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

Accepted Manuscript Development Of Antioxidant Active Films Containing Tocopherols To Extend The Shelf Life Of Fish Letricia Barbosa-Pereira, José Manuel Cruz, Raquel Sendón, Ana Rodríguez Bernaldo de Quirós Ana Ares, Mar Castro-López, Maria José Abad, Julio Maroto, Perfecto Paseiro-Losada PII: S0956-7135(12)00536-1 DOI: 10.1016/j.foodcont.2012.09.036 Reference: JFCO 2954 To appear in: Food Control Received Date: 23 April 2012 Revised Date: 17 September 2012 Accepted Date: 25 September 2012 Please cite this article as: Barbosa-PereiraL., CruzJ.M., SendónR., Ana AresA.R.B.d.Q., Castro- LópezM., AbadM.J., MarotoJ. & Paseiro-LosadaP., Development Of Antioxidant Active Films Containing Tocopherols To Extend The Shelf Life Of Fish, Food Control (2012), doi: 10.1016/ j.foodcont.2012.09.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Highlights • The natural compounds used could replace synthetic antioxidants in food industry. • The incorporation of antioxidant products into plastic matrices results in active films. • The addition of these compounds into LDPE extensively inhibited lipid oxidation of salmon muscle. • Active films containing tocopherols could be used to develop a promising active packaging to be applied in food preservation by food industry. 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 Letricia Barbosa-Pereiraa, José Manuel Cruz b, Raquel Sendón a*, Ana Rodríguez 4 Bernaldo de Quirós, Ana Aresc, Mar Castro-Lópezc, Maria José Abadc, Julio Marotod, 5 Perfecto Paseiro-Losadaa. 6 * [email protected]s 7 Phone number: +34881814964 8 Fax: +34 881815106 9 a Department of Analytical Chemistry, Nutrition and Food Science. Faculty of 10 Pharmacy. University of Santiago de Compostela, E-15782. Santiago de Compostela. 11 Spain. 12 b Department of Chemical Engineering. Industrial Engeneering School. University of 13 Vigo. E-36310 Vigo. Spain. 14 c Group of Polymers, University of A Coruña, CIT- Campus de Esteiro, s/n. 15403-15 Ferrol. 16 d Centro Tecnológico del Mar, Bouzas. Vigo. Spain 17 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 18 Abstract 19 20 With the purpose of develop active films for fish, five natural products with 21 antioxidant properties containing tocopherols were selected and their antioxidant 22 activity in vitro was tested using the DPPH method. In addition these antioxidants were 23 also tested directly on the salmon muscle using the TBARS method. Besides, thermal 24 degradation and differential scanning calorimetry tests allow us to select the products 25 for incorporation into the polymer matrix. Then, two natural products have been 26 selected and incorporated in low density polyethylene films. Film 2 and Film 3, which 27 contain product C at 1 and 5 % respectively, presented a reduction in lipid oxidation up 28 to 30 % for Film 2 and to 40 % for Film 3. Film 4, which contains 5 % of product D, 29 reduced lipid oxidation during storage around 30-35 %. The results showed that Film 3 30 was the most effective to salmon conservation at long-term storage. These films could 31 be used as future active packaging by the food industry for the salmon conservation to 32 extend its shelf life. 33 34 Keywords: Lipid oxidation, natural antioxidants, tocopherols, active films, LDPE, 35 salmon.36 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 1. Introduction 37 38 Seafood has recently received more attention from consumers as important 39 source of nutritional components (unsaturated fatty acids, minerals and vitamins) that 40 have positive benefits in nutrition and human health. The nutritional significance of fish 41 and fish oils intake is due to their high content of essential long-chain omega-3 42 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and 43 docosahexaenoic acid (DHA), which protect against heart disease, cancer, hypertension, 44 rheumatoid arthritis, depression, diabetes, among other health benefits (Richards & 45 Hultin, 2002; Sidhu, 2003). The high content of PUFAs in fish muscle, as well the 46 presence of relevant prooxidant compounds, promote the development of enzymatic and 47 non-enzymatic rancidity leading to organoleptic, physical and nutritional quality losses 48 in food (Frankel, 1993; McClements & Decker, 2000; Erickson, 1997). 