Introduction

The Fabaceae family, boasting 800 genera and 20,000 species, secures its place as the third-largest flowering plant family globally, following Orchids and Asteraceae [1]. Vicia faba var minor, a prominent member of this botanical cohort, claims the second spot among the world's primary food crops, yielding only to cereals [2].

Recent investigations underscore the nutritional significance of faba bean seeds, positioning them as not only a robust protein source but also rich in specific fibers, vitamins, and minerals—qualities shared with other legumes, earning them the reputation as “the meat of the poor” [3]. Given the pivotal role of legumes in providing fats for both animal and human nutrition [4], considerable attention has been directed toward unraveling the lipid profile of legumes. The nature and quantity of fatty acids in seed oils wield considerable influence over their physical, chemical, and nutritional attributes [5], motivating extensive scrutiny into the lipophilic composition of legumes [6].

Beyond lipid constituents, legumes emerge as reservoirs of bioactive compounds, encompassing phenolic or polyphenolic compounds, tocopherols, and triterpenic acids [7,8,9]. This diverse array includes phenolic acids, lignans, stilbenes, condensed and hydrolyzable tannins, and flavonoids [10]. Apart from their contribution to plant coloration, these compounds function as antioxidants, protecting against UV radiation, attracting pollinators, acting as signaling molecules for nitrogen fixation, and serving as defenses against microbial threats [11]. The chemical stability and widespread occurrence of flavonoids position them as taxonomic chemical markers for higher plants [12].

Epidemiological inquiries further affirm the protective effects of phenolic-rich foods against various chronic diseases [13]. Among these phenolic compounds, polyphenols stand out as extensively studied molecules, renowned for their manifold biological effects, including antimutagenic, anticarcinogenic, cytoprotective, and antioxidant activities [14]. Given the observed reduction in oxidative stress by natural antioxidants [15], specific flavonoids have been explored for their potential in mitigating ailments affecting the cardiovascular and nervous systems, as well as conditions predisposing to cancer [16].

Against the backdrop of South Tunisia, where traditional agricultural practices prevail, faba beans flourish as a versatile crop, cultivated through time-honored methods and natural inputs. This investigation unfolds within the framework of a genetic improvement program for faba beans in arid environments, spearheaded by progenitors of plant breeding programs at the Institute of Arid Regions of Medenine (IRA). The imperative lies in harnessing the genetic diversity of Vicia faba L. var minor collections in arid regions to enhance grain yield and nutritional quality, addressing challenges posed by climate change, water deficits, drought, salinity, and rising temperatures.

Noteworthy is the absence of data pertaining to the phytochemical composition of faba bean seeds cultivated in the southern regions of Tunisia. Consequently, this study meticulously explores the sugar, fatty acids, phenols, flavonoids, and condensed tannins in faba bean seeds, concurrently evaluating their antioxidant activities. The ambition is twofold: to illuminate the nutritional significance of faba bean seeds sourced from diverse southern Tunisian locales and to underscore their potential as a newfound source of natural antioxidants and functional foods.

Material and Methods

Plant Material and Cultivation Conditions

Twenty-three genotypes of Vicia faba L. var. minor were collected from the southern oases of Tunisia by researchers from the Arid Lands Institute. These genotypes were cultivated in the experimental field of the Arid Lands Institute of Medenine, following a randomized complete block design (RcBD) with three replications. Sowing took place on October 12th, 2015, with each genotype represented by ten plants per row in each block, spaced at 60 × 60 cm. The soil, classified as loamy sand with low organic matter, required immediate irrigation post-sowing and subsequent bi-weekly irrigation throughout the trial period, supplemented with mineral fertilizer (NPK 20-20-20). Weed control was manual, with chemical pesticides used as needed. Climate data recorded during the trial period indicated rare and irregular monthly precipitation, with a cumulative precipitation not exceeding 120 mm from October 2015 to May 2016, characteristic of arid conditions. Soil composition, sandy to loamy, featured minimal organic matter and limited water retention, often with alkaline properties and high pH levels. Seed samples, collected at maturity (MS) for drying, underwent a drying process at 65°C for 48 h before being milled into fine powder for chemical composition analysis.

