Accep ed Manusc ip
De elopmen O An ioxidan Ac i e Films Con aining Tocophe ols To Ex end The
Shel Li e O Fish
Le icia Ba bosa-Pe ei a, José Manuel C uz, Raquel Sendón, Ana Rod íguez
Be naldo de Qui ós Ana A es, Ma Cas o-López, Ma ia José Abad, Julio Ma o o,
Pe ec o Pasei o-Losada
PII: S0956-7135(12)00536-1
DOI: 10.1016/j. oodcon .2012.09.036
Re e ence: JFCO 2954
To appea in: Food Con ol
Recei ed Da e: 23 Ap il 2012
Re ised Da e: 17 Sep embe 2012
Accep ed Da e: 25 Sep embe 2012
Please ci e his a icle as: Ba bosa-Pe ei aL., C uzJ.M., SendónR., Ana A esA.R.B.d.Q., Cas o-
LópezM., AbadM.J., Ma o oJ. & Pasei o-LosadaP., De elopmen O An ioxidan Ac i e Films
Con aining Tocophe ols To Ex end The Shel Li e O Fish, Food Con ol (2012), doi: 10.1016/
j. oodcon .2012.09.036.
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Highligh s
• The na u al compounds used could eplace syn he ic an ioxidan s in ood
indus y.
• The inco po a ion o an ioxidan p oduc s in o plas ic ma ices esul s in ac i e
ilms.
• The addi ion o hese compounds in o LDPE ex ensi ely inhibi ed lipid
oxida ion o salmon muscle.
• Ac i e ilms con aining ocophe ols could be used o de elop a p omising ac i e
packaging o be applied in ood p ese a ion by ood indus y.
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DEVELOPMENT OF ANTIOXIDANT ACTIVE FILMS CONTAINING 1
TOCOPHEROLS TO EXTEND THE SHELF LIFE OF FISH 2
3
Le icia Ba bosa-Pe ei aa, José Manuel C uz b, Raquel Sendón a*, Ana Rod íguez 4
Be naldo de Qui ós, Ana A esc, Ma Cas o-Lópezc, Ma ia José Abadc, Julio Ma o od, 5
Pe ec o Pasei o-Losadaa. 6
* [email p o ec ed]s 7
Phone numbe : +34881814964 8
Fax: +34 881815106 9
a Depa men o Analy ical Chemis y, Nu i ion and Food Science. Facul y o 10
Pha macy. Uni e si y o San iago de Compos ela, E-15782. San iago de Compos ela. 11
Spain. 12
b Depa men o Chemical Enginee ing. Indus ial Engenee ing School. Uni e si y o 13
Vigo. E-36310 Vigo. Spain. 14
c G oup o Polyme s, Uni e si y o A Co uña, CIT- Campus de Es ei o, s/n. 15403-15
Fe ol. 16
d Cen o Tecnológico del Ma , Bouzas. Vigo. Spain 17
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18
Abs ac 19
20
Wi h he pu pose o de elop ac i e ilms o ish, i e na u al p oduc s wi h 21
an ioxidan p ope ies con aining ocophe ols we e selec ed and hei an ioxidan 22
ac i i y in i o was es ed using he DPPH me hod. In addi ion hese an ioxidan s we e 23
also es ed di ec ly on he salmon muscle using he TBARS me hod. Besides, he mal 24
deg ada ion and di e en ial scanning calo ime y es s allow us o selec he p oduc s 25
o inco po a ion in o he polyme ma ix. Then, wo na u al p oduc s ha e been 26
selec ed and inco po a ed in low densi y polye hylene ilms. Film 2 and Film 3, which 27
con ain p oduc C a 1 and 5 % espec i ely, p esen ed a educ ion in lipid oxida ion up 28
o 30 % o Film 2 and o 40 % o Film 3. Film 4, which con ains 5 % o p oduc D, 29
educed lipid oxida ion du ing s o age a ound 30-35 %. The esul s showed ha Film 3 30
was he mos e ec i e o salmon conse a ion a long- e m s o age. These ilms could 31
be used as u u e ac i e packaging by he ood indus y o he salmon conse a ion o 32
ex end i s shel li e. 33
34
Keywo ds: Lipid oxida ion, na u al an ioxidan s, ocophe ols, ac i e ilms, LDPE, 35
salmon.36
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1. In oduc ion 37
38
Sea ood has ecen ly ecei ed mo e a en ion om consume s as impo an 39
sou ce o nu i ional componen s (unsa u a ed a y acids, mine als and i amins) ha 40
ha e posi i e bene i s in nu i ion and human heal h. The nu i ional signi icance o ish 41
and ish oils in ake is due o hei high con en o essen ial long-chain omega-3 42
polyunsa u a ed a y acids (PUFAs), such as eicosapen aenoic acid (EPA) and 43
docosahexaenoic acid (DHA), which p o ec agains hea disease, cance , hype ension, 44
heuma oid a h i is, dep ession, diabe es, among o he heal h bene i s (Richa ds & 45
Hul in, 2002; Sidhu, 2003). The high con en o PUFAs in ish muscle, as well he 46
p esence o ele an p ooxidan compounds, p omo e he de elopmen o enzyma ic and 47
non-enzyma ic ancidi y leading o o ganolep ic, physical and nu i ional quali y losses 48
in ood (F ankel, 1993; McClemen s & Decke , 2000; E ickson, 1997). 49
