Physicochemical, Biochemical, Microbiological, and Sensory Characterization of a Fermented Lima Bean (Phaseolus lunatus) Probiotic Beverage During Refrigerated Storage

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Background: Growing public health concerns regarding the consumption of conventional dairy products, coupled with the increasing prevalence of plant-based and vegan dietary preferences, have stimulated considerable scientific interest in the development of innovative fermented legume-based beverages. The lima bean (Phaseolus lunatus) represents a particularly promising raw material in this context, due to its high protein content, the presence of prebiotic α-galactosides, and its notable endogenous antioxidant compounds. Aims: This study aimed to evaluate the probiotic viability, physicochemical and biochemical properties, sensory characteristics, and antioxidant potential of fermented lima bean-based beverages subjected to refrigerated storage at 4°C for 28 days. Material and Methods: Four lima bean-based beverages (Lbs) were formulated by incorporating a 1% inoculum of either the single-strain probiotic Lactobacillus acidophilus La5 (LbLa5), Bifidobacterium animalis subsp. lactis Bb12 (LbBb12), a co-culture of both strains (Lb coculture), or no bacterial inoculum, serving as a non-fermented control beverage. Results: The probiotic Lbs exhibited a high crude protein content (3.15% – 3.34%) comparable to that of soy-based and bovine milk, and were characterized by a substantial carbohydrate fraction (2.08% – 2.32%). Viable counts of L. acidophilus La5 and B. lactis Bb12 were maintained at 6 – 7 log CFU/mL throughout the shelf-life period, with B. lactis Bb12 demonstrating enhanced survival under co-culture conditions. High-performance liquid chromatography (HPLC) analysis revealed significant reduction in the prebiotic oligosaccharide raffinose throughout storage with final concentrations ranging from 2.17 to 3.35 g/L. Probiotic metabolic activity induced a progressive decline in pH (4.00 – 4.30) accompanied by an increase in titratable acidity (0.70 – 0.89%). Chromatographic profiles demonstrated that both selected strains were proteolytically active during cold storage, resulting in a reduction of free amino acid content and the generation of bioactive low-molecular-weight peptides (≤ 10 kDa). Fermentation promoted an increase in total phenolic compound (TPC) content over the first seven days, whilst DPPH radical scavenging activity increased over the initial three weeks of storage. The co-culture treatment yielded the highest TPC content (1.71 – 2.03 mg GAE/mL) and DPPH radical scavenging activity (0.86 – 1.52 mg TE/mL) throughout the entire storage period. Sensory flavor attributes and overall acceptability were enhanced across all fermented beverage formulations relative to the non-fermented control. Conclusions: The incorporation of L. acidophilus La5 and B. lactis Bb12 into lima bean-based beverages yields a nutritionally dense, functionally enriched, and sensorially acceptable plant-based alternative well-suited to vegan and health-conscious dietary regimens. These findings support the valorization of Phaseolus lunatus as a viable substrate for the development of probiotic fermented beverages. Keywords: Lima Bean-Based Beverage; Probiotics; Raffinose; Organic Acids; Free Amino Acids; Bioactive Peptides; Antioxidant Capacity.

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