Spontaneously fermented rennet-coagulated whey from milk of different ruminants: from microbiota characterisation to isolation of lactic acid bacteria with proteolytic potential.

Cheese whey is a highly nutritious by-product of cheesemaking, whose microbiota has been insufficiently studied, particularly in spontaneous fermentation systems. This study aimed to characterize the microbiota of rennet-coagulated whey obtained from raw cow, goat and sheep milks, collected from artisanal farms differing in altitude and sampling season, and to explore spontaneous fermentation as a strategy to recover bacteria with technological potential for whey valorisation. Culturomic and metataxonomic approaches were combined, and spontaneous fermentations at different temperatures were performed to promote the selection of distinct bacterial populations. In addition, metataxonomic analysis revealed that the microbiota composition of basal whey varied according to animal source, farm location and season of the year, although some patterns, particularly those involving sheep whey, should be interpreted cautiously. Nevertheless, members of the genera Lactococcus, Streptococcus, Enterococcus, Leuconostoc, Staphylococcus and Pseudomonas were commonly detected. Spontaneous fermentation promoted marked microbial shifts depending on incubation temperature and was associated with increased proteolytic activity in several whey samples. Correlation analyses showed positive associations between specific lactic acid bacteria (LAB), particularly Lactococcus and Streptococcus, and high OPA values, although these associations do not establish a causal contribution to whey proteolysis. A total of 420 bacterial isolates were recovered, of which 161 were preliminarily screened for proteolytic activity in milk. Seven LAB isolates showing high proteolytic potential were selected for genome sequencing and preliminary safety characterization. Overall, spontaneously fermented whey represents a valuable source of LAB with promising technological properties and potential application in whey valorisation.