Lipid oxida ion is he main cause o spoilage o ish du ing i s p ocessing and 50
s o age, being a key ac o in he shel li e o ood (F ankel, 1993; F ankel 1998b; 51
F ankel, 1998a). As a consequence o his de e io a ion p ocess, hyd ope oxides a e 52
o med (p ima y oxida ion) which a e uns able and decompose ela i ely quickly in o 53
aldehydes, ke ones, alcohols, acids, es he s o hyd oca bons (seconda y oxida ion). 54
These seconda y oxida i e p oduc s a e esponsible o changes in colo , ex u e, odo 55
and la o in ood (McClemen s & Decke , 2000; E ickson, 1997). 56
The use o an ioxidan s is an e ec i e way o p e en o minimize lipid 57
oxida ion in ood p oduc s, delaying he o ma ion o oxic oxida ion p oduc s 58
main aining he nu i ional quali y and p olonging hei shel li e (Huang, Ou, & P io , 59
2005). An ioxidan s ac by inhibi ing he oxida ion o lipids and o he molecules 60
a oiding he damaging e ec s o oxida ion in animal issues. The majo mechanisms o 61

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ac ion in p o ec ing he ood om he oxida ion p ocess include he ee adical 62
sca enging and chain b eaking by inhibi ing he ini ia ion o p opaga ion phases o ee 63
adical chain eac ions o lipid oxida ion, me al chela ion and single oxygen quenching 64
(Huang, Ou, & P io , 2005; Aidos, Masbe na -Ma inez, Lu en, Boom, & an de Pad , 65
2002). 66
An ioxidan s may be p esen na u ally as ood componen s as in he case o 67
ocophe ols o added du ing ood p ocessing (Choe, & Min, 2009). In many si ua ions, 68
ood p ocessing is esponsible o he loss o hese endogenous compounds ac i i y and 69
equi ed he addi ion o syn he ic an ioxidan s such as 2 (3)- e -bu yl-4-me hoxyphenol 70
(BHA), bu yla ed hyd oxy oluene (BHT ) and p opyl galla e, which a e widely used in 71
he ood indus y. In ecen yea s, se e al s udies ha e shown e idence o some oxici y 72
by syn he ic an ioxidan s such BHT and BHA ha may possess ca cinogenic e ec s a 73
high le els (Wa enbe g, 1986; Ka l, 1984) (EFSA, 2012; EFSA, 2011). Fo his eason, 74
and o human heal h bene i s, he use o na u al an ioxidan s such as ocophe ols, 75
asco bic acid o na u al ex ac s wi h high le els o polyphenols a e inc easingly used as 76
an ioxidan s in ood. Besides, hese na u al compounds a e being e ec i e in he 77
p e en ion and/o ea men o diseases caused by ee adicals (Shahidi, & Zhong, 78
2010; F ankel, 1996). 79
Tocophe ols a e an ioxidan s ex ensi ely used as ood addi i es by being 80
e ec i e, a soluble and inhibi o s o oxida ion eac ions because hei e icien chain-81
b eaking capaci y, demons a ing e ec i eness agains lipid pe oxida ion and o he 82
oxida i e si ua ions caused by he p esence o ee adicals (Ohka su, Kajiyama, & A ai, 83
2001; B igelius-Flohé, & T abe , 1999). To p e en and/o delay he ood spoilage, 84
an ioxidan s a e di ec ly added as ood addi i es o could be added indi ec ly h ough 85
he packaging ma e ial, using wha is known as ac i e packaging. 86
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Ac i e packaging ep esen s a new packaging concep wi h g ea in e es in ood 87
indus y. This sys em allows he elease o he ac i e componen s in o he ood and has 88
shown g ea po en ial in he p ese a ion o he ood quali y being an al e na i e o 89
adi ional packaging. An ioxidan s such as BHT, BHA and α- ocophe ol ha e been 90
inco po a ed in plas ic ma e ials in o de o p o ec he polyme ma ix om deg ada ion 91
du ing p ocessing. The α- ocophe ol has been used in di e en si ua ions o s abilize 92
plas ic ma e ials such as low densi y polye hylene (LDPE) and polyp opylene (PP) 93
p o ing o be a good p ese a i e o hese ma e ials (Wessling, Nielsen, & Giacin, 94
2000). 95
Ne e heless, he possibili y o an ioxidan compounds mig a ing om he 96
plas ic ma e ial being in con ac wi h ood has been obse ed. A s udy pe o med by 97
Wessling, Nielsen, Leu én, and Jäge s ad, (1999) sugges s ha LDPE spiked wi h α-98
ocophe ol can be used as ac i e packaging, o oods wi h high a con en , eleasing 99
he an ioxidan and ex ending he shel li e o he p oduc . 100
The aim o his s udy is o e alua e and compa e he an ioxidan e ec i eness o 101
di e en na u al an ioxidan p oduc s ob ained comme cially which con ains 102