Preparation of Methanolic Extracts

For each genotype, 0.5 g of the dried seeds was milled and dissolved in 5 mL of methanol. This mixture underwent continuous stirring for 6 h in the absence of light and was then filtered through Whatman paper (3 mm). The resultant extract was centrifuged at 3000 rpm for 20 min at 25°C, and the resulting dried extracts were stored at 4°C in the dark for subsequent analyses. The filtrate, representing the methanolic extract, was earmarked for the determination of the chemical profile.

Analysis of Phenolic Compounds

Total Polyphenols Content

The determination of total polyphenols content involved the application of a modified Folin–Cicalteu reagent method [17]. In this procedure, a 125-μL aliquot of the extract, suitably diluted five times in methanol, was combined with 500 μL of distilled water and 125 μL of the Folin-Ciocolteu reagent. After thorough mixing and a subsequent 3-minute incubation, 1250 μL of a 5% solution of CO3(Na2) was introduced to the mixture. The total volume was adjusted to 3 mL with distilled water. Following a 90-min incubation period at room temperature in the absence of light, the absorbance was measured at 760 nm. Each sample underwent triplicate measurements, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (DW) (mg GAE/g DW).

Total Flavonoid Content

The determination of total flavonoids content followed the colorimetric assay outlined by Dewanto [18]. In this method, 250 μL of the methanolic extract (diluted five times) was combined with 75 μL of a 5% NaNO2 solution. After a 6-min incubation at room temperature, 150 μL of freshly prepared 10% aluminum chloride (AlCl3) was added, and the mixture underwent a 5-min incubation period at room temperature. Subsequently, 500 μL of 1M NaOH was introduced, and the final volume was adjusted to 2500 μL [18]. Following a 10-min incubation at room temperature, the absorbance of the resulting pink-colored mixture was measured at 510 nm and compared to a control tube. The total flavonoid content of the various extracts was expressed as milligrams of quercetin equivalent per gram of dry weight of plant material (mg QE/g DW).

Condensed Tannins Content

To quantify the condensed tannins content, we employed the vanillin method as delineated by Broadhurst and Jones [19]. This method exploits the conversion of tannins into red anthocyanidols through their interaction with vanillin. In a streamlined procedure, 250 μL of each methanolic extract underwent amalgamation with 3 mL of 4% vanillin and 150 μL of concentrated HCl. Following a 20-minute incubation at 30°C, the concoction experienced additional 15-minute incubation in the absence of light, after which the resulting absorbance was measured at 500 nm using a spectrophotometer, with a control tube as a reference. The condensed tannins content for diverse extracts was quantified in milligrams of catechin equivalents (CE) per gram of the dry weight of plant material (mg CE/g DW).

ABTS (ABTS Radical Scavenging Activity)

The evaluation of ABTS radical-scavenging activity, employing the Trolox antioxidant equivalent capacity test (TEAC), adhered to the methodology delineated by Elfalleh et al. [20]. This method hinges on the oxidation of ABTS (7 mM ABTS solution) triggered by potassium persulfate (2.45 mM K2S2O8), leading to the formation of the ABTS cation radical. The relative antioxidant capacity of the samples was established with Trolox serving as the benchmark for comparison. The assessment specifically focused on quantifying the prowess of antioxidants in neutralizing the ABTS cation radical. To facilitate the evaluation, the ABTS cation solution underwent careful dilution with methanol until an absorbance of 0.700 was attained. Following a 20-s vortexing, meticulous absorbance measurements were taken at 734 nm over a 5-min interval. The conclusive TEAC value for each antioxidant compound was deduced by rigorously comparing the ABTS cation discoloration with the reaction induced by Trolox. This comprehensive approach not only yielded valuable insights into the antioxidant potential of the plant extracts but also underscored the reliability of the results, with all measurements executed in triplicate for enhanced robustness.

High-Performance Liquid Chromatography (HPLC) Analysis of Phenolic Compounds

The LC–ESI-MS separation of phenolic extracts was conducted using a Shimadzu UFLC XR system (Shimadzu, Kyoto, Japan), which included a SIL-20AXR auto-sampler, a system controller SCL-10A, a CTO-20 AC column oven, an LC-20ADXR binary pump, and a quadripole 2020 detector system. A Discovery BIO Wide Pore C18 column (S250×4.0 mm id; 5 μm) was employed with a column temperature set at 40 °C. The injection volume was 5 μL, and the flow rate was maintained at 0.5 mL/min.

