3.1. Human neonates
Importantly, it has been demonstrated that hLf benefits the nascent gut health and immune development and functioning in preterm and neonate infants. These effects are due to Lf favors the decrease of its permeability and increase of its maturation 
. The impact of Lf on the colonization of intestinal microbiota has also been addressed in neonate populations, since ongoing intestinal maturation puts them at risk for life-threatening diseases, including necrotizing enterocolitis 
. Buccigrossi et al. (2007) found that both hLf and bLf exert a mucosal trophic effect on enterocytes (Caco2 cells) that is related to its concentration; at high Lf concentrations, it was promoted a more rapid proliferation of these cells, whereas at low Lf concentration it was induced their differentiation 
The role of Lf on the colonization of microbiota has been addressed during the early stages of intestinal maturation in neonates fed maternal milk and an infant formula diet 
. In early trials, a relationship between the bLf contained in an infant formula diet and the fecal microbiota in neonates was not seen 
. In current studies, the abundance of fecal bifidobacteria and lactobacilli was significantly associated with the levels of Lf in feces from breastfed newborns 
. These discoveries suggest that hLf levels in neonates are beneficial for contributing to the establishment of the gut microbiota.
Regarding the microbiota composition and its relation with Lf intake, Mastromarino et al. (2014) measured the content of Lf and the microbiota of breast milk and of feces of infants at birth and one month after delivery 
. Interestingly, in preterm infants, higher concentrations of fecal Lf at birth and 30 days after delivery were observed than in full-term infants; also, the amount of fecal bifidobacteria and lactobacilli were significantly associated with the concentration of fecal Lf. These results suggested that Lf promotes a bifidogenic microflora in the gut in neonate and preterm infants. High levels of fecal Lf in in the first days of life contribute to a strong early host-microbe interaction that could be important for the composition of the neonatal gut microbiota and the development of these microorganisms, in addition to the antimicrobial activity of this milk glycoprotein. This interaction is critical for having a healthy immune system and a correct metabolic program in newborns.
A clinical trial in VLBW (<1500 g) babies tested the effect of a rrhLf, administered at 150 mg/kg/day every 12 h by a nasogastric route from day one to 28 of life. Proteobacteria
were the major Phyla
in feces from babies treated or untreated (placebo) with rhLf. It should be noted that the fecal abundance of the pathogenic species Enterobacter
was decreased, while Citrobacter
abundance was increased, but it was not associated with infections 
. In breastfed babies, the abundance of fecal bifidobacteria was predominant, and that of the facultative anaerobes was poor; by contrast, in babies fed formula containing bLf (10 or 100 mg/ml), obligate anaerobes (Clostridium
) were predominantly seen in regard to bifidobacteria 
. A delicate balance of microbiota members is prone to the disturbance in preterm infants 
; thus, findings indicate that the effect of bLf dosage on promoting anaerobic growth must be kept in mind to prevent potential risks in dysbiosis in newborns.
In a retrospective cohort study in preterm babies fed bLf in combination with LGG, the incidence of severe necrotizing colitis was significantly reduced, and the resolution of this disease was improved. Notably, bLf had no collateral effects, but a severe case of sepsis by LGG was found 
. These results indicate that due to the extreme fragility of very low birth weight neonates, stringent precautions should be taken to avoid the risk of severe cases of neonatal sepsis.
Currently, several recommendations of these therapies using Lf and probiotics against NEC and other gut diseases have been addressed. For example, the therapy with Lf must be as early in the life as possible, more than 100 mg/day is the recommended dosage, Lf is apparently more effective in preterm than in term infants, and the efficacy versus Gram-negative bacteria could be limited. Important gaps in the knowledge exist concerning dosages, schedule, duration of treatment, most effective probiotic strains, and interactions of probiotics with human and bovine milk 
. As stated below, experimental findings in preterm piglets support these claims 
3.2. Piglet Model
Given their physiological and anatomical resemblance to humans, piglets have been used in experimental studies as a model for the neonatal gastrointestinal tract to provide insights about the presumable mechanisms underlying the role of Lf and its derivatives on intestinal homeostasis in neonates 
A recombinant Lf fusion peptide consisting of Lfcin and lactoferrampin (Lfampin) expressed in Pichia pastoris
has been shown to exhibit potent probiotic effects on bifidobacteria and lactobacilli throughout the gastrointestinal tract in weaned piglets 
. Additional trials in healthy full-term piglets demonstrated that bLf, in combination with probiotics increased the richness of microbiota in the small and large intestine. Interestingly, bLf reversed the effects of probiotics on the increase or decrease of ferric or ferrous iron transport system abundance, respectively 
. Thus, the iron-binding ability of bLf on ferric ions seems to affect the role of probiotics on microbiota modulation.
