## 3 Results and Discussion
### Analysis of Bacterial Diversity  
Relative abundance of bacterial communities at phylum, genus, and species level are shown in Figure 2. A total of five identified phyla were found in fresh milk, being _Proteobacteria_ dominant (about 70%), followed by _Bacteroidetes_ (less than 20%), and to a lesser extent by _Firmicutes_, _Actinobacteria_, and _Verrucomicrobia_ (Figure 2A). In a recent work, the bacterial composition of donkey milk from different farms was analysed by high-throughput sequencing, revealing the occurrence of the same phyla [16]. These authors found a higher concentration of _Proteobacteria_ (more than 85%) and a lower composition in _Bacteroidetes_ (less than 1%) in almost all samples [16], suggesting that the microbial milk composition could be strictly related to the different breeding conditions. In the present work, a high incidence of Gram-negative psychrotrophic bacterial population was found in fresh milk, with _Pseudomonas_ spp. being the most representative (more than 45%). The remaining bacteria were associated with species belonging to the genera _Chryseobacterium_, _Stenotrophomonas_, _Splingobacterium_, _Citrobacter_, and _Delftia_, and to a lesser extent by _Azospirillum_, _Massilia_, and _Serratia_ (Figure 2B). These results partially agree with the previous metagenomic analysis, which reported a dominance of _Pseudomonas_ spp., and the occurrence at different levels of further Gram-negative bacteria belonging to the genera _Ralstonia_, _Acinetobacter_, and _Cupriavidus_[16]. Figure 1: Rarefaction curves of bacterial population at genera taxonomic level for fresh (**A**) and cold stored (**B**) milk samples. Venn diagram (**C**) of the number of unique OTUs in fresh (M_0/yellow) and cold stored milk (M_3/blue). Shared OTUs are in red. Figure 2: Cont. It is widely reported that Gram-negative psychotrophic bacteria in raw milk are of special concern to the dairy industry because they can produce extracellular enzymes, mainly proteases and lipases, that contribute to the spoilage of dairy products [27,28]. Worldwide, species of _Pseudomonas_ are the most common contaminants isolated from cold raw milk [16], while other genera, including _Chryseobacterium_, _Citrobacter_, and _Serratia_, have been reported as critical post-pasteurization spoilers [29, 30, 31]. Although Gram-negative bacteria are frequently identified in the milk of healthy animals, their abundance could be linked to mastitis and poor health conditions of the livestock, thus reducing the quality of raw milk [32,33]. However, _Salmonella_ and _Staphylococcus aureus_, the main etiological agents for mastitis, were not found in our samples, and neither were the main foodborne pathogens in milk and the dairy environment (i.e., _Camyylobacter jejuni_, Shiga-toxin producing _Escherichia coli_, _Listeria monocytogenes_, _Yersinia enterocolitica_, and _Salmonella_ spp.). Nonetheless, the genera detected in fresh donkey milk included potential pathogenic species. For example, _Pseudomonas putida_, _Stenotrophomonas multiophilia_, and _Citrobacter braakii_, which together accounted for about 15% of the microbial population, are classified as risk group 2 biological agents, and reported as opportunistic human pathogens [34, 35, 36]. Furthermore, it is interesting to underline that in our samples we detected a level of _Serratia_ spp. lower than other genera rarely documented in milk or dairy ecosystems. Besides, _Sphingobacterium_ spp. has been isolated from different habitats, including raw cow and yak milk [37, 38, 39]. _Massilia_ spp. was found in goat milk and fermented yak milk [12,40], while _Azospirillum_ was only recently described as an inhabitant of fermented bovine products [41]. Concerning the protechnological microbes, in our samples of fresh milk, we detected reads for the lactic acid bacteria (LAB) lower than 5%, with _Lactococcus_ spp. (2.7%) being the most abundant, followed by species of the genera _Streptococcus_ spp. (1%), and _Enterococcus_ spp. (0.5%) (Figure 2B).