`331
`332
`333
`334
`335
`336
`33?
`338
`339
`340
`
`
`
`4
`
`

`
`
`
`Y.-C. Chen er al. J Food Control xxx (2007) xx.r—xx.r
`
`5
`
`341
`
`342
`
`3-13
`344
`345
`346
`34'?
`348
`349
`350
`35 I
`352
`353
`354
`355
`356
`35'?
`358
`359
`360
`36]
`362
`363
`364
`365
`366
`367
`368
`369
`370
`3'5’ I
`3'32
`333
`374
`335
`376
`37'?
`3'.-'3
`
`3. Results and discussion
`
`3.}. Moisture ondfzrr contents of tr-outfitters
`
`No (P 5 0.05) interaction between flaxseed oil (F0) and
`ct-tocopheryl acetate (oi—TA) for moisture and fat contents
`were found for fillets stored at abusive-temperature refrig-
`erated storage (10 °C) (Tables 3 and 4}. In addition, there
`were no (P > 0.05} main effects (F0 and ct-TA) for mois-
`ture and fat contents in the stored fillets. Regardless of sup-
`plementing basal diet with F0 and or-TA, vacuum packed
`fillets had higher (P < 0.05) moisture content
`than the
`non—vacuum packed ones
`for 6-day storage, but not
`{P>0.0S} for the 8-day storage. Although the difference
`for the 6-day storage was statistically significant, it proba-
`bly was oflimited practical meaning due to low magnitude.
`When muscle proteins denature during storage, they also
`partially lose their fnnctionalities including water holding
`capacity, which may account
`for
`the differences
`in
`moisture.
`
`F0, 0!-TA, and packing method did not affect (P > 0.05}
`fat content of stored fillets (Table 4). Lin, Lin, and Kuo
`(2002) reported no differences for moisture and fat contents
`in chicken frankfurters supplemented with different levels
`of fish oil (0%, 2%, and 4%) and subjected to refrigerated
`storage. Therefore, our data shows similar
`trend for
`omega—3-enhanced trout
`fillets during storage at 10°C.
`Wang et al.
`(2005) demonstrated that
`feeding cobia
`(Racfiycem‘i'on crmadum canodtmr) diets with increased con-
`centration of lipids resulted in higher concentration of fat
`in the fish; however in contrast, Regost, Arzel, Cardinal,
`Laroche, and Kaushik (2001) reported that the level of die-
`tary fat did not affect the lipid concentration in the fillets
`obtained from trout. Probably there are several factors that
`affect lipid concentration in fish fillets as a function of die-
`tary fat level. In our research, supplementing trout diet
`with F0 and or-TA did not (P ? 0.05) affect the lipid con-
`centration of trout fillets during storage. Therefore, our
`data is similar to those reported by Regost et al. (200l).
`Similar to our data, Chaiyapechara, Casten, Hardy, and
`
`Table 3
`
`Dong (2003} and Jittinandana, Kenney, Slider, Kamireddy,
`and Hankins (2006) demonstrated that dietary supplemen-
`tation of trout with at-TA did not show any effects on mois-
`ture and fat contents in the fillets during storage. Based on
`our results and those available from the literature, we con-
`cluded that moisture and fat contents in trout fillets are not
`
`altered during storage due to dietary supplementation of
`fish with llaxseed oil (F0) and or-tocopheryl acetate (or-
`TA) as well as packing method of the fillets.
`
`3.2. or-Tocopherof content and z’ipr'd oxidation
`
`Stored fillets from non-F0 and ci—TA—supplemented
`groups had higher (P< 0.05) ct-tocopherol contents than
`F0 and non—ci—TA supplemented ones, respectively (Table
`5). Vacuum packed fillets also showed higher {P< 0.05)
`Ot-lIOC0]3l'l€'.1‘0l content than the non—vacuum packed ones
`(Table 5). However, the opposite results were determined
`for thiobarbituric acid-reactive substances (TBARS} values
`in stored fillets [Table 6}.
`Increased levels of dietary Gt-TA have been demon-
`strated to elevate ct-tocopherol content in meat systems.
`In a long-terrn feeding trial with beef cattle supplemented
`with three levels of oi-TA, tissue -:t—tocopherol concentra-
`tion was significantly increased [A1-nold, Scheller, Arp,
`Williams, & Schaefer,
`1993). When
`supplementing
`200 mg/kg of Qt-TA into non-fat diet, olive oil and sun-
`flower oil formulated diets, higher cetocopherol contents
`in rabbit muscle were determined (Lopez-Bote, Rey, Sana,
`Gray, & Buckley, 1997). Akhtar, Gray. Cooper, Garling,
`and Booren H999} fed rainbow trout diets supplemented
`with <r—TA at 500 mg/kg, which increased at-tocopherol
`content in fillets. These results are similar to our data. Jitt-
`
`inandana et al. [2006] investigated elTects of supplementa-
`tion of rainbow trout with a~TA at 200 and 5000 mg/kg
`on storage stability of trout fillets. The high vitamin E sup-
`plemented group maintained higher oi—tocopherol content
`in stored fillets than that in the low vitamin E supple-
`mented ones after 6-month frozen storage and 3-day refrig-
`erated storage.
