# Analysis of Potential Markers of Pork Freshness Based on Volatile Organic Compounds
## 3 Results and Discussion
### Selection of Indicators for Pork Freshness  
By applying the Benjamini-Hochberg method to adjust for multiple comparisons (\(p<0.05\)), the \(t\)-test identified 32 VOCs with significant differences between 'fresh' and'spoiled' pork samples. FC analysis revealed 30 compounds with FC values above 2, indicating an increase, and 10 compounds with FC values less than 0.5, indicating a decrease. These results prompted the creation of a volcano plot (Figure 4), which illustrates the statistically significant compounds and the magnitude of their expression changes. Integrating these methodologies enabled the identification of 21 VOCs that were significantly elevated due to spoilage, while 10 were found to notably decline (\(p<0.05\), BH-corrected). According to the PCA results displayed in Figure 5A, PC1 explains 31.4% of the total variance, whereas PC2 explains 11.9%. This analysis clarifies the pattern of sample changes over time and highlights the significant impact of storage duration on VOC characteristics. Fresh pork samples are located in the negative quadrant of PC1 and exhibit a gradual transition towards the positive quadrant with increasing storage time. The considerable overlap observed for the results from day 2 and day 3 indicates that their VOC profiles are highly similar. Based on this foundation, variables that significantly contribute to distinguishing pork samples by storage duration were further extracted using PLS-DA. The PLS-DA score plot in Figure 5B illustrates the distinction between samples. However, the classification ability of PLS-DA was found to be limited, likely due to the high similarity of VOC components in fresh pork samples. In the early stages of storage, fresh samples are minimally influenced by microbial metabolism and enzymatic activities, resulting in negligible changes in the types and concentrations of VOCs. This high degree of consistency within the group complicates the ability of PLS-DA to extract clear classification features, thus diminishing its effectiveness in differentiating between groups. Conversely, samples stored for longer periods are characterized by more marked differences, enhancing the model's classification capabilities. The model fitting metrics, R\({}^{2}\) and Q\({}^{2}\), were 0.96 and 0.90, respectively, indicating strong predictive performance, as an R\({}^{2}\) value greater than 0.7 typically signifies strong predictive capability. Cross-validation confirmed the stability and reliability of the PLS-DA model (Figure 5C). Sixteen VOCs with scores exceeding one were identified by the analysis of VIP scores. The dynamic patterns of these VOCs are clearly illustrated by the VIP score heatmap, with significant concentration fluctuations over various storage periods being demonstrated (Figure 5D). In this study, 15 VOCs were initially identified through univariate and multivariate analysis methods (Table 1). Six VOCs--2,4-dimethyl-3-heptanone, azetidine, ether, pentan-2-one, pentane, and pentanol--were excluded as potential markers due to their lack of a consistent correlation with freshness or storage time. Among the remaining nine VOCs, most were observed to appear or disappear by day 5 or day 6 of storage. This behavior may be attributed to protein degradation and lipid oxidation, where initial concentrations of these VOCs remain below the detection threshold and gradually increase to detectable levels. Conversely, some VOCs may diminish due to chemical reactions or metabolic activities. While these VOCs can serve as indicators of spoilage, they are limited in providing precise timing information regarding the spoilage process. Notably, hexanal was found to lack a reliable freshness signal due to its inability to induce a visible color change, thereby restricting its applicability in rapid meat freshness assessment. Figure 4: Differentially expressed compounds during pork storage (\(\,\mathrm{log_{2}FC}\,\mathrm{|}\,>\,2\,\mathrm{\textit{p}}\,<\,0.05\)).