49 Lipid oxidation is the main cause of spoilage of fish during its processing and 50 storage, being a key factor in the shelf life of food (Frankel, 1993; Frankel 1998b; 51 Frankel, 1998a). As a consequence of this deterioration process, hydroperoxides are 52 formed (primary oxidation) which are unstable and decompose relatively quickly into 53 aldehydes, ketones, alcohols, acids, esthers or hydrocarbons (secondary oxidation). 54 These secondary oxidative products are responsible for changes in color, texture, odor 55 and flavor in food (McClements & Decker, 2000; Erickson, 1997). 56 The use of antioxidants is an effective way to prevent or minimize lipid 57 oxidation in food products, delaying the formation of toxic oxidation products 58 maintaining the nutritional quality and prolonging their shelf life (Huang, Ou, & Prior, 59 2005). Antioxidants act by inhibiting the oxidation of lipids and other molecules 60 avoiding the damaging effects of oxidation in animal tissues. The major mechanisms of 61 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 action in protecting the food from the oxidation process include the free radical 62 scavenging and chain breaking by inhibiting the initiation or propagation phases of free 63 radical chain reactions of lipid oxidation, metal chelation and singlet oxygen quenching 64 (Huang, Ou, & Prior, 2005; Aidos, Masbernat-Martinez, Luten, Boom, & van der Padt, 65 2002). 66 Antioxidants may be present naturally as food components as in the case of 67 tocopherols or added during food processing (Choe, & Min, 2009). In many situations, 68 food processing is responsible for the loss of these endogenous compounds activity and 69 required the addition of synthetic antioxidants such as 2 (3)-tert-butyl-4-methoxyphenol 70 (BHA), butylated hydroxytoluene (BHT ) and propyl gallate, which are widely used in 71 the food industry. In recent years, several studies have shown evidence of some toxicity 72 by synthetic antioxidants such BHT and BHA that may possess carcinogenic effects at 73 high levels (Wattenberg, 1986; Karl, 1984) (EFSA, 2012; EFSA, 2011). For this reason, 74 and for human health benefits, the use of natural antioxidants such as tocopherols, 75 ascorbic acid or natural extracts with high levels of polyphenols are increasingly used as 76 antioxidants in food. Besides, these natural compounds are being effective in the 77 prevention and/or treatment of diseases caused by free radicals (Shahidi, & Zhong, 78 2010; Frankel, 1996). 79 Tocopherols are antioxidants extensively used as food additives by being 80 effective, fat soluble and inhibitors of oxidation reactions because their efficient chain-81 breaking capacity, demonstrating effectiveness against lipid peroxidation and other 82 oxidative situations caused by the presence of free radicals (Ohkatsu, Kajiyama, & Arai, 83 2001; Brigelius-Flohé, & Traber, 1999). To prevent and/or delay the food spoilage, 84 antioxidants are directly added as food additives or could be added indirectly through 85 the packaging material, using what is known as active packaging. 86 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 Active packaging represents a new packaging concept with great interest in food 87 industry. This system allows the release of the active components into the food and has 88 shown great potential in the preservation of the food quality being an alternative to 89 traditional packaging. Antioxidants such as BHT, BHA and α-tocopherol have been 90 incorporated in plastic materials in order to protect the polymer matrix from degradation 91 during processing. The α-tocopherol has been used in different situations to stabilize 92 plastic materials such as low density polyethylene (LDPE) and polypropylene (PP) 93 proving to be a good preservative of these materials (Wessling, Nielsen, & Giacin, 94 2000). 95 Nevertheless, the possibility of antioxidant compounds migrating from the 96 plastic material being in contact with food has been observed. A study performed by 97 Wessling, Nielsen, Leufvén, and Jägerstad, (1999) suggests that LDPE spiked with α-98 tocopherol can be used as active packaging, for foods with high fat content, releasing 99 the antioxidant and extending the shelf life of the product. 