Exploring the nutritional potential and antioxidant capacities of improved haricot bean (Phaseolus vulgaris L.) varieties cultivated in Ethiopia

Abstract

Despite the significant nutritional value they offer, unexplored improved haricot bean varieties from Ethiopia were the focal point of this study, which aimed to exclusively investigate the nutritional potential and antioxidant capacities of six varieties. The analysis revealed notable differences in moisture (9.53%–10.4%), ash (3.83%–5.14%), crude fibre (5.29%–9.09%), crude fat (0.760%–1.37%), crude protein (18.3%–25.6%), and utilisable carbohydrate (51.2%–61.0%). Calcium, iron, and zinc levels (mg/100 g) fell within ranges of 73.5–110, 4.64–9.07, and 2.56–4.49, respectively. Phytate (6.29–17.4 mg/g) and tannin (9.84–21.7 mg/g) contents were also assessed. All varieties exhibited significant total phenolics (245–622 mg gallic acid equivalent/100 g) and total flavonoids (107–216 mg catechin equivalents/100 g), reflecting robust antioxidant capacities through DPPH (42.2%–82.8%) and ferric reducing antioxidant power (17.8–41.5 μmol Fe(II)/g) assays. DAB 96, Tafach, and BZ 2 varieties stood out as promising choices for developing nutritionally dense and value-added food products.

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Introduction

Haricot beans, locally known as “Boleqe” in Ethiopia, are a significant leguminous plant with substantial nutritional value (Katungi et al., 2009). This legume species is widely cultivated globally, covering over 35 million hectares of land and producing >28 million metric tons annually, with Africa contributing 26.8% of this yield (FAOSTAT, 2022).

In Ethiopia, haricot beans hold historical importance as a staple food in human consumption (Alemu, 2017). They are versatile food ingredients used in various dishes like kurkufa and fossese, forming an essential part of the local diet (Teamir et al., 2003). These beans are multipurpose food constituents and are used in the preparation of various foods, such as ready-to-eat bakery foods, meat analogues, canned foods, and bakery products (Mecha et al., 2021).

Haricot beans offer multiple benefits in terms of nutrition and food security, particularly in developing countries like Ethiopia (Wafula et al., 2020). They are rich in proteins, minerals, starch, and dietary fibre, making them a valuable and affordable source of essential nutrients (Celmeli et al., 2018; Heredia-Rodriguez et al., 2019). Despite their nutritional advantages, haricot beans contain antinutritional factors such as tannins, lectins, and phytic acid, which can hinder mineral absorption (Carbas et al., 2020; Kan et al., 2017). On the other hand, they are also rich in phenolic compounds, known for their antioxidant and anti-inflammatory properties (Carbas et al., 2020).

Research has shown that different haricot bean varieties exhibit variations in nutritional content, antinutrient content, and antioxidant properties (Wodajo et al., 2021; Wodajo & Emire, 2022). These attributes play a crucial role in determining their overall health benefits and consumer acceptance, emphasising the importance of selecting suitable varieties for specific purposes.

In Ethiopia, recent research efforts have predominantly focused on yield, maturity period, and disease resistance when selecting haricot bean varieties, neglecting nutritional quality and antioxidant properties (Amsalu et al., 2018). Current data on the nutritional, antinutritional, and antioxidant properties of improved haricot bean varieties released between 2015 and 2017 are limited. This study aims to bridge this gap by exploring the nutritional, anti-nutritional, and antioxidant profiles of these haricot bean varieties, providing valuable insights to promote their consumption and enhance food security in Ethiopia.

Materials and methods

Sample collection and preparation

Six improved haricot bean (Phaseolus vulgaris L.) varieties, DAB 372, Tafach, DAB 96, BZ 2, Ado, and BLS 5 as indicated in Figure 1, sourced from Melkassa Agricultural Research Centers in Ethiopia, were selected for their qualities such as high yield, rapid ripening, disease resistance, and adaptability to various agro-ecologies. The bean varieties were grown under the same agro-ecological conditions and received the same agronomic practices. The variation in their compositional attributes was assumed to be due to differences in the variety. The seeds underwent manual cleaning to remove impurities, damaged beans, faded beans, and irregular shapes. Approximately 2.5 kg of clean seeds from each variety were milled into fine flour using an electric grinder (KARLKOLB D-6072 Dreich, West Germany), sieved through a 0.5 mm mesh, packed in airtight polyethylene bags, and stored at 4 °C for subsequent analysis.

