Environmentally, legumes are highly sustainable, requiring lower water and nitrogen inputs compared to animal or cereal-based protein sources, making them key candidates for addressing both nutritional and ecological concerns [1, 2]. Legumes offer a rich nutritional profile, comprising high-quality proteins (20–45%), dietary fibre (5–37%), complex carbohydrates (65–70%), 85% of which is resistant starch, polyunsaturated fatty acids (66.1–85.3% of total lipids), and polyphenolic antioxidants [3–5]. However, the nutritional potential of legumes is frequently compromised by the presence of anti-nutritional factors (ANFs), such as lectins, phytates, tannins, trypsin inhibitors, and oligosaccharides of the raffinose family. These compounds reduce protein digestibility, hinder mineral absorption, and cause gastrointestinal discomfort [6]. Moreover, the characteristic “beany” flavour of legumes may limit consumer acceptance, necessitating processing techniques that enhance both nutritional bioavailability and sensory appeal [7]. Fermentation, particularly with lactic acid (LA) bacteria (LAB) such as Leuconostoc, Lactiplantibacillus, Lacticaseibacillus, and Limosilactobacillus, has emerged as an effective strategy to overcome these limitations, facilitating the enzymatic hydrolysis of complex carbohydrates, degrading ANFs, and synthesizing bioactive peptides [7–9]. LAB can further synthesize essential B-complex vitamins, especially riboflavin (B₂), folate (B₉), and cobalamin (B₁₂) [10], addressing key micronutrient limitations often associated with plant-based substrates.

Among fermentation modalities, solid-state fermentation (SSF) is particularly promising for legume processing. SSF enables microbial growth on moistened solid substrates with minimal water content, reducing energy and downstream processing demands while promoting nutrient preservation [11]. Previous studies have shown that LAB-mediated SSF can enhance the formation of functional metabolites across a range of substrates. In Spirulina, LAB-SSF increases protein-derived bioactive compounds such as GABA, L-glutamic acid, and biogenic amines [12]. Similarly, the SSF of A. argyi leaves elevated flavonoid, polyphenol, and related antioxidant activities [13]. Building upon these findings, the present study explored the application of LAB-mediated SSF to legume-based substrates. Legumes are widely consumed worldwide. Crops such as soybeans (SBs), peas, chickpeas, and beans are staple components of Mediterranean and other traditional plant-based dietary patterns, which are widely associated with improved metabolic and cardiovascular health outcomes [14] and represent promising fermentation matrices due to their favourable nutritional composition, including protein (e.g., 36.5 g/100 g in soybean), dietary fibre (e.g., 4.2 g/100 g in black beans), and carbohydrate content (e.g., 60.4 g/100 g in chickpeas) [15]. The global shift toward sustainable plant-based diets has created a strong demand for bioprocess strategies that enhance the nutritional and functional properties of legumes [16].

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, produced through LAB-mediated fermentation, play vital roles in gut health by serving as energy sources for colonocytes, reducing inflammation, and improving epithelial integrity [17, 18]. The prebiotic carbohydrates present in legumes can stimulate SCFA production during gut fermentation, and this effect is increased when legumes undergo LAB-mediated pre-fermentation [19, 20]. Concurrently, LA synthesis ensures inhibition of common foodborne pathogens and spoilage-causing microorganisms. In addition to acidification, LAB fermentation yields bioactive antimicrobials such as hydrogen peroxide and bacteriocins [21–23].

Despite the growing interest in bioprocessing, the majority of studies have emphasized submerged fermentation or the enhancement of a single metabolite [10, 21]. There remains a significant knowledge gap regarding the comparative evolution of multiple legume matrices under SSF, particularly using LAB consortia for multifunctional transformation. Specifically, the synergistic enhancement of SCFAs, vitamin biosynthesis, antimicrobial activity, and probiotic viability within a single SSF system has rarely been addressed. Furthermore, while fermentation is inherently dynamic, most research relies on endpoint analysis, leaving the systematic, time-dependent evolution of metabolites in SSF systems insufficiently explored. In view of this, eleven legumes, including SB, yellow pea (YP), white kidney bean (WKB), mung bean or green gram (GM), cowpea (BEB), black kidney bean (BKB), lima bean (LB), black gram (BG), black chickpea (KC), field bean (FB), and horse gram (HG), were initially screened in the present study to evaluate their suitability as substrates for LAB-mediated SSF. Selected matrices demonstrating favourable fermentation performance were further investigated in detail. The consortium comprising L. acidophilus (Lactobacillus acidophilus), L. plantarum [Lactobacillus plantarum (Lactiplantibacillus plantarum)], and L. rhamnosus [Lactobacillus casei var. rhamnosus (Lacticaseibacillus rhamnosus)], selected here, is based on prior evidence of its superior synergistic antimicrobial activity against a wide range of pathogens compared to individual strains [24]. This consortium also demonstrates enhanced efficacy in the presence of prebiotic co-substrates [25] and exhibits multi-enzyme activities, including anti-proteolytic, anti-collagenase, and anti-elastase effects, and significant anti-angiogenic properties [26]. The results would contribute to the development of sustainable, functional legume-based fermentations with applications in both food biotechnology and health-oriented biomanufacturing.