100 The aim of this study is to evaluate and compare the antioxidant effectiveness of 101 different natural antioxidant products obtained commercially which contains 102 tocopherols, on the stability of salmon during refrigerated storage. Besides, the most 103 suitable antioxidant for their incorporation into LDPE is selected. With the results 104 obtained, the products to be used in the development of active packaging with 105 antioxidant properties were selected and studies were conducted for assessment of their 106 effectiveness in the delay of lipid oxidation in the salmon muscle. 107 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 108 2. Materials and methods 109 110 2.1. Chemicals 111 112 2(3)-tert-butyl-4-hydroxyanisole (BHA) (98 %, CAS No. [25013-16-5]); 113 butylated hydroxytoluene (BHT) (99.0 %, CAS No. [128-37-0]); sodium azide (99.0 %, 114 CAS No. [26628-22-8]); 2-thiobarbituric acid (TBA) (≥98 %, CAS No. [504-17-6]) and 115 trichloroacetic acid (TCA) (puriss. p.a. 99.5 %, CAS No. [76-03-9]) were purchased 116 from Sigma-Aldrich (Steinheim, Germany). 1,1,3,3-tetraethoxypropane (TEP) (purum 117 ≥95 % (GC), CAS No. [122-31-6]) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) 118 (TECHN ≥85 %, CAS No. [1898-66-4]) were supplied by Fluka Chemie AG (Buchs, 119 Switzerland). Methanol (GC≥ 99.9%, CAS No. [67-56-1]); orthophosphoric acid (85 % 120 GR for analysis, CAS No. [7664-38-2]) and ethanol (absolute for analysis, CAS No. 121 [64-17-5]) were provided by Merck (Darmstadt, Germany). Irganox® 1076 (CAS No. 122 [2082-79-3]) and Irgafos® 168 (CAS No. [31570-04-4]) were kindly supplied by Ciba-123 Geigy, Switzerland. 124 125 2.2. Commercial products containing natural antioxidants 126 127 The natural products with antioxidant activity used, TOCOBIOL®, 128 TOCOBIOL® GL, NUTRABIOL®-T90, TOCOBIOL® PV, NUTRABIOL®-T50 PV, 129 were supplied by Biotecnologías Aplicadas, S.L., BTSA, (Madrid, Spain). The 130 presentation of the products TOCOBIOL ®, TOCOBIOL ® GL, NUTRABIOL ®-T90 131 was in oil form and products TOCOBIOL ® PV, NUTRABIOL ®-T50 PV were in 132 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 powder form. Table 1 shows the composition of the different natural antioxidants tested. 133 This information was provided by the company. 134 135 2.3. Materials 136 137 2.3.1. Fish sample 138 Salmon (Salmo salar) was selected to be used in the antioxidant effectiveness 139 assays. The salmon was supplied to the laboratory, in frozen slices with a thickness 140 between 1 and 1.5 cm, by the company Elaborados FREIREMAR SA. 141 For the antioxidant effectiveness assessment, salmon was defrosted at room 142 temperature and its skin and bones removed from the flesh. To prevent microbial 143 spoilage during storage, a small quantity of sodium azide (approx. 1 mg) was added on 144 the surface of salmon. 145 146 2.3.2. Processing of polymer formulations 147 Several formulations of the antioxidant compounds with LDPE matrix were 148 prepared. The recipes of formulations are shown in Table 2. The matrix used was a low 149 density polyethylene (LDPE), provided by Repsol YPF (MFI; 20 g min-1; density; 919 150 kg m-3). Only the antioxidants compounds with a suitable thermal stability were mixed 151 with the polymer. 152 Blends with LDPE and antioxidants were prepared in an internal mixer 153 Brabender W50EHT at 150 ºC. Besides the natural antioxidants, the commercial 154 antioxidants IRGAFOS® 168 (secondary antioxidant) and IRGANOX® 1076 (primary 155 antioxidant) were also added in the usual proportions in industry in order to prevent the 156 oxidation of polymer matrix. 