`
`379
`380
`38l
`382
`383
`384
`385
`386
`387
`
`388
`
`389
`390
`39!
`392
`393
`394
`395
`396
`39?
`393
`399
`400
`40 I
`402
`403
`404
`405
`406
`40'?
`408
`409
`4 l 0
`4l 1
`412
`4] 3
`414
`4] 5
`416
`
`Moislure content in trout fillets as affected by feed supplementation tllaxseed oi! (F0) and ct-tocopheryl acetate to-TA}) and packing method during
`temperature-abused refrigerated storage“ (10 “Cl
`Storage period (d)
`% F0 supplementation
`u—TA [ppm]
`Packing method
`0
`l 5
`0
`Vacuum
`
`Non—vacuum
`
`900
`
`6
`8
`
`% Moisture”
`71.1 :i: 0.35
`'.l'l.2i0.60
`
`P-value
`
`Tl .5 :: 049
`7l.4:.:0.46
`
`TI .5 i 0.45
`'?'l.l :: 0.45
`
`7| .2 :l: 0.4|
`7l.5 i0.60
`
`32.4 :: 0.23‘
`7l.'?:l:0.48
`
`'i"| .3 :I: 0.30
`'i"0.9d:0.55
`
`
`F0
`ot—TA
`F0 >< or-TA
`Packing method
`6
`0.34
`0.44
`0.43
`0.00
`8
`0.32
`0.63
`0.1]
`0.28
`
`“ Mean values in the main ellecl {F0 or or-TA) and blocking effect (packing method) with * indicate significant differences {P 4. 0.05) within the same
`storage period.
`1’ Data are given as mean i SEM { main and blocking effect: it = I2; interaction effect: n = 6].
`
`
`
`5
`
`

`
` 6
`
`Y.—C. Chen er al. I Food Control xxx (200?) xxx-xxx
`
`Table 4
`in trout fillets as atlected by feed supplementation (flaxseed oil {F0} and u—tocopheryl aoetate (oi-TA)) and packing method during
`Fat content
`temperature—abused refrigerated storage“ [10 °C)
`Storage period [cl]
`% F0 supplementation
`at-TA (ppm)
`Packing method
`0
`15
`0
`Vacuum
`
`Non-vacuum
`
`900
`
`
`
`% Fat”
`
`6
`8
`
`5.53 :l: 0.69
`6.83 i 0.43
`
`6.78 i 0.49
`6.90 :: 0.59
`
`6.26 :: 0.69
`6.98 :i: 0.70
`
`6.05 :l: 0.56
`6.26 :I: 0.28
`
`6.37 :i: 0.75
`7.09 i 0.60
`
`5.95 :l: 0.4?
`6.65 :l-. 0.45
`
`3
`8
`
`P—value
`
`F0
`
`0.13
`0.93
`
`
`
`at-TA
`
`0.31
`0.’.-'8
`
`F0 X or-TA
`
`0.33
`0.24
`
`Packing method
`
`0.55
`0.59
`
`a Mean values in the main clTect {F0 or or-TA] and blocking cliect [packing method] with I indicate significant differences (P C 0.05) within the same
`storage period.
`
`Table 5
`
`ct-tocopheml content in trout fillets as affected by feed supplementation {flaxseed oil [F0] and ct-tocopheryl acetate (:x—'I'A)] and packing method during
`temperature—abused refrigerated storage" (10 °C)
`Storage period [d]
`% F0 supplementation
`r:t—TA (ppm)
`Packing method
`
` 0 15 0 900 Vacuum Nonwacuum
`
`
`
`
`
`
`
`
`6
`8
`
`mg of ot-tocopherolfkg of filletsb
`TL9 :|: 3.45
`87.0 :I: 5.03’
`81.3 :l: 4.53’
`'r'l.5 :l: 3.96
`86.9 :|: 4.95’
`372.0 :i: 3.60
`82.3 :i: 3.55°
`60.! :l: 4.42
`67.? :l: 3.92
`80.? :l: 4.59‘
`83.5 d: 324'
`64.8 :l: 4.20
`P-value
`
`
`Packing method
`F0 x nt—TA
`ct—TA
`F0
`6
`0.00
`0.00
`0.98
`0.00
`
`8
`€0.00
`0.00
`0.14
`<0.00
`
`“ Mean values in the main efiect (F0 or or-TA) and blocking effect (packing method] with * indicate significant differences {P < 0.05) within the same
`storage period.
`b Data are given as mean :: SEM {main and blocking efiect: rt = 12; interaction effect: it = 6).
