Influence of controlled fermentation on the nutritional composition, antinutritional factors, and protein digestibility of Bambara groundnut flour

Introduction

Bambara groundnut (Vigna subterranea L. Verdc.) is an indigenous African legume crop widely cultivated across sub-Saharan Africa, characterized by its exceptional drought tolerance, nitrogen-fixing symbiosis, and ability to produce nutritious seeds on marginal soils where improved legumes fail to establish [1,2] . The seeds are a nutritionally dense food source, containing 15-23% crude protein with a balanced essential amino acid profile, 4-7% fat predominantly as oleic and linoleic acids, 55-65% carbohydrate, and appreciable levels of calcium, iron, zinc, and B-vitamins [2,3] ; however, despite this impressive nutritional profile, Bambara groundnut remains chronically underutilized and undervalued in formal food systems relative to cowpea and groundnut, primarily because of its hard seed coat that prolongs cooking time, its high content of antinutritional factors (ANFs), and its lower consumer familiarity compared with widely marketed legumes [1,4] . Antinutritional factors including phytic acid (phytate), condensed tannins, oxalates, and protease inhibitors are inherently present in unprocessed Bambara groundnut and function to limit the bioavailability of protein, iron, zinc, and calcium through chelation, enzyme inhibition, and precipitation mechanisms [5,6] . Phytic acid, in particular, forms insoluble complexes with divalent mineral cations (Fe²⁺, Zn²⁺, Ca²⁺) and binds to storage proteins, substantially reducing their digestibility and the nutritional value of the protein consumed [5,7] . Trypsin inhibitors directly impair proteolytic digestion by inhibiting trypsin and chymotrypsin International Journal of Agriculture and Food Science https://www.agriculturaljournals.com activity in the small intestine, while condensed tannins form indigestible tannin-protein complexes that reduce amino acid availability [6,8] . Fermentation is among the oldest and most widely practiced food processing technologies globally, and has been consistently demonstrated to reduce antinutritional factors in legumes through the enzymatic and metabolic activities of fermenting microorganisms (predominantly lactic acid bacteria, yeasts, and Bacillus spp.) that produce phytases, tannase, protease, and organic acids capable of degrading and inactivating ANF molecules [9, 10, 11] . Additionally, fermentation generates free amino acids and short peptides through partial proteolysis, creates a more favourable protein conformation for enzymatic digestion, and produces organic acids that lower the pH to levels that further inactivate protease inhibitors [10,12] . While the benefits of fermentation for improving the nutritional quality of widely studied legumes such as cowpea, soybean, and common bean are extensively documented [11,13] , comparative investigations of the effects of controlled temperature and duration parameters of natural fermentation specifically on Bambara groundnut flour composition, ANF levels, and in vitro protein digestibility under conditions relevant to West African food processing remain limited [14,15] . The present study was therefore designed to systematically evaluate the effects of controlled natural fermentation at two temperatures (30 °C and 37 °C) and three durations (24, 48, and 72 h) on the proximate composition, ANF profile (phytic acid, tannins, oxalate, trypsin inhibitor activity), and in vitro protein digestibility of Bambara groundnut flour from cream-seeded landrace varieties cultivated in Ghana, hypothesizing that fermentation at 37 °C for 72 h would achieve the greatest ANF reductions and highest protein digestibility improvements relative to unfermented control flour [16,17,18,19] .

Materials and Methods

Materials

Cream-seeded Bambara groundnut (Vigna subterranea L. Verdc.) seeds of the Accra landrace, sourced from three smallholder farms in the Northern Region of Ghana, were cleaned, sorted to remove damaged and shrivelled seeds, and pooled to form a homogeneous bulk sample. Seeds were washed three times with distilled water, soaked for 18 h at room temperature (25 ± 2 °C), and dehulled manually by gentle rolling and winnowing to minimize hull contamination of fermented flours. Dehulled cotyledons were washed, drained on sterile muslin cloth, and subjected to natural fermentation (without inoculation of starter cultures) at controlled incubation temperatures of 30 °C and 37 °C for 24, 48, and 72 h periods in pre-sterilized polypropylene containers lined with sterile gauze. Unfermented dehulled cotyledons constituted the control. Following fermentation or control processing, all samples were oven-dried at 60 °C for 12 h, milled using a Retsch SM 100 mill to pass a 425 μm sieve, and stored at -20 °C in airtight containers until analysis. All chemical reagents used for proximate and ANF analyses were of analytical grade (BDH/VWR Chemicals, UK; Sigma-Aldrich, Germany). Porcine pepsin (800-900 units mg⁻¹, Sigma-Aldrich) and pancreatin (8× USP specification, Sigma-Aldrich) were used for in vitro protein digestibility determination.