The mobile phases consisted of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). The analysis involved a linear gradient program: 0–14 min (10 to 20% B), 14–27 min (20 to 55% B), 27–37 min (55 to 100% B), 37–45 min (100% B), and 45–50 min (10% B). Mass spectrometry (MS) conditions utilized electrospray ionization (ESI) with a dissolving line temperature of 280 °C. Nitrogen was employed as the drying gas at a flow rate of 15.0 L/min, and the nebulizing gas flow was set at 1.50 L/min. LC ESI (−) MS mass spectra [M-H]- were acquired through Lab Solutions software (Shimadzu) [21].

Quantification and identification of phenolic compounds in the samples were achieved by comparing retention time and mass spectra with known standards analyzed under the same conditions [21].

Fatty Acid Profile

For the analysis of fatty acids through Gas Chromatography (GC), the initial step involves their conversion into non-polar, low molecular weight derivatives known as methyl esters. This transformation is essential for the subsequent identification of fatty acids by GC, where the retention time is compared to that of a control mixture of fatty acids. The method employed for extracting and methylating total fatty acids follows the analytical methods for the determination of fatty acid composition described in the European Economic Community Regulation [22]. In this process, a known amount (0.1 g) of the sample is introduced into a glass tube, followed by the addition of 3 mL of hexane and 0.5 mL of a 2 N solution of KOH. After vortexing, 1 μL of the upper phase containing the methyl esters is recovered using a syringe and then injected into the GC.

The GC–MS analysis of fatty acids involves the conversion of fatty acids into fatty methyl esters (FAMES) using potassium hydroxide in methanol. A Shimadzu GC2010 equipped with a fused silica capillary column (Supelcowax Tm10; 30 m × 0.25 mm × 0.25 μm film thickness) is employed for the analysis. The column temperature is programmed to increase from 50 to 230°C at a rate of 20°C/min, followed by 50–200°C at 25°C/min, and 200–230°C at 3°C/min, with a hold time of 21 min at 230°C. The inlet temperature is maintained at 250°C.

Helium serves as the carrier gas with a constant pressure of 68.3 kPa and a flow rate of 1.20 mL/min. In the split mode, 1.0 μL of each sample is injected, and the injector temperature is set at 230 °C. Mass spectrometry conditions include an ionization voltage of 70 eV, interface and ion source temperatures of 200 and 220°C, respectively, and a full scan mode in the m/z range of 35–500.

The polar stationary phase of the capillary column allows for the separation of methyl esters of fatty acids based on both the length of their carbon chain and their degree of unsaturation. Each fatty acid is represented by a peak on the chromatogram, and the identification of these peaks is achieved by injecting a control mixture of fatty acids under the same chromatographic conditions, considering the law of James and Martin [23]. The identification and estimation of fatty acid composition are facilitated using the GC MS solution program and the NIST11 and WILEY8 libraries.

Sugar Contents

To extract sugars, 6 g of each sample underwent a 2-h reflux with 100 mL of ultrapure water/ethanol (80/20, v/v). The resulting filtrate, obtained under reduced pressure, was evaporated to dryness. Subsequently, each sample was diluted and adjusted with ultrapure water to a final volume of 50 mL.

The mixture underwent centrifugation at 6000 rpm for 20 min, followed by filtration through a 0.45 μm membrane. High-performance liquid chromatography (HPLC) at room temperature was employed to analyze the sugar composition. Solvents, pre-filtered through a 0.45 μm membrane, were degassed in an ultrasonic bath Cleaner Model SM 25E-MT (Branson Ultrasonics Corporation, Danbury, CT) for 15 min.

The analysis utilized a Eurospher NH2 column (pore size: 100 Å, particle size: 7 mm, I.D.:250mm 4.6 mm) from Knauer, Germany. Acetonitrile and ultrapure water (80/20, v/v) served as the mobile phases. The liquid chromatography was equipped with a refractive index (RI) Detector K-2301 from Knauer, Germany. Throughout the experiment, the flow rate was maintained at 1 mL/min, with an injection volume of 2 mL [24].

Statistical Analyses

All analyses were performed in triplicate for the 23 genotypes. Statistical analyses were executed using SPSS for Windows (version 26.0). The dataset was assessed for normality using the Shapiro-Wilk test (α=0.05) and for homogeneity of variances using Levene’s test (α=0.05) before applying One-Way ANOVA (α=0.05). Subsequently, post hoc multiple comparison tests were conducted, with differences among genotypes deemed significant at the p < 0.05 level based on Duncan’s new multiple range test. Mean values and standard deviations were calculated, and data were expressed as mean ± SD. Multivariate analyses utilized the Pearson correlation approach, and results were presented as Heatmaps and PCA biplot performed by XlStat v2018.

