## 3 Results
### Monitoring the Process of SPC Hydrolysis  
Taking into account the ranking of enzymes suitable for soybean protein hydrolysis, as well as the number of peptide bitter-tasting indicators produced, the next step was to produce soybean hydrolysates to compare in silico and in vitro results. Five samples representing 0-SPC, B-SPC, F-SPC, P-SPC, and PK-SPC were subjected to RP-HPLC separation to observe the progress in SPC hydrolysis (see Figure 1 and Table 2). Three major time segments could be distinguished in all chromatograms, namely: 0.00-13.99, 14.00-39.99, and 40.00-60.00 min. The first time segment contained the highest peak eluting for about 10 min (not shown in Figure 1). It was an injection peak that could contain non-retained substances like the buffers used for protein/hydrolysate solutions and low molecular weight compounds present in protein concentrates [9].  
Three major time segments could be distinguished in all chromatograms, namely: 0.00-13.99, 14.00-39.99, and 40.00-60.00 min. The first time segment contained the highest peak eluting for about 10 min (not shown in Figure 1). It was an injection peak that could contain non-retained substances like the buffers used for protein/hydrolysate solutions and low molecular weight compounds present in protein concentrates [9]. In our previous experiment [9], the shape and area of an injection peak were approximately the same in all chromatograms. Therefore, peaks that could be observed within in 0.00 and 13.99 min were not considered for further interpretation. Peaks eluting between 40.00 and 60.00 min corresponded to high molecular weight compounds, like, e.g., proteins [9,22]. On the other hand, the chromatogram of the unhydrolyzed SPC sample contained much material with a short retention time (Table 2) as compared with the unhydrolyzed milk protein concentrate analyzed in our previous experiment [9].  
Figure 1: RP-HPLC (Reversed-Phase High Performance Liquid Chromatography) chromatograms of 0-soy protein concentrate (SPC) (**a**), B-SPC (**b**), F-SPC (**c**), P-SPC (**d**), and PK-SPC (**e**). Retention time range displayed in the Figure is between 14 th and 60 th minute. Chromatograms were acquired at \(\lambda=220\) nm as recommended by [PERSON] et al. [21]. Abbreviations: 0-SPC-soy protein concentrate, B-SPC, F-SPC, P-SPC and PK-SPC: bromelin-, ficin-, papain-, and proteinase K-soybean protein hydrolysate, respectively.  
The area of peaks within the range of 14.00-39.99 min and 40.00-60.00 min was almost equal in this experiment (see Table 2), whereas for milk protein concentrate, the ratio of total peak area within a shorter t\({}_{\text{R}}\) range to a longer t\({}_{\text{R}}\) range was only ca. 0.05 [9]. Taking the above into account, we introduced the parameter C (Equation (1)) to compare results obtained during experiments performed with soybean proteins (this experiment) and milk proteins [9].  
All chromatograms differed when looking at the time interval between 14.00 and 39.99 min. It was also reflected in the differences between the percentages of total peak areas observed in these time segments (Table 2). The order of the C parameter value was as follows: ficin > papain > proteinase K > bromelin, which suggested that ficin was the most efficient among the proteolytic enzymes used for hydrolysis of the soybean protein concentrate. The order of susceptibility of milk protein concentrate (MPC) [9] was reported to be as follows: proteinase K (C = 19.6) > ficin (C = 18.4) > papain (C = 7.8) > bromelin (C = 1.6). Ficin seemed to be sufficient for hydrolysis of both MPC and SPC. Proteinase K hydrolyzed MPC more extensively than SPC. There was a relatively high amount of unhydrolyzed proteins after the hydrolysis of both protein preparations by bromelin.