Piglet trials documented the substantive effects of bLf or recombinant hLf on stimulating the abundance of a wide array of microbiota members and on improving body weight gain 
. Recombinant Lfcin-Lfampin expressed in Photorhabdus luminescens
also enhanced the growth of bifidobacteria and lactobacilli and body mass gain 
. Glycan degradation results in the formation of SFCA, which in turn are a source of energy for the epithelial cells necessary for ongoing gut maturation 
. Thus, underlying mechanisms seem to be associated with the ability of Lf or its derivatives to favor the diversity of microbiota members with a pivotal role in breakdown glycans.
The microbiome has a critical role in intestinal maturation by providing signals for the development of innervations of the enteric nervous system connected in turn with the central nervous system (CNS); conversely, CNS and enteric nerves modulate intestinal maturation via microbiome signaling pathways 
. Notably, the effect of bLf on the abundance and diversity of microbiota in piglets seems to affect the intestinal expression of neurotransmitters such as ileal vasoactive intestinal peptide released by enteric nerve fibers 
and the expression of parameters associated with the maturation of enteric nerves such as brain-derived neurotrophic factor (BDNF) and ubiquitin carboxy-terminal hydrolase 1 [ubiquitin thiol esterase (UCHL1)] 
. These results suggest a presumable mechanism for the role of bLf on the interplay between the gut-brain axis and the microbiome, resulting in the maturation of enteric nerve fibers.
The impact of bLf on intestinal maturation has also been demonstrated with the increased activity of brush-border enzymes, such as jejunal lactase, as found in piglets fed formula containing probiotics and bioactive milk components, including bLf 
. The intestinal alkaline phosphatase activity was enhanced in piglets fed a sow milk replacement containing bLf 
. Benefits on epithelial architecture have been evidenced in the small intestine by larger crypt area, depth and width and thinner lamina propria, as found in piglets fed bLf, recombinant hLf or Lfcin-Lfampim from P. luminescens 
. bLf also enhanced jejune crypt proliferation, depth, area, and the crypt mRNA expression of β-catenin mRNA, as documented in colostrum-deprived piglets fed formula containing bLf 
. The upregulation of bLf on intestinal growth may result from the elicitation of the β-catenin-Wnt signaling pathway 
. β-Catenin mRNA encodes a cytosolic protein expressed at crypts that is regarded as a key effector of Wnt signaling; the latter drives the self-renewal and proliferation of stem cells and their concomitant differentiation to other cell components of the epithelial monolayer 
. The findings provide evidence that supports the role of Lf in growth and maturation in the neonatal intestine.
Maternal bLf supplementation increased the pregnancy rate, litter size, and survival, and the levels of IgA antibodies in the serum of gilts and their litter 
. These findings indicate that bLf consumption provided benefits on survival and immunity during pregnancy and lactation. In piglets fed daily for the first seven or 14 days of life with a formula containing bLf (367 or 1300 mg/kg body weight), the serum IgG antibodies increased; however, bLf did not affect the cellularity of the lymphoid populations (B cells and T cells), NKCs and neutrophils. In supernatants from lipopolysaccharide (LPS)-primed mesenteric lymph node cell cultures treated with bLf at 367 mg/kg of body mass, the IL-6 and IL-10 levels were enhanced, whereas the tumor necrosis factor (TNF)-α level was unaffected 
. The consumption of transgenic milk containing hLf had significant effects on the decrease in circulating neutrophils and the increase in lymphocytes without affecting cytokine expression 
, whereas in piglets fed transgenic milk containing recombinant human lysozyme and rhLf, the number of peripheral blood cells was increased without affecting the expression of TNF-α, IL-6, TGF-β and TLR4 
. Accordingly, these results indicate that natural or recombinant Lf products display a tendency to either not affect or downmodulate the markers of inflammation.
By contrast, piglets fed the chimera Lfcin-Lfampin showed enhanced serum antioxidant enzymes such as glutathione peroxidase and peroxidase, as well as the effectors of the adaptive immunity branch including IgA, IgG, and IgM antibodies and the components of the innate response involved in protection from the deleterious effects of inflammation . This finding agrees with the observation that rhLf-cow milk increased TLR-2 mRNA expression in the ileum and the levels of colonic IgG and nuclear factor-κB (NF-κB) p65, concomitant with the increase in spleen IL-2, -4, and -5 and plasmatic IgG, IgA and IL-12, and IL-10 . Although the assessment of parameters was systemic, the compartmental modulatory action on inflammatory and immune components underlies the protective role of recombinant Lf derivatives in the intestinal milieu. According to the above results, some presumable mechanisms that account for the impact of Lf on intestinal microbiota growth are depicted in Figure 2.
Figure 2. The impact of Lf on the promotion of the growth and diversity of intestinal microbiota may entail (i) strengthening of the permeability of the epithelial cell monolayer; (ii) favoring of the microbial antagonism that discourages the colonization and proliferation of enteric pathogens, enhancing the growth and maturation of (iii) cell-monolayer components and (iv) gut nerve fibers; and (v) providing signals to balance the anti- and proinflammatory responses resulting in homeostasis.