ocophe ols, on he s abili y o salmon du ing e ige a ed s o age. Besides, he mos 103
sui able an ioxidan o hei inco po a ion in o LDPE is selec ed. Wi h he esul s 104
ob ained, he p oduc s o be used in he de elopmen o ac i e packaging wi h 105
an ioxidan p ope ies we e selec ed and s udies we e conduc ed o assessmen o hei 106
e ec i eness in he delay o lipid oxida ion in he salmon muscle. 107
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108 2. Ma e ials and me hods 109
110
2.1. Chemicals 111
112
2(3)- e -bu yl-4-hyd oxyanisole (BHA) (98 %, CAS No. [25013-16-5]); 113
bu yla ed hyd oxy oluene (BHT) (99.0 %, CAS No. [128-37-0]); sodium azide (99.0 %, 114
CAS No. [26628-22-8]); 2- hioba bi u ic acid (TBA) (≥98 %, CAS No. [504-17-6]) and 115
ichlo oace ic acid (TCA) (pu iss. p.a. 99.5 %, CAS No. [76-03-9]) we e pu chased 116
om Sigma-Ald ich (S einheim, Ge many). 1,1,3,3- e ae hoxyp opane (TEP) (pu um 117
≥95 % (GC), CAS No. [122-31-6]) and 2, 2-diphenyl-1-pic ylhyd azyl (DPPH) 118
(TECHN ≥85 %, CAS No. [1898-66-4]) we e supplied by Fluka Chemie AG (Buchs, 119
Swi ze land). Me hanol (GC≥ 99.9%, CAS No. [67-56-1]); o hophospho ic acid (85 % 120
GR o analysis, CAS No. [7664-38-2]) and e hanol (absolu e o analysis, CAS No. 121
[64-17-5]) we e p o ided by Me ck (Da ms ad , Ge many). I ganox® 1076 (CAS No. 122
[2082-79-3]) and I ga os® 168 (CAS No. [31570-04-4]) we e kindly supplied by Ciba-123
Geigy, Swi ze land. 124
125
2.2. Comme cial p oduc s con aining na u al an ioxidan s 126
127
The na u al p oduc s wi h an ioxidan ac i i y used, TOCOBIOL®, 128
TOCOBIOL® GL, NUTRABIOL®-T90, TOCOBIOL® PV, NUTRABIOL®-T50 PV, 129
we e supplied by Bio ecnologías Aplicadas, S.L., BTSA, (Mad id, Spain). The 130
p esen a ion o he p oduc s TOCOBIOL ®, TOCOBIOL ® GL, NUTRABIOL ®-T90 131
was in oil o m and p oduc s TOCOBIOL ® PV, NUTRABIOL ®-T50 PV we e in 132
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powde o m. Table 1 shows he composi ion o he di e en na u al an ioxidan s es ed. 133
This in o ma ion was p o ided by he company. 134
135
2.3. Ma e ials 136
137
2.3.1. Fish sample 138
Salmon (Salmo sala ) was selec ed o be used in he an ioxidan e ec i eness 139
assays. The salmon was supplied o he labo a o y, in ozen slices wi h a hickness 140
be ween 1 and 1.5 cm, by he company Elabo ados FREIREMAR SA. 141
Fo he an ioxidan e ec i eness assessmen , salmon was de os ed a oom 142
empe a u e and i s skin and bones emo ed om he lesh. To p e en mic obial 143
spoilage du ing s o age, a small quan i y o sodium azide (app ox. 1 mg) was added on 144
he su ace o salmon. 145
146
2.3.2. P ocessing o polyme o mula ions 147
Se e al o mula ions o he an ioxidan compounds wi h LDPE ma ix we e 148
p epa ed. The ecipes o o mula ions a e shown in Table 2. The ma ix used was a low 149
densi y polye hylene (LDPE), p o ided by Repsol YPF (MFI; 20 g min-1; densi y; 919
150
kg m-3). Only he an ioxidan s compounds wi h a sui able he mal s abili y we e mixed 151
wi h he polyme . 152
Blends wi h LDPE and an ioxidan s we e p epa ed in an in e nal mixe 153
B abende W50EHT a 150 ºC. Besides he na u al an ioxidan s, he comme cial 154
an ioxidan s IRGAFOS® 168 (seconda y an ioxidan ) and IRGANOX® 1076 (p ima y 155
an ioxidan ) we e also added in he usual p opo ions in indus y in o de o p e en he 156
oxida ion o polyme ma ix. 157
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ob ained o he abili y o DPPH adical sca enging by na u al an ioxidan p oduc s 302
(Table 1) was compa ed wi h e e ence an ioxidan s BHA and BHT, usually used in 303
ood indus y. The EC50 alues ob ained o na u al p oduc s A, B, C, D, E we e 1.49 ± 304
0.075, 0.435 ± 0.050, 0.401 ± 0.062, 3.25 ± 0.095 and 0.704 ± 0.047 espec i ely, 305
whe eas o he syn he ic an ioxidan s BHT and BHA we e 2.53 ± 0.085 and 0.273 ±
306
0.025 g L-1 espec i ely. Resul s o he an ioxidan ac i i y de e mined by DPPH 307
me hod a e shown in Table 3.The EC50 alues ob ained o he syn he ic an ioxidan s 308
used as e e ence, BHA and BHT, a e in ag eemen wi h alues ound in he li e a u e 309
(C uz, Moldes, Bus os, To ado & Dominguez, 2007). 310
The esul s ob ained indica e ha all an ioxidan ex ac s es ed p esen ed 311
