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Article

Fermented Navy Bean (Phaseolus vulgaris) Products with Improved Nutritional, Antioxidant, and Antihypertensive Potential

by
Franco Van de Velde
1,2,*,
Micaela Albarracín
1,2,
Raúl E. Cian
1,2 and
Silvina R. Drago
1,2,†
1
Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química, Universidad Nacional del Litoral (UNL), Santa Fe 3000, Argentina
2
Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Santa Fe 3000, Argentina
*
Author to whom correspondence should be addressed.
†
Deceased author.
Fermentation 2026, 12(5), 238; https://doi.org/10.3390/fermentation12050238
Submission received: 24 March 2026 / Revised: 17 April 2026 / Accepted: 12 May 2026 / Published: 13 May 2026
(This article belongs to the Special Issue Fermented Cereals and Legumes: Innovation for the Development and Characterization of Functional Foods)
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Abstract

This study evaluated the impact of lactic acid fermentation on microbiological and nutritional quality, bioactive compound profile, and bioactive properties of mashed navy beans (MNB). Lactic Acid Bacteria (LAB) viability and microbiological quality of fermented mashed navy beans (FMNBs) were maintained for up to 28 days at 4 °C. Fermentation improved protein quality while reducing trypsin inhibitor activity. Additionally, fermentation enhanced the extractability of phenolic compounds, especially of bound forms. Proteolytic activity during fermentation generated low-molecular-weight peptides enriched in hydrophobic residues. Although antioxidant capacity remained comparable between samples, fermented samples exhibited higher angiotensin-converting enzyme inhibitory (ACE-I) activity (IC50 ACE-I = 0.635 ± 0.043 and 0.413 ± 0.002 mg solids mL−1 for MNBs and FMNBs, respectively). Simulated gastrointestinal digestion enhanced both antioxidant (ABTS•+) and antihypertensive potential. ECA-I inhibition was higher in the fermented sample dialysates (D), with IC50 values of 0.160 ± 0.005 and 0.117 ± 0.003 mg solids mL−1 for MNB-D and FMNB-D, respectively, due to the increased dialyzability of phenolic compounds and the presence of hydrophobic low-molecular-weight peptides in FMNB-D. Furthermore, FMNB-D exhibited competitive ACE-I inhibition. These findings demonstrate that lactic fermentation is an effective strategy to enhance the nutritional and health-promoting properties of legume-based foods.

1. Introduction

Beans (Phaseolus vulgaris) along with other pulses are widely consumed all over the world [1]. From a nutritional perspective, beans represent an inexpensive source of macronutrients, containing up to about 40% protein on a dry-weight basis and serving as a rich source of dietary fiber, with levels typically around 20–25 g per 100 g. Their carbohydrate fraction (55–65%) is largely composed of slowly digestible and resistant starch, contributing to lower glycemic responses [2,3,4]. Pulses are also an important source of phenolic compounds, which are associated with various bioactive effects such as antioxidant, anti-inflammatory, and antidiabetic activities [5,6].
In recent years, the consumption of beans and other pulses has risen, largely driven by the rapid expansion of the plant-based food market and the increasing demand for healthier and more sustainable dietary options among health-conscious consumers [2]. In parallel, the development of pulse-based beverages and yogurt-like products has been explored as potential non-dairy alternatives [7,8,9].
Despite their well-balanced nutritional composition, pulse consumption has been limited by the presence of antinutritional factors (ANFs). ANFs include phytic acid and enzyme inhibitors, such as trypsin, chymotrypsin, and α-amylase inhibitors, among others [10,11]. These compounds may interfere with nutrient bioavailability and protein digestibility, hence their reduction or removal in pulses before consumption is desirable [12]. Various processing methods, including soaking, thermal treatments, germination, and fermentation, among others, can reduce or eliminate ANFs in pulses while also improving protein digestibility and functional properties, thereby enhancing their suitability for diverse food applications. Particularly, fermentation is one of the oldest food processing techniques and involves the transformation of food through the metabolic activity of microorganisms such as bacteria, molds, and yeasts. Among bacteria, LAB have traditionally been involved in pulse fermentation, as they are naturally associated with legume seeds [13]. During fermentation, microbial and endogenous enzymatic activities promote hydrolytic transformations that can degrade antinutritional components while improving the sensory and nutritional attributes of pulses [14]. Additionally, fermentation enhances food safety and stability by inhibiting the growth of spoilage and pathogenic microorganisms through acidification and the formation of antimicrobial metabolites [15]. Interestingly, microbial metabolism during fermentation can increase the extractability of phenolic compounds and release bioactive peptides from storage proteins, improving the bioactive properties of fermented pulses [6,9,16,17]. In this context, recent studies have reported antihypertensive effects associated with bioactive peptides derived from pulses and fermented pulses. These effects are mainly related to the inhibition of the angiotensin-converting enzyme (ACE-I), a key enzyme in the renin–angiotensin–aldosterone system that regulates blood pressure [18].
This study aimed to evaluate the effects of mashing and fermentation of navy bean flour on its microbiological and nutritional quality, antinutritional factors, and the profiles of phenolic compounds and peptides, as well as its antioxidant and antihypertensive properties, assessed through ACE-I inhibitory activity. In addition, the impact of in vitro gastrointestinal digestion on bioactive compounds and the antioxidant capacity of the resulting products was examined. Finally, the ACE-I inhibitory activity of the digested samples was further evaluated, and the underlying mechanism of inhibition was characterized.

2. Materials and Methods

2.1. Reagents

Amino acid standard solution, pepsin from porcine gastric mucosa (EC Number: 3.4.23.1; P-7000; 250 U mg−1 solid), pancreatin from porcine pancreas (EC Number: 232-468-9; P-1750; 4X USP), HPLC peptide standard mixture (H2016), o-phthaldialdehyde (P1378), chlorogenic acid (PHR2202) caffeic acid (C0625), ferulic acid (W518301), p-coumaric acid (C9008), p-sinapic acid (D7927), 2,20-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+) (A1888), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (238813), 2,4,6-Tris(2-pyridyl)-s-triazinewere (TPTZ) (T1253), and N-Hippuryl-His-Leu hydrate (859052) were all obtained from Merck (St Louis, MO, USA). Acetonitrile HPLC grade was purchased from Panreac (Barcelona, Spain). Other reagents were of analytical grade and obtained from Cicarelli Laboratorios (San Lorenzo, Santa Fe, Argentina).

