Development of analytical methods for phenotypic characterization of antioxidant compounds and antioxidant activity of milk

Free radicals are unstable and reactive molecules, with one ore more unpaired electrons in the outer orbit and adverse effects on plant, animal, and human cells. In particular free radicals are responsible of lipids peroxidation, proteins oxidative damages, and DNA cleavage, resulting in increased risk of mutation. Antioxidants represent an important defence against these injuries. Vegetable derived foods are known as important sources of dietary antioxidants such as phenols, anthocyanin, tocopherols, tocotrienols, carotenoids, retinol precursors, and ascorbate. With this background, the aims of this Ph.D. project were: i) to develop and validate analytical methods for quantification of tocopherols, thiols, and total antioxidant activity (TAA) of milk; ii) to describe the phenotypic variation of thiols in milk of dairy and dual-purpose cow breeds; iii) to describe the phenotypic variation of TAA in milk of cow, buffalo, goat, and sheep; iv) to evaluate the feasibility of mid infrared spectroscopy to predict TAA of cow milk; v) to evaluate the effect of skimming and heat treatments on milk tocopherols and TAA. In the 1st Chapter, a simplified saponification protocol, followed by cheap HPLC method based on methanol elution and sensible fluorescence detection was developed and validated for the quantification of α-tocopherol and γ-tocopherol, in different types of commercial fluid milk. Chromatograms showed two analytical peaks, corresponding to α-T and γ-T. The method was able to detect the adverse effects of skimming and UHT treatment on α-T and γ-T concentrations. The proposed method could be usefully adopted in future for large scale studies, aiming to investigate phenotypic variation of tocopherols in milk. The 2nd Chapter represent a first contribution to the characterization of low molecular weight thiols in milk of different cattle breeds. Thiols were extracted from the soluble fraction of milk, and following a derivatization protocol they were separated by reverse phase HPLC and detected fluorimetrically. Six thiol species were detected and two of them, glutathione (GSH) and cysteine-glycine (Cys-Gly), were identified and quantified. The average concentration of Cys-Gly in milk was greater than that of GSH, and milk from dual-purpose breeds was richer in thiols than milk from dairy cows. The 3rd Chapter deals with the development and the validation of a robust and fast spectrophotometric method for the determination of TAA of different types of milk. The method was linear, and highly repeatable and reproducible. Preservative added on raw milk had negligible effects on TAA measurement. The greatest TAA was measured on skimmed pasteurised milk, followed by skimmed UHT milk, raw milk, whole pasteurised milk, and whole UHT milk. The 4th Chapter is the first contribution to the phenotypic characterization of TAA of bovine milk. This phenotype exhibited an exploitable variability, similar to that of other quality traits. Favourable phenotypic correlations of TAA with fat, protein, and casein percentages were observed, as well as with somatic cell score. Total antioxidant activity of milk increased across lactation. Mid-infrared prediction models developed to predict milk TAA were not enough accurate for analytical purposes. The 5th Chapter deals with the phenotypic characterization of TAA of buffalo, goat, and sheep milk. Sheep milk showed the greatest TAA. This is probably due to its relatively high content in fat, protein, and casein percentages, that are known as compounds contributing to milk antioxidant capacity. Accordingly, buffalo and goat milk had lower TAA as well as lower fat, protein and casein percentages. Milk TAA was unfavourably correlated with milk yield, but the relationship was significant only for buffalo, whereas protein and casein percentages were positively correlated with TAA of goat milk.

I radicali liberi sono molecole reattive e instabili, con uno o più elettroni spaiati nell'orbitale esterno, con possibili effetti negative a livello di cellule vegetali e animali. In particolare, i radicali liberi sono responsabili della perossidazione dei lipidi, dell'ossidazione delle proteine e del danneggiamento del DNS, che causa un aumento del rischio di mutazioni. Gli antiossidanti rappresentano un'importante difesa contro questi danni. Gli alimenti di origine vegetale sono noti come risorse primarie di antiossidanti, come i fenoli, gli antociani, i tocoferoli, i carotenoidi, i precursori del retinolo e l'ascorbato. In questo contesto, gli scopi della presente tesi di dottorato sono: i) lo sviluppo e la validazione di metodi analitici per la quantificazione dei tocoferoli, dei tioli e della capacità antiossidante totale nel latte; ii) la descrizione della variazione fenotipica dei tioli in vacche da latte e a duplice attitudine; iii) la descrizione della variabilità della capacità antiossidante totale (TAA) nel latte di vacca, di bufala, pecora e capra; iv) la valutazione della capacità del medio infrarosso nella predizione della TAA del latte vaccino; v) la valutazione dell'effetto della scrematura e del trattamento al calore sulla concentrazione in tocoferoli e sulla TAA.