an ioxidan ac i i y and he na u al p oduc wi h highes adical sca enging ac i i y was 312
p oduc C ollowed by B, E, A and D which had he lowes an ioxidan ac i i y among 313
he p oduc s s udied. 314
Excep o D, all he p oduc s es ed demons a e highe an ioxidan ac i i y 315
compa ed o BHT, syn he ic an ioxidan commonly used in ood indus y. 316
An ioxidan s p oduc s B and C ha e displayed highe an ioxidan ac i i y 317
p esen ing EC50 alues o 0.435 and 0.401 g L-1 espec i ely, compa able o he 318
e e ence syn he ic an ioxidan BHA which p esen ed an EC50 o 0.273 g L-1. 319
Compa ing he h ee TOCOBIOL® p oduc s, he mos ac i e was B ollowed by 320
A and inally D. 321
The highes an ioxidan capaci y p esen ed by p oduc B may be due o he ac 322
ha in i s composi ion in addic ion o 25 % o TOCOBIOL® p oduc also includes 25 % 323
o he syn he ic an ioxidan p opyl galla e, used in he ood indus y due o i s high 324
an ioxidan ac i i y (Ma inez-Tome, Jimenez, Ruggie i, F ega, S abbioli, & Mu cia, 325

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2001), and he e o e showed g ea e an ioxidan ac i i y han he p oduc A (EC50 o 326
1.49 g L-1). 327
P oduc D was less e ec i e in adical sca enging because i is composed by 328
only 65 % o TOCOBIOL® p oduc which is he esponsible o i s an ioxidan ac i i y. 329
Compa ing bo h NUTRABIOL® p oduc s, C p oduc p esen s he highes adical 330
sca enging capaci y o ee adicals because is he one which con ains highes 331
pe cen age o ocophe ols. 332
O he wo p oduc s es ed in powde o m, an ioxidan E showed highes 333
an ioxidan capaci y wi h an EC50 alue o 0.704 g L-1 because o i s high pe cen age o 334
ocophe ols (50.3 %). The same was obse ed o he oils, he p oduc wi h highes 335
p opo ion o ocophe ols, p oduc C (90.2 %), was he mos e ec i e, i.e. p esen ed he 336
highes an ioxidan ac i i y wi h an EC50 alue o 0.401 g L-1. 337
338
3.2. An ioxidan e ec i eness o comme cial p oduc s di ec ly on salmon 339
To e alua e he e ec i eness o hese compounds di ec ly on ood, salmon was 340
selec ed and lipid oxida ion was e alua ed du ing s o age a 4 °C. 341
The s udy was di ided in o wo pa s and samples we e analyzed a di e en 342
days. In he i s pa o his s udy he an ioxidan p oduc s in he oil o m we e es ed 343
and in he second one an ioxidan p oduc s in powde o m. In each assay he e was a 344
con ol es p epa ed as samples bu wi hou he addi ion o comme cial p oduc s
345
an ioxidan p oduc s. 346
347
3.2.1. E alua ion o an ioxidan e ec i eness o he oil p oduc s 348
The an ioxidan e ec i eness o he di e en p oduc s es ed in he salmon 349
muscle was de e mined by he TBARS me hod. The da a ob ained wi h he 3 oils 350
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applied a 2 di e en concen a ions used on he salmon muscle a e shown in Table 3
351
4a. 352
Lipid oxida ion o salmon muscle samples inc eased du ing he s o age in 353
di e en deg ees being he con ol es (wi hou addi ion o an ioxidan ) which showed 354
high TBARS alues. 355
All an ioxidan s applied in his assay educed lipid oxida ion in salmon 356
compa ed o he con ol es . A he lowes concen a ion o he s udy (30 µg·dm-2), 357
p oduc B showed be e e ec i eness, educing no ably lipid oxida ion compa ed o 358
con ol assay. Fo he C an ioxidan , also a highe concen a ion o an ioxidan 359
employed in he salmon muscle (125 µg·dm-2) lowe an ioxidan e ec was obse ed. 360
A e 10 days he an ioxidan e ec p esen ed by he wo concen a ions o p oduc C is 361
app oxima ely he same, wi h TBARS alues a ound 2.4 mg MDA kg-1 sample, which 362
co esponds o a educ ion in lipid oxida ion a ound 40 % espec o con ol assay. 363
Analyzing he da a ob ained, i can be seen ha he oil, ha which p esen ed be e 364
an ioxidan e ec in salmon is B p oduc B, and o he wo concen a ions e alua ed, 365
he highe is he concen a ion used highe he an ioxidan e ec i eness. B1 and B2 366
concen a ions educed lipid oxida ion abou 70 % being B2 sample sligh ly mo e 367
e ec i e. An ioxidan s A and B applied a he highes concen a ion, i.e. 125 mg dm-2 368
(A2 and B2), emain oxida ion alues below 1 mg kg-1 MDA o sample which is 369
equi alen o a de e io a ion educ ion in ood a ound 75 % compa ed o con ol assay. 370
Fo compounds A and B i can be seen ha he highe he concen a ion used he g ea e 371