2.2. Raw Materials

Navy beans (Phaseolus vulgaris) were purchased from a local market in Santa Fe, Argentina. The grains were first thermally inactivated by steam treatment at 100 °C for 10 min and subsequently dried in a forced-air oven (Bioelec®, Santa Fe, Argentina) at 50 °C until a moisture content of 11 g 100 g−1. The thermal treatment was sufficient to inhibit lipoxygenase activity, which was reduced to nearly zero after processing.
The grains were then dehulled and ground using a roll mill (Bühler-Miag, Uzwil, Switzerland) to obtain flour. Then, flour was further milled using a cyclone sample mill (Belt Drive UD3010, UDY Corporation, Fort Collins, CO, USA) equipped with a 1 mm sieve. Finally, the samples were stored in plastic bags at 4 °C until further use and analysis. The chemical composition of flour in g 100 g−1 dry weight (dw) was protein = 26.50 ± 0.50, lipid = 1.63 ± 0.01, ash = 4.10 ± 0.04, total dietary fiber = 19.22 ± 0.51, and carbohydrates (calculated by difference) = 48.55. The phytic acid content of flour was 1.63 g 100 g−1 dw. The trypsin inhibitor content of flour was 1.88 ± 0.20 trypsin inhibitor units (TIU) mg−1 sample.

2.3. Fermented Product Obtention and Characterization

Mashed navy bean product (MNB) flours dispersion was prepared according to [19] by heating at 75 °C for 40 min, with the addition of α-amylase (ALPHALASE®, Novozymes, Bagsværd, Denmark), following manufacturer instructions. Then, enzyme was inactivated and flour mashed pasteurized by heating for 10 min at 90 °C and aseptically aliquoted by transferring to sterile plastic containers (50 g). Each aliquot was inoculated at 0.02 g 100 g−1 with the starter YF-L812 (CHR Hansen, Hørsholm, Denmark), containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus, and incubated for 6 h at 43 °C without aeration. The pH of samples was measured using a glass puncture electrode coupled to a digital pH meter (FC200B; Hanna Instruments, Pittsburgh, PA, USA).
MNB and fermented mashed navy bean product (FMNB) samples were stored at 4 °C for 28 days. Samples were subjected to microbiological analysis at days 0, 14, and 28, and subsequently stored at −20 °C before lyophilization (Flexy-dry freeze dryer, SP Scientific, Gardiner, NY, USA) and further analysis.

2.3.1. Microbiological Analysis

Microbiological analyses were conducted on MNB and FMNB samples. Inoculated MNB samples were also analyzed before fermentation to assess the initial LAB levels. LAB counts were performed at 37 °C according to [20] on Man, Rogosa & Sharpe (MRS) acidified and M17 broths, with incubation times of 72 h (anaerobically) and 48 h (aerobically), for L. bulgaricus and S. thermophilus, respectively. Total coliforms were analyzed by the method [21]. Total mesophilic aerobic (TMA) and total psychrotrophic aerobic (TPA) bacteria, and total yeasts and molds were analyzed by FIL/IDF 94B:1990 [22]. Results were expressed as log CFU mL−1 product.

2.3.2. Amino Acid Profile

Total amino acid composition was determined after acid hydrolysis of samples with 6 N HCl at 110 °C for 20 h. The resulting amino acids were quantified by RP-HPLC, using an LC-20AT pump coupled to an SPD-M20A diode-array detector (Shimadzu, Kyoto, Japan), following derivatization with diethyl ethoxymethylenemalonate, using D,L-α-aminobutyric acid as an internal standard, in accordance with the procedure described by [23]. Separation was conducted in a 300 × 3.9 mm i.d. reversed-phase column (Novapack C18, 4 μm; Waters, Milford, MA, USA). Amino acid content was expressed as g 100 g−1 protein using a concentration-response curve of 0–120 y 0–200 µmol mL−1 for Cys and the rest of the amino acids, respectively.

2.3.3. Peptide Analysis by FPLC and HPLC

Product powders (1 g) were added to 10 mL of water and shaken for 80 min on an orbital shaker (Decalab, Buenos Aires, Argentina). Then, samples were sonicated (Ultrasonic Cleaner Model PS-10A, Arcano, Shanghai, China) for 20 min and finally centrifuged at 10,000× g (Centrifuge Cavour Model 1675-D, Buenos Aires, Argentina). Supernatants were used for the peptide analysis. The protein content of supernatants was measured according to [24].
Fast peptide liquid chromatography (FPLC) was performed according to [25]. Fractionation was performed using a KNAUER AZURA system (Berlin, Germany) equipped with a Superdex 10/300 GL column (GE Life Sciences, Piscataway, NJ, USA). The molecular mass of the peptide fractions was estimated by comparison with molecular weight standards from 7 kDa to 100 Da. In addition, the relative peak area for each peak was calculated as the peak area divided by the total chromatographic area.
Peptide analysis of the products was performed by RP-HPLC as described in [25]. Chromatographic analyses were performed on a Gemini 110 Å C18 column (250 × 4.6 mm, 5 µm particle size; Phenomenex, Torrance, CA, USA). Fractions exhibiting absorbance maxima at 280 and 220 nm were selected for analysis. Peptide retention times and peak areas were recorded. Peptide hydrophobicity was assessed by dividing the chromatogram into three time intervals, 0–20 min (low hydrophobicity), 20–40 min (medium hydrophobicity), and 40–60 min (high hydrophobicity), and calculating the relative area of each interval with respect to the total chromatographic area. All analyses were conducted in triplicate.

2.3.4. Free Amino Groups

The concentration of free amino groups was determined following the procedure described by Nielsen et al. [26], using an L-serine calibration curve in the range of 0–0.95 mEq L−1. The degree of protein hydrolysis (DH) of MNBs and FMNBs, before and after the gastrointestinal process, was determined assuming a total peptide bond content of 7.60 mEq g−1 protein in the substrate, as estimated by Nielsen et al. [26].

2.3.5. Analysis of Organic Acids

Organic acids of samples were determined by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) as detailed in [27,28]. Briefly, 0.25 g of the sample was added to 2.5 mL of water. Subsequently, samples were acidified to pH 2.0 with 18 mol L−1 H2SO4, heated at 70 °C for 10 min, and then centrifuged at 3500× g using a VT 3216 centrifuge (Cavour, Buenos Aires, Argentina). Supernatants were used for analysis, and results were expressed as mg 100 g−1 (dry weight, dw).

