## 3 Results  
### Study of Growth of E. coli in HM and PIF Held at 22 \({}^{\circ}\)C for 4 h  
The results of the growth of _E. coli_ obtained after maintaining HM and PIF under neonatal nursery conditions, at 22 \({}^{\circ}\)C for 4 h, are presented below.  
Table 1 shows _E. coli_'s growth evolution in HM subjected to different treatments and maintained at 22 \({}^{\circ}\)C for 4 h, expressed as the number of generations that appeared at each time, and the generation time (time it takes for the _E. coli_ population to double) for each type of milk treated.  
The mixed ANOVA showed that there were significant differences between the number of generations obtained at different times and that these differences were not the same for each type of HM treated. Regarding the generation number obtained at different times, for the frozen milk (treatment A), significant differences were found after 2.5 h. In the case of frozen and pasteurized milk (treatment B), and frozen, pasteurized, and fortified milk (treatment C), differences in the generations were detected at 1.5 h and 2 h, respectively.  
For the generation time, the one-way ANOVA showed that there were significant differences between the generation times obtained in the HM subjected to the different treatments (Table 1). Specifically, there were differences between frozen milk and frozen and pasteurized milk, with the generation time being shorter in the case of frozen and pasteurized milk (126.65 min).  
Table 2 shows _E. coli_'s growth evolution in PIF maintained at 22 \({}^{\circ}\)C for 4 h, expressed as the number of generations that appeared at each time and the generation time for each brand.  
The mixed ANOVA showed that there were significant differences between the number of generations obtained at different times and that these differences were not the same for each brand of PIF. The times from which significant differences in the number of generations were obtained were 3 h for brands A, C, and D, and 1.5 h for brand B.  
Regarding the generation times, the one-way ANOVA showed that there were significant differences between the different milk brands, obtaining the greatest generation time for brand A (313.61 min) and the shortest for brand D (179.24 min).  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & & & & & & & & & & \\ \hline
**Treatment\({}^{1}\)** & **0.5 h** & **1 h** & **1.5 h** & **2 h** & **2.5 h** & **3 h** & **3.5 h** & **4 h** & \(p\) & **(Minutes)** \\ \hline A (n = 12) & \(-0.09\pm 0.15\) & \(-0.02\pm 0.16\) & \(0.14\pm 0.11\) & \(0.06\pm 0.13\) & \(0.39\pm 0.28\)\({}^{*}\) & \(0.45\pm 0.22\) & \(0.75\pm 0.30\) & \(1.04\pm 0.19\) & \(0.013\) & \(229.72\pm 37.84\)\({}^{*}\) \\ \hline B (n = 12) & \(-0.12\pm 0.49\) & \(0.13\pm 0.5\) & \(0.58\pm 0.46\)\({}^{*}\) & \(0.71\pm 0.59\) & \(0.93\pm 0.62\) & \(1.20\pm 0.73\) & \(1.79\pm 0.79\) & \(1.89\pm 0.71\) & \(0.001\) & \(126.65\pm 53.32\)\({}^{b}\) \\ \hline C (n = 12) & \(-0.17\pm 0.24\) & \(0.20\pm 0.44\) & \(0.15\pm 0.60\) & \(0.65\pm 0.69\)\({}^{*}\) & \(0.73\pm 0.54\) & \(0.90\pm 0.63\) & \(1.30\pm 0.46\) & \(1.53\pm 0.58\) & \(0.001\) & \(157.05\pm 66.17\)\({}^{Ab}\) \\ \hline \end{tabular} \({}^{1}\) A = freezing at \(-20\) \({}^{\circ}\)C for 3 months; B = pasteurization and freezing at \(-20\) \({}^{\circ}\)C for 3 months; C = pasteurization, freezing at \(-20\) \({}^{\circ}\)C for 3 months; and fortification. \({}^{2}\) Different superscripts (\({}^{a}\), \({}^{b}\)) in this column represent significant differences between treatments (_p_ = 0.007). \({}^{*}\) Indicates that from this time, significant differences were obtained in the number of generations that appeared (_p_ < 0.05). The _p_-values of these differences are shown in the corresponding column.  
\end{table}
Table 1: _Escherichia coli_ growth evolution in human milk (HM) maintained at 22 \({}^{\circ}\)C for 4 h, expressed as number of generations at different times and generation times (mean \(\pm\) standard deviation).