educ ion o oxida ion in ish was obse ed, while in he p oduc C his e ec was no 372
obse ed. Fo compounds A and B i can be seen ha he highe he concen a ion used, 373
he g ea e educ ion o oxida ion in ish was obse ed, while in he p oduc C his 374
e ec was no obse ed. This could be due o ha C was he compound wi h highes 375
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concen a ion o ocophe ols (see Table 1) and p esen ed he highes EC50 in he DPPH 376
me hod (EC50 = 0.401 g L-1), and subsequen ly, when applied o he ood a he same 377
concen a ions ha we e used wi h o he p oduc s, i could ha e a p ooxidan e ec 378
caused by his high amoun o ocophe ols. Nume ous au ho s ha e desc ibed his 379
p ooxida i e e ec o α- ocophe ols a e applying his p oduc a high concen a ions 380
(F ankel, 1996; B igelius-Flohé, & T abe , 1999; Huang, F ankel, & Ge man, 1994). A 381
e y high concen a ions, he p ooxidan e ec o α- ocophe oloccu s du ing he 382
induc ion pe iod o p ima y oxida ion whe e hyd ope oxides a e o med in la ge 383
amoun s. This happens because a high concen a ions, α- ocophe ol loses i s ac i i y o 384
inhibi he o ma ion o hese hyd ope oxides demons a ing a low e ec i eness in he 385
p ima y oxida ion inhibi ion. Howe e his e ec was no obse ed in he seconda y 386
oxida ion whe e a signi ican ly delaying o seconda y p oduc s o ma ion is e i ied 387
(Huang e al., 1994; Ta azoli, W igh , & O’B ien, 2005; Tong, Sasaki, McClemen s, & 388
Decke , 2000). Thus, he esul s ob ained using he C an ioxidan p oduc could be due 389
o a possible loss o ac i i y o he compound as esul o he la ge amoun o 390
ocophe ols p esen in high concen a ions because only a small amoun o α-391
ocophe ols consumed in he oxida ion eac ion and a la ge pa emains a ailable o 392
u he eac ions ha occu simul aneously and leads o an oxida ion enhancemen 393
(Fus e , Lampi, Hopia, & Kamal-Eldin, 1998; Decke , Wa ne , Richa ds, & Shahidi, 394
2005). 395
396
3.2.2. E ec i eness o p oduc s in powde o m 397
TBARS alues ob ained om salmon addi i a ed wi h wo comme cial p oduc s 398
in powde o m wi h an ioxidan p ope ies, D and E, a wo di e en concen a ions a e 399
displayed in Table 3 4b. 400
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Analyzing he da a ob ained in his assay and, aking in o accoun ha he 401
con ol was no much oxidized, which makes di icul he compa a i e, imp o emen s 402
we e obse ed in he conse a ion o ish addi i a ed wi h an ioxidan p oduc s in he
403
concen a ions a he concen a ions 1 and 2 (30 and 125 mg dm-2 espec i ely), excep 404
o he D2. An ioxidan e ec i eness o p oduc E in ish muscle inc eased wi h he 405
inc easing o he concen a ion employed. A e 3 days o s o age, he an ioxidan 406
e ec i eness was highe han 50% compa ed o con ol es . 407
On he se en h day o ial, he deg ee o inhibi ion emained o he D1, E1 and 408
E2 bu in less pe cen age (52, 22 and 40 % espec i ely). Powde E showed an EC50 o 409
0.70 g L-1 in he DPPH me hod indica ing he s ong an ioxidan / adical sca enge 410
ac i i y as well ha he p oduc had an ioxidan ac i i y in i o. This an ioxidan e ec 411
was con i med when applied di ec ly in o he salmon muscle and consequen ly migh be 412
conside ed as a possible ex ac o be used in ish conse a ion. This an ioxidan e ec 413
was con i med when applied di ec ly in o he salmon muscle and consequen ly could be 414
conside ed as a possible an ioxidan p oduc o be used in ish conse a ion. 415
An ioxidan D displayed he highes EC50 o he i e compounds es ed being he 416
p oduc which showed lowe an ioxidan ac i i y in DPPH assay. Howe e , added a 417
concen a ion o 30 mg dm-2 (D1) on he sample o salmon demons a ed an ioxidan 418
e ec in he 7 days o s o age, while a highe concen a ion (D2) a possible p ooxidan 419
e ec was obse ed because lipid oxida ion did no dec ease. Compound D seems o be 420
an example o an ioxidan ha al hough i did no demons a e g ea an ioxidan ac i i y 421
by he indi ec me hod (EC50 EC50= 3.25 g L-1), i was e ec i e agains lipid 422
pe oxida ion o ood (Table 34b). The e o e, me hods o de e mine ee adical 423
sca enging such as DPPH me hod a e e y use ul o make a sc eening o he 424
