## 2 Materials and Methods
### Data Analysis  
In the study of the growth of _E. coli_ in HM and PIF, after obtaining _E. coli_ counts at each time point, the number of _E. coli_ generations (n) appearing at a given time was calculated in the different sample types. One generation is considered a microorganism count doubling; thus, the calculation was as follows: n = (log N \(-\) logN\({}_{0}\))/0.301, where N is the count (CFU/mL) at that given time and N\({}_{0}\) is the baseline microorganism count. The generation time (g), or the time it takes for a microbial population to double, was also calculated for each type of milk: g = t/n, where t is the time elapsed (240 min in our study) and n is the generation number that appeared during that time [25].  
In the study of different thawing methods, _E. coli_ counts were obtained for each of the thawed samples according to the four methods used. The results were obtained in CFU/mL and were transformed to log10 CFU/mL.  
The statistical analysis of the results was performed using IBM SPSS software (Version 27). The differences were considered statistically significant at \(p\) < 0.05. A mixed analysis of variance (ANOVA) was used to study the effect of maintaining milk in the neonatal room conditions on the generations that appeared at the different times, and to determine whether any change in the number of _E. coli_ generations was the result of the interaction between the type of milk (different treatments or different brands) and time. For HM, the two factors considered were the milk treatment (between-subjects factor), with three levels for HM (frozen milk; pasteurized and frozen milk; pasteurized, frozen and fortified milk), and the time (within-subjects factor), with eight levels (0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h). For PIF, the two factors considered were the milk brand (between-subjects factor), with four levels, and the time (within-subjects factor), with eight levels (0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h).  
To compare the results of the number of generations obtained in HM to PIF, a mixed ANOVA was also used. One factor was the milk type (between-subjects factor), with two levels (powdered infant formula and human milk), and the other factor was time (within-subjects factor), with eight levels (0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h).  
To determine the differences between the groups, a post hoc analysis was used with the Bonferroni adjustment test. To verify the data normality, the Kolmogorov-Smirnov test was used (_p_ > 0.05). The homogeneity of the variance assumption within the subjects and between the subjects was examined using [PERSON]'s test of sphericity (_p_ > 0.05) and [PERSON]'s test (_p_ > 0.05), respectively. In the cases where the sphericity hypothesis was not met, the multivariate adjustment was applied.  
For the comparison of _E. coli_ generation times in the HM subjected to different treatments, a one-way ANOVA was used. Similarly, a one-way ANOVA was performed to compare the generation times obtained in the different brands of PIF.  
To evaluate the thawing methods, a one-way repeated-measures ANOVA with four levels (thawing at room temperature (20 \({}^{\circ}\)C) for 2 h; in a thermostatic bath at 35 \({}^{\circ}\)C for 30 min; in a thermostatic bath at 25 \({}^{\circ}\)C for 40 min; and refrigated at 4 \({}^{\circ}\)C for 12 h) was performed.