2.3.6. Phenolic Compound Analysis

Free phenolic compounds were extracted from samples according to [29] with modifications. 0.2 g of sample was added to 5 mL of a solvent mixture composed of 80% methanol, 20% water, and 0.5% acetic acid, and the suspension was sonicated for 20 min. The extracts were subsequently centrifuged at 3500× g for 20 min, and the supernatants were transferred to 15 mL centrifuge tubes. The extraction of the residual pellets was repeated with an additional 5 mL of solvent. After centrifugation, the supernatants were pooled and kept for further analysis. The remaining pellets were then subjected to alkaline hydrolysis to recover bound phenolic compounds. For this purpose, the pellets were treated with 5 mL of 2 mol L−1 NaOH for 3 h, acidified to pH 1 with 6 mol L−1 HCl, and extracted twice with ethyl acetate. The organic phase was evaporated to dryness, and the residues were redissolved in methanol and stored for subsequent analysis. All extractions were made in triplicate. Phenolic compound HPLC analysis was conducted as reported in [29] on a 100 mm × 3.0 mm, 2.7 μm particle size Poroshell 120 column (Agilent, Santa Clara, CA, USA). Compounds were identified by matching their retention times and spectral profiles with those of standard compounds. Quantification was carried out using external calibration curves prepared with chlorogenic acid (5–70 mg L−1) and with caffeic, p-coumaric, ferulic, and p-sinapic acids (0.2–10 mg L−1). Data were reported as mg 100 g−1 dw.

2.3.7. Analysis of Antinutrients: Phytic Acid and Trypsin Inhibitor Content

The phytic acid of the products was determined according to the AOAC method 986.11 [30]. Results expressed as g 100 g−1 dw. Trypsin inhibitory activity (TIA) was measured according to the Method 22-40.01 [31]. Results were expressed as trypsin inhibitor units (TIU) mg−1 sample.

2.4. Bioaccessibility of Biotive Compound Analysis

Simulated gastrointestinal digestion of samples, with gastric and intestinal phases, was performed as described by [29]. To simulate intestinal absorption, dialysis bags (molecular weight cut-off: 6–8 kDa, Spectra/Por, Spectrum, CA, USA) were employed. Following digestion, the dialysate fractions corresponding to the in vitro intestinal phase of MNBs and FMNBs, designated as MNB-D and FMNB-D, respectively, were collected, transferred to flasks, weighed, and subjected to analysis. All experimental procedures were performed in triplicate (n = 3).
The protein and free amino group contents of dialysates were determined as above. The phenolic compound profile of dialysates was determined as previously described, and phenolic dialyzability was calculated as reported in [29]. The total free phenolic compound/free amino groups (mg/g−1) ratio was calculated for dialyzed samples. In addition, peptide analysis of the dialysates was performed as described earlier. In addition, the DH of fermented products after simulated gastrointestinal digestion was measured as mentioned before.

2.4.1. Antioxidant Properties

Antioxidant activity of MNBs, FMNBs, MNB-D, and FMNB-D was evaluated through the ABTS and the ferric reducing antioxidant power (FRAP) assays. ABTS•+ radical scavenging capacity was determined following the method reported in [32]. A Trolox calibration curve (0–2.5 mmol L−1 in 0.01 mmol L−1 PBS, pH 7.4) was constructed, and absorbance was measured at 734 nm after 6 min of reaction using an Asys UVM340 microplate reader (Biochrom Ltd., Cambridge, UK). Results were expressed as mmol Trolox equivalent L−1. The FRAP method was assessed according to [33]. A standard curve was prepared with Trolox (0–1 mmol L−1 in 75% ethanol), and absorbance was recorded at 620 nm after 5 min of incubation. FRAP values were expressed as mmol Trolox L−1.

2.4.2. Antihypertensive Properties

The antihypertensive properties of MNBs, FMNBs, MNB-D, and FMNB-D were evaluated by the inhibition of the angiotensin-converting enzyme-I (ACE-I), according to Hayakari et al. [34]. Results were expressed as ACE-I inhibition (%). The sample concentration that inhibits 50% of enzyme activity was defined as the IC50 value. To determine the IC50 value, serial dilutions of samples from 0.005 to 0.1700 mg of solids mL−1 were prepared. The IC50 value was calculated according to Albarracín et al. [27].
The kinetic analysis of enzymes was performed using the Michaelis–Menten equation [35]. Different substrate concentrations (1–30 mmol L−1) were incubated with the enzyme solution with and without samples.

2.5. Statistical Analysis

Results were expressed as mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance (ANOVA) with STATGRAPHICS Centurion XV version 15.2.06 (Statpoint Technologies, Inc., Warrenton, VA, USA). Differences between samples were considered significant based on Tukey’s honestly significant difference (HSD) test. IC50 and kinetic analysis were fitted with GraphPad Prism software version 6.07 (GraphPad Software, La Jolla, San Diego, CA, USA). For kinetic analysis, the Michaelis–Menten equation was used. Vmax and Km parameters were provided by the same software, considering all the experimental plots.

3. Results and Discussions

3.1. Chemical Characterization and Microbiological Quality of Samples

The chemical composition of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products did not differ significantly from each other or from that of the flour.
The initial pH value of MNBs (Table 1) agreed with those reported for navy bean mashed products (6.09–6.50) [7,9]. Upon fermentation with LAB (S. thermophilus and L. bulgaricus), the pH of FMNBs decreased rapidly, reaching values around 4.8 after 6.0 h of incubation. This acidification, which was not observed in non-fermented samples (MNB), was associated with lactic acid production during fermentation, since the inoculated LAB strains are considered homofermentative microorganisms that convert one molecule of glucose into two molecules of lactate [36].
The lactic acid content of FMNBs (Table 1), around 0.9 g 100 g−1 dw (0.16 g 100 g−1 wet basis), was the only organic acid detected under the conditions evaluated, a value in the range of that reported for fermented navy bean with Lactobacillus paracasei CBA L74 after 16 h of incubation (0.19 g 100 mL−1). In addition, other fermented pulses, such as chickpea, presented 0.13 g 100 mL−1 of lactic acid after fermentation with S. thermophilus and L. bulgaricus for 10 h at 40 °C. Differently, the content of lactic acid produced by starters in milk-based yogurt typically ranged from 0.67 to 0.86 g 100 g−1 [37]. This higher content in lactic acid can be explained by the different buffer capacity of milk due to its protein and mineral composition. Thus, a greater amount of lactic acid is required to achieve the same pH decrease in milk compared to pulse-based products [38]. However, in both cases, the decrease in pH can act as a bio-preservative, inhibiting the growth of spoilage and pathogenic microorganisms [9].
As shown in Table 2, fermentation increased S. thermophilus counts in FMNBs by about 2 log units relative to the inoculum level. These counts remained approximately 1.5 log units above the initial level after 28 days of refrigerated storage. The count of L. delbrueckii subsp. bulgaricus was maintained at the same inoculation level after incubation. Then, the load count decreased by about 0.7 log cycles after 14 days and by less than 1 log cycle after 28 days of refrigerated storage. The limited growth of L. delbrueckii subsp. bulgaricus in FMNBs may be attributed to its strong adaptation to milk substrates, which are characterized by the presence of lactose and caseins. In contrast, the navy bean-based matrix lacks lactose and contains different protein and carbohydrate profiles. Under these conditions, S. thermophilus, which exhibits greater metabolic flexibility, is better able to adapt to this environment and consequently shows improved growth [39]. Other works also reported LAB development on fermented pulse products. The fermentation of lupin and pea water extracts with L. acidophilus ATCC 4356, Lm. Fermentum DSM 20052, and Lc. paracasei DSM 20312 showed a marked increase in the cell counts, which reached values close to 8 log CFU mL−1 after 28 days of storage at 4 °C [40].
L. delbrueckii subsp. bulgaricus and S. thermophilus are common starter cultures that can exert beneficial effects when present at ≥107 CFU g−1 [41]. Therefore, maintaining viable counts above this threshold in fermented foods is essential for their functional impact. Therefore, mashed navy beans may serve as a suitable matrix for delivering LAB in fermented foods, supporting the viability of S. thermophilus at levels ≥107 CFU g−1 for at least 28 days of storage at 4 °C.
As previously noted, fermentation acts as a bio-preservation strategy by inhibiting the growth of spoilage and potentially harmful microorganisms. Accordingly, as shown in Table 2, counts of total mesophilic aerobes (TMA), total psychrotrophic aerobes (TPA), total coliforms, and molds and yeasts in FMNB products remained below the detection limit of the method (<1 log CFU mL−1) throughout storage. These results indicate that fermentation enhances microbiological safety and reinforces the preservative effect of the prior pasteurization step conducted during MNB preparation.