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an ioxidan s, howe e o e i y hei e ec i eness in ood, me hods o e alua e he ood 425
de e io a ion mus be used. 426
The an ioxidan e ec i eness in ood depends on a a ie y o ac o s such as 427
pola i y, solubili y and me als quenching ac i i y as well as he in e ac ion wi h o he 428
ood componen s (Roginsky, & Lissi, 2005; Decke e al., 2005). An ioxidan D 429
p esen s a e y simila beha io o ha obse ed wi h he an ioxidan oil C in e ms o a 430
possible p o-oxidan e ec o inhibi ion o an ioxidan ac i i y when applied a high 431
concen a ions. 432
433
3.3. The mal s abili y and an ioxidan e ec i eness o compounds 434
435
The he mal s abili y o he i e an ioxidan s was e alua ed and he mos sui able 436
ones, ha is, which do no su e he mal deg ada ion a he LDPE p ocessing 437
empe a u e, we e chosen o blending wi h polyme ma ix. 438
Figu e 1 displays he he mog ams ob ained om he TGA expe imen s. The A 439
and B compounds ha e a lowe he mal s abili y han o he s. The deg ada ion p ocess o 440
A and B an ioxidan s s a om 100 ºC, whe eas in he o he ones (C, D and E), i 441
begins a 250 ºC (see onse in each cu e) app oxima ely. Keeping in mind ha LDPE 442
p ocessing empe a u e is a ound 150 ºC (indica ed by he a ow in he g aph), he A
443
and B an ioxidan s an ioxidan s A and B we e uled ou o blend wi h he polyme . 444
Then, he an ioxidan ac i i y o he di e en LDPE / an ioxidan blends was e alua ed 445
measu ing he OIT by DSC. The oxida ion induc ion ime inc eases wi h he inc ease o 446
an ioxidan amoun . This e ec is obse ed wi h h ee an ioxidan s, C (Blends LDPE 1-447
3), D (Blends LDPE 4-6) and E (Blends LDPE 7-9) (Figu e 2). Compa ing same 448

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o mula ions, he an ioxidan ac i i y is qui e simila in blends wi h an ioxidan C and E 449
( he maximum ime assayed) bu i is much lowe wi h D compound. 450
On he o he hand, hese esul s e eal a beha io o s udied an ioxidan s as long- e m 451
an ioxidan . The e o e, OIT alues inc ease wi h inc easing na u al an ioxidan le els, 452
cha ac e is ic beha io o phenolic compounds. Howe e phospha e s abilize s alone do 453
no inc ease he OIT o polye hylene in he empe a u e ange o 180-210 ºC (Phease, 454
Billingham, & Bigge , 2000). 455
To p e en he ma ix oxida ion du ing he ex usion and he p ema u e 456
consump ion o he na u al an ioxidan s, he wo comme cial an ioxidan s (IRGANOX® 457
1076 and IRGAFOS® 168) we e added o he polyme blends. The OIT da a o samples 458
LDPE 10 and LDPE 11 ( he maximum ime assayed) shown an a ailable le el o 459
an ioxidan in he ilm o be eleased o ish. 460
Based on he esul s ob ained, he na u al an ioxidan s ha should be selec ed o 461
inco po a e in he ilms o be es ed in he salmon packaging assay would be C and E. 462
Once hese wo p oduc s a e cons i u ed by he same ac i e p inciple Nu abiol® bu in 463
di e en p opo ions, and o he pu pose o es ing wo di e en p oduc s wi h di e en 464
composi ions in an ioxidan ac i e compounds, he p oduc s selec ed o inco po a e o 465
LPDE ilms we e C and D. Thus, wo di e en o mula ions in ocophe ols can be 466
e alua ed as well as hei e ec i eness agains lipid pe oxida ion ge ing mo e 467
in o ma ion abou he possible applica ion o hese an ioxidan p oduc s as ing edien s 468
o ac i e ilms. 469
In summa y, he C and D an ioxidan s oge he wi h he comme cial an ioxidan s 470
(I ga os® 178 and I ganox® 1076 a 0.2 and 0.4 % espec i ely) we e chosen o p epa e 471
he LDPE ac i e ilms. The ac i e ilms o mula ions selec ed we e: Film 2 and 3 472
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inco po a ed wi h na u al p oduc C a 1 % and 5 % espec i ely; Film 4 wi h na u al 473
p oduc D a 5 %. Film 1 wi h only comme cial an ioxidan s was used as con ol. 474
475
3.4 E alua ion o an ioxidan e ec i eness o he ac i e ilms. 476
477
When inco po a ed in o he plas ic ma ix, compounds wi h an ioxidan ac i i y 478
will no be a ailable as in p e ious assays, which we e added di ec ly in o ish muscle. 479
In addi ion, du ing ilms p ocessing, ha occu s a high empe a u es and agg essi e 480
condi ions, he ac i e compounds could be educed and i s an ioxidan capaci y be 481