Results

Total Phenolic, Flavonoids, and Condensed Tannin Contents

Table 1 displays the comprehensive phytochemical profiles of Vicia faba L. var minor genotypes. Remarkable diversity was observed in the total phenolic, flavonoid, and condensed tannin contents. The total phenolic content ranged from 10.90 (Vf3) to 19.86 mg GAE/g DW (Vf21), with an average of 16.46 mg GAE/g DW. Concurrently, the total flavonoid content exhibited variation, ranging from 5.25 to 6.96 mg CTE/g DW for Vf1 and Vf21, with an average of 6.27 mg CTE/g DW. Notably, condensed tannin levels showed significant diversity, with Vf10 recording the highest content (0.67 mg CE/g DW) and Vf22 the lowest (0.27 mg CE/g DW), averaging approximately 0.47 mg CE/g DW.

Table 1 Total phenols, flavonoids, condensed tannins contents, and ABTS radical scaving activity of Vicia faba var minor seeds
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ABTS Radical Scavenging Activity

The antioxidant capacity of V. faba seed extracts, assessed through the ABTS test (Table 1), demonstrated variability, ranging from 0.077 (Vf15) to 0.206 mM TEAC (Vf20), with an average of 0.146 mM TEAC.

Soluble Sugar Compositions

Examining the soluble sugar composition (Table 2), significant variations (p < 0.05) among genotypes were observed. Sucrose content ranged from 6.73 (Vf2) to 18.25 mg/g DW (Vf1), while the average glucose content was 3.521 mg/g DW.

Table 2 Sugar composition of Vicia faba var minor seeds
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Fatty Acid Composition

Detailed in Table 3, the fatty acid composition of the twenty-three V. faba genotypes revealed variations. Vf22 exhibited the highest percentage of total saturated fatty acids (SFA) at 27.71%, while monounsaturated fatty acids (MUFA) ranged from 23.22 to 38.78% for Vf23 and Vf4, respectively. Major fatty acids, including palmitic, stearic, oleic, linoleic, and linolenic acids, constituted about 93.57% of the total fatty acids.

Table 3 Fatty acid composition (%) of Vicia faba var minor seeds
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Phenolic Acid Composition

Table 4 presents the concentrations of individual phenolic compounds revealed by LC-MS analysis. Statistically significant differences (p < 0.05) among genotypes were observed. Quinic acid emerged as the most abundant phenolic acid, averaging 438.97 μg/g DW, with Vf9 recording the highest amount at 1636.31 μg/g DW. Other identified phenolic acids included p-coumaric acid, transfrulic acid, and syringic acid.

Table 4 Phenolic acids contents identified of Vicia faba var minor seeds
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Additionally, flavonoids such as catechin, epicatechin, quercetrin, quercetin, cirsiliol, and apigenin-7-o-glucoside were identified (Table 5). Notably, epicatechin and catechin were the most abundant flavonoids.

Table 5 Flavonoids contents identified of Vicia faba var minor seeds
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Multicriteria Analysis and Clustering

Multivariate analysis incorporating principal component analysis (PCA) and Heatmap (Figs. 1 and 2) provides insights into the relationships among assessed traits and distinct genotypic clusters. PCA revealed 45.20% variability, identifying four distinct groups among populations. Heatmap-based clustering identified three clusters, highlighting correlations between genotypes, phenolic acids, antioxidant activities, and sugar content.

Fig. 1
figure 1

PCA Biplot: Multivariate classification of the studied Vicia faba var minor populations based on the most discriminative traits

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Fig. 2
figure 2

Heatmap and hierarchical clustering for phenolic contents, antioxidant activities, and chemical composition of Vicia faba var minor. Genotypes names are presented on the horizontal axis; green color presents the highest contents of, total polyphenols (PT), phenolic acids (quercetin, quercetrin, epicatechin, transfrulic acid, α-coumaric acid, catechin (+) and quinic acid), antioxidant activities (ABTS), Fatty acids (MUFA and SFA), Soluble sugar (sucrose and glucose)

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The Heatmap (Fig. 2) allowed us to distinguish between three clusters: the first cluster was represented by Vf5, Vf6, Vf20, Vf9, and Vf14, correlated with high levels of phenolic acids, especially with catechin (+); genotypes Vf3, Vf4, Vf10, Vf21, Vf1, Vf12, Vf15, Vf16, Vf18, Vf11, Vf7, and Vf8 presented high sucrose and glucose contents, composing the second cluster; while the third cluster gathered the genotypes Vf23, Vf13, Vf17, Vf19, Vf2, and Vf22, correlated with high antioxidant activities and quercetrin content.

