Investigating the potential of proteolytic Enterococcus faecium strains to produce probiotic whey beverage with improved aromatic attributes

• Cheese whey repurposed into probiotic beverages, minimizing dairy waste. • E. faecium strains exhibit no hemolytic activity or risks of biogenic amines. • A fermentation potential of 8.27 log CFU/ml confirms strong activity. • Acetaldehyde and diacetyl enhance the aroma of the fermented whey drink. The dairy industry generates significant waste like cheese whey, which contains 55% of milk’s nutrients and 20% of its proteins, making it suitable for fermented probiotic beverages. This study aimed to isolate proteolytic lactic acid bacteria from traditional cheeses and assess their probiotic properties for use in whey-based beverage with desirable flavor. From 21 isolates, three E. faecium strains (HBUAS70116 (Co1), VRE111 (Co6), AALTL (B4)) with the highest proteolytic activity were chosen. They were assessed for safety (antibiotic resistance, hemolysis, biogenic amines, and virulence gens) and probiotic properties (hydrophobicity, auto/co-aggregation, acid/bile salt tolerance, and antibacterial activity). The strains showed no hemolysis or biogenic amine production, had acceptable antibiotic resistance, and lacked the esp and ace virulence genes. Hydrophobicity was 55.98% (Co6), 76.44% (Co1), and 83.99% (B4). Auto-aggregation was 6.88% (B4), 15.98% (Co1), and 22.55% (Co6). Their inhibition zones against pathogens ranged from 61.71 mm (B4 vs. S. thyphimurim ) to 77.53 mm (Co6 vs. S. aureus ). In beverage production, fermentation for 24 h decreased pH from 6.24 to 5.25, and increased acidity from 0.12% to 1.12% (Co6). Viable cell count reached 8.33 log CFU/m (Co1), and B4 was a significant rise in the content of free amino groups. Some flavor compounds included acetaldehyde (Co6: 2.15 ppm), diacetyl (B4: 29.54 ppm), and acetic acid (B4: 23.71 ppm). The selected strains demonstrate strong potential as starter cultures for producing flavorful, probiotic whey beverages.