a ec ed. The e o e, based on he esul s o di ec adding es s, comme cial p oduc s 482
which showed e idences o g ea e ec i eness in inhibi ion o lipid oxida ion in salmon 483
we e selec ed. Bo h o he p oduc s exhibi ed be e esul s a lowe concen a ions and a 484
possible p o-oxidan e ec when applied a he highes concen a ion demons a ing 485
hei high ac i i y. 486
Compound C showed a high an ioxidan ac i i y applied an ioxidan ac i i y 487
when applied a low concen a ion, a high an ioxidan ac i i y by DPPH me hod and in 488
i s composi ion con ains a high pe cen age o ocophe ols. The e o e i was inco po a ed 489
in o he plas ic ma ix a wo di e en concen a ions (1 % and 5 %). Compound D was 490
inco po a ed in o he plas ic ma ix only a he highes concen a ion o 5 % because 491
showed less an ioxidan ac i i y by DPPH me hod (EC50 = 3.25 g L-1). 492
The an ioxidan e ec o ilms addi i a ed wi h selec ed p oduc s was 493
de e mined by he capaci y o inhibi lipid oxida ion in salmon muscle du ing s o age a 494
4 °C by TBARS me hod. 495
Figu e 3 shows he malondialdehyde alues (TBARS) TBARS alues, exp essed 496
as mg MDA Kg-1 o sample, ob ained in he salmon muscle packed wi h he h ee ilms 497
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addi i a ed wi h he na u al p oduc s con aining ocophe ols compa ed o con ol ilm 498
(Film 1). Resul s showed ha lipid oxida ion inc eased in all samples du ing s o age bu 499
a di e en a es. Con ol es p esen ed a maximum o oxida ion o app oxima ely 5.8 500
mg MDA kg-1, while he samples packed wi h ac i e ilms p esen ed TBARS alues 501
ha a ied o e a ange o anging wi hin 3-4 mg MDA kg-1. 502
Salmon packed wi h ilms spiked wi h an ioxidan p oduc C in he wo 503
concen a ions o ac i e ing edien applied a 1 and 5 % (Film 2 and Film 3 504
espec i ely), expe imen ed an impo an lipid oxida ion dec ease, and he g ea es 505
an ioxidan e ec was e i ied a highe concen a ion used es ed. 506
In he i s days o assay, he e we e no impo an di e ences be ween he wo 507
concen a ions used bu a e he 7 h day di e ences we e mo e ema kable. A he i s 508
days o assay, no main di e ences be ween he wo concen a ions es ed was obse ed, 509
howe e , a e he 7 h day, di e ences we e mo e ema kable. A 7 h day, lipid 510
oxida ion was educed a ound 25 % o Film 3, while o Film 2 his dec ease was less 511
han 10 % compa ed o con ol (Film 1). A e day 11 o assay, Film 3 showed an 512
oxida ion dec ease o a ound 40 %. In he same pe iod less educ ion, a ound 15 %, was 513
obse ed o he Film 2, which a he end o he assay oxida ion was dec eased by 30 %. 514
TBARS alues o ish packed wi h Film 4 which con ains 5 % o an ioxidan p oduc D 515
also showed a signi ican educ ion in lipid oxida ion o salmon h oughou en i e assay 516
compa ed o con ol, inc easing ood s abili y. Du ing he i s 7 days o s udy, a 517
educ ion a ound 40 % was obse ed o Film 4, and a e ha , oxida ion dec easing 518
emained cons an a ound 30-35 % un il he end o he assay. 519
Compa ing he cu es o he ilms spiked wi h he wo p oduc s applied in his 520
s udy a he same concen a ion (5 %), i can be poin ed ou ha bo h ilms showed 521
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impo an an ioxidan e ec i eness and he e we e no impo an di e ences no main 522
di e ences be ween he wo compounds. 523
The e was a small di e ence in he i s days o e he i s days o analysis 524
whe e Film 4 (which con ains TOCOBIOL®® PV) was mo e e ec i e han Film 3, 525
while a he end o he assay Film 3 is he mos e ec i e ilm. This may be due o he 526
high amoun s o ocophe ols p esen s in Film 3 (p oduc C). 527
Ac i e ilms used had shown an inhibi o y e ec on he lipid oxida ion p ocess 528
compa able o he e ec obse ed wi h di ec addi ion o an ioxidan s o he ish 529
muscle. 530
Resul s ob ained wi h Films 2 and 3 con i m he e ec i eness o compound C as 531
well as he highe concen a ion inco po a ed in o he ilms g ea e he e ec i eness o 532
hem. 533
Resul s also con i med he high an ioxidan e ec o compound D, which 534
al hough did no showed high an ioxidan ac i i y in he ee adical sca enging me hod 535
(DPPH), i showed g ea e ec i eness agains salmon lipid oxida ion.536
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Figu e cap ions
Figu e 1.
