## 3 Results and Discussion  
### Determination of the Microbial Count Evolution  
A progressive increase (\(p\) < 0.05) in the total aerobe counts (\(r\) = 0.94, quadratic fitting) was detected in the fish muscle as the storage time progressed (Table 2). However, even at the end of the storage period (9 d), the aerobe counts did not reach values above 6 log CFU units, and therefore, they did not pass over the 7 log CFU value considered the acceptable limit [40]. In contrast, the psychotroph counts exhibited important increases which led to concentrations above 7 log CFU units at the end of the storage time (Table 2). However, the psychotroph counts on day 6 did not reach 6 log CFU units, and as in the case of the aerobes, this may be regarded as a satisfactory result. As in the case of the aerobe mesophiles, the psychotroph bacteria steadily increased (\(p\) < 0.05) during refrigerated storage (\(r\) = 0.92, quadratic fitting). \begin{table}
\begin{tabular}{c c c c c} \hline
**Descriptor** & **Highest Quality** & **Good Quality** & **Fair Quality** & **Objectional Quality** \\  & **(E)** & **(A)** & **(B)** & **(C)** \\ \hline \hline \multirow{3}{*}{Skin} & Highly intense pigmentation; transparent mucus & Milky mucus; insignificant pigmentation losses & Slightly grayish mucus; pigmentation & Widely opaque mucus; important \\  & & & without shine & pigmentation losses \\ \hline \multirow{3}{*}{Eyes} & Convex; transparent cornea; bright and black pupil & Convex and slightly sunken; slightly opalescent cornea; black and cloudy pupil & Flat; opalescent cornea; opaque pupil & Concave and milky cornea; internal organs blurred \\ \hline \multirow{3}{*}{Gills} & Bright red; without odor; lamina perfectly separated & Rose-colored; without odor; lamina adhered in groups & Slightly pale; incipient fishy odor; lamina & Gray-yellowish color; intense ammonia odor; lamina totally adhered \\ \hline \multirow{2}{*}{External odor} & Sharp seaweed and shellfish smell & Weak seaweed and shellfish smell & Incipiently putrid and rancid & Putrid and rancid \\ \hline \end{tabular}
\end{table} 
Table 1: Scale used for freshness degree evaluation of pouting fish. \begin{table}
\begin{tabular}{c c c c} \hline
**Microbial** & & & \\
**Parameters** & & & \\ \hline  & **0** & **3** & **6** & **9** \\ \hline Total aerobes & 3.90 \(\pm\) 0.35 a & 3.94 \(\pm\) 0.42 a & 4.36 \(\pm\) 0.32 a & 5.52 \(\pm\) 0.07 b \\ \hline Psychotrophs & 4.38 \(\pm\) 0.03 a & 5.55 \(\pm\) 0.65 b & 5.89 \(\pm\) 0.13 b & 8.54 \(\pm\) 0.36 c \\ \hline _Enterobacteriaceae_ & 1.23 \(\pm\) 0.40 a & 2.09 \(\pm\) 0.16 b & 2.49 \(\pm\) 0.20 c & 3.10 \(\pm\) 0.95 c \\ \hline \end{tabular} \({}^{*}\)Average values \(\pm\) standard deviations of three independent determinations (\(n\) = 3). \({}^{**}\)In each row, different letters (a, b and c) indicate differences (\(p\) < 0.05) with chilling time.  
\end{table} 
Table 2: Determination of microbial activity (log CFU:g\({}^{-1}\) muscle) \({}^{*}\) in pouting muscle subjected to chilling storage \({}^{**}\). Similar to the results observed for the other microbial parameters evaluated, a substantial (\(p<0.05\)) increase in the _Enterobacteriaceae_ counts was observed after 3 and 6 days of storage (Table 2). Interestingly, and although an average increase was also determined at the end of the storage time (day 9), this increase was not found significant. Notably, the _Enterobacteriaceae_ counts showed a good relationship with the storage time (\(r=0.94\)). Interestingly, the presence of _Enterobacteriaceae_ in the pouting muscle did not reach unsatisfactory levels (4 log CFU units) [40] even after 9 days of refrigerated storage. To the best of our knowledge, no previous studies have evaluated the metrics of the microbial breakdown of pouting muscle during chilled storage.