## 3 Results and Discussion  
In this study, non-commercial extracellular serine protease produced by _Yarrowia lipolytica_ yeast was applied for casein degradation and production of casein chelate with iron chelating activity. The additives were applied to yoghurts production, and their usefulness as bioactive compounds with pro-health effects in yoghurt fortification was evaluated. In our previous research, the enzyme was also successfully applied for the degradation of proteins and the production of bioactive peptides of different activities [29; 30; 31; 32; 33]. The progress of casein degradation was analyzed by the degree of hydrolysis [DH], which gradually increased with the time of the process (Figure 1). The highest DH was determined in the hydrolysate after 3 h of the reaction and its level was 19.8%, while in the control sample it remained unchanged at the level of 4.3%. The high susceptibility of casein to serine protease from _Yarrowia lipolytica_ yeast was also confirmed by [PERSON] et al. [29], who showed the gradual degradation of the same substrate, starting at the beginning of the process, and correlated with the determined DH increase. Additionally, other authors [30; 34], who investigated the proteolytic activity of extracellular protease from _Yarrowia lipolytica_ against other substrates, showed that, among the analyzed proteins,casein, particularly its \(\beta\) fraction, was the most susceptible to degradation with the protease produced by utilized 30 _Y. lipolytica_ strains. The progress of hydrolysis was also monitored by determining the content of FAG soluble in water and in phosphotungstic acid (PTA) (Figure 2). It was found that the amount of water-soluble FAG increased during the hydrolysis. The content of low-molecular nitrogen fractions-soluble in PTA pointed to an intensive degradation of the analyzed protein. After 3 h of the process, FAG were determined at the level of 1493.0 umol Gly/g for water-soluble compounds and 363.0 umol Gly/g for PTA-soluble compounds, respectively. Many authors have shown that the enzymatic degradation of casein is an effective way of producing bioactive peptides [35]. Application of noncommercial protease isolated from _Yarrowia lipolytica_ may result in the formation of new or enhanced activities, including those with metal ions binding and transporting activity. Therefore, the obtained casein hydrolyses were also characterized by the iron chelating capacity (Fe\({}^{2+}\)). The chelating activity was correlated with the DH and it increased over time, although the highest changes were observed at the beginning of the process (Figure 3). Increased chelating activity in comparison to casein was detected in the hydrolyses after 1 h of  
Figure 1: Hydrolysis degree (%DH) of casein degraded with the extracellular serine protease preparation from _Yarrowia lipolytica_ yeast, at the dose of 500 U/g [blue], and in control sample: non-hydrolyzed isoelectric casein [orange]. Figure 2: Free amino group’s content (FAG) in casein hydrolyses obtained with the extracellular serine protease preparation from _Yarrowia lipolytica_ yeast, at the dose of 500 U/g soluble in water [blue] and in PTA [orange], and in the control sample—non-hydrolyzed isoelectric casein [grey]. the hydrolysis process (64.4 \(\upmu\)g Fe\({}^{2+}\)/mg protein), while the highest chelating activity was determined after 3 h of enzymatic degradation (66.1 \(\upmu\)g Fe\({}^{2+}\)/mg protein, Figure 3). The hydrolysate obtained after 3 h of hydrolysis and characterized by the highest Fe\({}^{2+}\) chelating activity was further subjected to FeCl\({}_{2}\) chelation. After spray drying both preparations, the 3 h hydrolysate and its chelate, were used as the bioactive additives in yoghurt production for dry matter (DM) elevation. The characteristics of analyzed additives showed that the levels of main components differed significantly (Table 1).