In this study, legumes were selected as fermentation substrates as they are low-cost, protein-rich matrices containing intrinsic prebiotic carbohydrates (resistant starches, oligosaccharides) that facilitate microbial colonization and metabolic flux toward SCFA and vitamin biosynthesis. Organic acid production is often monitored over extended fermentation periods, typically up to five days. The temporal dynamics of vitamin biosynthesis have been less frequently investigated, with most studies reporting vitamin levels only at selected time points, such as 24, 48, or 72 h [36–38]. Therefore, a comprehensive time-dependent evaluation is essential to better understand metabolite progression and to determine optimal fermentation intervals for simultaneous enhancement of multiple functional compounds. SSF of legume flours with Lactobacillus consortium represents a scalable bioprocessing platform that integrates microbial growth support up to 7 log₁₀ CFU/g with substrate valorisation, improving protein accessibility, reducing antinutrient load, and enhancing metabolite recovery yields [39, 40]. Multiple additional legumes, including HG, FB, KC, WKB, GM, BEB, BKB, LB, and BG, were also screened, demonstrating that the LAB consortium maintains functional robustness across diverse legume matrices. Maximum LAB viability was observed at a legume-to-water ratio of 1:3 (~67% moisture), consistent with previous findings that 60–65% moisture is ideal for nutrient diffusion and microbial metabolic activity [41]. Viable counts ranged from 11.8 to 12.4 log₁₀ CFU/g across most of the screened legumes at 1:3 moisture, demonstrating strong microbial adaptability and substrate compatibility. Although YP exhibited the highest viability (16.04 log₁₀ CFU/g), several other legumes, such as BKB, GM, FB, and HG, also supported substantial LAB proliferation. Among the legumes studied, YP and SB were selected for detailed analysis because of their contrasting macronutrient compositions, as mentioned in Table 1.

Fermentation kinetics revealed that YP supported more rapid LAB proliferation, 15.52 log₁₀ CFU/g and pH drop to 4.9, reaching within the first 24 h, whereas SB exhibited delayed growth peaking at 14.6 log CFU/g by 72 h with a more gradual pH decrease. Previous studies on LAB reported 8–9 log₁₀ CFU/g growth of L. casei and L. plantarum on lentils [42], while Śliżewska et al. (2020) [27] observed proliferation of Lactobacillus strain up to 10.28 log₁₀ CFU/g using wheat, barley, rye, and maize flours. These findings indicate that the higher fermentable carbohydrate content of YPs promotes rapid and efficient fermentation. Conversely, SBs support slower and more gradual microbial growth and acidification, likely due to their limited availability of fermentable sugars and high concentrations of ANFs such as trypsin inhibitors and phytic acid, which may hinder microbial metabolism by chelating essential nutrients or inhibiting enzymatic pathways critical for LAB activity [43].

LA production also exhibited clear substrate-dependent variability. In the present study, LA accumulation reached 4.2 g/L in YP at the end of 96 h and 7.28 g/L in SB at 72 h, with SB producing higher LA despite lower LAB counts, suggesting enhanced per-cell metabolic activity. This may result from acid stress-induced amino acid catabolism, which buffers the intracellular pH while sustaining LA synthesis [44]. In YP, a rapid decline in pH was observed within the first 24 h, which may be due to its distinct carbohydrate composition (Table 1), with a high content of complex carbohydrates. Oligosaccharides such as verbascose, raffinose, and stachyose are utilized in mixed-acid pathways that generate organic acids like formic and succinic, along with carbon dioxide, lowering the pH during early fermentation [45]. In contrast, SB contains a higher proportion of readily fermentable soluble sugars, particularly hexoses (Table 1), supports rapid homolactic fermentation, resulting in earlier and higher accumulation of LA. Thus, the substrate-specific sugar profile strongly governs acid production patterns and explains the contrasting pH–LA dynamics observed between YP and SB during the early fermentation. At later fermentation stages (≥ 120 h), a modest increase in pH was observed for both substrates. This apparent decoupling of pH and LA levels can be attributed to the shift in metabolism of microorganisms to consume organic acids after readily available sugars are depleted, leading to the production of alkaline metabolites, such as ammonia, that partially neutralize the accumulated organic acids in the medium [46]. Previous studies have also demonstrated the influence of substrate composition on LA yield. For example, SB yielded 136.2 mg/kg of LA using L. sakei in 24 h [21], while oil palm empty fruit bunches (EFBs) produced 6.49 g/L at 120 h via L. acidophilus [47]. In the present study, LA accumulation in YP and SB reached 4.14 and 5.38 g/L at the end of 120 h respectively. Moreover, LAB strains, particularly L. plantarum, have been shown to increase LA output by up to 94.9%, correlating with enhanced protease and cellulase activity that promotes the synergistic degradation of complex biomass, such as SB meal [48].