Methods

A 2 × 3 factorial randomized complete block design (RCBD) was employed, with two fermentation temperatures (30 °C and 37 °C) and three fermentation durations (24, 48, and 72 h), yielding six fermented treatments plus an unfermented control (seven treatments total), replicated three times (21 experimental units). Proximate composition (moisture, crude protein, crude fat, crude fibre, ash, and carbohydrate by difference) was determined by standard AOAC 2019 Official Methods [16]: moisture by oven-drying at 105 °C to constant mass (AOAC 925.10); crude protein by Kjeldahl (N × 6.25, AOAC 978.04); crude fat by Soxhlet extraction in petroleum ether (AOAC 920.39); ash by muffle furnace at 550 °C for 6 h (AOAC 942.05); crude fibre by successive acid-alkali digestion (AOAC 985.29). Phytic acid was determined by the Wade reagent method [5] ; condensed tannins by the vanillin-HCl colorimetric method [6] ; oxalate by titration with KMnO₄ (AOAC 2012.15); and trypsin inhibitor activity (TIA) by the N-α-benzoyl-dlarginine-p-nitroanilide (BAPNA) substrate method [8] . In vitro protein digestibility (IVPD, %) was assessed by the two-enzyme pepsin-pancreatin method [17]: samples were digested sequentially with pepsin (pH 2.0, 37 °C, 3 h) then pancreatin (pH 7. [18,19] . [2,3,16] .

Results

Fermentation significantly (p < 0.01) altered all proximate composition parameters of Bambara groundnut flour, with the magnitude of change increasing with both temperature and duration (Table 1). Crude protein content increased progressively from 19.8% (control) to 25.6% (37 °C/72 h), representing a 29.3% improvement, attributable to concentration of protein on a dry weight basis following fermentative carbohydrate catabolism and microbial protein biosynthesis, consistent with findings of Nwokolo [2] and Fasoyiro et al. [3] for Bambara groundnut and related legumes. Crude fat increased from 5.4% (control) to 6.8% (37 °C/72 h), reflecting microbial lipolytic activity producing more extractable free fatty acids [10] . Ash content improved marginally (3.1 to 3.9%) across treatments, while crude fibre decreased from 4.8% to 3.7% at 37 °C/72 h, consistent with cellulolytic microbial enzyme activity degrading structural cell wall polysaccharides [9,12] . Carbohydrate content declined significantly with fermentation duration (61.3% in control to 50.8% at 37 °C/72 h), primarily reflecting consumption of simple sugars during fermentative metabolism by resident microorganisms [10, 11] . [5,6,7,8] .

All antinutritional factors were progressively and significantly reduced by increasing fermentation duration at both temperatures, with the 37 °C regime consistently producing greater reductions than 30 °C at equivalent durations (Table 2). Phytic acid the most abundant ANF in unfermented flour (8.42 mg g⁻¹) was reduced by 76.4% at 37 °C/72 h (1.99 mg g⁻¹), primarily attributed to the elevated phytase activity of fermenting lactic acid bacteria and Bacillus spp. at higher incubation temperatures, which catalyse phytate dephosphorylation and release mineralbound phosphate [5,7] . Condensed tannins were reduced from 4.86 to 1.54 mg CE g⁻¹ (68.2% reduction at 37 °C/72 h) through tannase-mediated ester bond hydrolysis by fermenting microorganisms and direct pH-driven tanninprotein complex dissolution [6,12] . Oxalate decreased from 128.4 to 36.2 mg 100 g⁻¹ (71.8% reduction), while trypsin inhibitor activity (TIA) fell from 14.8 to 2.8 TIU mg⁻¹ an 81.3% reduction at 37 °C/72 h attributed to heat-labile proteinaceous inhibitor denaturation and proteolytic cleavage by microbial proteases [8,10] . These ANF reductions are consistent with values reported by Vadivel and Janardhanan [14] for fermented underutilized legumes and exceed those achieved by boiling alone, confirming the superior efficacy of fermentation for ANF elimination in Bambara groundnut [13,15] . [17,18,19] .

