The chemical composition of donkey and sheep milk showed significant differences, with donkey milk containing less fat and protein and a higher level of lactose, while sheep milk had more dry matter and a higher proportion of casein and saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids. This study investigated the biological effects of in vitro-digested donkey and sheep milk, and their blends at different ratios, on differentiated human intestinal Caco-2 cells. Gene expression analysis showed that treatment with digested donkey milk (100%) significantly altered the expression of the autophagy-related gene (SQSTM1), tight junction (CLDN4), and innate defense genes (DEFB1, MUC2, and MUC5), showing a non-cytotoxic response and a profile consistent with a barrier-related transcriptional response. In contrast, sheep milk (100%) and certain milk blends (70% and 50% sheep’s milk) down-regulated genes involved in the autophagy process (ULK1, AMBRA, BECN1, ATG5, GABARAP, and SQSTM1), tight junction genes (OCLN and CDH1), and innate defense genes (DEFB1 and MUC2), suggesting a distinct epithelial response that warrants further confirmation. Metabolomic profiling revealed clear compositional and functional distinctions among the digested milks, identifying 166 metabolites that were differentially regulated (136 compounds were down-regulated and 30 up-regulated). Donkey milk digestion yielded a metabolite profile enriched in polar peptides and compounds with reported antioxidant associations, whereas sheep milk digestion showed a different pattern dominated by hydrophobic peptides and lipid-related metabolites. These findings indicate that, under the tested in vitro conditions, donkey milk digestion is associated with gene expression and metabolomic patterns consistent with epithelial barrier support.

Equid milk, specifically from donkeys (Equus asinus) and mares (Equus caballus), has garnered significant interest due to its unique nutritional and therapeutic properties. This study aimed to compare the chemical composition and microbiological quality of donkey and mare milk, focusing on their potential as functional foods and hypoallergenic alternatives for sensitive populations, such as infants with Cow Milk Protein Allergy (CMPA). Samples were collected from donkeys and mares at different lactation stages under semi-extensive farming conditions in Serbia. Donkey milk was characterized by a low fat content (0.3-1.8%), high lactose levels (6.0-7.4%), and rich concentrations of bioactive compounds such as lysozyme, lactoferrin, and lactoperoxidase. These bioactive components contribute to its antimicrobial properties and prolonged shelf life. The milk's low somatic cell count (SCC) and total bacterial count (TBC) further support its potential for therapeutic use and safety for CMPA patients. In contrast, mare milk had a higher fat content (1.5-2.0%), providing increased energy density. It also exhibited elevated levels of polyunsaturated fatty acids (omega-3 and omega-6), which support cardiovascular health, along with significant amounts of calcium and phosphorus beneficial for bone health. Despite its higher nutritional density, mare milk showed slight variability in protein and lactose content, likely influenced by breed, lactation stage, and feeding conditions. The microbiological quality of mare milk was also found to be high, with moderate SCC and TBC levels. Both donkey and mare milk possess valuable and complementary nutritional, functional, and therapeutic benefits. While donkey milk is particularly beneficial for immune protection and hypoallergenic uses, mare milk offers a broader range of health benefits, particularly for bone and cardiovascular health. The findings highlight the need for continued research to optimize milk production methods and ensure microbiological safety for these products' broader use in functional foods.