## 4 Discussion  
Various guidelines [10; 11] and authors [5; 12; 13; 26] recommend that the maximum residence time of milk administered to infants under nursery conditions (20-22 \({}^{\circ}\)C) should be 4 h. However, the results obtained in the present study showed that under conditions of _E. coli_ contamination of 10\({}^{4}\) CFU/mL, there were differences in the counts at earlier time points. In the case of frozen HM, count differences were obtained after 2.5 h, and for frozen and pasteurized milk, the times were reduced to 1.5 h. It must be considered that authors such as [PERSON] [26], who is used as a bibliographic reference in some guides, such as the British Dietetic Association [10], carried out a study with fresh HM and frozen HM for six weeks and their counts were only performed from natural contamination. However, in the present study, for frozen HM, the count differences were obtained after 2.5 h, but the freezing time was longer (3 months), and _E. coli_ inoculation was also performed to obtain homogeneity in the contamination of the samples. In addition, it was of interest to study the microbial behavior in milk under conditions of potential contamination. The obtained results are consistent with the United States CDC recommendation [14], which states that HM should not be kept thawed for more than 1-2 h at a temperature of 25 \({}^{\circ}\)C. Otherwise, [PERSON] [27] found a significant increase in the bacterial count of HM that was frozen for 7 d at \(-\)20 \({}^{\circ}\)C, thawed, refrigerated for 24 h, heated, and kept at room temperature for 4 h; thus, these authors propose reducing this time to 3 h and recommend re-evaluating the possibility of keeping milk at room temperature. This is consistent with the results obtained in the present study, as we also obtained increases in the milk counts before 4 h.  
Moreover, it is important to consider the freezing time, as longer times (3 months) affect the antimicrobial capacity of the HM [8] and could allow for faster microbial growth under NICU conditions. [PERSON] [6] also obtained a significant increase in _E. coli_ counts in HM that was previously frozen and maintained at 37 \({}^{\circ}\)C for 24 h. At 6 h and 24 h, _E. coli_ increased its number 10\({}^{7}\)-fold and 10\({}^{9}\)-fold, respectively. In this case, microbial growth was very high at 6 h, but it must be taken into account that the holding temperature was 37 \({}^{\circ}\)C.  
In HMBs, milk is frozen for long periods for a maximum of two times (before and after pasteurization), but it is also pasteurized. According to the results of the present work, the _E. coli_ generation times were significantly reduced (\(p\) = 0.012) when the milk was frozen and pasteurized; thus, the time at which the differences in the counts were obtained was reduced to 1.5 h. Various authors [7; 8] have shown that pasteurization significantly reduces the bactericidal capacity of HM, and according to the studies by [PERSON] [28] and [PERSON] [29], pasteurization and freezing/ thawing processes cause a significant loss of lactoferrin, lysozyme, IgG, and IgA, components with important antibacterial activity, which could explain the reduction of this capacity in HM subjected to these treatments. It is evident that the pasteurization process ensures the absence of pathogenic microorganisms in the milk that is supplied to the infant, but if milk contamination occurs during subsequent handling, microorganisms could multiply when the milk supply is kept at room temperature. In addition, feeding tubes can contain high amounts of potentially pathogenic and antibiotic-resistant bacteria, which could develop under NICU feeding conditions [2; 30]. It is important, therefore, to maximize hygiene conditions throughout milk processing and administration and to re-evaluate the timing of the milk supply. It seems key, therefore, to reduce the time of milk administration to less than the 4 h recommended in several guidelines. Regarding fortification, in the present study, no significant differences were found in the _E.