## 2 Materials and Methods
### Protein and Amino Acid Analysis of Soybeans and Pea Beans
#### 2.3.1 Protein Dosage by the Bradford Method  
The absorbances of the samples were measured using a Perkin Elmer Life and Analytical Science Molecular Absorption Spectrophotometer LAMBDA 25 (\(\lambda\) = 595 nm). The method is reproducible, has little interference (sodium, potassium and sugars do not interfere, only strongly alkaline solutions or detergents, such as sodium lauryl sulfate or detergents in cleaning products) and is also fast (the reaction is practically complete after 2 min, and the coloration is stable for 1 h) [38]. Preparation of the Bradford reagent: 0.0125 g of Coomassieie Brilliant Blue G-250\({}^{\text{\textregistered}}\) (Sigma-Aldrich, Darmstadt, Germany) in a Berzelius beaker was weighed on the analytical balance, and 5 mL of ethanol was added. After dissolution, 10 mL of phosphoric acid was added. The mixture was transferred to a 100 mL volumetric flask, and the Berzelius beaker was rinsed several times with ultrapure Milli-Q\({}^{\text{\textregistered}}\) water. It was brought to the mark with the same solvent and was kept for 24 h in the refrigerator, for stabilization. The reagent was fresh for one week. The calibration curve was obtained according to the method first published in 1976; the standard used was bovine serum albumin (BSA) [38]. Two stock solutions of 1 mg/mL were prepared, in Tris-glycine or water, from which successive dilutions were performed to obtain the calibration curve. BSA stock solutions: 0.0250 g of BSA was introduced into a 25 mL volumetric flask. Then, it was dissolved in solvent (Tris-glycine buffer or ultrapure water) and was made up to the mark using the same solvent. The BSA standards were prepared from the stock solution, according to Table 3, at a final volume of 1.25 mL. Then, the solution was made up to 5 mL using the Bradford reagent (in the volumetric flask). The analyzed samples were prepared by mixing 1.25 mL (\(V_{\text{P}}\)) protein extraction solution with Bradford reagent in a 5 mL volumetric flask. It was brought up to the mark with Bradford reagent and was read in the spectrometer at \(\lambda\) = 595 nm after 15 min. The coloration was stable for 1 h. The analyses were carried out in triplicate. Protein Determination Using the Kjeldahl Method  
Sample digestion: 0.5000 g sample and 4.0000 g disaggregation mixture (100 g potassium sulfate, 5.0 g copper sulfate, 2.5 g selenium) were weighed on the analytical balance. Then, 15 mL of concentrated sulfuric acid was added. Next, the mixture was introduced into the Digesdahl 23130-21 apparatus. The mixture was subjected to digestion (mineralization) at 440 \({}^{\circ}\)C by periodic stirring. The temperature was maintained until the sample became clear after approximately 15-20 min (the sample became clear, transparent, or colored green, the shade varying depending on the sample). Then, it was cooled to room temperature. \begin{table}
\begin{tabular}{c c c} \hline
**Concentration of BSA (\(\mu\)/mL)** & **Stock Solution Volume of BSA (\(\mu\)L)** & **Solvent Volume (\(\mu\)L)** \\ \hline
50 & 62.5 & 1187.5 \\ \hline
100 & 125 & 1125 \\ \hline
200 & 250 & 1000 \\ \hline
400 & 500 & 750 \\ \hline
600 & 750 & 500 \\ \hline \end{tabular}
\end{table} 
Table 3: Preparation of BSA standard solutions for protein analysis using the Bradford method. The resulting mixture after digestion was transferred into a 500 mL distillation flask. The digestion vessel was rinsed several times with water. A total of 50 mL of 4% boric acid solution and a few drops of indicator mixture were introduced into the collection vessel, and 50 mL of 30% NaOH solution (prepared by diluting a 50% sodium hydroxide solution) was added to the funnel. The water tap was turned on to constantly cool the descending refrigerant. The alkaline solution was allowed to drip slowly into the still mixture. Water was introduced successively into the funnel and allowed to flow up to half the capacity of the distillation vessel. A small amount of water was kept in the funnel.