characterization of donkey milk proteins by a proteomic approach

The increased incidence of allergies to cow’s milk proteins (CMP) in newborns enhanced the production of infant formula based on either soy proteins and/or hydrolysates of CMP. These formula, although having a good nutritional value, can be affected by the presence of peptides with a size stimulating the immunological response in predisposed subjects and/or an unpleasant taste for the infant consumers. Recent clinical studies indicated that patients with CMP allergy and also intolerant to these formula, showed a high tolerance towards the donkey milk, due to its greater quantitative similarities to human milk than cow’s milk. Specifically, although the mechanism of this tolerance has not yet been clarified, it is reasonable to hypothesize that the reduced allergenic properties of donkey milk can be related to structural differences of its protein components with respect to cow’s milk. At present, compared with cow’s milk, the characterization of donkey milk proteins (whey proteins and mainly caseins) is at a relatively early stage of progress, and only limited data are related to their heterogeneity. In this PhD thesis the complexity and specifically the qualitative and quantitative heterogeneity of donkey milk proteins of 77 animals reared in Italy was investigated using a proteomic approach. This analytical procedure for structural analysis of protein fractions is based on the combination of highly efficient separation techniques, such as one-dimensional electrophoretic techniques (PAGE pH 8.6, UTLIEF, PAGE-SDS) or, more efficiently, the two dimensional (2-DE) electrophoresis, coupled either to Coomassie Brilliant Blue staining or immunospecific staining with polyclonal antibodies, to chromatography analysis (RP-HPLC), interfaced to structural Mass Spectrometry analysis (LC/ESI/MS, MALDI/TOF/MS, ESI q-TOF/MS). For donkey caseins, these combined methodologies allowed the contemporary identification of the four casein fractions (αs1-, αs2-, β- and κ-CN) in donkey milk together with their related heterogeneity due to post-translational phenomena such as the different phosphorylation degree of the caseins (αs1-, αs2-, β-CN) and the high glycosylation level of κ-CN, incorrect splicing of primary transcript in mRNA (non allelic deleted forms of αs1-, αs2-, β-CN) and genetic polymorphism of αs1- and β-CN (one or more variants in individual samples). At this regard, for the first time the primary structure of a new αs1-CN variant was determined, as well as and the amino acid sequences of three new genetic variants of β-CN (B, C, D) were characterized, using the known common αs1- and β-CN phenotype from donkey as reference, respectively. From a quantitative analysis, β-CN was always present in all analyzed donkey milk samples, as in human milk, representing the most abundant casein fraction followed by αs-CN complex. The αs1-CN showed an high variability in its expression level (0.2-2 g/L), confirming a quantitative polymorphism at this locus as in goat milk. The minor expression of αs1-CN together with the absence of αs2-CN in some donkey milk samples, as in the human milk, confirms the compositional similarity of two milks and represent a scientific basis for donkey milk’s use in nutrition of infants with CMP allergy. The screening carried out on the individual whey protein samples by the proteomic approach revealed a monomorphism either for α-La (A) and for β-Lg I (B). β-Lg II was instead polymorphic, for the occurrence of four variants (A, B, C, D) already known in literature and for the identification of a new variant E. The primary structure of β-Lg II E was determined and differs from β-Lg II D for D Asp2/E Asn2, D Arg18/E Lys18, D Val25/E Ala25 amino acid substitutions with a Mr18256 Da. Moreover a quantitative polymorphism seemed to affect the donkey β-Lg II locus since different phenotypes, with a relative quantitative percentage respect to total whey protein ranging from 0% to 3.34%, were detected by RP-HPLC. This quali-quantitative polymorphism at donkey β-Lg II locus seems to affect donkey milk composition and therefore, its allergenic properties. Moreover, the achieved results also highlighted that all examined samples have a high lysozyme content (2 g/L) which together with lactoferrin is important from an immunological point of view and for the shelf life of the milk. Finally, the donkey milk samples were shown to be poor in the total proteins (1.48%) as human milk, and specifically they were characterized by a lower casein content (35-40%) with allergenic properties and a higher whey protein content (50-60%) with biological activities, compared to bovine milk, with a CN/WP ratio <1. However, in analyzed donkey milk samples, an individual variability either of each casein or whey protein amounts suggests further studies in this direction aimed at a selection of animals producing milk with a composition “qualitatively and quantitatively” more similar to human milk, and “functionalized” for infant feeding.