Determination of the proximate composition

The proximate compositions, moisture, total ash, crude fibre, fat, and crude proteins were determined according to the Association of Official Analytical Chemists (AOAC, 2000). The utilisable carbohydrate content was calculated by difference, as reported in a previous study (Onwuliri & Obu, 2002).

Determination of mineral content

Mineral contents haricot bean flour samples were determined using the atomic absorption spectrophotometer (210 VGP). A 2 g of flour samples was digested using a concentrated HNO₃ solution (65%, v/v) followed by a 37% HCl solution (2.5 ml). Mineral contents were expressed as mg of a mineral per 100 g of the flour sample (mg/100 g) (AACC, 2000).

Determination of antinutritional factors

Phytate

Phytate content of haricot bean flour was determined by the method of Vaintraub and Lapteva (1988). Results were expressed as mg phytate per 100 g of the flour sample on dry matter basis.

Tannin

The tannin content of haricot bean flour sample was determined by the vanillin-HCl assay of Burns (1971) by extracting flour samples (2 g) with 10 ml 1% HCl in methanol for 24 hr with a mechanical shaker at room temperature. Catechin hydrate (200–1,000 μg/ ml) was used for plotting of a calibration curve. Results were expressed as milligram of catechin equivalents per 100 g of the flour sample (mg CE/100 g db).

Determination of phytate:mineral molar ratio

Molar ratios (MR) of phytatae to mineral: phytate to iron (Phy:Fe), phytate to zinc (Phy:Zn), phytate to calcium (Phy:Ca), and phytate^*calcium to zinc (Phy^*Ca:Zn) as an indicative intestinal mineral absorption were calculated after dividing the mass of phytate and minerals with their molecular and atomic mass (phytate = 660 g/mol; Fe = 56 g/mol; Zn = 65 g/mol; Ca = 40 g/mol) and dividing the MR of phytate with the individual mineral MR (Zhang et al., 2020).

Determination of phenolic contents and antioxidant capacity

Sample extraction

Sample extraction was carried out as described in the procedure of Ferreira et al. (2007). Flour sample (10 g) was mixed with 100 ml of methanol (99.8%) and incubated in an incubator shaker (ZHWY-103B) at 150 rpm for 24 hr at room temperature. Following the incubation, the supernatants were filtered with Whatman No. 1 paper. The filtered extracts were stored at 4 °C and used to determine the total phenolic contents (TPCs), the total flavonoid contents (TFCs), and antioxidant capacity.

Total phenolic content

The TPC was determined using the Folin–Ciocalteu colorimetric method as described by Xu and Chang (2007) using gallic acid as the standard. The absorbance was measured at 765 nm using a UV–vis spectrophotometer (T80, UK) against a distilled water blank. Total phenolic content was expressed as mg of gallic acid equivalent (GAE)/100 g of extract.

Total flavonoid content

Total flavonoid content of the haricot bean flour sample was determined by the aluminium chloride (AlCl₃) colorimetric assay as previously described Zhishen et al. (1999) using catechin as the standard. Total flavonoid content was expressed as mg catechin equivalents per hundred grams of sample (mg CE/100 g).

DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of the methanolic extract was evaluated following the method described by Kirby and Schmidt (1997). A 0.004% DPPH solution in methanol was prepared, and 4 ml of this solution was combined with 1 ml of the extract at varying concentrations (0.20–0.75 mg/ ml) in methanol. The mixtures were incubated in the dark at room temperature for 30 min. The radical scavenging activity was assessed using a UV–vis spectrophotometer (T80, UK) by measuring the reduction in absorbance at 517 nm (AS). The absorbance of the freshly prepared DPPH solution served as the control (AC). Butyl hydroxytoluene (BHT) was used as positive control. The extract concentration required to achieve 50% inhibition of DPPH radicals (IC50) was determined from the plot of DPPH inhibition percentage against extract concentration.

where Abc is the control absorbance (DPPH radical + methanol) and Abs is the sample absorbance (DPPH radical + sample).

