**3. Results**

The antioxidant packaging was firstly evaluated using the methodology described by Pezo et al. [3], based on the exposure of the packaging material to an atmosphere enriched in free radicals. This method, where the free radical scavenging system is applied directly on the film, without requiring a previous extraction of the polymer, allows the evaluation of real antioxidant performance of the packaging. This method was necessary, as Vera et al. [13] demonstrated that the free radical diphenylpicrylhydrazyl (DPPH) was too big to cross the LDPE layer and arrive at the adhesive where the antioxidants, the SeNPs, are placed. Thus, the antioxidant performance can be considered as the actual one corresponding to the film. The material under study, containing SeNPs, was not previously tested on food.

As the aim of this work was to demonstrate the efficiency of this antioxidant packaging to protect the food versus oxidation processes, two series of tests were designed, the first series at laboratory scale and the second one at industrial level.

Several methodologies were applied and compared to objectively measure the oxidation of fats.

#### *3.1. Monitorization of Hexanal Released by the Nuts*

Hexanal is the main product of linoleic acid oxidation. Therefore, it is an indicator of lipid oxidation, which has been used as a good marker to monitor many foods: Potato crisps [27], Rapeseed oil [28], Iberian dry-cured loin (Muriel et al. 2007) meat and meat derivatives [29]. Nonanal and hexanal were measured in olive oil [30] and also in nuts [31].

The SPME fiber selected was Carboxen/Polydimethylsiloxane (CAR/PDMS) with 75 μm thickness, since according Pastorelli et al. [32] it had the highest sensitivity for this compound. The limit of detection for hexanal was 5 ng mL−1.

In our case, hexanal did not result in a good marker. Since hexanal is a small molecule, it was able to pass through the laminate that formed the bags. Hexanal was measured after 9, 27, 63, and 105 days of storage. It was observed that the amount of hexanal was gradually decreasing throughout the study, probably due to its permeation throughout the multilayer. Therefore, the hexanal value could not be considered as reliable in this case. This also demonstrates that the presence of nanoSe in the multilayer does not enhance the barrier properties of the multilayer versus organic vapors, such as aldehydes. It is the first time that this permeation of hexanal in the presence of nanoSe in a multilayer is described.

Subsequently, this methodology for monitoring the oxidation versus time was discarded.

#### *3.2. Fatty Acids Oxidation Study*

It has been demonstrated that the fatty acid composition of oils is a strong indicative factor that could predict the oxidative state [14–16]. Owing to this, the determination of fatty acids was selected here to study their oxidation rate.

Derivatization was the selected methodology for the fatty acid analysis by GC, since the use of ester derivatives is recommended. The methyl ester derivatives are much more volatile than the corresponding fatty acids. Moreover, they are much less polar, avoiding peak tailing and peak asymmetry on GC [33].

Methyl palmitate, methyl linoleate, methyl oleate, and methyl stearate were the esters determined in this work, since palmitic acid, linoleic acid, oleic acid, and stearic acid are the most abundant fatty acids in hazelnuts and walnuts [34].

Table 1 shows the amount of these fatty acids expressed as ng/g hazelnuts and as ng/g walnuts in the bags made with laminate A (made with adhesive alone) and laminate B (made with adhesive + 10% of nanoSe). Fatty acid values were around 50% higher in the bags made with laminate B, which contained nanoSe at both day 21 and day 42, respectively. The highest differences were found for walnuts. An improvement on the amount of fatty acids from 50 to 66% at day 42 was found in the active bag. It can be seen that 11.3 ng/g of methyl oleate were found in the bag made with laminate B and 3.88 ng/g were found in the bag made with laminate A. In the case of hazelnuts, the differences on the fatty acid amount between the active bag and the blank ranged from 24 to 53% at day 42. Therefore, these results demonstrate the antioxidant effect on food and the consequent shelf life extension of the packaged nuts, since pristine fatty acids remain for much more time in nuts.


**Table 1.** Concentration of fatty acids expressed as ng/g of hazelnuts and walnuts obtained for the laminates A (control, without nanoselenium particles) and laminates B (with nanoselenium particles) expressed as ng/g food.

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

Unsaturated fatty acids are oxidized to form odor-free, tasteless hydroperoxides. Then, they are decomposed to flavorful secondary oxidation products, which are mainly aldehydes, such as hexanal, 4-hydroxynonenal (HNE), and malondialdehyde (MDA) [35]. The most common method to determine MDA in foods is the spectrophotometric measurement of the colored adduct of MDA with 2-thiobarbituric acid (TBA). This methodology is not specific, but it has been reported to be a more accurate and sensitive parameter in assessing the oxidative deterioration than other methods [36].

Figure 2 shows the TBARS results of hazelnuts and walnuts packaged for 21 and 42 days, respectively, in both types of laminates, control and active. It can be observed that the hazelnuts packaged in active bags (made with laminate B) released around 10% less MDA than the blanks (laminate A) after 21 days of being packaged, and up to 20% when they were measured after 42 days. In the case of the walnuts study, the bags made with laminate B (active) released 5% less MDA than the blanks (laminate A) after 21 days and around 25% less after 42 days. The values of MDA were higher in walnuts than in hazelnuts. This can be attributed to the fact that walnuts contain a higher amount of PUFA [25], which are more vulnerable to oxidation. Student T-test was performed, and the results were 0.005 for hazelnuts and 0.016 for walnuts. Thus, it could be confirmed that the results with laminate A and B had significant differences with a 95% confidence level.

The results also demonstrated that the active packaging based on nanoSe prevents the oxidation of nuts and significantly extends the shelf life of the nuts.

