## 3 Results and Discussion
### NGS Analysis
Cluster I consisted of samples stored for extended periods: OM refrigerated for 10 and 14 days and stored on ice for 14 and 21 days, and DM refrigerated for 10 and 14 days and stored on ice for 21 days. Cluster II included OM and DM samples from SC for 37 and 50 days, respectively. _Pseudomonas_ (Cluster B) was the dominant genus in both clusters. Cluster I also contained relatively large populations of _Acinetobacter_, _Brochotrhix_, and _Shewanella_ (Cluster A). Cluster III included samples stored for shorter periods: days 0 (OM and DM), 3 (ice-stored OM and DM), and 3 (refrigerated DM). Cluster IV comprised samples from days 6 (refrigerated OM and DM), 27 (SC OM and DM), and 14 (ice-stored DM). Cluster V included samples stored for 7 days under various storage conditions (refrigerated OM and DM, stored on ice, and SC), 3 days (refrigerated OM), and 17 days (SC OM and DM). In Cluster III, the bacterial flora was more diverse, with _Pseudomonas_ and other genera, such as _Staphylococcus_ (Cluster C), _Acinetobacter_, and _Psychrobacter_ (Cluster D), which were abundant. Regardless of the initial bacterial diversity, _Pseudomonas_ consistently dominated at all storage temperatures as the storage progressed. However, spoilage-associated bacterial communities varied between refrigerated and ice-stored samples and between SC samples. These findings were consistent with the VCC data for _B. thermosphacta_, _Pseudomonas_ spp., and H2S-producing bacteria (_Shewanella_ spp.). When it was assumed that the shelf life was the shortest period of storage, in which both muscle sites belonged to clusters I and II, spoilage occurred after 10 (refrigeration), 14 (ice storage), and 37 (SC) days. By reaching 7 Log CFU/g of mesophilic bacteria, spoilage was observed at 14, 21, and 50 days for the refrigerated, ice-stored, and SC samples, respectively. The spoilage period of yellowtail meat estimated from the results of cluster analysis and VCC was slightly shorter in the cluster analysis than in the VCC analysis, suggesting that shelf life may be evaluated more accurately using NGS. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c c c c c c c} \hline \multirow{3}{*}{} & \multirow{3}{*}{**Muscle**} & \multirow{3}{*}{**0 Day**} & \multicolumn{6}{c}{**3\({}^{\circ}\)C**} & \multicolumn{6}{c}{**0\({}^{\circ}\)C**} & \multicolumn{6}{c}{**-3\({}^{\circ}\)C**} \\ \cline{3-14}  & & \multicolumn{3}{c}{**Storage Periods (Day)**} & \multicolumn{6}{c}{**Storage Periods (Days)**} & \multicolumn{6}{c}{**Storage Periods (Days)**} & \multicolumn{6}{c}{**Storage Periods (Days)**} \\ \cline{3-14}  & & & & & & & & & & & & & & & & & & & & & \\ \cline{3-14}  & & & & & & & & & & & & & & & & & & & & & & & & \\ \hline \multirow{3}{*}{Rancidity} & OM & 1.4 & 1.4 & 1.8 & * & 1.9 & * & 2.4 & * & 1.4 & 1.7 & 2.3 & * & 2.4 & * & 1.7 & 2.0 & * & 2.2 & * & 2.3 & * & 2.4 & * & 2.7 & * \\  & DM & 1.5 & 1.6 & 2.0 & 2.3 & * & 2.5 & * & 1.6 & 2.2 & * & 2.6 & * & 2.5 & * & 1.9 & 2.3 & * & 2.3 & * & 2.4 & * & 2.4 & * \\ \cline{3-14}  & & & & & & & & & & & & & & & & & & & & & & \\ \cline{3-14}  & & & & & & & & & & & & & & & & & & & & & & & \\ \cline{3-14}  & & & & & & & & & & & & & & & & & & & & & & & & \\ \hline \multicolumn{14}{c}{The values represent the means of triplicate determinations (\(n=3\)). OM, ordinary muscle in dorsal part, DM, dark muscle. Asterisks indicate a significant difference compared to day of the storage for the same flash type and analysis item (\(p<0.05\)).  
\end{table} 
Table 4: Changes in sensory scores of raw yellowtail muscles stored at different temperatures.