# Analysis of Potential Markers of Pork Freshness Based on Volatile Organic Compounds
## 3 Results and Discussion
### 165 rRNA Gene Sequencing Analysis  
Sequence analysis of the 165 rRNA gene revealed that 3093 operational taxonomic units (OTUs) clustered with 97% similarity. The Chao1 index (Figure 2A), indicating high species richness, peaked on the second day of storage. Similarly, the Shannon index (Figure 2B) reached high levels, demonstrating significant evenness and diversity within the microbial community. However, these indices later showed a downward trend, suggesting a simplification in community structure and a decrease in microbial richness. All samples achieved a coverage index of 1.000, indicating that the sequencing depth was sufficient to encompass all species and ensure the completeness of the sequencing.  
The composition of the microbial population underwent significant changes throughout the storage period. Despite the presence of various species, the spoiling process was dominated by a few microbial species. The ten principal bacteria depicted in Figure 2C, such as _Brochotlrix, Pseudomonas, Listeria_, and _Acinetobacter_, are considered key to the degradation of air-stored pork [35; 38]. Initially, _Listeria_ spp. were predominant, comprising up to 80% of the total microbial population. However, during the storage period, the quantity of _Listeria_ spp. progressively decreased, nearly reaching extinction by day 10. _Pseudomonas_ spp., reaching a peak relative abundance of 78.7% on day 10, demonstrated a significant competitive advantage, becoming the predominant microbe in the later stages of storage.  
During the mid-phase of storage, _Acinetobacter_ spp. and _Macrococcus_ spp. exhibited certain advantages, yet _Pseudomonas_ spp. eventually surpassed them. Similarly, the relative abundance of _Brochotlrix_ spp. increased from the middle to late stages, peaking at 6.53% on  
Figure 2: Microbial diversity and composition during pork storage: Alpha diversity index (**A**), Shannon diversity index over time (**B**), and relative abundance of dominant bacterial genera (**C**).  
day 6. Despite their low abundance, _Psychrobacter_ spp. remained quite stable throughout the storage period.  
_Pseudomonas_ spp. are frequently isolated from rotten meat due to their heightened activity in aerobic environments [39; 40]. Initially, these bacteria metabolize meat glucose, a precursor to VOCs such as ethanol, 3-methyl-1-butanol, and acetoin. In aerobic conditions, _Pseudomonas_ spp. convert substrates into lactate and pyruvate, whereas in anaerobic conditions, they transform them into pyruvate and gluconate [41]. When nutrients are scarce, _Pseudomonas_ spp. secrete extracellular proteases to degrade connective tissues between muscle fibers. This enables access to nutrients and provides a competitive edge in the microbial community [42]. Besides accelerating the spoilage process, the activity of these proteases also softens the meat and generates TVB-N [35; 40]. Although _Acinetobacter_ spp. are the primary spoilage bacteria in refrigerated seafood, they are not major contributors to rotting in other contexts, as they do not produce extracellular lipases, hydrogen sulfide, or trimethylamine. However, through quorum-sensing signaling molecules, _Acinetobacter_ spp. can still accelerate the spoilage process by promoting the growth of other spoilage bacteria [43].  
_Brochottrix thermosphacta_ is commonly found as a spoilage bacterium in fresh meat stored under modified atmosphere packaging and refrigeration conditions. It is frequently observed in both low-oxygen and high-oxygen environments [44]. Ribose, glycerol, and amino acids are utilized by this bacterium as energy sources [45], resulting in the production of VOCs, such as 3-hydroxy-2-butanone, acetic acid, and alcohols, which are responsible for the generation of unpleasant odors in meat [43].