# Hake Fish Preservation Using Plant-Based Impregnated Polylactic Acid Food Films as Active Packaging
## 2 Materials and Methods
### Monitoring and Control of the Shelf Life of the Packaged Product  
#### 2.7.1 Physical and Chemical Analysis of the Packaged Product  
Water activity (a\({}_{w}\)) was measured in triplicate using a hygrometer (Aqualab Serie 3, Decagon Devices, Pullman, WA, USA) calibrated with standard salt solutions and an internal temperature control to measure the a\({}_{w}\) at 25 \({}^{\circ}\)C (\(\pm\)0.2 \({}^{\circ}\)C). Samples of the fish fillets were measured in triplicate using a hygrometer (Aqualab Serie 3,  
(\(\sim\)1.0 g) were placed in measurement capsules (Decagon Devices, Pullman, WA, USA), ensuring complete coverage of the capsule's base.  
The pH values of the fish fillets were determined in triplicate using a digital pH meter (Micro pH 2001, Crison Instruments, penetration pH electrode for solids and semi-solids SensION(tm), Barcelona, Spain). The pH meter was calibrated every 10 measurements using buffer solutions at pH 4.0 and 7.0.  
The rate of drip loss of the fish fillets during storage was calculated as percent water lost compared to the initial sample weight according to Equation (4):  
\[\textit{Drip loss}\ (\%)=(W_{1}-W_{2})/W_{1}\times 100 \tag{4}\]  
where \(W_{1}\) is the weight of the fish fillet at a predetermined time and \(W_{2}\) is the initial weight of the fish fillet [34].  
The muscle and skin color of the fish fillets were measured through a Minolta colorimeter CM-2600d (Minolta Co., Osaka, Japan) equipped with a standard D65 illuminant. The calibration was performed with both white and black standard plates. SpectraMagicTM NX software (ve. 1.9, Pro USB) was used to record the measurements and analyze color. The \(a^{*}\), \(b^{*}\), and \(L^{*}\) parameters were determined corresponding to the red-green component (red for \(a^{*}\) > 0 and green for \(a^{*}\) < 0), the yellow-blue component (yellow for \(b^{*}\) > 0 and blue for \(b^{*}\) < 0), and lightness (black for \(L^{*}\) = 0 and white for \(L^{*}\) = 100), respectively. Color change (\(\Delta\)E) was calculated on each sampling day in comparison to day 0, according to Equation (5) [35]:  
\[\Delta\mathrm{E}=\sqrt{[(\Delta L^{*})^{2}+(\Delta a^{*})^{2}+(\Delta b^{*})^{ 2}]} \tag{5}\]  
which indicated the overall color difference between the fish fillet after storage and the original sample at day 0. The determination of \(\Delta\)E allowed us to predict differences to the naked eye by analyzing the variation in the CIELab parameters. According to [PERSON] et al. [36], \(\Delta\)E < 1.5 represents slight differences, 1.5 \(<\)\(\Delta\)E <3.0 is considered different to a trained eye, and when \(\Delta\)E > 3.0, the samples are statistically different.  
The contents of total volatile base nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N) in the fish fillet samples were measured by employing Conway Microdiffusion Units Bel-Art(tm) Conway Diffusion Cells, SP Scienceware(tm) (Fisher Scientific S.L., Madrid, Spain), according to a previously reported method by [PERSON] et al. [37]. Each analysis was repeated in duplicate, and the concentration was also expressed as mg 100 g\({}^{-1}\).