157 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 14 obtained for the ability of DPPH radical scavenging by natural antioxidant products 302 (Table 1) was compared with reference antioxidants BHA and BHT, usually used in 303 food industry. The EC50 values obtained for natural products A, B, C, D, E were 1.49 ± 304 0.075, 0.435 ± 0.050, 0.401 ± 0.062, 3.25 ± 0.095 and 0.704 ± 0.047 respectively, 305 whereas for the synthetic antioxidants BHT and BHA were 2.53 ± 0.085 and 0.273 ± 306 0.025 g L-1 respectively. Results of the antioxidant activity determined by DPPH 307 method are shown in Table 3.The EC50 values obtained for the synthetic antioxidants 308 used as reference, BHA and BHT, are in agreement with values found in the literature 309 (Cruz, Moldes, Bustos, Torrado & Dominguez, 2007). 310 The results obtained indicate that all antioxidant extracts tested presented 311 antioxidant activity and the natural product with highest radical scavenging activity was 312 product C followed by B, E, A and D which had the lowest antioxidant activity among 313 the products studied. 314 Except for D, all the products tested demonstrate higher antioxidant activity 315 compared to BHT, synthetic antioxidant commonly used in food industry. 316 Antioxidants products B and C have displayed higher antioxidant activity 317 presenting EC50 values of 0.435 and 0.401 g L-1 respectively, comparable to the 318 reference synthetic antioxidant BHA which presented an EC50 of 0.273 g L-1. 319 Comparing the three TOCOBIOL® products, the most active was B followed by 320 A and finally D. 321 The highest antioxidant capacity presented by product B may be due to the fact 322 that in its composition in addiction to 25 % of TOCOBIOL® product also includes 25 % 323 of the synthetic antioxidant propyl gallate, used in the food industry due to its high 324 antioxidant activity (Martinez-Tome, Jimenez, Ruggieri, Frega, Strabbioli, & Murcia, 325 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 15 2001), and therefore showed greater antioxidant activity than the product A (EC50 of 326 1.49 g L-1). 327 Product D was less effective in radical scavenging because it is composed by 328 only 65 % of TOCOBIOL® product which is the responsible for its antioxidant activity. 329 Comparing both NUTRABIOL® products, C product presents the highest radical 330 scavenging capacity to free radicals because is the one which contains highest 331 percentage of tocopherols. 332 Of the two products tested in powder form, antioxidant E showed highest 333 antioxidant capacity with an EC50 value of 0.704 g L-1 because of its high percentage of 334 tocopherols (50.3 %). The same was observed for the oils, the product with highest 335 proportion of tocopherols, product C (90.2 %), was the most effective, i.e. presented the 336 highest antioxidant activity with an EC50 value of 0.401 g L-1. 337 338 3.2. Antioxidant effectiveness of commercial products directly on salmon 339 To evaluate the effectiveness of these compounds directly on food, salmon was 340 selected and lipid oxidation was evaluated during storage at 4 °C. 341 The study was divided into two parts and samples were analyzed at different 342 days. In the first part of this study the antioxidant products in the oil form were tested 343 and in the second one antioxidant products in powder form. In each assay there was a 344 control test prepared as samples but without the addition of commercial products 345 antioxidant products. 346 347 3.2.1. Evaluation of antioxidant effectiveness of the oil products 348 The antioxidant effectiveness of the different products tested in the salmon 349 muscle was determined by the TBARS method. The data obtained with the 3 oils 350 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 16 applied at 2 different concentrations used on the salmon muscle are shown in Table 3 351 4a. 352 Lipid oxidation of salmon muscle samples increased during the storage in 353 different degrees being the control test (without addition of antioxidant) which showed 354 high TBARS values. 355 All antioxidants applied in this assay reduced lipid oxidation in salmon 356 compared to the control test. At the lowest concentration of the study (30 µg·dm-2), 357 product B showed better effectiveness, reducing notably lipid oxidation compared to 358 control assay. For the C antioxidant, also at higher concentration of antioxidant 359 employed in the salmon muscle (125 µg·dm-2) lower antioxidant effect was observed. 