## 3 Results and Discussion
### Proteolytic and ACE-Inhibitory Activity (%) During Ripening of Dutch-Type Cheese Models
The study results indicate that the proteolytic transformations occurring during cheese model ripening are significantly influenced by the adjunct cultures which intensify casein hydrolysis by releasing peptides responsible for ACE inhibition from its chains. Considering the proteolytic activity of the analyzed LAB strains established in the cheese models by determining the number of free amine groups, being a measure of the degree of proteolysis during ripening, its values were observed to vary and increase throughout the ripening of cheese models depending on the adjunct LAB strain used in cheesemaking. There were no statistically significant differences in the cheese models with the addition of _Lb. acidophilus_ 2499, _Lb. rhamnus_ 489, and _Lb. casei_ 2639 cultures. The IC\({}_{50}\) values of the studied cheese models determined throughout the ripening period are presented in Table 1. Immediately after cheesemaking, the lowest IC\({}_{50}\) value (0.7 mg mL\({}^{-1}\)) was determined in the cheese models with the addition of _Lb. acidophilus_ 2499 and _Lb. rhamnus_ 489. The addition of these two adjunct strains during cheesemaking had a significant effect on the produce of ACE inhibitors. An inconsiderably higher IC\({}_{50}\) value was determined in the cheese models with the addition of _Lb. delbrueckii_ 490 and _Lb. casei_ 2639. In turn, the highest concentration of peptides needed to obtain 50% inhibition of ACE activity was determined in the control cheese models (0.84 mg mL\({}^{-1}\)). The lowest IC\({}_{50}\) values were noted in the cheese models containing lactobacilli after 5 weeks of ripening. The results above indicate that the lactobacilli produced ACE inhibitors from the beginning of the ripening process. After 5 weeks of ripening, the lowest IC\({}_{50}\) value among all cheese models with the addition of LABs from the genus _Lactobacillus_ was determined in those containing _Lb. delbrueckii_ 490 (0.39 mg mL\({}^{-1}\)), and the highest value was determined in those with _Lb. casei_ 2639 (0.47 mg mL\({}^{-1}\)) (Table 1). IC\({}_{50}\) values were not significantly different between the control cheese model and the other three models with the addition of _Lb. acidophilus_ 2499, _Lb. rhamnus_ 489, and _Lb. casei_ 2639. Therefore, the low value of IC\({}_{50}\) in the control cheese model may indicate a crucial role of the starter cultures in the formation of peptides with antihypertensive properties during the ripening of the examined cheese models. An increase in the ACE-inhibitory activity in cheeses containing various adjunct strains of LAB was reported by [PERSON] and [PERSON] [22]. A comparative analysis of the ACE-inhibitory activity of the same cheese models in our study revealed the IC\({}_{50}\) parameter to be a better indicator when comparing enzyme inhibition effectiveness because it takes account of the concentration of peptides and dissolved proteins in a sample. The results of investigations reported in the literature point to vast differences in the ACE-inhibitory activity among various cheese species and to the usability of in vitro studies in the identification of cheese samples with a high ACE-inhibitory activity. The ability to identify the stage of cheese ripening at which the concentration of bioactive peptides is the highest may help in establishing the point in the ripening process when the cheeses exhibit the greatest health-promoting properties [23,24]. The literature data indicate that the presence of ACE inhibitors is affected to a greater extent by the cheesemaking technology (including the heat treatment of milk) [10,25], starter culture and adjunct LABs used [21,22,25], and ripening conditions (period and temperature) [1,9,26,27,28,26], than by cheese species. The shelf-lives of the majority of the Dutch-type cheeses produced and consumed across the globe are not long. The recommended minimal ripening period is 5 weeks or preferably even shorter.