Development of donkey milk fermented with Lactococcus lactis subsp. lactis CECT 31096 and characterization of the...

Donkey milk (DM) is a nutritionally rich source of bioactive compounds, essential vitamins and minerals. However, its potential for developing functional fermented products remains unexplored. To address this gap, we investigated microbial dynamics during spontaneous fermentation of raw Zamorano-Leonese DM to isolate lactic acid bacteria (LAB) suitable for dairy applications. Raw milk from Zamorano-Leonese breed donkeys, raised on an organic farm in Salamanca (Spain), was spontaneously fermented at 37 °C until reaching pH 4.6. Metataxonomic analysis revealed a clear shift from environmental bacteria dominating the raw milk to an increased prevalence of LAB at the end of the fermentation process. Through culturomics, 34 bacterial strains were isolated and identified by 16S rRNA gene sequencing, including ten potential LAB candidates. Among these, five Lactococcus lactis isolates were selected to ferment pasteurized DM, which showed promising technological features and were subsequently subjected to whole-genome sequencing. L. lactis subsp. lactis CECT 31096 was selected due to its broad enzymatic repertoire linked to fermentation and the absence of antibiotic resistance or virulence-related genes. When used to ferment pasteurized DM, this strain improved both the physicochemical and nutritional profile of the resulting product. Fermentation significantly enhanced the bioaccesibility of bioactive compounds, as demonstrated by a higher inhibition of angiotensin-converting enzyme (the fermented sample showed IC₅₀ values of 3.17 g/L compared with IC₅₀ 7.23 g/L from pasteurized milk), doubling after digestion (IC₅₀ 1.25 g/L). Additionally, fermentation with L. lactis subsp. lactis CECT 31096 significantly increased soluble free phenols and peptides, boosting the reducing power and antioxidant capacity, and thereby enhancing the functionality of the final product.