Discussion

Total Phenolic, Flavonoids, and Condensed Tannin Contents

The measured values for total phenolic, flavonoid, and condensed tannin contents in V. faba seeds align with established literature, emphasizing the prevalence of these bioactive compounds. Phenolic compounds, widespread in plant species, are notably concentrated in legumes, contributing significantly to their antioxidant capacity [9, 25]. Our findings reinforce the crucial role of these phenolic compounds in enhancing the health-promoting properties of V. faba seeds. However, the significant variation in flavonoid content observed in our study, diverging from existing research, underscores the complex relationship between genetic and environmental factors shaping flavonoid production in Vicia faba L. var. minor seeds. This variability initiates a detailed exploration into the regulatory mechanisms governing flavonoid biosynthesis, particularly in response to ecological stimuli. Notably, the findings of Abo Gamar et al. [26] align with our observations, shedding light on how interactions among climate change components significantly influence biochemical parameters, including flavonoid content. Their study contributes to our understanding of the relationship between environmental stressors, such as elevated temperatures and water scarcity, and the biosynthesis of flavonoids.

Moreover, the observed lower condensed tannin content compared to certain varieties underscores the need for genotype-specific selection, considering the significant impact of condensed tannins on protein digestibility [27, 28]. Beyond quantification, ecological factors such as soil composition and climate can significantly impact condensed tannin levels in plants. Additionally, genetic variations within plant species can also influence the production of condensed tannins. Understanding these determinants is crucial for developing strategies to enhance tannin levels for ecological and agricultural purposes [29].

Recent studies, such as those by Karataş Çalışkantürk et al. [30], further emphasize the necessity of identifying low-tannin genotypes for both nutritional and agronomic purposes, but the specific genetic markers contributing to this trait in V. faba remain an open avenue for exploration. In fact faba beans serve as a significant dietary source of various minerals and proteins. However, their nutritional value may be impacted by anti-nutritional factors, such as condensed tannins found in the seed testa [31]. These tannins are known to impede protein digestion and hinder the absorption of essential micronutrients in monogastric animals [32]. Consequently, low-tannin faba bean varieties are considered the most suitable option for consumption by both humans and animals alike [33].

ABTS Radical Scavenging Activity

The antioxidant capacity of V. faba seeds, as determined by ABTS radical scavenging activity, aligns with the well-established redox properties of phenolic compounds. These compounds play a pivotal role in reducing oxidative stress induced by reactive oxygen species [34, 35]. The observed values, consistent with previous research, underscore the potential application of V. faba as a natural source of antioxidants [36, 37]. However, a more profound exploration of the specific phenolic compounds contributing to the observed antioxidant activity is crucial. Recent discussions in the literature highlighted the therapeutic potential of individual phenolic compounds, emphasizing the need to elucidate the mechanistic underpinnings of their antioxidative effects [38, 39]. Notably the antioxidant capacity holds significant implications for various applications, particularly in food preservation and the development of antioxidant-rich dietary supplements. Extensive research has underscored the crucial role of antioxidants in preserving food quality by regulating rancidity, inhibiting the formation of harmful oxidation products, and extending shelf life [40, 41]. Among the diverse array of phenolic compounds, flavonoids, such as quercetin and catechin, exhibit notable antioxidant activity, effectively preventing fats and oils from oxidizing and preserving the freshness of foods [40, 41]. Understanding these effects is essential for optimizing food preservation techniques and enhancing the nutritional value of food products. Furthermore, harnessing the antioxidant potential of Vicia faba L. var. minor seeds and other natural sources can offer promising avenues for promoting human health and preventing food spoilage.

Soluble Sugar Compositions

The measured sucrose and glucose content in V. faba seeds not only corresponds with prior studies but also opens avenues for discussions on contemporary dietary concerns. Our results, consistent with the findings of Duc et al. [42] and Gdala and Buraczewska [43], highlight the importance of V. faba seeds as a low glycemic index food option. In the context of modern health considerations, the emphasis on reducing sugar consumption is a critical aspect of public health [44, 45].

