# Ripening of Hard Cheese Produced from Milk Concentrated by Reverse Osmosis
## 3 Results and Discussion
### Volatile Compounds
#### 3.2.1. Carboxylic Acids  
The total amount of volatile carboxylic acids increased during the ripening of RO-cheese for six months, with a slightly higher content in 4-month-old than in 6-month-old cheese (Figure 5). The relative content of carboxylic acids was the highest in the 4-month-old cheese (46.3%TIC), but also retained high levels toward the end of the ripening period. Acetic acid was the most abundant compound with a very high relative content (1.3-41.0%TIC) during all stages of cheese maturation. The content of acetic acid increased greatly after one month of ripening (Table 2). Acetic and propanoic acids may have a microbial origin and could have been formed as a product of lactose metabolism [44]. Hexanoic acid was present in relatively low levels, exhibiting an ascending tendency solely in the middle of RO-cheese ripening until disappearing after the fourth month of ripening. Butanoic acid was present in comparable amount at the beginning of ripening and showed a substantial increase in amounts up to the fourth month of maturation and disappeared thereafter. Short-chain fatty acids and butanoic and hexanoic acids could be produced as a result of the enzymatic hydrolysis of triglycerides, which is also one of the biochemical pathways essential for cheese flavor development [35]. It is likely that the linear-chain fatty acids could have been transformed into esters [49] since their content was higher at the end of RO-cheese ripening (Figure 5). The main branched-chain fatty acids in RO-cheese--2- and 3-methylbutanoic acid--were present in substantial amounts after four months of ripening (Table 2) and could provide sweaty, sour, fruity, and buttery flavor notes [47; 49]. Methylated acids as well as methylated aldehydes and methylated alcohols have a proteolytic origin and, obviously, are produced by the catabolism of branched-chain amino acids (Leu, Ile, and Val) by aminotransferase [47; 49]. The prompt decreased in the relative content of Leu in RO-cheese from the fourth month of ripening could be associated with the higher content of 3-methyl-butanoic acid, 3-methyl-1-butanol, and 3-methyl-butanal in ripened 4- and 6-month-old RO-cheeses. In addition, the relative content of Val did not increase much after the second month of ripening, which correlated to the appearance of 2-methylpropanoic acid. Volatiles 2-methylbutanoic acid, 2-methyl-1-butanol, and 2-methyl-butanal originate from Ile, but we could not relate the occurrence of these components in the ripened RO-cheeses to the content of Ile, as it still steadily increased during the RO-cheese maturation. All volatile carboxylic acids identified in RO-cheese (except for propanoic acid) have been previously reported as aroma-active in a wide range of young, medium, and aged traditional Gouda-type cheeses by SPME-GC-Olfactometry [50]. In addition, the same acids (except for acetic and propanoic acid) have been detected in 6-week-old Gouda-type cheeses, while aged cheeses have not shown them at all [51].  
Figure 5. Changes in the content of the main chemical groups of the volatile compounds (AU \(\times\) 10\({}^{4}\)) identified during RO-cheese ripening. AU: arbitrary units (peak area).  
Acetic, butanoic, and hexanoic acids, as determined by SPME-GC-Olfactometry, have been found to contribute greatly to the characteristic cheesey sharp and mild to strong savory aroma of typical hard Parmesan and Grana Padano cheeses [52]. Along with the mentioned carboxylic acids, octanoic and decanoic acids were among the abundant aroma compounds in Parmigiano-Reggiano of different ages reported by [PERSON] et al. [53].