Ferric reducing antioxidant power assay method

The ferric reducing antioxidant power (FRAP) assay was performed as previously described in Benzie and Strain (1996). Extract or standard solution (100 μl) with a FRAP reagent (25 ml of acetate buffer:2.5 ml of FeCl₃-6H₂O:2.5 ml of TPTZ solution). The mixture was incubated for 30 min in the dark, and the absorbance of the extract solution was read at 593 nm using a UV–vis spectrophotometer (T80, UK). Different concentrations of ferrous sulphate heptahydrate (0.1–1.0 μg/ml (FeSO₄.7H₂O) was used to construct a calibration curve, and the results were presented as μmol Fe(II)/g.

Statistical analysis

A one-way analysis of variance (ANOVA) was performed to evaluate the effect of variety on the nutritional composition, phytochemical content and antioxidant capacity of the haricot bean varieties using Minitab Statistical Software (version 21). Significant differences among the means were detected with the Tukey test at p ≤ .05. Pearson correlation was computed to study the relationships between different response variables.

Results and discussion

Proximate compositions

The proximate compositions of the haricot bean varieties exhibited significant differences (p ≤ .05), as presented in Table 1.

Moisture content

The moisture content of the studied haricot bean varieties ranged from 9.53% to 10.39%, which is within the acceptable range for safe storage. Previous studies by Ketema et al. (2019) and Kan et al. (2017) reported similar moisture content values, confirming the consistency of our results with previous research. However, de Barros and Prudencio (2016) observed slightly higher moisture levels (12.09%–14.47%). Moisture content is a crucial factor in determining the shelf life and storage stability of flour and other bean-based products. Keeping moisture content <10% is particularly important for preventing microbial growth and spoilage (Singh et al., 2005). This finding is applicable in food storage and processing industries, as low-moisture beans can reduce postharvest losses and enhance product stability.

Total ash content

The Tafach variety exhibited the highest ash content (5.14%), while DAB 96 had the lowest (3.83%). Ash content represents the total mineral composition in food products, indicating the presence of essential minerals such as calcium, iron, and zinc. The observed values slightly exceeded those reported by Ketema et al. (2019), Minuye and Bajo (2021), and Shimelis and Rakshit (2005) for Ethiopian haricot bean varieties (2.86%–4.70%). Higher ash content indicates a greater deposition of essential minerals, which are crucial for various biochemical functions in the human body (Chimphepo et al., 2021). The findings suggest that haricot bean varieties with higher ash content can serve as valuable ingredients in food fortification programs aimed at addressing mineral deficiencies, particularly in regions with high prevalence of malnutrition.

Crude fat content

The crude fat content varied significantly among the studied haricot bean varieties, ranging from 0.760% (BLS 5) to 1.37% (BZ 2). These values are consistent with the findings of Celmeli et al. (2018) but slightly lower than those reported by Ketema et al. (2019) and Minuye and Bajo (2021) (0.84%–2.86%). Legumes, including haricot beans, generally have low-fat content since they primarily store energy in the form of starch rather than lipids (Iwe et al., 2016). Low-fat content in beans implies better storage stability, as high-fat foods are more susceptible to oxidative rancidity (Syed, 2016). However, varieties with relatively higher fat content can contribute to enhanced flavour and palatability (Yegrem, 2021). The findings highlight the potential application of specific haricot bean varieties in different food products, where either low-fat or high-fat content is preferred, such as in flour blends or bean-based snacks.

Crude fibre content

Crude fibre content varied from 5.29% (DAB 96) to 9.09% (Tafach), slightly exceeding previously reported values for Ethiopian haricot bean varieties (4.07%–8.89%) (Ketema et al., 2019; Minuye & Bajo, 2021; Shimelis & Rakshit, 2005). The differences in fibre content may be attributed to variations in the seed coat thickness among the studied varieties (Sreerama et al., 2010). Dietary fibre plays a crucial role in digestive health, promoting bowel regularity and reducing the risk of constipation (Nwadike et al., 2018). Higher fibre content in haricot beans enhances their applicability in functional food formulations, such as high-fibre diets for individuals managing diabetes, obesity, or cardiovascular diseases. These results indicate that high-fibre haricot bean varieties can play a beneficial role in dietary interventions designed to enhance gut health and overall wellness.