360 After 10 days the antioxidant effect presented by the two concentrations of product C is 361 approximately the same, with TBARS values around 2.4 mg MDA kg-1 sample, which 362 corresponds to a reduction in lipid oxidation around 40 % respect to control assay. 363 Analyzing the data obtained, it can be seen that the oil, that which presented better 364 antioxidant effect in salmon is B product B, and for the two concentrations evaluated, 365 the higher is the concentration used higher the antioxidant effectiveness. B1 and B2 366 concentrations reduced lipid oxidation about 70 % being B2 sample slightly more 367 effective. Antioxidants A and B applied at the highest concentration, i.e. 125 mg dm-2 368 (A2 and B2), remain oxidation values below 1 mg kg-1 MDA of sample which is 369 equivalent to a deterioration reduction in food around 75 % compared to control assay. 370 For compounds A and B it can be seen that the higher the concentration used the greater 371 reduction of oxidation in fish was observed, while in the product C this effect was not 372 observed. For compounds A and B it can be seen that the higher the concentration used, 373 the greater reduction of oxidation in fish was observed, while in the product C this 374 effect was not observed. This could be due to that C was the compound with highest 375 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 17 concentration of tocopherols (see Table 1) and presented the highest EC50 in the DPPH 376 method (EC50 = 0.401 g L-1), and subsequently, when applied to the food at the same 377 concentrations that were used with other products, it could have a prooxidant effect 378 caused by this high amount of tocopherols. Numerous authors have described this 379 prooxidative effect of α-tocopherols after applying this product at high concentrations 380 (Frankel, 1996; Brigelius-Flohé, & Traber, 1999; Huang, Frankel, & German, 1994). At 381 very high concentrations, the prooxidant effect of α-tocopheroloccurs during the 382 induction period of primary oxidation where hydroperoxides are formed in large 383 amounts. This happens because at high concentrations, α-tocopherol loses its activity to 384 inhibit the formation of these hydroperoxides demonstrating a low effectiveness in the 385 primary oxidation inhibition. However this effect was not observed in the secondary 386 oxidation where a significantly delaying of secondary products formation is verified 387 (Huang et al., 1994; Tafazoli, Wright, & O’Brien, 2005; Tong, Sasaki, McClements, & 388 Decker, 2000). Thus, the results obtained using the C antioxidant product could be due 389 to a possible loss of activity of the compound as result of the large amount of 390 tocopherols present in high concentrations because only a small amount of α-391 tocopherols consumed in the oxidation reaction and a large part remains available for 392 further reactions that occur simultaneously and leads to an oxidation enhancement 393 (Fuster, Lampi, Hopia, & Kamal-Eldin, 1998; Decker, Warner, Richards, & Shahidi, 394 2005). 395 396 3.2.2. Effectiveness of products in powder form 397 TBARS values obtained from salmon additivated with two commercial products 398 in powder form with antioxidant properties, D and E, at two different concentrations are 399 displayed in Table 3 4b. 400 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 18 Analyzing the data obtained in this assay and, taking into account that the 401 control was not much oxidized, which makes difficult the comparative, improvements 402 were observed in the conservation of fish additivated with antioxidant products in the 403 concentrations at the concentrations 1 and 2 (30 and 125 mg dm-2 respectively), except 404 for the D2. Antioxidant effectiveness of product E in fish muscle increased with the 405 increasing of the concentration employed. After 3 days of storage, the antioxidant 406 effectiveness was higher than 50% compared to control test. 407 On the seventh day of trial, the degree of inhibition remained for the D1, E1 and 408 E2 but in less percentage (52, 22 and 40 % respectively). Powder E showed an EC50 of 409 0.70 g L-1 in the DPPH method indicating the strong antioxidant/radical scavenger 410 activity as well that the product had antioxidant activity in vitro. This antioxidant effect 411 was confirmed when applied directly into the salmon muscle and consequently might be 412 considered as a possible extract to be used in fish conservation. This antioxidant effect 413 was confirmed when applied directly into the salmon muscle and consequently could be 414 considered as a possible antioxidant product to be used in fish conservation. 