#### *3.4. Sensory Evaluation of Packaged Nuts*

The tasting was done by an expert panel composed of 10 tasters. They scored four parameters from 1 (worse) to 5 (better): appearance, aroma, taste, and rancidity after 42 days of storage of the bags.

Figure 3a,b show the results of the tasting. It can be observed that aroma, flavor, and rancidity were scored at higher values in both hazelnuts and walnuts in laminate B (active) than in laminate A (blank). Rancidity obtained a difference of score of one point between hazelnuts packaged in active bags after 42 days of storage. Moreover, in walnuts the difference was even higher for rancidity (1.3 points of difference).

**Figure 3.** (**a**) Sensory evaluations of hazelnuts packaged in laminate A (control) and laminate B (actives with nanoselenium particles) after 42 days; (**b**) Sensory evaluations of walnuts packaged in laminate A (control) and laminate B (actives with nanoselenium particles) after 42 days.

This data were of grea<sup>t</sup> importance, since the consumer will judge the usefulness of the new active packaging. In addition to this, it can be underlined here that the tasting data matched with the analytical data found above by the two methods used.

#### *3.5. Industrial Tests*

Once different methodologies to study the oxidation were tested at laboratory scale in nuts, the active packaging was produced at industrial scale and used in other foods. TBARS method in combination with tasting (for ready-to-eat foods) were the methodologies selected for this work. The analysis of fatty acids was discarded, since TBARS offered sufficient results to compare active and blank packaged food, and TBARS was also simple and quicker.

To begin with, a laboratory scale study was done with the industrial multilayer to conduct an additional study of the active packaging with real food prior the industrial scale. The industrial active multilayer was prepared following the same steps as in the laboratory. Firstly, nanoparticles were prepared and incorporated into the adhesive. This active adhesive was then applied between the two plastic films to form the multilayer. This preliminary test was designed to save money and food coming from a large test at industrial scale in a food company. Once the antioxidant properties of the industrial multilayer were confirmed, the industrial tests with different food produce were carried out. In the preliminary study, potato chips, which are susceptible to oxidation, were selected. Once the results of this study were obtained, industrial trials were prepared with ham, vegetables seasoned with butter, and fresh chicken.

#### *3.6. Potato Chips (Laboratory Scale Study)*

Potato chips were selected for this study as they have a high amount of unsaturated fats, since they were fried in olive oil, according to the information provided by the suppliers. This kind of product is susceptible to rancidity, and it turns into a perfect target for the antioxidant packaging study.

These potato chips did not contain preservatives or antioxidants, as was mentioned in the experimental section. The time for the study was established according to the shelf-life of the product. The TBARS study and the tasting were done 21 days after the packaging.

The TBARS results (Figure 4) showed a high amount of malonaldehyde. This result was due to the high amount of fat contained in the product. A significant difference of 22% was found between the active laminates and the blanks. This data demonstrated that potato chips were protected from oxidation in this packaging, and therefore this antioxidant packaging could be a good target for its implementation in the market. Student T-test was performed, and the result was 0.045. It was confirmed that the results with laminate A and B were statistically different with a 95% confidence level.

**Figure 4.** TBARS results expressed as mg of MDA per kg of potato chips, ham, chicken, and ready-to-eat-vegetables packaged in laminate A (control) and laminate B (actives with nanoselenium particles) for 21 days.

The tasting results (Figure 5) showed that the detection of rancidity was almost two out of five points higher in laminate B (blank) than in laminate A (with nanoSe). Therefore, aroma and taste were consequently better in laminate A (nanoSe).

**Figure 5.** Sensory evaluations of potato chips packaged in laminate A (control) and laminate B (actives with nanoselenium particles) after 21 days.

#### *3.7. Industrial Study: Cooked Ham, Chicken and Ready-to-Eat Vegetables Dressed with Butter*

Cooked ham, chicken, and ready-to-eat vegetable mixture seasoned with butter were the foods studied in the industrial trial, since producers of these kind of foods expressed their interest in the antioxidant packaging for their products. A large series of each food was packaged in their industrial packaging lines, using the same procedure, packaging speed, and system as the conventional multilayer. Blank and active samples were also packaged in each case and the same procedures above mentioned and optimized, were applied to monitor the behavior of the packaged food. Half the amount of the tested food was evaluated by the food companies and half by the research team at the University. All the results arrived at the same conclusions about the active nanoSe material. Drawbacks related to the packaging lines were not found, as the material behaved the same as the conventional one, because the active agen<sup>t</sup> was in the middle of the multilayer, in sandwich mode, and consequently it did not affect the performance of the plastic.

Figure 4 shows the TBARS results for all of them packaged for 21 days. In the four types of foods under study, a significantly lower amount of MDA, around 50% for ham, released in the food packaged with the nanoSe were found. Therefore, this means a considerable improvement in the quality of the market produce, since less rancid products are released. This also turns into an extended shelf-life of food produce and demonstrates that the active packaging with nanoSe is a successful option. This is the first time that an industrial active multilayer containing Se nanoparticles as an antioxidant agen<sup>t</sup> was studied. This study opens the door for further active materials, where nanoparticles can be introduced in a skilled manner without affecting the behavior of the material, whilst harnessing the properties and advantages of the packaging materials. Student T-test was performed, and the result was 0.04 for ham, 0.01 for chicken, and 0.06 for vegetables, respectively. The results with laminate A and B were significantly different with a 95% confidence level, for ham and chicken. Vegetables packed with laminate A and B were not significantly different.
