Changes of Milk Metabolomic Profiles Resulting from a Mycotoxins-Contaminated Corn Silage Intake by Dairy Cows

Abstract

In this study, an untargeted metabolomics approach based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used for investigating changes in chemical profiles of cow milk considering diets based on mycotoxins-contaminated corn silages. For this purpose, 45 milk samples were classified into five clusters according to the corn silage contamination profile, namely (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites, and subsequently analyzed by UHPLC-HRMS followed by a multivariate statistical analysis (both unsupervised and supervised statistical approaches). Overall, the milk metabolomic profile highlighted potential correlations between the quality of contaminated corn silages (as part of the total mixed ration) and milk composition. Metabolomics allowed to identify 628 significant milk metabolites as affected by the five levels of corn silage contamination considered, with amino acids and peptides showing the highest metabolite set enrichment (134 compounds). Additionally, 78 metabolites were selected as the best discriminant of the prediction model built, possessing a variable importance in projection score >1.2. The average Log Fold-Change variations of the discriminant metabolites provided evidence that sphingolipids, together with purine and pyrimidine-derived metabolites were the most affected chemical classes. Also, metabolomics revealed a significant accumulation of oxidized glutathione in milk samples belonging to the silage cluster contaminated by emerging Aspergillus toxins, likely involved in the oxidative imbalance. These preliminary findings provide new insights into the potential role of milk metabolomics to provide chemical indicators of mycotoxins-contaminated corn silage feeding systems.

Screening of Regulated and Emerging Mycotoxins in Bulk Milk Samples by High-Resolution Mass Spectrometry

“…Additionally, cluster 5 (i.e., high levels of fumonisins and their metabolites) showed the most differential and exclusive profile. The heat map reported in Figure 2 allowed us to observe an indirect correlation between the contamination profile of corn silages and milk samples, as also reported in our previous work evaluating the global changes of the major chemical classes because of the contaminated feeding systems [16]. Interestingly, cluster 5 was outlined as the most discriminant also in our previous work, thus highlighting the potential of high levels of fumonisins and their metabolites in the corn silages to potentially drive chemical differences in cow milk.…”

“…Samples of bulk tank milk (500 mL) ( n = 45) were taken in the period January–June 2018 from dairy farms located in the Po Valley (Italy). These latter farmed Holstein Friesian housed in free-stall barns without pasture access [ 16 ]. The herds were characterized by having on average 38.3% ± 1.9 of primiparous on lactating dairy cows and 2.4 ± 0.2 lactations before culling.…”

“…The lactating dairy cows were fed with the same corn silages, previously analyzed by Gallo et al [ 5 ], from at least four weeks, thus avoiding collecting milk in the period in which corn silage bunkers were changed. Additional information regarding herd composition, milk yield of lactating groups and milk quality, dry matter intake (DMI) as well as TMR formulation characteristics are reported in our previous published works [ 5 , 16 ]. The corn silages were grouped in five clusters according to the mycotoxin contamination profiles, as reported in our previous work [ 5 ], namely: cluster 1 (corn silages contaminated by low levels of both Aspergillus - and Penicillium -produced mycotoxins); cluster 2 (corn silages contaminated by low levels of fumonisins, and other Fusarium -produced mycotoxins); cluster 3 (corn silages contaminated by high levels of Aspergillus -mycotoxins); cluster 4 (corn silages contaminated by high levels of Fusarium -produced mycotoxins); cluster 5 (corn silages contaminated by high levels of fumonisins and their metabolites; number of samples: 3).…”

“…Starting from these background conditions, in this work, a high-resolution UHPLC-Orbitrap mass spectrometry approach was used to evaluate the mycotoxins profile of bulk milk samples collected from dairy farms using corn silages as the main ingredients of the total mixed ration (TMR) (i.e., 30.51 ± 5.84% on a dry matter, DM, basis) [ 16 ]. The corn silages were classified in five main clusters according to the mycotoxin contamination, as previously reported in Gallo et al [ 5 ].…”

“…The corn silages were classified in five main clusters according to the mycotoxin contamination, as previously reported in Gallo et al [ 5 ]. On the same milk samples, we have previously demonstrated the potential of untargeted metabolomics to evaluate the impact of contaminated corn silages on the most important chemical classes, thus providing evidence that sphingolipids, together with purine and pyrimidine-derived metabolites, are the most affected groups of metabolites [ 16 ]. Therefore, the aim of this work was to provide new insights into the ability of a retrospective screening based on HRMS to assess the mycotoxin profile of bulk milk samples, thus potentially evaluating food safety issues as related to both regulated and emerging toxins.…”