Isolation, Characterization, and Probiotic Evaluation of Lactic Acid Bacteria from Traditional Non-Dairy Fermented Beverages in Kano, Nigeria

Abstract

Non-dairy fermented beverages represent a largely untapped reservoir of functionally diverse lactic acid bacteria (LAB) with considerable probiotic potential. The growing global demand for plant-based probiotic alternatives, driven by rising rates of lactose intolerance, dyslipidemia, and veganism, underscores the importance of exploring indigenous fermented beverages as novel probiotic sources. A total of six samples comprising zobo (Hibiscus sabdariffa beverage), ginger drink, and kunun-zaki (fermented millet beverage) were collected from open markets in Kano Metropolis, Nigeria. Physicochemical parameters including pH, temperature, and titratable acidity (TTA) were determined. LAB were isolated using de Man, Rogosa, and Sharpe (MRS) agar under anaerobic conditions, and presumptively grouped using colony morphology, Gram staining, spore staining, and catalase testing; these phenotypic tests can only confirm broad membership of the lactic acid bacteria group and cannot reliably resolve genus, so any genus-level names applied below are tentative pending molecular confirmation. Probiotic characteristics evaluated included antibiotic susceptibility, haemolytic activity, bile salt tolerance, and cellular auto-aggregation. Zobo samples exhibited the lowest pH values (1.7–2.7), inhibiting microbial growth, while ginger drink and kunun-zaki supported LAB proliferation. Five LAB isolates were recovered; cocci and rod-shaped (lactobacilli) morphology were present. Four isolates (1a, 2a, 3a, and 4a) advanced to probiotic safety and functional screening while 5a isolate was lost. The majority showed susceptibility to clinically relevant antibiotics. Haemolytic testing showed that isolates 2a and 3a were non-haemolytic (γ-haemolysis), while isolates 1a and 4a exhibited α-haemolysis, a mild and non-disqualifying pattern under FAO/WHO probiotic safety criteria, in contrast to the strongly disqualifying β-haemolytic phenotype, which none of the isolates displayed. Recalculated bile salt tolerance data (see Table 7) indicate that survival after three hours in 0.3% bile ranged more widely across isolates than initially reported, with isolate 3a showing the strongest tolerance; this discrepancy from the originally reported figures is addressed in the Discussion and should be verified against the underlying raw colony-forming-unit counts before final submission. Auto-aggregation capacities ranged from 80% to 95% after one hour of static incubation. The LAB isolates recovered from ginger drink and kunun-zaki demonstrated multiple probiotic-associated properties and represent promising, though still preliminary, candidates for further development as functional food ingredients. Genus-level identification, molecular confirmation via 16S rRNA gene sequencing, in vivo safety validation, and technological characterization are strongly recommended before any further health-related claims are advanced.