3.2. Changes in the Phenolic Compound Content of Products

Table 3 presents the changes in the phenolic compound profile of MNBs and FMNBs, as analyzed by HPLC, following fermentation and subsequent refrigerated storage at 4 °C for 28 days. As seen, five major phenolic acids belonging to the hydroxycinnamic acid derivative family (chlorogenic, caffeic, p-coumaric, ferulic, and p-sinapic acids) were detected in the samples. In agreement, these phenolic acids were mostly identified in the cotyledon of common beans [42]. Additional minor phenolic compounds were observed in the chromatogram, but they were excluded from the profile due to the unavailability of reference standards. The main phenolic compounds were detected in both free and bound forms, except for chlorogenic acid, which was exclusively present in the free fraction. Among the free phenolic acids, chlorogenic acid showed the highest concentration, accounting for more than 33% of the total phenolic compounds analyzed by HPLC in MNBs at day 0 (Table 3). Chlorogenic acid was also only detected in free form in cooked black, pinto, and ruviotto cotyledon beans, with a concentration ranging from 1.23 to 1.36 mg 100 g−1 (dw) [43]. In contrast, p-coumaric, ferulic, and p-sinapic acids were mainly found in the bound fraction, with ferulic acid representing approximately 45% of the total phenolic content of MNBs. Fermentation did not produce an increase in the total free phenolic compound fraction, resulting in no significant difference between MNBs and FMNBs at day 0. Fermentation is indicated as a treatment that can enhance the level of phenolics in pulses [42]. However, LAB can metabolize phenolic compounds as an energy source [44], potentially counteracting the anticipated increase in phenolics after fermentation, as observed in this work for FMNBs. Conversely, the bound fraction of phenolics of FMNBs was 1.23 times higher than in MNBs. This increase was primarily due to higher levels of the bound forms of p-coumaric, ferulic, and p-sinapic acids following fermentation (Table 3). LAB strains can display enzymatic activities, such as de-esterification, decarboxylation, and demethylation of phenolic compounds, thereby promoting their release and extractability from the food matrix [13,42]. In this context, a prior fermentation step may facilitate the extraction of bound phenolic compounds during alkaline hydrolysis, ultimately enhancing phenolic recovery.
Regarding the effect of storage, the chlorogenic acid content in MNV was 1.6- and 1.4-fold higher at days 15 and 28, respectively, compared with day 0. This increase, despite the absence of a detectable bound fraction, may be attributed to a progressive release from soluble conjugated forms or to an enhanced extractability associated with structural changes in the food matrix over time. No changes were observed for this phenolic acid in FMNBs over time (Table 3). Although chlorogenic acid increased in MNBs and remained stable in FMNBs, the total phenolic content (free and bound forms) at days 15 and 28 was approximately 50% lower than on day 0 for both samples. This result may be attributed to the lower recovery of bound phenolic compounds in these samples, which were likely degraded or metabolized by bacteria during refrigerated storage. Phenolic acids can act as antioxidant agents during storage and may undergo oxidation in the process [45]. Moreover, despite refrigeration, residual LAB activity could contribute to the metabolism of phenolic compounds, which may, in turn, be transformed or degraded [39]. Although the total phenolic content decreased during storage, free ferulic acid levels in FMNBs were higher at days 15 and 28 than at day 0 (1.9- and 1.7-fold, respectively), likely because of its release from the bound fraction. Therefore, fermentation can enhance the extractability of phenolic compounds but can also promote their degradation during storage through microbial consumption. Consistently, fermentation of cowpea and voandzou flours with Lactobacillus plantarum led to a two-fold increase in total phenolic content after 30 h, followed by a decrease at 48 h. This behavior was explained by cell wall breakdown and release of bioactive compounds at early stages, and by microbial detoxification and consumption of phenolics at later stages [17].

