# Analysis of Potential Markers of Pork Freshness Based on Volatile Organic Compounds
## 3 Results and Discussion
### Analysis of VOCs by HP-SPME-GC-MS  
A total of 109 VOCs were detected using HP-SPME-GC-MS. After excluding data with high variability, 51 VOCs were retained for further analysis (Table S1). Among these, there were 7 (cyclo)alkanes, 14 aldehydes and ketones, 2 esters, 2 alcohols, 6 unsaturated hydrocarbons, 2 ethers, 7 nitrogen-containing compounds, and 5 other types of compounds. Figure 3A,B depict the proportional composition and concentration changes of VOCs during pork storage.  
Figure 3: Proportions of different categories of volatile organic compounds (VOCs) in pork during storage (**A**) and a heat map of VOC concentration changes during storage (**B**). Red indicates an increase in relative content during storage, while green indicates a decrease in relative content during storage.  
Aldehydes are regarded as the primary volatile compounds formed during the oxidative degradation of meat. These compounds are predominantly produced via the hydrolysis of triglycerides and the metabolic pathways of fatty acids, and they can also be generated through the transamination of amino acids. Common aldehydes detected in meat include hexanal, nonanal, heptanal, and 3-methylbutanal. Among these, hexanal is considered a key contributor to meat flavor development [46]. In agreement with the findings of [PERSON] et al. [47], the concentration of hexanal was initially high at the beginning of storage but then decreased significantly over time. In the later stages of storage, the degradation of pork proteins becomes closely associated with a rapid increase in the synthesis of 3-methylbutanal, which can be detected as early as day 5 [48]. This suggests that 3-methylbutanal could serve as a potential marker for identifying non-fresh pork [23].  
Ketones are recognized as byproducts of the Strecker degradation [49]. Among all ketones detected, acetoin exhibited a significant change in concentration during the storage period, increasing from 29.61 ng/g on day 5 to 168.03 ng/g. Acetoin can be formed through the oxidation of 2,3-butanediol, enzymatic decarboxylation of 2-acetolactate, or as a byproduct of glucose metabolism and microbial degradation [41; 47]. Numerous studies have proposed acetoin as a reliable indicator of spoilage in stored chicken breast and pork [22; 23; 50; 51; 52].  
Lipoxygenase and peroxidase convert linoleic acid in muscle into alcohols, which are believed to contribute to the volatile flavors of the meat [46]. During storage, the concentration of 3-methylbutanal increases, whereas the levels of known alcohols, such as 1-octen-3-ol, heptanol, and pentanol, decrease. Studies have demonstrated that 1-hexanol and 1-octanol, volatile compounds characteristic of fresh pork, are entirely absent in spoiled meat [47]. Notably, 1-octen-3-ol, which imparts a distinct mushroom-like aroma and plays a critical role in enhancing pork's flavor, is predominantly present during the early storage stages. This compound is formed via the enzymatic breakdown of eicosapentaenoic acid by 15-lipoxygenase and arachidonic acid by 12-lipoxygenase [53]. However, its concentration markedly decreases by day 5 of storage [30; 54]. [PERSON] et al. suggested that pentanol and 1-octen-3-ol could serve as potential spoilage markers for lamb [51]. Similarly, [PERSON] et al. identified 3-methylbutanal as a promising volatile spoilage marker for chicken breast [55], while [PERSON] and [PERSON] proposed that both 3-methylbutanal and 1-octen-3-ol could serve as reliable indicators of spoilage in grouper [56].