In vitro protein digestibility improved significantly with fermentation, increasing from 62.4% in the unfermented control to 74.8% (30 °C/72 h) and 81.7% (37 °C/72 h) (Table 3), corresponding to a 30.9% improvement under the optimal treatment. This improvement was attributable to the cumulative effects of phytate reduction which eliminated phytate-protein complex formation that sterically hinders protease access tannin degradation that reduced tannin-protein complex precipitation, and direct partial proteolysis by microbial proteases during fermentation that pre-digested protein into smaller, more digestible polypeptide fragments [7, 10, 12] . Essential amino acid concentrations increased with fermentation at both temperatures, with all indispensable amino acids in the 37 °C/72 h treatment meeting or exceeding the FAO/WHO/UNU (2007) reference requirements for school-age children. The essential amino acid score (EAAS) improved from 0.84 (control) to 1.02 (37 °C/72 h), with methionine + cysteine remaining the first limiting amino acid across all treatments a characteristic common to all legume proteins [2,18] . Pearson correlation analysis revealed highly significant negative correlations between phytic acid level and IVPD (r = -0.94, p < 0.01) and between TIA and IVPD (r = -0.91, p < 0.01), confirming that ANF reduction is the primary driver of digestibility improvement during fermentation [5,7,17] .

Discussion

The significant and temperature-and duration-dependent improvements in nutritional composition, ANF reduction, and protein digestibility of Bambara groundnut flour observed in this study confirm the effectiveness of controlled natural fermentation as a processing strategy for enhancing the nutritional quality of this chronically underutilized West African legume [1,13] . The 29.3% increase in crude protein content following 37 °C/72 h fermentation is consistent with the concentration effect reported by Fasoyiro et al. [3] for fermented Bambara groundnut and Dankwa et al. [9] for fermented cowpea, whereby microbial catabolism of carbohydrates as energy substrates concentrates protein as a proportion of remaining dry matter. The progressive reduction in phytic acid with fermentation duration (from 8.42 to 1.99 mg g⁻¹ at 37 °C/72 h) exceeds the 60-70% phytate reduction rates reported in fermented common bean [11] and approaches the 80% reductions documented in fermented soybean, attributable to the induction of endogenous seed phytase activity by fermentation-associated pH changes and the secretion of extracellular phytases by lactic acid bacteria and Bacillus spp. that proliferate under the conditions applied in this study [7,10] . The 81.3% reduction in TIA at 37 °C/72 h substantially exceeded the typical 40-60% reduction achieved by boiling alone, as reported by Vadivel and Janardhanan [14] for Bambara groundnut, confirming that fermentation engages both denaturation and proteolytic degradation of the inhibitory proteins that boiling alone cannot completely eliminate [8,15] . The strong negative correlation between phytic acid and IVPD (r = -0.94) provides compelling mechanistic evidence that phytate is the primary nutritional constraint on protein digestibility in unfermented Bambara groundnut flour, a finding that aligns with the theoretical framework proposed by Sandberg [5] for the role of phytate-protein complexes in limiting pepsin and pancreatin access to substrate proteins. The achievement of an EAAS of 1.02 in the 37 °C/72 h fermented flour exceeding unity for the first time across all treatments suggests that fermented Bambara groundnut flour can fully meet the essential amino acid requirements of school-age children without complementation, which represents a significant nutritional advancement for a traditionally foodinsecure population group in Ghana and West Africa [18,19] . The methionine + cysteine limitation observed across all treatments is characteristic of legume seed proteins [2] and indicates that complementation of fermented Bambara groundnut flour with cereal-based ingredients which are relatively rich in sulfur amino acids would produce nutritionally superior complementary food formulations for young children [13,16,17] .