SCFA production was similarly influenced by pH dynamics. In SB, AA and PA reached maximum concentrations (AA: 11.73 g/L at 24 h; PA: 15.95 g/L at 48 h) at pH 5.16–5.61. In contrast, YP’s rapid acidification curtailed the window for secondary acid biosynthesis. When the pH dropped below 4.5, AA and PA levels declined due to the inhibition of acetyl-CoA and propionyl-CoA pathways [49]. BA levels in SB were 1.17-fold higher than in YP but remained ~60% lower than in unfermented SB. This reduction likely reflects a LAB-driven metabolic shift prioritizing LA synthesis, along with the possible enzymatic conversion of BA into volatile esters, such as ethyl butyrate. Additionally, BA’s cytotoxicity may restrict its accumulation during extended fermentation [50, 51]. Fermentations using L. plantarum on jicama extract have demonstrated that LAB can produce all three major SCFAs, including 1.58 mg/mL PA, 1.31 mg/mL AA, and 1.05 mg/mL BA [52]. L. acidophilus cultivated in MRS supplemented with 2% salt produced 4.23 g/L AA, 0.0246 g/L PA, and 0.024 g/L BA after 24 h [49], while skim milk-based fermentation after 48 h reported AA production of 2.243 g/L with 1% glucose, with BA concentrations of 0.111 g/L, and PA production up to 0.019 g/L in skim milk alone [53]. Compared to MRS broth and dairy-based fermentation systems, the elevated SCFA production observed in legume-based SSF in the present study highlights the strong influence of substrate composition, particularly complex carbohydrates and oligosaccharides, on microbial metabolic flux and secondary acid biosynthesis. This highlights the ability of LAB to biosynthesize a full spectrum of SCFAs, underscoring the potential of diverse plant substrates to support SCFA biosynthesis even without butyrate-producing co-cultures. Given the health-promoting effects of SCFAs on energy metabolism, immune modulation, and gut integrity, fermented legumes represent a sustainable dietary source compared to chemical sources and monoculture fermentation approaches [54, 55].

Legume fermentation also enriches the B-vitamin content. B₂ accumulation was higher in YP (7.097 µg/mL) compared to SB (0.97 µg/mL) at the end of 96 h. Previous reports of L. plantarum isolated from rice-based foods demonstrated production of up to 0.7 µg/mL [10] and 0.481 µg/mL in soymilk fermentations [56]. B₂ enrichment following fermentation has been widely reported in traditional fermented foods. Tempeh fermented by R. oryzae and natto fermented by Bacillus natto contain approximately 3.58 µg/g and 1.9 µg/g B₂ [15], respectively, while idli batter fermentation has been reported to yield up to 5.9 µg/g B₂ [57]. B₉ production in both YP and SB reached similar levels (~3.6 μg/mL) at 24 h, indicating a strong and sustained biosynthetic capacity under these conditions. Tempeh and natto contain relatively low B₉ concentrations (0.24 µg/g and 0.08 µg/g, respectively) [15]. L. fermentum was earlier reported for B₉ concentration in chemically defined B₉-free medium, where production peaked at 0.092 μg/mL after 24 h [10], and L. rhamnosus K4E yielded 0.24 μg/mL after 12 h [38], highlighting the high biosynthetic potential of select strains under legume-based SSF. These vitamins serve as nutraceuticals and key metabolic cofactors. B₂-derived coenzymes (FMN and FAD), for instance, are crucial in redox reactions and energy metabolism, enhancing the biosynthesis of LA and SCFAs during fermentation [10]. SB supported higher yields (2.187 µg/mL) of B₁₂ than YP (0.96 µg/mL), as observed at 72 h. This difference likely reflects strain-specific interactions with matrix components, as certain LAB (e.g., L. plantarum, L. casei, L. acidophilus) exhibit enhanced vitamin synthesis in response to precursor availability and matrix complexity. In comparison, plant-based fermented foods typically contain negligible amounts of vitamin B₁₂, with tempeh reported to contain approximately 0.0008 µg/g and natto showing undetectable levels [15]. Acetobacter malorum in sourdough systems has been reported to produce up to 0.018 µg/mL in 48 h [58], while Das et al. (2021) [38] reported B₁₂ production up to 0.084 µg/mL in soymilk fermented after 12 h with L. fermentum. This indicates a better yield of vitamins in legume-based SSF systems. The decrease in certain vitamin levels after an initial increase during Lactobacilli fermentation is generally caused by the consumption of these vitamins by the bacteria for their own growth and metabolism, as well as the degradation of specific vitamins due to the highly acidic environment [59].