Crude protein content

Protein is one of the key nutritional components of haricot beans, and its content among the studied varieties ranged from 18.3% (DAB 96) to 25.6% (BZ 2). This variation may be linked to differences in nitrogen-fixing abilities among the bean varieties (Oliveira et al., 2017). The protein content observed aligns with previously reported values for Ethiopian haricot bean varieties (17.96%–25.98%) (Ketema et al., 2019; Minuye & Bajo, 2021), although our highest recorded value was slightly lower than that of Carbas et al. (2020) (22.0%–31.3%). Protein-rich haricot beans can serve as a vital plant-based protein source, particularly in regions where animal protein is scarce or expensive. Moreover, high-protein varieties can be incorporated into composite flours, which are commonly used in formulating fortified foods for vulnerable populations, such as children and pregnant women. The significance of these findings extends to food security initiatives, where protein-rich legumes are recommended to combat protein-energy malnutrition.

Utilisable carbohydrate

The utilisable carbohydrate content ranged from 51.2% (Tafach) to 61.0% (DAB 96). Carbohydrate content in Ethiopian haricot bean varieties has been reported within the range of 58.2%−66.3% (Ketema et al., 2019). The variations observed in this study can be attributed to genetic differences influencing carbohydrate composition, particularly in terms of starch and sugar levels. Carbohydrates are the primary source of energy in human diets, and sufficient intake ensures that proteins are utilised for their primary functions rather than as an energy source (Awuchi et al., 2020). High-carbohydrate haricot bean varieties may be particularly beneficial in the preparation of weaning formulas and energy-rich breakfast meals aimed at combating protein-energy malnutrition. The results of this study are relevant for food industries producing ready-to-eat meals, flour blends, and infant nutrition formulations.

Mineral composition

The mineral contents of the mentioned varieties exhibited significant differences (p ≤ .05), as detailed in Table 2. Calcium content varied among the haricot bean varieties, with the highest content observed in Tafach (110 mg/100 g) and the lowest in DAB 96 (66.5 mg/100 g). Sahasakul et al. (2022) reported comparable or slightly higher results ranging from 31.97 to 242.17 mg/100 g for various bean cultivars. Differences in calcium content across varieties are likely influenced by the proportions of cotyledon and seed coat fractions, with seeds having higher seed coat percentages tending to possess elevated calcium levels (Zeffa et al., 2021).

The iron and zinc concentrations varied significantly, ranging from 4.64 to 9.07 mg/100 g for iron and 2.56 to 4.49 mg/100 g for zinc. BLS-5 exhibited the highest iron and zinc content, while DAB 96 had the lowest levels. Variability in iron and zinc contents among the varieties may be due to differences in genes related to mineral accumulation (Gunjača et al., 2021) and the mineral uptake efficiency of the seeds (Moraghan et al., 2002). Interestingly, higher iron and zinc levels were obtained compared to previously reported values (iron: 0.01−8.58 mg/100 g, zinc: 0.05−3.77 mg/100 g) by Anino et al. (2019) and Ketema et al. (2019). The results suggest that haricot beans are a significant source of zinc, potentially aiding in alleviating zinc deficiency in sub-Saharan Africa, where low nutritional zinc foods are prevalent (WHO, 2018). These varieties could play a vital role in enhancing the iron and zinc content of bean cultivars by identifying key genes responsible for mineral translocation.

Antinutritional factors

This study evaluated phytate and tannin contents in haricot bean varieties as shown in Table 3 to address their impact on nutrient bioavailability, particularly in populations with protein and micronutrient deficiencies. Significant variation (p ≤ .05) was observed, with phytate ranging from 6.29 (BLS 5) to 17.40 mg/g (DAB 96) and tannins from 9.84 (Tafach) to 21.7 mg/g (DAB 96). These differences are likely due to genotypic and environmental factors, as noted by Chávez-Mendoza et al. (2018). Phytate levels were slightly higher than those reported by Carbas et al. (2020) (5.42–15.8 mg/g) but lower than Wafula et al. (2022) (14.8–23.4 mg/g). Tannin content aligned with Wafula et al. (2022) (0.00–44.3 mg/g).

The findings highlight the importance of selecting varieties with lower antinutrient levels, such as BLS 5 and Tafach, to mitigate malnutrition risks. High phytate and tannin levels can inhibit nutrient absorption, worsening deficiencies in vulnerable populations. These results are valuable for breeding programs aiming to improve nutritional quality and for dietary recommendations in regions with high malnutrition rates.

