## 4 Discussion
coli_ generation time between HM with and without fortifier. These results are consistent with those of [PERSON] [31], who found no differences in microbial growth at 22 \({}^{\circ}\)C for 6 h between fortified milk (with iron-rich and iron-poor fortifiers) and unfortified milk. However, these authors did not observe significant microbial growth during those 6 h in any of the milk types studied. This difference, as compared to our results, may be due to the fact that the HM used was fresh, not frozen or pasteurized, and they also inoculatedanother microorganism (_Cronobacter sakazaki_). As indicated above, milk treatments seem to be determinants of possible microbial growth. Different results were obtained by [PERSON] [23], who investigated the growth of different strains of _C. sakazaki_ in HM previously frozen at \(-80\)\({}^{\circ}\)C, fortified (1.44 mg of iron/100 mL), and unfortified, kept at 23 \({}^{\circ}\)C for 24 h. In this case, the obtained results differed depending on the investigated strain; therefore, differences in growth were obtained for some strains but not for others. This indicates that the type of microorganism also plays a role, which may justify the differences compared to this study, since in the present study, _E. coli_ was used. In addition, recent studies have shown that lactoferrin's antibacterial capacity is not totally dependent on its iron saturation. This capacity is rather related to direct interaction between lactoferrin and bacteria [32]. In addition, in HM, there are many other components with antimicrobial capacity, such as oligosaccharidesadriades, lipids (e.g., glycerol monolaurate), other proteins (casein201), peptides, and enzymes (e.g., lysozyme, peroxid oxidases, and xanthine oxidase) [33; 34; 35; 36; 37]. For PIF and with regard to the time at which differences appear in _E. coli_ generations, variable results were obtained; these times ranged between 1.5 and 3 h, depending on the milk brand. [PERSON] [6] obtained similar results in PIF with \(10^{3}\) CFU/mL of _E. coli_ added, although in this study, the incubation temperature was 37 \({}^{\circ}\)C. At this temperature, the authors detected rapid microorganism growth, whose number was 10 times and \(10^{4}\) times greater at 2 h and at 4 h, respectively. In our case, the microbial growth was not as fast, but the holding temperature was also lower (22 \({}^{\circ}\)C). If the generation times obtained in PIF are compared with those of HM, it can be observed that there are no significant differences between them. This is a possible consequence of human milk antimicrobial capacity loss due to the applied treatments. Otherwise, [PERSON] [6] observed significant differences in the _E. coli_ counts obtained in PIF and HM maintained at 37 \({}^{\circ}\)C for 24 h at all the evaluated time points. In this case, the HM used was frozen at \(-20\)\({}^{\circ}\)C (no freezing time was indicated) but not pasteurized, which, as shown above, seems to affect the HM antimicrobial capacity. In summary, in both the treated HM and PIF, the results obtained show that differences in _E. coli_ counts are obtained before 4 h. Consequently, new studies are needed to evaluate microbial growth in milk under NICU administration conditions, considering different microbial loads and with other microorganisms of interest in this food, such as _C. sakazaki_. This bacterium has posed an emerging danger in recent years in the feeding of infants, both in PIF and improperly handled HM [38; 39]. The thawing process is also an important phase of HM processing before neonate administration, as it provides another opportunity for contamination or microbial growth. The obtained results showed that there were significant differences between the evaluated thawing methods, obtaining the smallest count increase for thawing at 4 \({}^{\circ}\)C for 12 h. Of the studied methods, it was the only one in which the milk remained at refrigeration temperatures that inhibit microbial growth throughout.