Investigation of kidney bean protein and its phenolic interaction

In recent years, the increasing consumer demand for sustainable, functional, and health-promoting food products has brought plant-based proteins to the forefront. However, limitations in solubility, digestibility, and oxidative stability restrict the functional applications of plant-based proteins in food systems. To overcome these limitations, enhancing the functional properties of proteins through interactions with natural phenolic compounds has emerged as a significant research area. In this context, kidney bean (Phaseolus vulgaris L.), which has a high protein content and a functional amino acid profile, stands out as an alternative plant-based protein source. In this study, non-covalent complexes were formed between kidney bean protein isolate (KPI) and gallic acid (GA), and their structural and functional properties were investigated. From 1 kg of raw kidney beans, an average of 104.45 g of protein isolate was obtained, corresponding to an approximate 3.15-fold increase in protein content, with a yield of 37.05%. The protein purity of KPI extracted via alkaline extraction and isoelectric precipitation was determined as 85.56% (dry basis). KPI solutions were prepared at a concentration of 20 mg/mL in 10 mM sodium phosphate buffer (pH 7.0) and stirred overnight at 4 °C. GA solutions at varying concentrations (0, 250, 500, 750, 1000 µmol/g) were mixed with KPI at a 1:1 ratio and incubated at 25 °C for 2 hours. The resulting complexes were purified in dialysis bags (6–8 kDa cut-off) for 24 hours, frozen at –18 °C, and lyophilized. To ensure a pure and controlled matrix for the complexes, the physical and chemical compositions of both defatted kidney bean meal and KPI were analyzed. While the moisture content was 11.23% in raw beans, it decreased to 0.45% in the isolate. Similarly, on a dry basis, the fat content decreased from 1.21% to 0.47%, while the ash content increased from 3.71% to 5.63%. The protein content increased from 27.16% in defatted beans (dry basis) to 85.56% in the isolate. In the complex samples, possible changes in protein content due to interaction were also measured: protein content decreased from 83.81% in the control group to 81.96% in the group 1000. Comprehensive functional analyses were conducted to examine the effects of GA on the structural and functional properties of KPI. With increasing GA concentration, surface hydrophobicity decreased from 765.72 to 444.57, and disulfide bonds decreased from 2.82 to 1.56 µmol/g, while free sulfhydryl groups increased from 9.90 to 27.47 µmol/g (p < 0.05). The water holding capacity increased from 24.56% to 32.49% with higher GA concentration, whereas the oil holding capacity decreased from 92.67% to 54.08%; the least gelation concentration decreased from 7.50% to 5.00%. Solubility showed a significant increase from 82.04% to 99.39% (p < 0.001). Emulsion activity increased from 73.55% to 100%, and emulsion stability improved from 36.30% to 76.61%. Foam capacity increased from 30.76% to 46.15%, and foam stability rose from 17.65% to 26.31% (p < 0.001). The complexes also showed significant effects on total phenolic content and DPPH antioxidant activity during in vitro extraction and digestion phases (p < 0.05). The total phenolic content in the extractable fraction increased from 4.54 to 31.75 mg GAE/g, while it rose from 36.54 to 47.40 mg GAE/g in the intestinal phase. DPPH radical scavenging capacity increased from 10.42% to 57.49% initially and from 33.96% to 89.13% in the intestinal phase. Structural analyses using FTIR and intrinsic fluorescence spectroscopy revealed shifts in peak intensities and a decrease in fluorescence intensity from 36115 to 28604 a.u., confirming structural modifications. DSC analyses indicated that the denaturation temperature increased from 86.02 °C to 94.89 °C, while enthalpy decreased from 218.29 to 171.68 J/g. Finally, SEM microstructure analysis showed that in group 750, protein particles were more tightly bound and formed a more compact structure. The non-covalent interaction between GA and KPI led to structural changes such as an increase in free sulfhydryl groups, a decrease in disulfide bonds, and strengthened hydrogen bonding. These structural modifications enhanced hydrophilicity and compactness on the protein surface, resulting in significant improvements in solubility, foaming, emulsion stability, and gelation properties. Thermal stability improved by approximately 10%. In vitro digestion tests confirmed that the KPI-GA complexes exhibited over 30% higher antioxidant activity compared to the control throughout the digestion process (p < 0.05).