Investigation on donkey milk protein fractions: in vitro antimicrobial, antiviral and anti-proliferative activities and casein separation by cation exchange chromatography

Abstract

This thesis was aimed to study donkey milk protein fractions and their biological properties. In the first Chapter the donkey¡¯s milk gross composition and the related hygienic-sanitary aspects are introduced. The main use of donkey milk in human nutrition, especially in infancy and in patients with cow milk protein allergy (CMPA), is also discussed. The second Chapter ¡°Antimicrobial compounds in milk¡± was drafted after an intensive course at the University of Copenhagen: ¡°Functional milk compound ¨C with focus on milk proteins¡±, and presented as the final report of the first year of PhD school. It deals with a review of the antimicrobial activity of milk components, with particular attention to whey proteins, as lactoferrin and lysozyme and their derived bioactive peptides, such as lactoferricin, which play a crucial role in human health and nutrition. The detailed description of the donkey whey proteins, which exhibit antimicrobial activity is presented in third chapter. Moreover, the antimicrobial activity of fresh, powdered, digested and fermented donkey milk has been discussed. The Fourth Chapter reports the studies on the antiviral activity of donkey milk and its fractions (skimmed milk, digested milk, casein, whey and low molecular weight whey fraction) that has been tested in vitro on an enterovirus, echovirus type 5, in two experimental trials. Among the others, we found that whey proteins showed the strongest inhibitory effects, probably thanks to the synergic actions of lactoferrin, lysozyme and lactoperoxidase. A preliminary investigation of anti-proliferative effect of skimmed donkey milk and its main protein fractions (casein and whey proteins), carried out through two assay performed in vitro on Human Neuroblastoma Cell Lines (SK-N-BE) is presented in the fifth Chapter. In our experimental conditions only the whey proteins, at the highest concentration, have shown anti-proliferative effect on cell growth. Finally, in the sixth Chapter, the preliminary results obtained from the analysis of the donkey¡¯s casein fraction by ion exchange chromatography, are described. By coupling chromatographic and mass spectrometry techniques, we obtained three different fractions from the separation of donkey¡¯s casein: pure ¦Â-casein, ¦Ás-caseins and a pool containing ¦Ê-casein and ¦Ás-caseins. The survey was carried out with the aim to obtain protein standards to use in quantification analysis.