Conclusion

This investigation provides systematic and statistically robust evidence that controlled natural fermentation, particularly at 37 °C for 72 h, is a highly

References

1. Bamshaiye OM, Adegbola JA, Bamshaiye EI. Bambara groundnut: an under-utilized nut in Africa. Adv Agric Biotechnol. 2011;1:60-72.
2. Nwokolo E. Bambara groundnut. In: Nwokolo E, Smartt J, editors. Food and feed from legumes and oilseeds. London: Chapman & Hall; 1996. p. 216-220.
3. Fasoyiro SB, Ajibade SR, Omole AJ, Adeniyan ON, Farinde EO. Proximate, minerals and anti-nutritional factors of some underutilized grain legumes in southwestern Nigeria. Nutr Food Sci. 2006;36(1):18-23.
4. Brough SH, Azam-Ali SN. The effect of soil moisture on the proximate composition of Bambara groundnut (Vigna subterranea) at harvest. J Sci Food Agric. 1992;60(2):197-203.
5. Sandberg AS. Bioavailability of minerals in legumes. Br J Nutr. 2002;88(Suppl 3):S281-S285.
6. Deshpande SS, Deshpande US, Salunkhe DK. Nutritional and health aspects of dry beans. In: Mattews RH, editor. Legumes: chemistry, technology, and human nutrition. New York: Marcel Dekker; 1989. p. 443-558.
7. Greiner R, Konietzny U. Phytase for food application. Food Technol Biotechnol. 2006;44(2):125-140.
8. Liener IE, Kakade ML. Protease inhibitors. In: Liener IE, editor. Toxic constituents of plant foodstuffs. 2nd ed. New York: Academic Press; 1980. p. 7-71.
9. Dankwa K, Plahar WA, Annan NT. Nutritional quality and acceptability of fermented dried cowpea flour. Plant Foods Hum Nutr. 1996;49(4):303-315.
10. Nout MJ, Sarkar PK, Beuchat LR. Indigenous fermented foods. In: Doyle MP, Beuchat LR, editors. Food microbiology: fundamentals and frontiers. 3rd ed. Washington (DC): ASM Press; 2007. p. 817-835.
11. Vidal-Valverde C, Frías J, Valverde S. Changes in the carbohydrate composition of legumes after soaking and cooking. J Am Diet Assoc. 1993;93(5):547-550.
12. Sanni AI. The need for process optimization of African fermented foods and beverages. Int J Food Microbiol. 1993;18(2):85-95.
13. Chukwu MN, Ejidike BN. Antinutritional contents and their reductions in cowpea seeds with differently boiling temperatures. Pak J Nutr. 2010;9(6):567-571.
14. Vadivel V, Janardhanan K. Nutritional and antinutritional characteristics of seven South Indian wild legumes. Plant Foods Hum Nutr. 2005;60(2):69-75. 15. Mubarak AE. Nutritional composition and antinutritional factors of mung bean seeds (Phaseolus aureus) as affected by some home traditional processes. Food Chem. 2005;89(4):489-495.
15. AOAC International. Official methods of analysis of AOAC International. 21st ed. Gaithersburg (MD): AOAC International; 2019.
16. Akeson WR, Stahmann MA. A pepsin-pancreatin digest index of protein quality evaluation. J Nutr. 1964;83(3):257-261.
17. FAO/WHO/UNU. Protein and amino acid requirements in human nutrition: report of a joint WHO/FAO/UNU expert consultation. WHO Technical Report Series No. 935. Geneva: World Health Organization; 2007.
18. Boye J, Wijesinha-Bettoni R, Burlingame B. Protein quality evaluation twenty years after the introduction of the protein digestibility-corrected amino acid score method. Br J Nutr. 2012;108(S2):S183-S211.