3.3. Changes in the Amino Acid Content and Peptides of the Products

The amino acid profile and chemical score (CS) of MNBs and FMNBs are presented in Table S1. As shown, FMNBs exhibited slightly lower arginine content (≈7%) and higher methionine and cysteine levels, which were 1.1- and 1.5-fold higher, respectively, than in MNBs. Microbial proteolysis during fermentation, together with a redistribution of the amino acid profile resulting from the selective utilization of certain amino acids by LAB, may contribute to the observed changes in sulfur-containing amino acids [46]. Accordingly, the chemical score (CS) of FMNBs was 1.25-fold higher than that of MNBs, suggesting an improvement in the protein quality of the fermented product. Since legumes are typically deficient in sulfur-containing amino acids, such as methionine and cysteine, fermentation may contribute to improving the contribution of these limiting amino acids [47].
Figure 1a,b show the FPLC gel filtration profiles of MNBs and FMNBs, respectively. Both samples exhibited six main peaks in their molecular weight (MW) distributions. MNBs were characterized by peaks at >7 kDa, 4 kDa, 790 Da, 370 Da, 190 Da, and <100 Da (Figure 1a), whereas FMNBs showed peaks at >7 kDa, 4 kDa, 700 Da, 430 Da, 180 Da, and <100 Da (Figure 1b). As can be seen, the chromatographic profiles of MNBs and FMNBs were very similar. However, the relative contribution of each peak area to the total chromatogram area differed markedly between the two products. In this regard, the proportion of the >7 kDa fraction was higher in MNBs than in FMNBs (30.1 ± 0.6 vs. 23.0 ± 0.5%, respectively). It is worth noting that this fraction corresponds to proteins and oligopeptides. These results suggest that fermentation promoted the degradation of proteins and oligopeptides, leading to the generation of lower-MW peptides. As is well known, LAB can metabolize substrate proteins and oligopeptides through a proteolytic system that includes protein degradation, peptide transport, peptide hydrolysis, and amino acid catabolism [34]. Through these processes, LAB can release low-MW peptides with potential bioactive properties, as discussed below. In this context, the proportion of low-MW peptides (700–180 Da) was higher in FMNBs than in MNBs (63.3 ± 1.3 vs. 26.2 ± 0.5%, respectively). In contrast, the proportion of free amino acids (<100 Da) was higher in MNBs than in FMNBs (44.5 ± 0.9 vs. 10.1 ± 0.2%, respectively). This difference may be attributed to the consumption of free amino acids during fermentation, although this was not reflected in the total amino acid content (Table S1). Although evident proteolysis occurred in FMNB samples, the DH values of MNBs and FMNBs were not significantly different (7.1 ± 0.3 and 6.6 ± 0.3% for MNBs and FMNBs, respectively). The concomitant increase in low-MW peptides (700–180 Da), together with the reduction in the proportion of free amino acids in FMNB samples, did not lead to higher levels of free amino groups, as measured by the OPA method, compared with the unfermented samples (MNB).
On the other hand, RP-HPLC analysis of MNBs and FMNBs showed that the fermented sample had a higher proportion of hydrophobic peptides than the unfermented sample (15.8 ± 2.4 vs. 0.56 ± 0.00%, respectively). Consequently, the proportion of low-hydrophobicity peptides was higher in MNBs than in FMNBs (96.2 ± 0.3 vs. 89.9 ± 2.5%, respectively). Overall, fermentation increased the proportion of hydrophobic peptides in the final product, a trend that has also been reported for LAB fermentation in other legumes [48,49]. In this context, the increase in hydrophobic peptides during LAB fermentation is primarily attributable to the proteolysis of storage proteins such as vicilins and legumins. These proteins have globular structures in which hydrophobic amino acid residues are typically located in the internal regions of the molecule [50]. During fermentation, the proteolytic enzymes produced by LAB hydrolyze these proteins, exposing previously buried hydrophobic regions and releasing low-MW peptides enriched in hydrophobic residues. As a result, the proportion of low MW hydrophobic peptides increases in the fermented product [51,52].

3.4. Changes in the Antinutrients of the Products

The phytic acid content of MNBs and FMNBs are presented in Table 4. As seen, results did not differ significantly among MNBs and FMNBs, with values 1 g 100 g−1. As observed, the mashing process reduced phytic acid by approximately 25% relative to the content in navy bean flour (Section 2.2), likely due to partial activation of endogenous phytases.
However, subsequent fermentation did not further affect the level of this antinutritional compound. L. delbrueckii subsp. bulgaricus and S. thermophilus are not considered significant sources of microbial phytases [53]. Thus, fermentation was not expected to reduce the phytic acid content. However, the phytic acid level in both MNBs and FMNBs was lower than the content of soaked and boiled navy beans (around 1.90 g 100 g−1 dw) [54].
In contrast, the trypsin inhibitor content in MNBs was already relatively low due to the thermal treatment applied during processing and was further reduced by fermentation, with significantly lower values observed in FMNBs compared to MNBs (Table 4). The more extensive proteolysis observed in FMNB samples during LAB fermentation may contribute to the reduction in this antinutritional factor. The combined action of microbial proteases and the acidic conditions generated during fermentation has been reported to promote the degradation of trypsin inhibitors in pulses [55]. However, the extent of this effect appears to depend on the legume matrix. For example, LAB fermentation of green pea has been shown to reduce trypsin inhibitor activity by 50%, whereas no significant reduction was observed in fermented lentil samples [56].

3.5. Bioaccessibility of Bioactive Compounds

MNBs and FMNBs (day 0) were subjected to in vitro gastrointestinal digestion, and the bioaccessibility of phenolics and peptides was estimated as their percentages of dialyzability. Results are shown in Table 5. The dialyzability (%) of chlorogenic and p-coumaric acids increased by 2.5- and 5.1-fold, respectively, in FMNBs compared with MNBs. This suggests that fermentation improved the bioaccessibility of these compounds. As previously discussed, fermentation facilitates the extractability of phenolic compounds and, in turn, their release during digestion, thereby increasing their concentration in intestinal digests and potentially improving their bioaccessibility and bioavailability [13].
Conversely, the dialyzability (%) of ferulic and p-sinapic acids decreased by about 50% in FMNBs compared with MNBs. This effect may be attributed to their greater sensitivity to gastrointestinal conditions, which leads to partial degradation, and to the higher extractability of the fermented matrix, which could facilitate their exposure to the digestive environment. Although these phenolic acids showed a reduced dialyzability (%) in FMNBs, the overall dialyzability of total phenolics was 1.5-fold higher in FMNBs than in MNBs (Table 5). Thus, fermentation can selectively improve the bioaccessibility of selected phenolic compounds. To the best of our knowledge, there are no reports addressing the dialyzability of phenolic compounds in fermented products derived from pulses. However, in line with our results, the fermentation of fruit purees (seriguela, mangaba, mango, and acerola) by L. acidophilus and/or Lacticaseibacillus casei improved the bioaccessibility of certain phenolic compounds. Acerola puree fermented with LAB showed an increased bioaccessibility of all phenolic compounds, except for cis-resveratrol and kaempferol-3-O-glucoside [57].

