## 3 Results and Discussion
In this work, the shifts at 1439 cm\({}^{-1}\) and 1655 cm\({}^{-1}\) were selected because they gave the highest values of the EF. According to calculations [49] (considering the refractive index of the fat as 1.46 [50], an average molecular weight of 272.27 Da, according to the fatty acid composition of pork meat [51], and a fat density of 0.91 g\(\cdot\)cm\({}^{-3}\)[52]), the dimensions of the hourglass-shaped sample volume were \(\sim\)2.6 \(\upmu\)m diameter spot in the focal plane with a depth of \(\sim\)30 \(\upmu\)m. By subtracting the volume of silver nanoparticles, the effective Raman sampling of fat was about 82 \(\upmu\)m\({}^{3}\). Considering the characteristics of the fat extract, the detection volume contained \(\sim\)0.88 pmol. \begin{table}
\begin{tabular}{c c} \hline
**Raman Shift [cm\({}^{-1}\)]** & **Functional Group** \\ \hline
3008 & C–H stretching of C=C–H \\
2850 & C–H stretching \(\
u\)(C–H) in \(\cdot\)CH\({}_{3}\) and \(-\)CH\({}_{2}\)– \\
1655 & stretching of C=C bonds \\
1439 & scissoring deformation \(\delta\)(CH\({}_{2}\)) of \(-\)CH\({}_{2}\)– \\
1301 & in-plane C=C–H deformation \\
1266 & in-phase \(-\)CH\({}_{2}\)– twisting \\ \hline \end{tabular}
\end{table} 
Table 1: Raman shifts responsible for lipid oxidation obtained by SERS. Figure 4: Spectrum of lipids extracted from ground pork after 0 days by SERS (red) and conventional Raman (green). Both spectra are shown on the same scale. The EFs were calculated from an analytical chemistry point of view [53; 54] according to the formula EF = (I\({}_{\text{SERS}}\)/N\({}_{\text{SERS}}\))/(I\({}_{\text{Raman}}\)/N\({}_{\text{Raman}}\)), where I\({}_{\text{SERS}}\) and I\({}_{\text{Raman}}\) were the signal intensities with and without silver substrate, respectively, N\({}_{\text{SERS}}\) the number of molecules responsible for the enhancement (first layer in contact with silver, \(\sim\)8.4 \(\times\) 10\({}^{6}\)) and N\({}_{\text{Raman}}\) the number of fat molecules in the detection volume (\(\sim\)5.3 \(\times\) 10\({}^{11}\)). The maximum EF values obtained were 1.64 \(\times\) 10\({}^{7}\) at 1439 cm\({}^{-1}\) and 8.58 \(\times\) 10\({}^{6}\) at 1655 cm\({}^{-1}\). These Raman shifts were chosen as reference for normalization (1439 cm\({}^{-1}\), CH\({}_{2}\) scissoring, which is relatively unaffected by oxidation) and for assessing oxidative fat degradation, which was mainly due to unsaturation (1655 cm\({}^{-1}\), C=C stretching). Taking into account possible interferences, MDA, one of the main end products of lipid peroxidation, was considered as a reference. In a recent study [55], the MDA signal by SERS with mixed Au/Ag nanoparticles in the vicinity of 1655 cm\({}^{-1}\), more precisely at 1663 cm\({}^{-1}\) used by the authors for the effective measurement of a complex between MDA and 4-aminophenylthiophen, was negligible. Since lipid peroxidation occurs mainly in unsaturated fatty acids, and double bonds between carbon atoms are lost in the process, the decrease in the peak at 1655 cm\({}^{-1}\) can be satisfactorily attributed to lipid peroxidation. Figure 5 shows the overlapped spectra on different days showing both bands, corresponding to samples stored with LDPE (left) and with active packaging (right). It can be clearly seen that the decrease in the 1655 cm\({}^{-1}\) band over time was much less pronounced in meat samples stored in active packaging. This demonstrates the protective effect of active agent by delaying fat oxidation. Figure 6 shows the graphical results of the SERS analysis, where a significant difference between conventional and active packaging was observed, as the error bars did not overlap. Therefore, the developed method can be successfully applied to monitor fat oxidation or to study the role of active packaging on fat and fatty products. Furthermore, a decreasing trend was observed from day 7 onwards.