Development Of Antioxidant Active Films Containing Tocopherols To Extend The Shelf Life Of Fish

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. This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 108 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 14 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 15 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 16 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 17 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 18 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 19 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 20 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 21 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 22 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 23 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 31 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). MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 33 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 35 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 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 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Figu e 3.