Vicia faba is a rich source of plant-based protein, fiber, and various essential nutrients. For individuals with diabetes or those at risk of metabolic disorders, incorporating Vicia faba into their diet can have several benefits [46]. The high fiber content of Vicia faba can help regulate blood sugar levels and improve insulin sensitivity, which is crucial for managing diabetes and preventing metabolic disorders [47]. Additionally, the protein and nutrient content of Vicia faba can contribute to overall health and well-being [48].

Recent research on the glycemic index and glycemic load of foods in relation to health outcomes could provide valuable context [49].

Moreover, considering the rising global prevalence of obesity and metabolic syndrome, a more extensive exploration of the role of V. faba seeds in weight management and metabolic health is warranted [50]. The unique combination of low glycemic index and soluble fiber content in V. faba seeds positions them as potential contributors to strategies combating diet-related chronic diseases.

The unique combination of low glycemic index and soluble fiber content in V. faba seeds positions them as potential contributors to strategies combating diet-related chronic diseases. This specific nutritional profile aligns with the broader scientific evidence supporting the inclusion of legumes in a healthy diet to reduce the risk of cardiometabolic diseases, as discussed in the review by Bouchenak and Lamri-Senhadji [4]. By examining the specific attributes of V. faba seeds in the context of the broader impact of legumes on cardiometabolic health, it becomes evident that these seeds exemplify the potential of legumes as a dietary intervention for reducing the risk of chronic diseases.

Fatty Acid Composition

The comprehensive assessment of fatty acids in V. faba seeds provides insights into their potential as sources of bioactive lipids. The preference for plant-based ingredients, evident in the increasing demand for “clean label” compounds, aligns with the observed fatty acid profile in V. faba [5, 51,52,53].

While our findings confirm previous reports, indicating substantial levels of unsaturated fatty acids in V. faba seeds [54].

Unsaturated fatty acids, specifically omega-3 and omega-6 fatty acids, are known for their various health benefits. V. faba seeds, also known as broad beans, contain substantial levels of these unsaturated fatty acids, which can contribute to overall health and well-being [54].

Omega-3 fatty acids, such as alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have been extensively studied for their potential health benefits. These fatty acids have been shown to have anti-inflammatory properties, which can help reduce the risk of chronic diseases such as heart disease, arthritis, and certain types of cancer. Additionally, omega-3 fatty acids are essential for brain health and cognitive function, and they have been linked to a reduced risk of depression and anxiety [54].

The presence of substantial levels of unsaturated fatty acids in V. faba seeds suggests that consuming these seeds may have potential health benefits. Incorporating V. faba seeds into a balanced diet may contribute to reducing inflammation, supporting heart and brain health, and promoting overall well-being [54]. Numerous research studies investigated the health advantages associated with the consumption of Vicia faba seeds and their rich unsaturated fatty acid content. For instance, Prabhu and Devi Rajeswari [55] assessed the potential of faba bean’s high lipid content to provide substantial energy calories for humans. Similarly, Mejri et al. [56] confirmed the potential health benefits of consuming faba beans, citing their hypocholesterolemic properties and their role in reducing the incidence of cardiovascular diseases. Moreover, Khalil et al. [57] emphasized that the high fatty acid content of V. faba seeds made them a valuable component of a healthy diet.

Recent debates in the literature emphasize the role of unsaturated fatty acids, particularly monounsaturated and polyunsaturated fatty acids, in cardiovascular health, brain function, and inflammation modulation [58,59,60].

Vicia faba is recognized for its acid content, predominantly α-linolenic acid (ALA). ALA has been associated with cardiovascular benefits. Angeles et al. [61] suggest that the consumption of ALA-rich foods, such as those derived from Vicia faba, may contribute to reduced triglyceride levels and improved cardiovascular health.

Historically, various species within the Vicia faba have been employed for medicinal purposes and to address a diverse array of health issues globally [56].

In conclusion, the observed fatty acid composition in Vicia faba, particularly the presence of omega-3 fatty acids like ALA, holds promise for promoting cardiovascular, cognitive, and overall health.

