*2.2. Laminates*

Laminates made with selenium nanoparticles were prepared. Firstly, selenium nanoparticles were synthesized using a solution-phase approach based on the reduction of selenite with ascorbic acid in the presence of 7% of concentration of 2,4,7,9-tetramethyl-5decyne-4,7-diol ethoxylate as stabilizer agen<sup>t</sup> (at pH 6.5). The final concentration of selenium nanoparticles in the solution were 1000 ppm. SeNPs between 50 and 70 nm were obtained. Figure 1 shows the SEM analysis, where the size and identification of SeNPs in the solution can be seen. The detailed description of the synthesis can be read in Vera et al. [13].

**Figure 1.** (**a**) SEM of 1500 ppm solution of nanoSe and (**b**) EDX analysis of SeNPs. Samples on Cu grid.

This nanoselenium solution was incorporated at 10% ( *w*/*w*) into a water-based adhesive dispersion. The formula of adhesive was provided under a confidential agreemen<sup>t</sup> and cannot be disclosed here.

Using the adhesive, the laminate structure [substrate 1-adhesive-substrate 2] was manufactured first at laboratory scale and later at industrial scale. Industrial conditions were optimized as well to provide a colorless multilayer, where nanoSe was homogeneously distributed. At laboratory scale, the laminates were prepared using an extender machine K control coater, RK printcoat instruments (RK PrintCoat Instruments Ltd, Litlington, UK) on a 20 × 30 cm of substrate 1 polyethylene terephthalate (PET, 12 μm), forming a uniform layer. The grammage of adhesive placed was 3 g/m2, which was gravimetrically measured. Afterwards, a second 20 × 30 cm of 60 μm thickness film of low density polyethylene (LDPE) was placed on top of it, and then the laminate was pressed at 80 ◦C at speed number 4 in a BiO 330 model Laminator (Laminator-BIO 330, Korea)

Blank laminates were also prepared as follows: PET (12 μm)/adhesive/PE (60 μm) (laminate B) using the same formula without nanoSe.

#### *2.3. Food Samples and Packagings*

Hazelnuts, walnuts, and potato chips were bought in bulk. Fresh hazelnuts and walnuts without the peel were used. Negret hazelnut and Hartley walnut variety, additive free, were selected. Fresh fried potato chips with the following composition: Sunflower oil 80% and olive oil 20% were chosen for the study, assuming that they were prone to oxidation. In the studies made at laboratory scale, 5 × 2.5 cm<sup>2</sup> thermosealed bags were made with the laminates using a thermal sealer. An LDPE layer was inside the bag in contact with the food. Nuts and potato chips were ground to increase the surface in contact with the packaging, and to increase the oxidation effect to see differences in the oxidation of blanks and actives.

Industrial samples were provided by several companies interested in the study. Their names are confidential. Breaded Wiltshire cured ham (British stile ham) was studied. No antioxidants were added to the ham. Ready-to-eat vegetable mixture seasoned with butter consisting of sweet corn, chopped broccoli, and chopped carrot were studied. Fresh raw chicken breast was also packaged by another food company. In all cases, their own industrial packaging line and their own format (shape and size) for the selected food were used, without intervention or participation of people involved in the nanoSe project and development.

#### *2.4. Monitoring the Hexanal*

Hexanal analysis was done by headspace-solid phase microextraction (HS-SPME) coupled to a gas chromatography mass spectrometry (GC-MS). The SPME selected was Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS). The fiber was introduced into the bags made with the laminates, which contained the nuts. The extraction time was 15 min at room temperature and the desorption was done at 250 ◦C in the GC-MS. The equipment used was an Agilent 6890N gas chromatograph with a mass spectrometer MS 5975B detector. All of them were from Agilent Technologies (Madrid, Spain).

The capillary column used was a HP-5MS (30 m × 0.25 μm × 250 μm) from Agilent Technologies (Madrid, Spain). The oven program was as follows: 40 ◦C for 2 min, with a rate of 10 ◦C/min up to 300 ◦C, maintained for 2 min. The injection type was splitless and the helium flow was 1 mL/min. The mass detector was set at SCAN mode (in the range *m*/*z* 45–350) for the identification of the compounds, and SIM mode for the quantification of the compounds in the migration extracts.

#### *2.5. Fatty Acids Oxidation Study*

In the fatty acids oxidation study, a derivatization of the fatty acids was performed, where 10 g of grounded nuts were added to a recipient and then 20 mL of DCM:cyclohexane (1:1) were added. The two phases were separated. The liquid phase was transferred to another recipient and dried at room temperature. Then, 4 mL of NaOH 0.5 M were added to a 100 mg of this extract. This mixture was kept boiling for 10 min. Then, 5 mL of boron trifluoride 14% in methanol were added and the mixture was kept boiling for 2 min. Finally, 4 mL of heptane were added and the mixture was kept boiling for 1 min. The mixture was allowed to cool for 2 min. Subsequently, 15 mL of NaCl were added and the mixture was shaken with a Vortex®.

The organic phase was analyzed by GC-MS. The equipment used was a CTC Analytics CombiPal autosampler coupled to an Agilent 6890N gas chromatograph with a mass spectrometer MS 5975B detector. All of them were from Agilent Technologies (Madrid, Spain).

The capillary column used was a HP-5MS (30 m × 0.25 μm × 250 μm) from Agilent Technologies (Madrid, Spain). The oven program was as follows: 40 ◦C for 2 min, with a rate of 10 ◦C/min up to 300 ◦C, maintained for 2 min. The injection type was splitless and the helium flow was 1 mL/min. The mass detector was set at SCAN mode (in the range *m*/*z* 45–350) for the identification of the compounds, and SIM mode for the quantification of the compounds in the migration extracts.

#### *2.6. TBARS (Thiobarbituric Acid Reactive Substances)*

The oxidation of lipids was checked using the thiobarbituric acid reactive substances (TBARS) method. The assay was carried out following the method developed by Pfalzgraf et al. [23]. Briefly, 10 g of food were mixed with 20 mL of aqueous solution of trichloroacetic acid at a concentration of 10 μg/g, and then homogenized with an Ultra-Turrax at 18,000 rpm till uniform slurry was obtained. The supernatant was filtered using qualitative paper filter. Then, 2 mL of the filtrate were mixed with 2 mL of aqueous solution of thiobarbituric acid at a concentration 20 mM. The mixture was kept in a thermostatic bath at 97 ◦C during 20 min. The absorbance of the solution was measured at 532 nm against the blank sample (instead of filtered aliquot, 2 mL of trichloroacetic acid solution was used to prepare the blank sample). The results were expressed in equivalent concentration of malondialdehyde (MDA; mg of malondialdehyde/kg of meat). The malondialdehyde solution was prepared from 1,1,3,3-tetraethoxypropane dissolved in 1N aqueous hydrochloric acid for the calibration curve.
