## 3 Results and Discussion
### \({}^{1}h\) NMR Low Molecular Weight Water Soluble Metabolites of IF  
The \({}^{1}\)H NMR spectroscopy, within a single experiment, is able to detect all the molecular components bearing a hydrogen atom in a complex mixture, given a MW < 1 kDa and a concentration >1 uM. By peak integration procedures it is possible to obtain the concentrations of the identified metabolites expressed in moles. A representative \({}^{1}\)H NMR spectrum of IF1 is shown in Figure S1. In the IF1 and IF2 samples the lactose concentrations, as previously determined (Table 3), were used to calibrate the relative concentrations of the other metabolites, and the results are shown in Table 6.  
IF1 samples showed a higher overall content of low molecular weight metabolites than IF2. Besides lactose, citric acid was the most abundant metabolite in both formulations. Among organic acids, hippuric acid can be considered a biomarker of grass pasture diets [40]. No traces of lactic acid or benzoic acid were detected, which could suggest a good microbiological quality of the formulations. The IF1 samples showed a higher free amino acid content than the IF2 samples (Table 6). Amino acids in their free form are more easily absorbed than those present in proteins and may have a beneficial role during early post-natal development [41]. The following free amino acids were identified: proline, glutamic acid, lysine, leucine, isoleucine, alanine, valine, and threonine, together with the bioactive peptide anserine. The most represented amino acid in the two formulations was proline which shows neuroprotective properties [8]. No resonances ascribable to aromatic amino acids were found. The amino acid profile found in IF1 and IF2 samples is compatible with the presence in the whey from cheese making, used to obtain retenate, of the bioactive caseinomacropeptide (CMP), which is formed by chymosion hydrolysis of milk \(\kappa\)-casein (\(\kappa\)-cn) [42]. Several health beneficial effects of CMP have been reported [42]. In the \({}^{1}\)H NMR spectra of the analyzed samples, signals ascribable to nucleotides, nucleosides, and sugar nucleotides, such as UDP-galactose, UDP-glucose, Uridine, UDP-GlcNAc and UDP-GalNAc were detected [11]. These compounds belong to the non-protein-nitrogen (NPN) fraction of milk. Nucleotides are believed to be active in the maturation of the gastrointestinal tract and the development of neonatal immune function [1]. They are also assumed to play an important role in carbohydrate, lipid, protein, and nucleic acid metabolism [1]. Sugar nucleotides usually occur intracellularly and are utilised as donors for glycosyltransferases that biosynthesize glycogenjugates, but their biological functions in milk or colostrum for suckling neonates are unknown [43]. Although a heterogeneity of sugar nucleotides among breeds exists [43], it has been shown that ovine milk contained the highest concentrations of nucleosides and nucleotides compared to goat's and cows' milk [11]. Nucleotides play key roles in many biological processes and currently, the addition of 5\({}^{\prime}\)-CMP, 5\({}^{\prime}\)-UMP, 5\({}^{\prime}\)-AMP, 5\({}^{\prime}\)-GMP, 5\({}^{\prime}\)-IMP to infant formulae is suggested but