3.6. Product Peptide Dialyzability After Gastrointestinal Digestion

Figure 1c,d show the FPLC gel filtration profiles of MNB-D and FMNB-D, respectively. As observed, the chromatographic profiles of the dialysates obtained from MNBs and FMNBs were very similar. Both MNB-D and FMNB-D exhibited peaks at 790 Da, 580 Da, 460 Da, 240 Da, and below 100 Da. Thus, digestive enzymes hydrolyzed the different protein species, generating digestion products with similar molecular size distributions. In this context, extensive degradation of proteins and oligopeptides (peak < 7 kDa), including 4 kDa peptides, was observed. This process resulted in the formation of novel peptide species not detected before the digestion (Figure 1a,b), with molecular weights of 580, 460, and 240 Da. In line with these observations, the degree of hydrolysis (DH) increased from ≈7% to 55.6 ± 0.6% and 48.2 ± 2.3% for MNBs and FMNBs, respectively, after simulated gastrointestinal digestion, confirming extensive additional proteolysis. In contrast to the molecular weight profiles obtained by FPLC, the RP-HPLC peptide profiles of MNB-D and FMNB-D were markedly different. FMNB-D displayed a significantly higher proportion of hydrophobic peptides compared to MNB-D (44.1 ± 1.5% vs. 13.7 ± 2.0%, respectively), reflecting the greater abundance of highly hydrophobic peptides previously observed in FMNBs. Conversely, the proportion of low-hydrophobicity peptides was higher in MNB-D than in FMNB-D (74.0 ± 2.3% vs. 69.3 ± 2.1%, respectively).
Overall, although the molecular weight distribution of the peptides generated after gastrointestinal digestion was similar for both dialyzed products, the hydrophobic profile of MNB-D and FMNB-D was different. This pattern is likely attributable to fermentation-induced proteolysis, which promotes the release of peptides containing hydrophobic amino acid residues that were previously buried within legume storage proteins [52]. These peptides subsequently acted as substrates for digestive enzymes, yielding new bioaccessible peptides. Consistently, peptide dialyzability was around 10% for both products (Table 5).

3.7. Bioactive Properties of Products After Gastrointestinal Digestion

Figure 2 shows the antioxidant capacity of mashed navy bean (MNB) and fermented mashed navy bean (FMNB), as well as their corresponding dialysates (MNB-D and FMNB-D), measured by the ABTS•+ (Figure 2a) and FRAP (Figure 2b) assays. Although fermentation increased the content of phenolic compounds in FMNBs, this effect was not reflected in the antioxidant capacity of the undigested samples, as no significant differences were detected between MNBs and FMNBs (Figure 2a,b). In contrast, the antioxidant capacity measured by ABTS•+ in the dialysates obtained after gastrointestinal digestion (MNB-D and FMNB-D) was almost three times higher than in the undigested samples, with FMNB-D showing slightly higher ABTS•+ values than MNB-D (Figure 2a). These results indicate that the gastrointestinal process released bioactive compounds with higher antioxidant activity, likely due to the generation of lower–molecular–weight oligopeptide fractions (Figure 1c,d) and the presence of phenolic compounds in the dialysates, which together contributed to the enhanced ABTS•+ response. Moreover, fermentation increased the dialyzability of phenolic compounds such as chlorogenic and p-coumaric acids (Table 3), further enhancing the antioxidant potential of FMNB-D.
In contrast, the antioxidant capacity of MNB-D measured by FRAP was approximately 25% lower than that of the undigested MNB, and no significant differences were observed between FMNB-D and FMNB (Figure 2b). This discrepancy between assays reflects their different mechanisms: ABTS•+ evaluates both hydrogen atom transfer (HAT) and single electron transfer (SET), whereas FRAP measures only SET-based reducing capacity [58]. In this context, the small peptides released during digestion, together with phenolic compounds, likely contributed more effectively to antioxidant activity via HAT than SET, resulting in higher ABTS•+ values. Additionally, fermentation improved the extractability of phenolic compounds, increasing their dialyzability and further supporting HAT-based antioxidant activity. These findings are consistent with previous reports showing that fermented soy, peanut, and chickpea milk released more phenolic compounds than unfermented counterparts during simulated digestion, leading to enhanced antioxidant activity measured by DPPH, which primarily reflects HAT mechanisms [59].
Regarding ACE-I inhibitory activity (Table 6), the FMNB product exhibited a lower IC50 value than MNBs (35% lower), indicating that the fermentation process enhances this bioactive property. As shown in Table 3, MNBs and FMNBs displayed the same levels of the main free phenolic acids detected in navy beans; hence, the difference in ACE-I inhibition is attributable to the distinct peptide profiles of the two products. In this regard, it has been reported that LAB fermentation of pulse storage proteins contributes to the generation of ACE-I inhibitory peptides [60]. As discussed above, FMNBs contained a higher proportion of low MW hydrophobic peptides compared to MNBs. Consistently, fermentation of navy beans with L. bulgaricus exhibited the highest ACE-I inhibitory activity among the LAB strains tested, which was attributed to the increased production of hydrophobic low-MW peptides [61]. Thus, the observed differences could be attributed to the distinct peptide profiles of both products.
As shown in Table 6, in vitro digestion increased ACE-I inhibitory activity, with the IC50 values of MNB-D and FMNB-D being lower than those observed for the original products (p < 0.05). Thus, the digestive enzymes promoted the release of new bioactive peptides that enhanced ACE inhibitory activity. Moreover, FMNB-D exhibited the lowest ACE-I IC50 value among the samples, indicating that this digestion product possesses the highest antihypertensive activity. This result can be attributed to the peptide composition of FMNB-D, which contained a higher proportion of hydrophobic peptides than MNB-D. Hydrophobic peptides have been linked to stronger ACE-I inhibitory activity [61,62]. Therefore, the high proportion of low MW hydrophobic peptides in FMNB-D likely enhances its ACE-I inhibitory properties. Furthermore, phenolic acids may inhibit ACE-I activity by interacting with the Zn2+ ion and key residues at the enzyme’s active site, including disulfide bridges, thereby impairing its catalytic function [63,64,65]. In this context, the ratio of total free phenolic compounds to free amino groups in FMNB-D was higher than that in FMN-D (0.69 ± 0.06 vs. 0.40 ± 0.02 mg g−1, respectively), indicating a greater relative proportion of phenolic compounds in FMNB-D. Accordingly, the higher dialyzability of specific phenolic acids, such as chlorogenic and p-coumaric acids (Table 5), may contribute to the ACE-I inhibitory activity observed in FMNB-D. Supporting this, chlorogenic acid has been shown to significantly reduce systolic blood pressure, heart rate, and ACE-I activity in cyclosporine-induced hypertensive rats [66].
Since FMNB-D exhibited the strongest ACE-I inhibitory activity, its mechanism of inhibition was investigated. The kinetic analysis of ACE-I inhibition showed that FMNB-D inhibited the enzyme in a competitive mode (Figure 3). As shown in Table 6, there was no significant difference in Vmaxapp without inhibitor or in the presence of FMNB-D, while the Kmapp value for ACE-I activity was increased with the addition of FMNB-D (p < 0.05). This result is consistent with the findings of Rui et al. [67], who reported that P. vulgaris protein hydrolysate, produced by hydrolysis with Alcalase followed by in vitro gastrointestinal digestion, inhibited ACE-I through a competitive mechanism. Furthermore, these authors reported that the peptide fraction showing the highest ACE-I inhibitory activity via a competitive mechanism was characterized by greater hydrophobicity and lower molecular weight, which aligns with the observations for FMNB-D. Furthermore, phenolic compounds such as p-coumaric acid have been reported to inhibit ACE-I by chelating the Zn2+ ion at the enzyme’s active site [68]. Consequently, the high concentration of this phenolic compound in FMNB-D may also contribute to its observed ACE-I inhibitory activity mechanism.

