# Ripening of Hard Cheese Produced from Milk Concentrated by Reverse Osmosis
## 3 Results and Discussion
### Proteolysis During RO-Cheese Ripening
Nevertheless, conversely to \(\alpha_{\text{s1}}\)-CN degradation, significantly less \(\beta\)-CN was hydrolyzed in RO-cheese (45% of intact \(\beta\)-CN hydrolyzed by the end of ripening) compared to the levels reported in Old Saare and even in Gouda-type cheeses (78% and 60-50% of \(\beta\)-CN hydrolyzed by six months of ripening, respectively) [37; 38; 39; 28]. The greater activity of plasmin on \(\beta\)-CN could be expected in RO-cheese, as high cooking temperature inactivates inhibitors of plasminogen activators, leading to the conversion of plasminogen to the active plasmin [43]. Both the higher curd scaling temperature (52 \({}^{\circ}\)C) and higher pH during ripening (5.3-5.5) of Old Saare [28], as well as in Vasterbottenost cheeses (5.3-5.6) [36], than that of RO-cheese could have exerted an impact on more intensive primary hydrolysis of \(\beta\)-CN in those cheeses [43]. Considerable lower pH values in RO-cheese were evidently decisive for the reduced activity of the alkaline proteinase, plasmin [43], which resulted in impaired hydrolysis of \(\beta\)-CN in RO-cheese. Large peptides obtained from the primary hydrolysis serve as substrates for subsequent generation of shorter peptides and FAA by complex action of different proteolytic enzymes from LAB [39]. Figures 1 and 2b show that the production rate of chymosin-derived peptide \(\alpha_{\mathrm{s1}}\)-I-CN was the highest during the first two months, while during the next four months of ageing, the degradation rate of that peptide became higher than the production. At the beginning of the ripening, the content of plasmin-derived peptides \(\gamma\)-CNs decreased moderately during the first month, suggesting that they could have been more rapidly degraded further to shorter peptides by the LAB enzymes. The \(\gamma\)-CNs accumulated during further months of aging, showing an increase in the production rate in the period between four and six months (Figures 1 and 2b). The final step of proteolysis is the release of FAA, those enzymatic and chemical conversions to volatile compounds lead to the development of the characteristic cheese flavor [39; 44]. The total and relative (mol%) content of FAA released during six months of RO-cheese ripening is shown in Figures 3 and 4, respectively. The total content of the FAA increased from \(4.3\pm 0.3\) to \(149.9\pm 3.2\) mmol kg\({}^{-1}\) of cheese during maturation. The use of a thermophilic starter in cheese production could have considerably increased the total FAA content [45; 46] in RO-cheese. Nevertheless, the level of total FAA in 6-month-old RO-cheese was three-fold lower than that in hard Old Saare cheese (450 mmol kg\({}^{-1}\) of cheese) [28], it is also made with both mesophilic and thermophilic starters but shows intensive degradation of \(\beta\)-CN, unlike the RO-cheese. Moreover, the total FAA content in RO-cheese was somewhat lower than that in 6-month-old extra-hard Vasterbottenost (150-260 mmol kg\({}^{-1}\)) and semihard Herrgard (190 mmol kg\({}^{-1}\)) cheeses manufactured only with mesophilic starters [36; 46]. This clearly reveals that the lower total FAA content in RO-cheese can be attributed to the lower degree of hydrolysis of one of the main casein--\(\beta\)-CN--on the first stage of proteolysis mediated by plasmin activity. Figure 3: The change in total free amino acids (TFAA) content during RO-cheese ripening. The quantitatively dominating amino acids in the ripened 6-month-old RO-cheese cheese were Lys, Pro, Glu, Leu, and \(\gamma\)-aminobutyric acid (GABA) (Figure 4). The first four were found at very similar concentrations (14.2\(-\)14.8 mmol kg\({}^{-1}\) of cheese). It is noteworthy that the same four AA have been shown to be among the quantitatively dominating ones in matured Old Saare cheese [28]. A large amount of Pro (14.4 mmol kg\({}^{-1}\) in ripened RO-cheese) obviously produced by peptidases of _Lb. helveticus_, may introduce the sweet note in the cheese flavor profile [47].