Phenolic Acid Composition

Phenolic acids, identified as major components in V. faba seeds, warrant detailed discussions given their diverse sources and potential health benefits. The observed variations in phenolic acid profiles, in contrast to some previous studies, prompt considerations about the influence of geographical factors, cultivation practices, and extraction processes [62,63,64]. Recent reports on the bioavailability and health benefits of phenolic acids in dietary sources provided a backdrop to interpreting our findings [65, 66].

A deeper exploration of the potential bioavailability of these phenolic acids within the human body, considering factors such as gut micro bio interactions and metabolic pathways, contributed to the ongoing discourse on the health implications of dietary phenolic acids [67]. Moreover, the contrasting findings regarding the major phenolic acid identified in V. faba seeds compared to other regions underscore the need for comprehensive profiling in diverse geographical contexts. Recent advancements in analytical techniques, such as mass spectrometry, have facilitated more accurate identification and quantification of phenolic compounds, providing an opportunity to expand discussions on the precision and reliability of analytical methodologies in this context [68].

Exploring the specific health benefits associated with individual phenolic acids identified in V. faba seeds, such as their potential in preventing lipid peroxidation or exerting chemo-preventive effects against colon cancer, is warranted [69]. Recent research delving into the molecular mechanisms underlying these bioactive effects provides valuable insights [70].

Multicriteria Analysis and Clustering of Faba bean Genotypes

The application of PCA as a tool for understanding the genetic structure of faba bean genotypes based on biochemical parameters aligns with contemporary discussions on leveraging advanced analytics in crop improvement. Our study not only contributes to the growing body of literature on the genetic characterization of crop varieties [71, 72] but also extends the discourse to the relevance of such insights for crop improvement strategies. Recent studies, such as those by Ceramella et al. [73], highlight the potential implications of such genetic insights for tailoring crop improvement strategies.

The potential applications of PCA-derived information in precision agriculture and targeted breeding programs are interestingly used to highlight the significance of the multicriteria decision-making regarding the crops performers selection [74]. Several authors have questioned the widely accepted idea in the literature that nutrient levels in faba bean seeds are influenced by agro-ecological conditions. Instead, they found that precipitation levels during the growing season played a significant role in increasing soluble sugar content while decreasing the concentration of tannins and crude fat. This was supported by a negative correlation between crude fat concentration and precipitation, as well as a positive correlation with soluble sugars [75].

Recent advancements in crop genomics and the integration of molecular tools in crop improvement underscore the transformative potential of such insights for enhancing crop resilience to environmental stressors and optimizing yield [73, 76]. Furthermore, considering the dynamic nature of climate change and its impact on crop production, discussions on the adaptability of faba bean genotypes to changing environmental conditions would be pertinent [77, 78].

Conclusions

In this exploratory study, we endeavored to unveil the nutritive potential of twenty-three genotypes of Vicia faba L. var. minor seeds sourced from southern Tunisia, with a focus on their role as a rich reservoir of natural antioxidants. Our investigation spanned various nutritional facets, encompassing total phenolic and flavonoid content, condensed tannins, antioxidant activity, sugar composition, fatty acid profiling (analyzed through GC-MS), and the identification of individual phenolic compounds (scrutinized via LC-ESI-MS). The outcomes underscored substantial diversity across V. faba genotypes, revealing intriguing variations in each scrutinized compound.

Of particular interest is the notable abundance of total phenolics and saturated fatty acids discerned in the diverse methanolic extracts, particularly exemplified by the remarkable performance of genotype Vf22, which exhibited remarkably low condensed tannin content. This distinctive phytochemical composition, starkly influenced by total phenolic content, profoundly impacted the antioxidant activity measured through the ABTS assay. The elucidation of this genotype-specific phytochemical makeup not only contributes to our understanding of Vicia faba L. var. minor's nutritional profile but also paves the way for future endeavors aimed at enhancing the populations of this legume cultivated in southern Tunisia.

The significant variability unveiled in this study accentuates the potential for targeted breeding programs and agricultural interventions to fine-tune the nutritional attributes of Vicia faba genotypes, aligning them with specific dietary and health-related needs. This exploration serves as a foundational step, prompting a more comprehensive investigation into the genetic, environmental, and agronomic factors influencing the phytochemical composition of Vicia faba seeds. The insights garnered herein beckon further research avenues, heralding a promising trajectory for the advancement and optimization of Vicia faba L. var. minor populations in the agrarian landscapes of southern Tunisia.