Estimated mineral bioavailability

The MR of Phy:Zn, Phy:Fe, Phy:Ca, and PhyCa:Zn for mineral bioavailability significantly varied (p ≤ 0.05) from 13.9 to 67.7, 5.88 to 31.7, 0.14 to 1.58, and 348 to 1,126, respectively (Table 4). Except for BLS 5, all haricot bean varieties exhibited Phy:Zn MR exceeding critical limits, indicating poor zinc bioavailability. Previous studies reported Phy:Zn ratios for haricot beans ranging from 39.2 to 118.2 (Teshome & Emire, 2012; Wafula et al., 2022). Phy:Fe MR for each variety were above critical limits, suggesting low iron bioavailability, consistent with previous ranges of 15.83−37.1 (Teshome & Emire, 2012; Wafula et al., 2022). Phy:Ca and PhyCa:Zn MR exceeded critical limits for all varieties except BLS5, indicating low calcium and zinc bioavailability. High-calcium foods have been linked to phytate precipitation and reduced zinc bioavailability (Olaniyii & Rufai, 2020). Ratios exceeding critical limits may point to potential mineral deficiencies due to phytic acid (Abdulwaliyu et al., 2019). Overall, most varieties displayed low estimated mineral bioavailability likely due to high phytate content, highlighting the need for processing techniques to enhance mineral bioavailability before consumption.

Total phenolic and flavonoid content of haricot bean varieties

The total phenolic and flavonoid contents of the haricot bean varieties were significantly different (p ≤ .05), as indicated in Figure 2.

Total phenolic content

The TPC ranged from 245 (Tafach) to 622 mg GAE/100 g (BLS-5). Genetic factors may contribute to the variation in TPC among these varieties (Yang et al., 2018). According to Marathe et al. (2011), all varieties fell into the high phenolic category (>200 mg GAE/100 g). Several authors (Anino et al., 2019; Carbas et al., 2020; Rodríguez Madrera et al., 2021) have reported TPCs of Haricot bean cultivars ranging from 11 to 540 mg GAE/100 g, with the current study showing slightly higher values. The rich phenolic content in these beans highlights their medicinal value, suggesting potential health benefits from consuming these phenolic-rich varieties.

Total flavonoid content

Haricot beans displayed TFCs ranging from 107 to 216 mg CE/100 g, with BLS-5 and Tafach varieties showing the highest and lowest values, respectively, reflecting differences in seed coat colours (Karadžić Banjac et al., 2019). The study’s findings align with Carbas et al. (2020), reporting a range of 80−433 mg CE/100 g, slightly above values of 10.6−132.5 mg CE/100 g as reported by Rodríguez Madrera et al. (2021). Ramírez-Jiménez et al. (2018) suggested that incorporating haricot bean flour into snack formulations enhances the bioactive profile, emphasising the advantage of utilising varieties with higher TFC as functional ingredients for food enrichment.

Antioxidant capacity of haricot bean varieties

The DPPH scavenging capacity, FRAP and IC50 values of the haricot bean varieties were significantly different (p ≤ 0.05), as indicated in Table 5.

DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay

The percentage of DPPH at various concentrations (0.20−0.75 mg/ml) was evaluated, and among the concentrations used, the medium concentration (0.48 mg/ml) was chosen to indicate the scavenging activity potential. The DPPH quenching capacity of haricot bean varieties ranged from 42.2% (Tafach) to 82.8% (BLS 5) at the medium concentration of 0.48 mg/ml, indicating variability in antioxidant capacity possibly due to differences in phenolic compound profiles (Aquino-Bolaños et al., 2021). Our results are consistent with Chávez-Mendoza et al. (2018), reporting values from 25.2% to 98.1% for Mexican haricot bean cultivars. The robust antioxidant capacity of the BLS5 variety suggests its potential in the food industry for producing safer, antioxidant-rich products that could offer protection against oxidative damage from both internal and dietary sources.

The IC50 values ranged significantly from 0.057 to 0.616 mg/ml. Alarcon Esposito et al. (2022) and Karadžić Banjac et al. (2019) reported IC50 values between 0.008 and 12.9 mg/ml, consistent with our findings. However, our values were lower than those reported by Ombra et al. (2016), which ranged from 1.5 to 55.2 mg/ml. The IC50 values in this study suggest that a small amount of extract was effective in inhibiting 50% of the DPPH radical activity in most sample extracts, indicating strong antioxidant effects.

