Artisanal cheeses are known as the source of beneficial lactic acid bacteria (LAB). Therefore, this study aimed to isolate and characterize LAB with different proteolytic activities from Iranian artisanal white cheeses. The isolates were classified into low, medium, and high proteolytic activity clusters via K-means clustering and identified as Lactiplantibacillus (Lpb.) pentosus L11, Lpb. plantarum L33, and Enterococcus faecium L13, respectively. Some safety tests (such as resistance to antibiotics, hemolytic activity, and biogenic amine production), probiotic properties (including cell surface hydrophobicity, auto/co-aggregation, and antibacterial activity), and production of volatile compounds were evaluated. These were non-hemolytic and non-biogenic amine producers, and showed no irregular antibiotic resistance. Lpb. plantarum L33 had the highest hydrophobicity (30.55%) and auto-aggregation (49.56%), and the highest co-aggregation was observed for Lpb. pentosus L11 with Staphylococcus aureus (61.51%). The isolates also showed a remarkable antibacterial effect against pathogenic bacteria. Moreover, Lpb. pentosus L11 and Lpb. plantarum L33 with low and medium proteolytic activity produced a wider range of volatile compounds in milk compared to the strain with a high proteolytic effect. The results showed that a probiotic strain with low or medium proteolytic activity could improve the flavor characteristics of fermented milk.

In this study, single and co-cultures of proteolytic Lactobacillus delberueckii subsp. bulgaricus ORT2, Limosilactobacillus reuteri SRM2 and Lactococcus lactis subsp. lactis BRM3 isolated from different raw milk samples were applied as starter cultures to manufacture functional fermented milks. Peptide extracts from fermented milk samples were evaluated after fermentation and 7 days of cold storage for proteolytic, angiotensin-converting enzyme (ACE) inhibitory and antioxidant activity by different methods including 2, 2′-diphenyl-1-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP), OH-radical scavenging, and total antioxidant (molybdate-reducing activity). The highest proteolysis was found in milk fermented by co-cultures of three strains. Fermentation with the mentioned bacteria increased ACE inhibitory and antioxidant activity of the final products which were dependent on peptide concentration. The crude peptide extract obtained from fermented milk with triple co-culture showed the highest ACE inhibitory activity (IC₅₀ = 0.61 mg/mL) which was reduced after 7 days of cold storage (IC₅₀ = 0.78 mg/mL). Similar concentration-dependent activities were found in antioxidant activity at different antioxidant assays. Overall, high proteolytic activity resulted in increased ACE inhibitory and antioxidant activities, but the highest activity was not necessarily found for the samples with the highest proteolytic activity. The results of this study suggest the potential of using co-cultures of L. delberueckii subsp. bulgaricus, L. reuteri and L. lactis subsp. Lactis to manufacture antihypertensive fermented milk.