## 2 Materials and Methods
### Biochemical Characterization of White, Red, Black Kidney Bean Protein and Hydrolysates
#### 2.2.6 Biological Activity of Hydrolysates  
Antioxidant Activity  
DPPH Assay  
The radical scavenging activity of BKH, RKH, and WKH levels (50, 100, 200, 400, and 800 \(\mu\)g/mL) was deduced from the ability to convert the purple color of DPPH' to yellow as compared to TBHQ as synthetic antioxidant according to [PERSON], et al. [51] with some modifications. An aliquot (100 \(\mu\)L) of each hydrolysate level was mixed with 1 mL ethanolic DPPH and incubated for 30 min in the dark. The absorbance (Abs) was estimated at 517 nm against the control. DPPH' antiradical activity (%) was calculated in the following equation:  
\[\text{Radical scavenging activity }(\%)=\frac{(\text{Abs.control}-\text{Abs.sample}) }{(\text{Abs.control})}\times 100 \tag{5}\]  
\(\beta\)-Carotene/Linoleic Acid Bleaching  
The ability of BKH, RKH, and WKH (50, 100, 200, 400, and 800 \(\mu\)g/mL) to prevent discoloration of \(\beta\)-carotene was determined following [PERSON], et al. [52]. In brief, 0.2 mg of \(\beta\)-carotene was homogenized in 1 mL of chloroform, 20 mg of linoleic acid, and 200 mg of Tween 20 were added in a round-bottom flask. The chloroform was evaporated, and 50 mL of distilled water was added, and the mixture was vigorously stirred. An aliquot (0.5 mL) of each hydrolysate suspension was mixed with 3 mL of the \(\beta\)-Carotene/linoleic emulsion, and the absorbance (Abs0) was measured at 470 nm was recorded. The other samples were incubated at 50 \({}^{\circ}\)C for 120 min, and the absorbance was similarly recorded at 470 nm (Abs120). A control without hydrolysate was similarly assayed. Antioxidant activity was calculated as follows:  
\[\text{Antioxidant activity }(\%)=1-\frac{\text{Abs0 sample }-\text{ Abs120 sample}}{\text{Abs0 control }-\text{ Abs120 control}}\times 100 \tag{6}\]  
where Abs\({}_{0}\) sample is the absorbance of sample at 0-time, Abs\({}_{120}\) sample is the absorbance after 120 min, Abs\({}_{0}\) control is the absorbance of control at 0-time, and Abs\({}_{120}\) control is the absorbance of control after 120 min. Antimicrobial Activity  
Antibacterial  
Antibacterial activity was estimated by disc diffusion assay according to [PERSON], et al. [53]. Muller Hinton agar (MHA) plates were inoculated with activatedbacterial isolates. Discs (6 mm) were saturated with protein hydrolysates concentrations (50, 100, 200, 400, and 800 ug/mL) then placed on the surface of MHA plates and incubated for 24 h at 37 \({}^{\circ}\)C. The discs were saturated with sterilized distilled water used a control. The ruler was used to measure the zones of inhibition surrounding the discs (mm). The least concentration-inducing growth inhibition of bacteria was considered the minimum inhibitory concentration (MIC). The MIC was estimated by adding 50 uL of each level of tested hydrolysates (50, 100, 200, 400, and 800 ug/mL) to 9 mL MHB inoculated with 100 uL pathogenic bacteria. The tubes were incubated for 24 h at 37 \({}^{\circ}\)C, and the turbidity was recorded at 600 nm [54]. The least concentration totally kill the tested bacteria was, the minimum bactericidal concentration (MBC) was estimated by spreading 100 uL of each hydrolysate MIC on MHA plates' surface then incubated at 37 \({}^{\circ}\)C for 24 h and the bacterial growth was observed. Antifungal  
The tested _Candida_ and fungi species were cultured on Sabouraud dextrose agar (SDA) plates, and were incubated at 37\({}^{\circ}\) and 30 \({}^{\circ}\)C for 2, and 5 days, respectively. The antifungal activities of BKH, RKH, and WKH (50, 100, 200, 400, and 800 ug/mL) were evaluated using the disc assay [15]. The SD broth tubes inoculated with _Candida_ spp., or fungal spore suspension were incubated at 37 \({}^{\circ}\)C and 30 \({}^{\circ}\)C for 2 and 5 day, respectively to get inoculum (10\({}^{5}\) CFU ml\({}^{-1}\)) concentration. An aliquot (100 uL) of _Candida_ and fungal inoculum was spread over SDA plates. The saturated paper discs (6 mm) with BKH, RKH, and WKH (50, 100, 200, 400, and 800 ug/mL) were placed on the sides of cultivated SDA plates.