4. Conclusions

Fermentation improved navy bean protein quality, increasing the chemical score (1.25-fold), while reducing trypsin inhibitor activity (from 1.74 to 1.32 TIU mg−1). Additionally, fermentation enhanced the extractability of phenolic compounds, especially of bound forms. LAB viability and microbiological quality of fermented products were maintained for up to 28 days under refrigerated storage at 4 °C, supporting product stability and shelf life. Proteolytic activity during fermentation generated low-molecular-weight peptides enriched in hydrophobic residues (from 0.56% to 15.8%). Although antioxidant capacity remained comparable between samples, fermented samples exhibited higher ACE-I activity inhibition, with a 35% lower IC50 value. Simulated gastrointestinal digestion enhanced both antioxidant (ABTS•+) and antihypertensive properties, with ACE-I IC50 25% lower in the digested fermented product. This effect was associated with increased dialyzability of phenolic compounds and the presence of hydrophobic low-molecular-weight peptides. Overall, lactic acid fermentation of mashed navy beans represents a promising technological approach for the development of plant-based fermented products with functional properties. These fermented flours could be used in bakeries, gluten-free formulations, and plant-based beverages. This strategy may be relevant for vegan populations and individuals with dairy intolerance, while contributing to the diversification and added value of legume-based functional foods. Sensory evaluation was not performed in the present study, and future research should address consumer acceptance and sensory attributes to support potential applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation12050238/s1. Table S1. Amino acid profile of mashed navy bean (MNB) and fermented mashed navy bean (FMNB), and chemical score (CS). Reference [69] is cited in Supplementary Materials.

Author Contributions

Conceptualization, F.V.d.V. and M.A.; Data curation, F.V.d.V., M.A. and R.E.C.; Formal analysis, F.V.d.V., M.A. and R.E.C.; Funding acquisition, S.R.D.; Investigation and Methodology, F.V.d.V., M.A. and R.E.C.; Project administration, S.R.D.; Resources S.R.D.; Supervision, F.V.d.V. and S.R.D.; Writing—original draft, F.V.d.V.; Writing—review and editing, F.V.d.V., M.A., R.E.C. and S.R.D. Author Silvina R. Drago passed away prior to the publication of this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Universidad Nacional del Litoral (Argentina) through the project grant CAI+D-2024-Tipo II-85520240100119LI and from Agencia de Promoción Científica y Tecnológica (Argentina) through the project PICT-2020-SERIE A-03116.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank Marilin Aquino and Erika Gingene for their technical support. During the evaluation of this manuscript, Silvina R. Drago sadly passed away. The authors dedicate this work to her memory and express their deep gratitude for her contributions.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ACE -IAngiotensin-Converting Enzyme-I
FPLCFast Protein Liquid Chromatography
FMNBFermented Mashed Navy Beans
FMNB-DDialysate of Fermented Mashed Navy Beans
FMNB-DiDigested Fermented Mashed Navy Beans
HHLHippuryl-Histidyl-Leucine
IC50Half-maximal Inhibitory Concentration
LABLactic Acid Bacteria
MNBMashed Navy Beans
MNB-DDialysate of Mashed Navy Beans
MNB-DiDigested Mashed Navy Beans
MWMolecular weight