415 Antioxidant D displayed the highest EC50 of the five compounds tested being the 416 product which showed lower antioxidant activity in DPPH assay. However, added at 417 concentration of 30 mg dm-2 (D1) on the sample of salmon demonstrated antioxidant 418 effect in the 7 days of storage, while at higher concentration (D2) a possible prooxidant 419 effect was observed because lipid oxidation did not decrease. Compound D seems to be 420 an example of antioxidant that although it did not demonstrate great antioxidant activity 421 by the indirect method (EC50 EC50= 3.25 g L-1), it was effective against lipid 422 peroxidation of food (Table 34b). Therefore, methods to determine free radical 423 scavenging such as DPPH method are very useful to make a screening of the 424 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 19 antioxidants, however to verify their effectiveness in food, methods to evaluate the food 425 deterioration must be used. 426 The antioxidant effectiveness in food depends on a variety of factors such as 427 polarity, solubility and metals quenching activity as well as the interaction with other 428 food components (Roginsky, & Lissi, 2005; Decker et al., 2005). Antioxidant D 429 presents a very similar behavior to that observed with the antioxidant oil C in terms of a 430 possible pro-oxidant effect or inhibition of antioxidant activity when applied at high 431 concentrations. 432 433 3.3. Thermal stability and antioxidant effectiveness of compounds 434 435 The thermal stability of the five antioxidants was evaluated and the most suitable 436 ones, that is, which do not suffer thermal degradation at the LDPE processing 437 temperature, were chosen for blending with polymer matrix. 438 Figure 1 displays the thermograms obtained from the TGA experiments. The A 439 and B compounds have a lower thermal stability than others. The degradation process of 440 A and B antioxidants start from 100 ºC, whereas in the other ones (C, D and E), it 441 begins at 250 ºC (see onset in each curve) approximately. Keeping in mind that LDPE 442 processing temperature is around 150 ºC (indicated by the arrow in the graph), the A 443 and B antioxidants antioxidants A and B were ruled out to blend with the polymer. 444 Then, the antioxidant activity of the different LDPE / antioxidant blends was evaluated 445 measuring the OIT by DSC. The oxidation induction time increases with the increase of 446 antioxidant amount. This effect is observed with three antioxidants, C (Blends LDPE 1-447 3), D (Blends LDPE 4-6) and E (Blends LDPE 7-9) (Figure 2). Comparing same 448 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 20 formulations, the antioxidant activity is quite similar in blends with antioxidant C and E 449 (the maximum time assayed) but it is much lower with D compound. 450 On the other hand, these results reveal a behavior of studied antioxidants as long-term 451 antioxidant. Therefore, OIT values increase with increasing natural antioxidant levels, 452 characteristic behavior of phenolic compounds. However phosphate stabilizers alone do 453 not increase the OIT of polyethylene in the temperature range of 180-210 ºC (Phease, 454 Billingham, & Bigger, 2000). 455 To prevent the matrix oxidation during the extrusion and the premature 456 consumption of the natural antioxidants, the two commercial antioxidants (IRGANOX® 457 1076 and IRGAFOS® 168) were added to the polymer blends. The OIT data of samples 458 LDPE 10 and LDPE 11 (the maximum time assayed) shown an available level of 459 antioxidant in the film to be released to fish. 460 Based on the results obtained, the natural antioxidants that should be selected to 461 incorporate in the films to be tested in the salmon packaging assay would be C and E. 462 Once these two products are constituted by the same active principle Nutrabiol® but in 463 different proportions, and for the purpose of testing two different products with different 464 compositions in antioxidant active compounds, the products selected to incorporate to 465 LPDE films were C and D. Thus, two different formulations in tocopherols can be 466 evaluated as well as their effectiveness against lipid peroxidation getting more 467 information about the possible application of these antioxidant products as ingredients 468 of active films. 