Ferric reducing antioxidant power

The FRAP assay revealed reducing power values ranging from 17.8 to 41.5 μmol Fe(II)/g, with Tafach and BLS5 showing the lowest and highest values, respectively. These findings are consistent with Rodríguez Madrera et al. (2021), who reported values of 6.1−42.3 μmol Fe(II)/g for haricot bean cultivars, but lower than the range of 12.1−99.4 μM Fe(II)/g noted by Kan et al. (2017). Higher FRAP values, as seen in BLS5, indicate stronger antioxidant capacity (Zhao et al., 2014), suggesting that this variety could be particularly beneficial for dietary applications aimed at enhancing antioxidant intake.

Correlation analysis

In Table 6, correlations among different response variables are presented. Tannin content exhibited a significant (p ≤ .05) negative correlation with protein (r = −0.54). Phytate content showed a significant (p ≤ .001) strong negative correlation with iron and zinc (r = −0.96 and r = −0.97) and a significant (p ≤ .05) negative correlation with calcium (r = −0.59), indicating strong chelation of cations such as calcium, iron, magnesium, and zinc by phytate (Kumar et al., 2021). Total flavonoid and phenolic contents displayed a significant (p ≤ .001) strong positive correlation with DPPH (r = 0.90 and 0.89) and FRAP (r = 0.91 and 0.97) values. Suttisansanee et al. (2021) also supported this, suggesting a strong positive correlation between phenolic compounds and antioxidant capacities.

Conclusion

The study on six different varieties of haricot beans cultivated in Ethiopia between 2015 and 2017 revealed significant variations in their nutritional, antinutritional, and antioxidant properties. These differences suggest opportunities for developing tailored food products, improving agricultural practices, and promoting healthier dietary choices in the future. The BZ-2 variety had the highest protein content, while the Tafach variety stood out for its high fibre, ash, and mineral contents. Tafach, BZ-2, and BLS 5 varieties showed low levels of phytate and tannin with high estimated mineral bioavailability, indicating their nutritional promise. Moreover, BLS 5 and DAB 96 varieties exhibited high phenolic contents and antioxidant properties, suggesting their potential as parents for developing new and improved haricot bean varieties through breeding. This comprehensive assessment of haricot bean varieties provides valuable insights for formulating nutritious food products in future studies.

Data availability

The data in this study are available upon request from the corresponding author.

Author contributions

Endris Hussen Ahmed (Conceptualisation, Methodology, Investigation, Formal analysis, Writing—original draft), Tilahun A. Teka (Data curation, Supervsion, Writing—original draft), Kumela Dibaba Tolera (Visualisation, Editing, Supervision), and Habtamu Fekadu Gemede (Supervision, Writing—review & editing). Endris Hussen Ahmed (Conceptualisation [lead], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [lead], Methodology [equal], Project administration [supporting], Resources [equal], Software [equal], Supervision [equal], Validation [equal], Visualisation [equal], Writing—original draft [equal], Writing—review & editing [equal]), Tilahun A. Teka (Conceptualisation [equal], Data curation [equal], Formal analysis [supporting], Funding acquisition [supporting], Investigation [equal], Methodology [equal], Resources [supporting], Software [supporting], Supervision [equal], Validation [equal], Visualisation [equal], Writing—original draft [equal], Writing—review & editing [equal]), Kumela Dibaba Tolera (Conceptualisation [supporting], Data curation [supporting], Formal analysis [supporting], Funding acquisition [equal], Investigation [supporting], Methodology [equal], Resources [supporting], Software [supporting], Supervision [equal], Validation [supporting], Visualisation [equal], Writing—original draft [supporting], Writing—review & editing [equal]), and Habtamu Fekadu Gemede (Conceptualisation [supporting], Data curation [supporting], Formal analysis [supporting], Funding acquisition [supporting], Investigation [supporting], Methodology [supporting], Project administration [supporting], Resources [supporting], Software [supporting], Supervision [equal], Validation [equal], Visualisation [equal], Writing—original draft [supporting], Writing—review & editing [supporting])

Funding

This work was supported by Samara University.

Conflicts of interest

The authors declare no conflicts of interest regarding the research, authorship, and/or publication of this manuscript.

Acknowledgements

The author would like extend heartfelt gratitude to their colleagues, advisors, and lab assistants for their invaluable support and contributions to this research work. Special thanks to the research team for their dedication and assistance throughout this project.

Ethics approval

This research does not require ethical approval.

References