The antimicrobial activity observed in LAB-fermented SB and YP extracts demonstrates the capacity of LAB to produce bioactive metabolites capable of inhibiting both Gram-positive and Gram-negative bacteria. Among the tested fractions, WCB exhibited the strongest inhibition (up to 27.93 mm) for E. coli, followed by CL (up to 22.98 mm) and CFS (up to 21.51 mm). This hierarchy suggests that intact cells retain greater antimicrobial potency through the combined effects of membrane-bound bacteriocins, intracellular enzymes, and sustained acid production [24, 60]. Comparatively less antagonistic effect on S. aureus may be attributed to its acid tolerance and thick peptidoglycan wall, and the damaging effect of LA on lipopolysaccharide (LPS) barrier [61, 62].

This is evidenced by earlier studies reporting effective suppression of pathogenic and spoilage organisms following LAB fermentation in legume seed sprouts, including reductions in mold counts by 1 Log₁₀ CFU/mL, E. coli and Klebsiella spp. by 2 Log₁₀ CFU/mL, and complete elimination of Salmonella spp., Shigella spp., and Staphylococcus epidermidis. Additionally, S. aureus and S. saprophyticus populations declined by approximately 3 Log₁₀ CFU/mL, frequently reaching undetectable levels [63]. The superiority of plant-based substrates is further supported by SSF studies, where LAB-fermented Spirulina exhibited inhibition zones ranging from 9–16 mm against S. aureus, significantly exceeding those observed under submerged fermentation conditions [12]. Similarly, under optimized SSF conditions, L. plantarum fermenting bean by-products achieved maximum antimicrobial activity, resulting in reductions of approximately 2 and 4 Log₁₀ CFU/g against Listeria monocytogenes and E. coli, respectively [64]. When compared with dairy-based systems, the antimicrobial activity observed in the present study was notably stronger against E. coli. LAB isolates from fermented milk have previously shown inhibition zones of up to 15 mm against E. coli and approximately 22 mm against S. dysenteriae [65]. These observations reinforce the potential of LAB, particularly under SSF conditions, to generate bioactive compounds with selective antimicrobial effects, which may be enhanced by specific substrates and strain–matrix interactions.

SEM analysis revealed significant matrix degradation and LAB colonization in YP. After 48 h, fermented samples exhibited smoother, eroded surfaces indicative of enzymatic breakdown of proteins, fibres, and lipids, consistent with previous studies [66]. Numerous bacilli-shaped LAB cells were visible adhering to the matrix, often in proximity to spherical EPS-like structures, suggestive of biofilm formation. These findings align with previous SEM reports of LAB colonization on olive surfaces during fermentation [67].

Observed clustering and EPS secretion support classic phases of biofilm development: initial adhesion, maturation, and dispersal, as supported by morphological clustering and EPS secretion [68]. The SEM images showing LAB entrapped in fibrous grooves under acid and bile stress further suggest stress-induced adaptation.

Collectively, these findings demonstrate that legume-based SSF using a LAB consortium is an effective strategy for enhancing the nutritional and functional properties of plant-derived substrates. Screening of multiple legumes confirmed the broad adaptability of the consortium, with YP and SB selected for detailed evaluation due to their contrasting compositional profiles. Fermentation influenced probiotic viability and metabolite production patterns, resulting in substrate-specific variations in LA, SCFAs, and B-complex vitamins. YP supported higher BA production, whereas SB exhibited greater LA, PA, and AA production compared to their respective controls. Additionally, fermented extracts demonstrated strong antimicrobial activity, and SEM analysis confirmed matrix degradation and microbial colonization. A consolidated summary of functional and biochemical changes in selected substrates before and after fermentation is presented in Table 2.

Despite these promising outcomes, certain limitations remain, including the lack of strain-specific metabolite attribution and the observation that different substrates preferentially enhanced specific metabolites rather than simultaneously maximizing all functional compounds. This indicates that substrate selection plays a critical role in directing metabolic outputs and may require tailored optimization depending on the targeted metabolite. Future studies should therefore focus on improving metabolite yield through selection or blending of complementary legume substrates, incorporation of targeted prebiotic supplementation, and optimization of fermentation parameters. Furthermore, advanced metabolomic and molecular investigations, along with in vivo validation, will be essential to confirm the health benefits and functional efficacy of fermented legume products. Overall, these findings highlight the potential of LAB-mediated SSF as a sustainable approach for developing nutrient-enriched plant-based functional foods.