2.7. Quantitative Evaluation of Starch and Protein Digestion Kinetics

Starch and protein digestion kinetics of the pulse samples were studied quantitatively by assessment of the released metabolites in the digestive supernatant (Section 2.7.1 and Section 2.7.2) and qualitative observing the undigested fraction under the light microscope.

Digested starch (%) was determined by measuring reducing sugars present in the digestive supernatant by mixing 2 mL diluted supernatant with 1 mL dinitrosalicylic color reagent for each digestion time [44]. After incubation at 100 °C for 15 min, 9 mL of MilliQ water was added, reactants were mixed and cooled to room temperature. The absorbance of the mixture was determined (540 nm), and reducing sugars were calculated using a maltose calibration curve (0.5–2.0 mg/mL). Duplicate measurements were performed on all samples. Multiplication of the maltose concentration with 0.95 resulted in digested starch. Digested starch (%) at different digestion time points was expressed as starch equivalents of reducing sugars in the digests divided by the initial amount of starch present in the sample, expressed in percentage, see Equation (1):

As protein digestion results in a mixture of heterogeneously sized peptides, different protein digestion evaluation approaches were used as detailed explained in our previous publication [31]. Protein digestion (%) was evaluated by measuring ‘readily bioaccessible’ and ‘digested soluble’ protein fractions [24,31] (free NH₂ groups) using a spectrophotometric method [45,46].

To determine the ‘readily bioaccessible’ fraction, trichloroacetic acid (TCA) (3.2% final concentration) was added to the digestive supernatant precipitating larger proteins and peptides. In this way, centrifugation separates larger fractions from amino acids and small oligopeptides, which represent the potentially readily bioaccessible fraction. To describe the digested protein by its monomeric constituents (amino acids), the digestive supernatant was hydrolyzed by HCl (6 N) at 110 °C for 16 h followed by rotary evaporation of the acid as explained in detail elsewhere (‘digested soluble protein’) [24]. This allows quantifying the degree to which protein has been solubilized and could leave the cell due to enzymatic degradation.

Free NH₂ groups in the protein fractions were determined by OPA spectrophotometric assay. To determine the total α-amino groups in the undigested sample (NH2(total)), the undigested sample (NH2(initial)) corresponding to the oral phase (2 min) or the digested soluble protein fraction (NH2(hydrolyzed)), respective amounts of the sample or digestive supernatant (5 mg, 0.5 mL, 0.5 mL) were hydrolyzed with 1 mL HCl (6 N) at 110 °C for 16 h, in duplicate. After acid removal, samples were diluted in Milli-Q water and filtered (0.25 µm pore size). OPA reagent was freshly prepared for each experiment, as described by Zahir et al. [46]. 3 mL OPA reagent were mixed with 400 µL sample (Milli-Q water (blank), L-serine (standards), or digestive supernatant). The mixture was incubated in the dark for 2 min at room temperature, and absorbance at 340 nm was evaluated. The concentration of free α-amino groups was calculated as L-serine equivalents (12.5–100 mg/L). Digested protein (%) was calculated and expressed as ‘digested soluble protein’ as shown in Equation (2) and ‘readily bioaccessible protein’ (Equation (3)). Measurements were performed in duplicates.