*3.2. Proteolytic and ACE-Inhibitory Activity (%) During Ripening of Dutch-Type Cheese Models*

Results of determinations of the proteolytic and ACE-inhibitory activities in the studied cheese models during their ripening are presented in Figure 2. After one week of ripening, the highest ACE-inhibitory activity was found in the cheese models with the addition of *Lb. acidophilus* 2499 (Figure 2). In other cases of cheese models, the ACE-inhibitory activity was slightly higher than in the control cheeses (69%), and ACE inhibition ranged from 74 to 79%. The control cheeses were also characterized by the lowest proteolytic activity (0.270). This slightly higher value, reaching ca. 0.305, was determined for the cheese models containing adjunct cultures *Lb. acidophilus* 2499 and *Lb. delbrueckii* 490. In turn, the highest proteolytic activity was determined for the cheese models manufactured with the addition of *Lb. rhamnosus* 489 and *Lb. casei* 2639. Similar dependencies, but with higher values, were observed in cheese models after 3 weeks of ripening, with the highest proteolytic activity being demonstrated in the cheese models with adjunct cultures *Lb. rhamnosus* 489.

**Figure 2.** Angiotensin-converting enzyme (ACE) inhibitory activity (%) and proteolysis of water soluble extract (WSE) from Dutch-type cheese models during ripening (mean values and standard deviations). a–c: homogenous groups ACE, n = 6; A–D: homogenous groups proteolysis, n = 6.

One of the main factors affecting the proteolytic activity in cheeses is the water content. Water content of the analyzed cheese models (48–49%, data not shown) was higher than the typical water content of Dutch-type cheeses (42–45%). This was due to two reasons: firstly, the analyzed cheese models were not subjected to pressing and their brining was relatively short, and secondly, the higher water content of the produced models contributed to greater enhancement of proteolysis under model conditions.

After 3 and 5 week storage, a similar ACE-inhibitory activity was observed in the cheeses with adjunct cultures, despite the presence of different homogeneous groups in the Tukey tests (HSD). Cheese models with the addition of *Lb. acidophilus* 2499 were characterized by the best ACE-inhibitory capability among all cheese variants both after 1 week and 3 weeks of ripening.

After 5 weeks of storage, all analyzed cheese models were characterized by a high capability for ACE inhibition, exceeding beyond 90%. Similar ACE inhibition was noted in the cheese models with adjunct cultures of *Lb. acidophilus* 2499, *Lb. rhamnosus* 489, and *Lb. casei* 2639, however it was considerably higher than in the control cheese models. The addition of strains from the genus *Lactobacillus* to cheese models influenced the effectiveness of ACE inhibition because the tested strains contributed to a higher ACE inhibition compared to that achieved in the control cheeses at the end of ripening. The adjunct LAB strains also determined the proteolytic activity of the analyzed cheese models after 5 weeks of their ripening. The lowest proteolytic activity was determined in the cheese models with *Lb*. *delbrueckii* 490 and in the control models (Figure 2). A significantly higher activity compared to control cheeses was assayed in the cheese models containing adjunct cultures *Lb*. *acidophilus* 2499 and *Lb. casei* 2639 (ca. 0.460). In turn, the highest activity was determined in the cheese models with the addition of *Lb. rhamnosus* 489, and it was 1.5-fold higher than that determined in the control cheeses. Ong et al. [21] showed an increase in the content of inhibitors within the first 24 weeks of ripening of probiotic and control Cheddar cheeses, which remained at a similar level within the 12 subsequent weeks.

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. rhamnosus* 489, and *Lb. casei* 2639 cultures.

The IC50 values of the studied cheese models determined throughout the ripening period are presented in Table 1. Immediately after cheesemaking, the lowest IC50 value (0.7 mg mL−1) was determined in the cheese models with the addition of *Lb. acidophilus* 2499 and *Lb. rhamnosus* 489. The addition of these two adjunct strains during cheesemaking had a significant effect on the produce of ACE inhibitors. An inconsiderably higher IC50 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<sup>−</sup>1). The lowest IC50 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 IC50 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). IC50 values were not significantly different between the control cheese model and the other three models with the addition of *Lb. acidophilus* 2499, *Lb. rhamnosus* 489, and *Lb. casei* 2639. Therefore, the low value of IC50 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 Ong and Shah [22]. A comparative analysis of the ACE-inhibitory activity of the same cheese models in our study revealed the IC50 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.


**Table 1.** IC50 values of ACE-inhibitory activity from control cheese models and with adjunct culture (mg mL<sup>−</sup>1).

Mean values and standard deviations; a–d: means with different letters in line are significantly different (*p* < 0.05, n = 6); A–D: means with different letters in column are significantly different (*p* < 0.05, n = 6).

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) [1,9,26–28], 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. For this reason, investigations of cheeses of this type but with a ripening period that is a few or even a dozen times longer concern a relatively low number of cheeses available in retail. In our study, the cheese models were analyzed for 5 weeks and these analyses demonstrated an increasing activity of ACE inhibitors. This confirms that the Dutch-type cheeses should be ripened for the period of 5 weeks at a minimum.

It is difficult to establish a close relationship between the ACE-inhibitory activity in vitro and the hypotensive effect in vivo. This arouses some doubts concerning the use of the ACE-inhibitory activity in vitro as the sole criterion in the identification of substances with a potentially hypotensive effect, owing to the possibility of their physiological transformations in vivo [29]. This has been confirmed in a study conducted by Bernabucci et al. [30], where the authors demonstrated that the in vitro ACE-inhibitory activity of naturally-formed bioactive peptides in Parmigiano Reggiano (PR) and Grana Padano (GP) cheeses caused no hypotensive effect in vivo. Hence, the in vitro ACE-inhibitory activity cannot be used as the sole criterion in the evaluation of potentially hypotensive substances. Therefore, it is necessary to examine the beneficial hypotensive properties of bioactive peptides in vivo considering the possibility of their enzymatic degradation or diminished absorption under these conditions.