469 In summary, the C and D antioxidants together with the commercial antioxidants 470 (Irgafos® 178 and Irganox® 1076 at 0.2 and 0.4 % respectively) were chosen to prepare 471 the LDPE active films. The active films formulations selected were: Film 2 and 3 472 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 21 incorporated with natural product C at 1 % and 5 % respectively; Film 4 with natural 473 product D at 5 %. Film 1 with only commercial antioxidants was used as control. 474 475 3.4 Evaluation of antioxidant effectiveness of the active films. 476 477 When incorporated into the plastic matrix, compounds with antioxidant activity 478 will not be available as in previous assays, which were added directly into fish muscle. 479 In addition, during films processing, that occurs at high temperatures and aggressive 480 conditions, the active compounds could be reduced and its antioxidant capacity be 481 affected. Therefore, based on the results of direct adding tests, commercial products 482 which showed evidences of great effectiveness in inhibition of lipid oxidation in salmon 483 were selected. Both of the products exhibited better results at lower concentrations and a 484 possible pro-oxidant effect when applied at the highest concentration demonstrating 485 their high activity. 486 Compound C showed a high antioxidant activity applied antioxidant activity 487 when applied at low concentration, a high antioxidant activity by DPPH method and in 488 its composition contains a high percentage of tocopherols. Therefore it was incorporated 489 into the plastic matrix at two different concentrations (1 % and 5 %). Compound D was 490 incorporated into the plastic matrix only at the highest concentration of 5 % because 491 showed less antioxidant activity by DPPH method (EC50 = 3.25 g L-1). 492 The antioxidant effect of films additivated with selected products was 493 determined by the capacity to inhibit lipid oxidation in salmon muscle during storage at 494 4 °C by TBARS method. 495 Figure 3 shows the malondialdehyde values (TBARS) TBARS values, expressed 496 as mg MDA Kg-1 of sample, obtained in the salmon muscle packed with the three films 497 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 22 additivated with the natural products containing tocopherols compared to control film 498 (Film 1). Results showed that lipid oxidation increased in all samples during storage but 499 at different rates. Control test presented a maximum of oxidation of approximately 5.8 500 mg MDA kg-1, while the samples packed with active films presented TBARS values 501 that varied over a range of ranging within 3-4 mg MDA kg-1. 502 Salmon packed with films spiked with antioxidant product C in the two 503 concentrations of active ingredient applied at 1 and 5 % (Film 2 and Film 3 504 respectively), experimented an important lipid oxidation decrease, and the greatest 505 antioxidant effect was verified at higher concentration used tested. 506 In the first days of assay, there were no important differences between the two 507 concentrations used but after the 7th day differences were more remarkable. At the first 508 days of assay, no main differences between the two concentrations tested was observed, 509 however, after the 7th day, differences were more remarkable. At 7th day, lipid 510 oxidation was reduced around 25 % for Film 3, while for Film 2 this decrease was less 511 than 10 % compared to control (Film 1). After day 11 of assay, Film 3 showed an 512 oxidation decrease to around 40 %. In the same period less reduction, around 15 %, was 513 observed for the Film 2, which at the end of the assay oxidation was decreased by 30 %. 