`
`Table 6
`TBARS values in trout fillets as affected by feed supplementation [fiaxseed oil {F0} and omocopheryl acetate (at-TA)] and packing method during
`temperature-abused refrigerated storage“ {I0 °C)
`Storage period (d)
`% F0 supplementation
`at—TA (ppm)
`Packing method
`
`0
`15
`0
`900
`Vacuum
`Non-vacu um
`
`6
`8
`
`mg of MDA!kg of filletsb
`1.97 d: 0.13
`3.25 :i: 0.40‘
`2.55 :1: 0.28
`4.44 :l: 0.20‘
`
`2.88 i 0.42‘
`3.81 :l: 0.64‘
`
`2.33 :t: 0.25
`3.18 :t: 0.56
`
`1.95 :l: 0.1 l
`1.94 :l: 0.16
`
`3.2? i 0.40‘
`5.05 :l: 0.52’
`
`6
`8
`
`P—va|ue
`
`F0
`
`0:00
`<0.00
`
`
`
`at-TA
`
`0.09
`0.08
`
`FO >< at-TA
`
`0.29
`0. I 6
`
`Packing method
`
`0.0
`<0.00
`
`“ Mean values in the main cfl'ect {F0 or or-TA) and blocking eFl'ect [packing method} with * indicate significant difierenoes (P < 0.05) within the same
`storage period.
`
`Ill?
`413
`419
`420
`42l
`422
`423
`
`Higher concentration of unsaturated FA in the stored
`fillets was determined for the fillets obtained from trout
`
`fed 15% of F0 supplemented diets compared with those fil-
`lets from trout without F0 supplementation (Table 7}. The
`rate of lipid oxidation in meat systems highly correlates
`with the proportion of unsaturated FA in the total FA
`(Tichivangana & Morrissey, 1985). Therefore, while fish fil-
`
`lets are usually more susceptible to lipid oxidation and sub-
`sequently dcveiop rancidity more readily than meat
`products derived from terrestrial animals, trout fillets with
`enhanced content of PUFA may be even more susceptible
`to lipid oxidation and may require more efficient antioxi-
`dant system. Antioxidants, such as vitamins E and C affect
`lipid oxidation by slowing this reaction in meat products
`
`424
`425
`426
`42?
`428
`429
`430
`
`
`
`6
`
`

`
`
`
`}’.—C. Chen er al. J’ Food Control’ xxx (200.7) xxx—xxx
`
`7
`
`Table 7
`Fatty acid profile of trout fillets as affected by feed supplementation with flaxseed oil (F0) and ct—tocophery1 acetate [ct-TA) during temperature-abused
`refrigerated storage“ [10 °C)
`
`Fatty acids
`
`Storage period (cl)
`
`Saturated
`
`Unsaturated
`
`ALA
`
`EPA
`
`DHA
`
`LA
`
`AN
`
`i to-3
`
`2 to-6
`
`.
`
`69
`SE
`
`60
`35
`
`6E_
`8"
`
`6”
`s
`
`6
`SE
`
`6”
`SE
`
`6‘?
`8’:
`
`6'3
`3E
`
`6D
`SE
`
`Treatment (‘I/o FO:ct—TA (pprI1]]B
`0:0
`0:900
`
`‘Va Fatty acid in total fatty acids‘:
`35.5 :: 4.06
`41.9 :|: 2.33
`38.4 :1: 0.993
`39."? :1: 2.i0a
`
`64.5 i 4.06
`61.6 ab 0.991)
`
`18.? :l: 3.14bc
`19.0 :1: 1.07::
`
`0.39:|:0.l6
`ND‘:
`
`15.2 i l.28ab
`l5.| :1: 0.402113
`
`6.99 :1: 3. I0
`|'i’.9:l:0.28
`
`0.61 :l:0.l0
`0.45 :: 0.1413
`
`34.8 :1: 3.76
`34.] :1: l.0'I'b
`
`23.? :l: 2.36
`34.5 d: 0.34ab
`
`58.2 i 2.83
`60.3 i 2. 10b
`
`|2.9 i l.?9c
`12.5 -._-
`|.0lt:-C
`
`0.50 :: 0.0?
`ND
`
`18.2 :: |.23a
`19.? :1: 0.653
`
`10.9 : 3.43
`11510.81
`
`0.40:l:0.15
`0.45 i 0.08b
`
`31.6 :1: 2.09
`32.2 -L-1.4113
`
`30.3 i 2.27
`38.4 :.E 1.34:!
`
`15:0
`
`151900
`
`37.3 :1; 2.58
`36.3 :I: 4.123
`
`64.6 :1: 6.99
`63.? :1: 4.1213
`
`25.7 :1: 3.29ab
`24.3 :t 2.95b
`
`0.64 :l:0.20
`ND
`
`14.3 :1: L411)
`|4.2 : |.91b
`
`no.5 :1: 4.51
`12.8 :: 4.03
`
`0.34 ::0.l0
`0.76 : 0.051)
`
`40.6 :: 3.12
`38.5 :1: 2.241)
`
`26.2 : 3.91
`28.8-l-3.41 bc
`
`33.03 :: 0.79
`21.22 5.: 5.201)
`
`69.? :: 3.03
`28.3 :1: 5.2011
`
`33.6 :: 2.263
`41.? i 4.85:]