# Ripening of Hard Cheese Produced from Milk Concentrated by Reverse Osmosis
## 3 Results and Discussion
### Proteolysis During RO-Cheese Ripening  
Figure 1 shows the electropherograms of the intact CN fractions (\(\alpha_{\mathrm{s1}}\)-CN (8P and 9P), \(\alpha_{\mathrm{s2}}\)-CN (11P, 12P, and nP), \(\beta\)-CN (genetic variants A\({}^{1}\), A\({}^{2}\), and B), and para-\(\kappa\)-CN), and their primary hydrolysis products (\(\alpha_{\mathrm{s1}}\)-I-CN (8P and 9P), \(\gamma_{\mathrm{1}}\)-CN (A\({}^{1}\) and A\({}^{2}\)), and \(\gamma_{\mathrm{2}}\)-CN and \(\gamma_{\mathrm{3}}\)-CN) identified in RO-cheese during ripening. Intact \(\alpha_{\mathrm{s1}}\)-CN was subjected to more rapid degradation than \(\beta\)-CN: approximately 60% of \(\alpha_{\mathrm{s1}}\)-CN breakdown was observed within the first month and 70% was hydrolyzed by the end of the sixth month of ageing (Figures 1 and 2a). The peaks corresponding to \(\beta\)-CN also showed obvious changes throughout ripening, but still \(\sim\)55% of the initial \(\beta\)-CN remained intact by the end of the ripening period. The rate of the hydrolysis of the initial CN fraction in RO-cheese was the highest during the first months of ripening (Figure 2). Due to the lack of published data on RO-cheeses and the large diversity of the manufacturing parameters of traditional cheeses, a direct comparison of the ripening characteristics of our RO-cheese to those of other cheeses was fairly complicated. Nevertheless, it was instantly evident that the course of primary proteolysis in RO-cheese was comparable with those of traditional cheeses. In traditional cheese varieties with similar manufacturing technology, primary proteolysis is characterized by a rapid breakdown of intact caseins to high molecular weight peptides by an activity of both the rennet (chymosin) retained in the curd and the milk native proteinase plasmin. The addition of rennet based on the initial amount of milk used for RO-milk production resulted in an adequate degree of primary degradation of \(\alpha_{\text{s1}}\)-CN in our RO-cheese (Figures 1 and 2a), which is consistent with results on proteolysis in RO-Cheddar cheese [23]. Quite similar results on the breakdown of \(\alpha_{\text{s1}}\)-CN have been reported in a traditional hard cheese Old Saare, with manufacturing and ripening conditions very similar to our RO-cheese (a high curd scaling temperature and both mesophilic and thermophilic LAB as starters), where the same share---70% of \(\alpha_{\text{s1}}\)-CN--was hydrolyzed during six months of ripening [28]. In 26-week-ripened traditional extra-hard Vasterbottenost cheese, manufactured with high scaling temperature and mesophilic starter, the initial peaks of both \(\alpha_{\text{s1}}\)- and \(\beta\)-CN had almost completely disappeared according to the CE profiles [36]. Proteolysis in a typical Gouda-type cheese is determined by an ~70-80% decrease in \(\alpha_{\text{s1}}\)-CN content, mainly by the action of chymosin, already within the first month of ripening [37; 38; 39], which is consistent with our results for \(\alpha_{\text{s1}}\)-CN degradation; during the production of RO-cheese, the curd scaling temperature was higher (48 \({}^{\circ}\)C) compared to Gouda-type cheeses, which could have caused a partial inactivation of chymosin that affected the degradation of \(\alpha_{\text{s1}}\)-CN [40]. Indeed, a rather high percentage of \(\alpha_{\text{s1}}\)-CN breakdown determined in our cheese can be associated with a rather low pH of the cheese environment (in the range of 5.10 to 5.23 during the ripening period) being more favorable for the activity of chymosin as well as also the reversible and incomplete thermal inactivation of chymosin after cooking the cheese curds at 48 \({}^{\circ}\)C [40; 41]. The substantial contribution of plasmin to the primary proteolysis in cheeses with a high curd cooking temperature is evident [42].