514 TBARS values of fish packed with Film 4 which contains 5 % of antioxidant product D 515 also showed a significant reduction in lipid oxidation of salmon throughout entire assay 516 compared to control, increasing food stability. During the first 7 days of study, a 517 reduction around 40 % was observed to Film 4, and after that, oxidation decreasing 518 remained constant around 30-35 % until the end of the assay. 519 Comparing the curves of the films spiked with the two products applied in this 520 study at the same concentration (5 %), it can be pointed out that both films showed 521 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 23 important antioxidant effectiveness and there were no important differences no main 522 differences between the two compounds. 523 There was a small difference in the first days over the first days of analysis 524 where Film 4 (which contains TOCOBIOL®® PV) was more effective than Film 3, 525 while at the end of the assay Film 3 is the most effective film. This may be due to the 526 high amounts of tocopherols presents in Film 3 (product C). 527 Active films used had shown an inhibitory effect on the lipid oxidation process 528 comparable to the effect observed with direct addition of antioxidants to the fish 529 muscle. 530 Results obtained with Films 2 and 3 confirm the effectiveness of compound C as 531 well as the higher concentration incorporated into the films greater the effectiveness of 532 them. 533 Results also confirmed the high antioxidant effect of compound D, which 534 although did not showed high antioxidant activity in the free radical scavenging method 535 (DPPH), it showed great effectiveness against salmon lipid oxidation.536 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 31 Figure captions Figure 1. TGA thermograms of antioxidant compounds described in Table 1. Figure 2. OIT of LDPE blends described in Table 2. Figure 3. Lipid oxidation (TBARS) in salmon packed with antioxidant films stored at 4 °C during 21 days (Film 1: control; Film 2: 1% antioxidant C; Film 3: 5% antioxidant C; Film 4: 5% antioxidant D). MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Tables Table 1. Antioxidant produc ts composition Oils with antioxidant properties Powders with antioxidant properties TOCOBIOL ® B – TOCOBIOL ® GL C – NUTRABIOL ® -T90 D – TOCOBIOL ® -PV E – NUTRABIOL ® -T50 PV Monoglycerides Rest: Vegetable oil 18.6 % 9.70 % 4.30 % 19.8 % Tocobiol: Tocopherols Sterols Squalene Monoglycerides Rest: vegetable oil Propyl gallate Excipients: Lecithin Propylene glycol 25.0 % 18.6 % 9.70 % 4.30 % 19.8 % 25.0 % 35.0 % 15.0 % Tocopherols Alpha-tocopherol Gamma and Beta-tocopherol Delta-tocopherol Vegetable oil 90.2 % 15.5 % 63.3 % 21.1 % 9.80 % Tocobiol Tocopherols Sterols Squalene Monoglycerides Rest: vegetable oil Excipient: Silica gel 65.0 % 18.6 % 9.70 % 4.30 % 19.8 % 35.0 % Tocopherols Alpha-tocopherol Gamma and Beta-tocopherol Delta-tocopherol Vegetable oil Excipient: Silica gel MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 33 Table 2. 1 LDPE formulations 2 Natural antioxidants Commercial antioxidants Formulations 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 Free radical scavenging activity 5 Antioxidant 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 data obtained from salmon muscle in contact with different concentrations (30 and 125 mg dm-2, for samples 1 and 2 respectively) of antioxidant oils (described in Table 1) during storage at 4 °C. TBARS (mg malondialdehyde kg-1 salmon muscle) Time (days) Control 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 values obtained from salmon muscle in contact with different concentrations (30 and 125 mg dm-2, for samples 1 and 2 respectively) of antioxidant powders (described in Table 1) during storage time at 4 °C. TBARS (mg malondialdehyde kg-1 salmon muscle) Time (days) Control 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 (%) Temperature (ºC) E D B C A Figure 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 effect D effect E effect Synergic effect OIT(min) Blend nomenclature Figure 2 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Figure 3.