## 3 Results and Discussion
### Analysis of Bacterial Diversity
These results are consistent with those reported in donkey [14,16], and goat milk [12,42] but significantly lower than the LAB composition reported in conventional cow milk. Indeed, _Streptococcus_, mainly belonging to _Lactococcus_ spp., and to a minor extent to _Streptococcus_ spp., was the most represented population  
Figure 2: Relative abundance of bacteria communities at phylum (**A**), genus (**B**), and species (**C**) level in fresh (M_0) and three-days cold stored (M_3) donkey milk. Phyla and genera occurring at \(<\)0.5% abundance, and species occurring at \(<\)1% are defined as “Others”. (about 57%) in raw cow milk [43]. However, different frequencies of LAB could be attributable to several factors, including diet, environment, season, and health status, as well as the method employed for identification [42]. Raw milk is often refrigerated for up to three or four days until it is processed. A recent review reported, in association with this matrix, the presence of _Bacillus_, _Clostidium_, _Corynebacterium_, _Microoccus_, _Streptococcus_, _Staphylococcus_, _Microbacterium_, _Lactococcus_, and _Lactobacillus_ (Gram-positive), and _Pseudomonas_, _Aerononas_, _Alcaligenes_, _Achromobacter_, _Acinetobacter_, _Flavobacterium_, _Chryseobacterium_, and _Enterobacteriaceae_ (Gram-negative) [31]. Therefore, we analysed the same samples after three days of storage at refrigeration temperature. In these samples, the bacterial biodiversity of donkey milk was strongly reduced, since about 93% of the bacterial communities were identified as different species of psychotrophic _Pseudomonas_. In particular, _Pseudomonas korensis_ was found to be the dominant species, increasing its level from 17% in fresh milk to 61% after storage. In this last sample, _Pseudomonas reinekei_, _Pseudomonas parlerniana_, and _Pseudomonas japonica_ were found at a concentration of about 8%. All the other Gram-negative genera decreased to about 4% of the total bacterial communities (Figure 2B,C). Microbiological analysis using culture-dependent methods showed a slight increase of the total aerobic microorganisms from \(3.1\times 10^{4}\) cfu mL\({}^{-1}\) to \(3.9\times 10^{4}\) cfu mL\({}^{-1}\). These results are in agreement with those found by [PERSON] et al. [44] in donkey milk samples after 4 days of storage at 4 \({}^{\circ}\)C. Thus, the observed reduction of some taxonomic groups during cold storage could reflect a higher capability of certain _Pseudomonas_ spp. to grow under refrigeration conditions, more than a reduction of viable cells. It is well known that during cold storage of non-pasteurized milk, psychotrophic microorganisms are the dominant microflora [45]. In particular, _Pseudomonas_ spp. was found as the predominant microbiota in raw milk samples after a few days of refrigeration [38; 46; 47]. Moreover, although different treatments for the microbiological control could differently modulate the dominant bacterial populations in milk, cold-tolerant bacteria, such as _Pseudomonas_, _Stentroplomonas_, and _Delftia_ were persistent after submitting milk to different treatments for the microbial control [48]. The spoilage potential of psychotrophic bacteria isolated from raw milk could be increased by the thermo-stability of their proteolytic and lipolytic enzymes [28], and by the ability to produce volatile organic compounds responsible of undesirable aromatic attributes [49]. Therefore, the metabolic activity of psychotrophic bacteria results in a variety of defects that negatively affect the suitability of such milk for further processing. However, the low casein content of donkey milk does not allow caseification, thus reducing its processing availability. Nonetheless, in the last few years, donkey milk has been proposed to produce functional fermented beverages [5], and technological attempts at producing cheese from donkey milk have been suggested with the addition of bovine chymosin, camel chymosin, goat milk, or calf rennet [6; 50; 51; 52].