TGA he mog ams o an ioxidan compounds desc ibed in Table 1.
Figu e 2.
OIT o LDPE blends desc ibed in Table 2.
Figu e 3.
Lipid oxida ion (TBARS) in salmon packed wi h an ioxidan ilms s o ed a 4 °C du ing
21 days (Film 1: con ol; Film 2: 1% an ioxidan C; Film 3: 5% an ioxidan C; Film 4:
5% an ioxidan D).

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Tables
Table 1.
An ioxidan p oduc
s composi ion
Oils wi h an ioxidan p ope ies
Powde s wi h an ioxidan p ope ies
TOCOBIOL
®
B – TOCOBIOL
®
GL C – NUTRABIOL
®
-T90 D – TOCOBIOL
®
-PV E – NUTRABIOL
®
-T50 PV
Monoglyce ides
Res : Vege able oil
18.6 %
9.70 %
4.30 %
19.8 %
Tocobiol:
Tocophe ols
S e ols
Squalene
Monoglyce ides
Res : ege able oil
P opyl galla e
Excipien s:
Leci hin
P opylene glycol
25.0 %
18.6 %
9.70 %
4.30 %
19.8 %
25.0 %
35.0 %
15.0 %
Tocophe ols
Alpha- ocophe ol
Gamma and Be a- ocophe ol
Del a- ocophe ol
Vege able oil
90.2 %
15.5 %
63.3 %
21.1 %
9.80 %
Tocobiol
Tocophe ols
S e ols
Squalene
Monoglyce ides
Res : ege able oil
Excipien :
Silica gel
65.0 %
18.6 %
9.70 %
4.30 %
19.8 %
35.0 %
Tocophe ols
Alpha- ocophe ol
Gamma and Be a- ocophe ol
Del a- ocophe ol
Vege able oil
Excipien :
Silica gel
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Table 2. 1
LDPE o mula ions 2
Na u al an ioxidan s Comme cial an ioxidan s
Fo mula ions C D E IRGAFOS 168 IRGANOX 1076
LDPE 1 5%
LDPE 2 1%
LDPE 3 0.5%
LDPE 4 5%
LDPE 5 1%
LDPE 6 0.5%
LDPE 7 5%
LDPE 8 1%
LDPE 9 0.5%
LDPE 10 0.2% 0.4%
LDPE 11 5% 0.2% 0.4%
3
Table 3. 4
F ee adical sca enging ac i i y 5
An ioxidan EC50 (g L-1)
A 1.49 ± 0.075
B 0.435 ± 0.050
C 0.401 ± 0.062
D 3.25 ± 0.095
E 0.704 ± 0.047
BHT 2.53 ± 0.085
BHA 0.273 ± 0.025
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Table 34a.
TBARS da a ob ained om salmon muscle in con ac wi h di e en concen a ions (30
and 125 mg dm-2, o samples 1 and 2 espec i ely) o an ioxidan oils (desc ibed in
Table 1) du ing s o age a 4 °C.
TBARS (mg malondialdehyde kg-1 salmon muscle)
Time (days) Con ol A1 B1 C1 A2 B2 C2
0 0.31±0.12 0.31±0.12 0.31±0.12 0.31±0.12 0.31±0.12 0.31±0.12 0.31±0.12
3 2.2±0.21 0.83±0.41 1.2±0.12 2.2±0.41 0.61±0.05 0.49±0.09 2.4±0.38
7 3.9±0.17 2.6±0.04 0.57±0.08 2.0±0.29 0.68±0.09 0.63±0.10 2.9±0.16
10 3.9±0.17 3.5±0.14 1.0±0.21 2.4±0.05 1.1±0.25 0.79±0.12 2.3±0.01
Table 34b.
TBARS alues ob ained om salmon muscle in con ac wi h di e en concen a ions
(30 and 125 mg dm-2, o samples 1 and 2 espec i ely) o an ioxidan powde s
(desc ibed in Table 1) du ing s o age ime a 4 °C.
TBARS (mg malondialdehyde kg-1 salmon muscle)
Time (days) Con ol D1 E1 D2 E2
0 0.21±0.19 0.21±0.19 0.21±0.19 0.21±0.19 0.21±0.19
3 2.4±0.90 0.66±0.27 0.96±0.32 0.68±0.43 0.52±0.01
7 2.5±0.43 1.2±0.56 2.0±0.11 5.2±0.48 1.5±0.64
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1
0
20
40
60
80
100
50 150 250 350 450 550 650
Loss mass (%)
Tempe a u e (ºC)
E
D
B
C
A
Figu e 1.
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LDPE 1 LDPE 2 LDPE 3 LDPE 4 LDPE 5 LDPE 6 LDPE 7 LDPE 8 LDPE 9 LDPE 10 LDPE 11
0
50
100
150
200
250
C e ec
D e ec
E e ec
Syne gic e ec
OIT(min)
Blend nomencla u e
Figu e 2

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Figu e 3.