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Fermentation 12 00238 g001
Figure 1. FPLC gel filtration profiles of mashed navy bean (MNB) (a) and fermented mashed navy bean (FMNB) (b), as well as their corresponding dialysates: MNB-D (c) and FMNB-D (d), obtained after gastrointestinal digestion.
Figure 1. FPLC gel filtration profiles of mashed navy bean (MNB) (a) and fermented mashed navy bean (FMNB) (b), as well as their corresponding dialysates: MNB-D (c) and FMNB-D (d), obtained after gastrointestinal digestion.
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Figure 2. Antioxidant capacity of mashed navy bean (MNB) and fermented mashed navy bean (FMNB), as well as their corresponding dialysates (MNB-D and FMNB-D) obtained after gastrointestinal digestion by ABTS•+ (a) and FRAP (b) methods. Different lowercase letters for a particular antioxidant assay mean significant differences among samples (p < 0.05) according to the Tukey test.
Figure 2. Antioxidant capacity of mashed navy bean (MNB) and fermented mashed navy bean (FMNB), as well as their corresponding dialysates (MNB-D and FMNB-D) obtained after gastrointestinal digestion by ABTS•+ (a) and FRAP (b) methods. Different lowercase letters for a particular antioxidant assay mean significant differences among samples (p < 0.05) according to the Tukey test.
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Figure 3. Kinetic analysis of angiotensin converting enzyme-I at different substrate concentrations (1–30 mmol L−1) with and without FMNB-D.
Figure 3. Kinetic analysis of angiotensin converting enzyme-I at different substrate concentrations (1–30 mmol L−1) with and without FMNB-D.
Fermentation 12 00238 g003
Table 1. Values of pH and lactic acid content of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
Table 1. Values of pH and lactic acid content of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
AttributeProductValue
 pH MNB6.28 ± 0.05 b
FMNB4.80 ± 0.20 a
Lactic acid content
(g 100 g−1 dw)		MNB-
FMNB0.93 ± 0.12
Different letters in the same column for the same attribute mean significant differences between samples (p < 0.05), according to the Tukey test.
Table 2. Microbiological quality of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products over storage time at 4 °C.
Table 2. Microbiological quality of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products over storage time at 4 °C.
Microorganisms
(Log CFU/mL)		 Storage (d)
ProductInoculum01428
S. thermophilusMNB----
FMNB7.5 ± 0.1 a9.4 ± 0.1 c9.0 ± 0.1 b9.0 ± 0.1 b
L. delbrueckii subsp. bulgaricusMNB----
FMNB2.0 ± 0.1 b1.8 ± 0.3 b1.3 ± 0.1 a<1 a
TMAMNB-1.6 ± 0.2 a3.3 ± 0.3 c2.1 ± 0.1 b
FMNB-1.5 ± 0.1<1 *<1 *
TPAMNB-<1 *<1 *<1 *
FMNB-<1 *<1 *<1 *
Total ColiformsMNB-<1 *<1 *<1 *
FMNB-<1 *<1 *<1 *
Molds and yeastsMNB-<1 *<1 *<1 *
FMNB-<1 *<1 *<1 *
TMA: total mesophilic aerobic bacteria, TPA: total psychrotrophic aerobic bacteria, CFU: colony-forming units. * Corresponds to the absence of bacteria in the analytical technique. Different letters in the same row mean significant differences between samples (p < 0.05), according to the Tukey test.
Table 3. Phenolic compound content of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products during 28 days of refrigerated storage.
Table 3. Phenolic compound content of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products during 28 days of refrigerated storage.
Phenolic Compound
(mg 100 g−1 dw)		 MNB
0 d		FMNB
0 d		MNB
15 d		FMNB
15 d		MNB
28 d		FMNB
28 d
Chlorogenic acidF1.96 ± 0.13 a2.21 ± 0.01 ab3.14 ± 0.36 c2.63 ± 0.52 abc2.87 ± 0.12 bc2.78 ± 0.12 bc
B------
Caffeic acidF0.12 ± 0.02 a0.14 ± 0.00 a0.15 ± 0.01 a0.12 ± 0.02 a0.13 ± 0.01 a0.13 ± 0.00 a
B0.05 ± 0.01 b0.03 ± 0.00 a----
p-coumaric acidF0.04 ± 0.00 a0.03 ± 0.00 a0.03 ± 0.00 a0.04 ± 0.01 a0.03 ± 0.00 a0.03 ± 0.00 a
B0.41 ± 0.03 b0.51 ± 0.02 c0.01 ± 0.00 a0.01 ± 0.00 a0.004 ± 0.000 a0.01 ± 0.00 a
Ferulic acidF0.38 ± 0.06 b0.18 ± 0.01 a0.40 ± 0.02 b0.34 ± 0.01 b0.33 ± 0.05 b0.31 ± 0.00 b
B2.65 ± 0.11 b3.27 ± 0.16 c0.04 ± 0.00 a0.05 ± 0.00 a0.02 ± 0.00 a0.05 ± 0.00 a
p-sinapic acidF0.05 ± 0.01 a0.04 ± 0.02 a0.04 ± 0.01 a0.05 ± 0.01 a0.04 ± 0.01 a0.04 ± 0.01 a
B0.22 ± 0.02 b0.27 ± 0.01 c0.004± 0.000 a0.005 ± 0.000 a0.003 ± 0.000 a0.005 ± 0.000 a
Total (T)F2.55 ± 0.10 a2.60 ± 0.03 a3.76 ± 0.40 b3.18 ± 0.60 b3.40 ± 0.19 b3.30 ± 0.01 b
B3.28 ± 0.15 b4.05 ± 0.19 c0.05 ± 0.00 a0.07 ± 0.00 a0.03 ± 0.00 a0.06 ± 0.00 a
T5.83 ± 0.24 b6.65 ± 0.16 c3.81 ± 0.40 a3.25 ± 0.60 a3.43 ± 0.19 a3.36 ± 0.02 a
F: free phenolic compounds, B: bound phenolic compounds, dw: dry weight. Different letters in the same row indicate significant differences (p < 0.05) according to the Tukey test.
Table 4. Values of phytic acid and trypsin inhibitor of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
Table 4. Values of phytic acid and trypsin inhibitor of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
AntinutrientProductValue
Phytic acid
(g 100 g−1)		MNB1.17 ± 0.16 a
FMNB1.21 ± 0.04 a
Trypsin inhibitor
(TIU mg−1)		MNB1.79 ± 0.16 b
FMNB1.32 ± 0.06 a
Different letters in the same column for the same attribute mean significant differences between samples (p < 0.05), according to the Tukey test.
Table 5. Dialyzability of phenolic compounds and peptides of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
Table 5. Dialyzability of phenolic compounds and peptides of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products.
Dialyzability (%)MNBFMNB
 Phenolic  compounds  
Chlorogenic acid18.2 ± 1.0 a46.3 ± 0.5 b
p-coumaric acid0.27 ± 0.03 a1.37 ± 0.09 b
Ferulic acid10.2 ± 0.8 b4.6 ± 0.9 a
p-sinapic acid12.6 ± 0.28 b6.9 ± 1.0 a
Total11.9 ± 0.8 a18.2 ± 0.3 b
Peptides10.0 ± 0.3 a9.9 ± 0.2 a
Different letters in the same row mean significant differences among samples (p < 0.05) according to the Tukey test.
Table 6. Angiotensin converting enzyme-I (ACE-I) inhibition of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products, and the corresponding dialysates (D) obtained after gastrointestinal digestion.
Table 6. Angiotensin converting enzyme-I (ACE-I) inhibition of mashed navy bean (MNB) and fermented mashed navy bean (FMNB) products, and the corresponding dialysates (D) obtained after gastrointestinal digestion.
IC50 Value (mg Solids mL−1)
MNBFMNBMNB-DFMNB-D
ACE-I0.635 ± 0.043 d0.413 ± 0.002 c0.160 ± 0.005 b0.117 ± 0.003 a
Enzyme Kinetic Parameters
  Sample Concentration
(mg solid mL−1)		Kmapp
(mmol L−1)		Vmaxapp
(µmol min−1 mL−1)
ACE-I -11.5 ± 0.8 a0.975 ± 0.078 a
ACE-I + FMNB-D 0.277932.6 ± 1.5 b0.965 ± 0.086 a
Kmapp: Michaelis constant. Vmaxapp: maximum reaction velocity. Results are expressed as mean value ± standard deviation. Different letters in the same row for IC50 value and different letters in the same column for the Enzyme kinetic parameters mean significant differences among samples (p < 0.05) according to the Tukey test.
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MDPI and ACS Style

Van de Velde, F.; Albarracín, M.; Cian, R.E.; Drago, S.R. Fermented Navy Bean (Phaseolus vulgaris) Products with Improved Nutritional, Antioxidant, and Antihypertensive Potential. Fermentation 2026, 12, 238. https://doi.org/10.3390/fermentation12050238

AMA Style

Van de Velde F, Albarracín M, Cian RE, Drago SR. Fermented Navy Bean (Phaseolus vulgaris) Products with Improved Nutritional, Antioxidant, and Antihypertensive Potential. Fermentation. 2026; 12(5):238. https://doi.org/10.3390/fermentation12050238

Chicago/Turabian Style

Van de Velde, Franco, Micaela Albarracín, Raúl E. Cian, and Silvina R. Drago. 2026. "Fermented Navy Bean (Phaseolus vulgaris) Products with Improved Nutritional, Antioxidant, and Antihypertensive Potential" Fermentation 12, no. 5: 238. https://doi.org/10.3390/fermentation12050238

APA Style

Van de Velde, F., Albarracín, M., Cian, R. E., & Drago, S. R. (2026). Fermented Navy Bean (Phaseolus vulgaris) Products with Improved Nutritional, Antioxidant, and Antihypertensive Potential. Fermentation, 12(5), 238. https://doi.org/10.3390/fermentation12050238

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