Antibacterial potential of donkey's milk disclosed by untargeted proteomics

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM. The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H₂O₂. κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins. Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SIGNIFICANCE: Donkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy. DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring. DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H₂O₂. In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits. Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population. Data of this study represent the largest inventory of proteins identified in Equidae milk, so far.