# Chemometric Classification of Feta Cheese Authenticity via ATR-FTIR Spectroscopy
## 4 Discussion
Additionally, freeze-drying improves sample homogeneity by removing structural water and reducing surface variability, which likely enhances the reproducibility and discriminative power of the FTIR measurements. Partial least squares discriminant analysis of FTIR data highlighted several spectral regions that contributed to distinguishing Feta from other white brined cheeses. Key features were associated with protein amide bands, carbohydrate linkages, phosphate groups, and lipid structures. All preprocessing methods identified strong discriminant bands near \(\sim\)2920 cm\({}^{-1}\) (C-H stretching in lipids), \(\sim\)1650 cm\({}^{-1}\) (Amide I from proteins), and within the 1300-900 cm\({}^{-1}\) region. Interestingly, the use of derivative spectra not only improved model performance but also emphasized different spectral regions as being most influential in distinguishing between classes. Derivative processing enhances the resolution of overlapping bands, making it easier to distinguish subtle features that are otherwise obscured in the raw spectrum. In complex samples like cheese, where signals heavily overlap, this can isolate smaller but relevant variations. Moreover, the derivative spectrum highlights rate-of-change in absorbance, which often corresponds to subtle changes in band shape--features that may be more diagnostic of compositional differences than absolute absorbance values. Second derivative spectral variables with VIP scores exceeding 2 were observed primarily in the lipid (C=O at \(\sim\)1740, CH\({}_{2}\) at \(\sim\)1450 cm\({}^{-1}\)) and protein-related regions (Amide III near \(\sim\)1650, \(\sim\)1540, and \(\sim\)1240 cm\({}^{-1}\)), indicating that differences in fat and protein composition were the most discriminative features. These results are consistent with known compositional variations across cheese types and milk species. Patterns reflect the underlying chemical composition of the samples and align with markers of milk origin and processing. In particular, the region between 750 and 770 cm\({}^{-1}\) can be attributed to aromatic C-H out-of-plane bending [34], likely originating from amino acids such as phenylalanine and tyrosine in casein and whey proteins [35]. The region around 880-930 cm\({}^{-1}\) can also represent signals from aromatic amino acids and but also glycosidic C-O-C linkages in lactose [36]. Another notable region lies between 990 and 1075 cm\({}^{-1}\), which is dominated by carbohydrate-related vibrations. This includes a prominent C-O stretching band near 1040 cm\({}^{-1}\), commonly attributed to lactose in dairy products. In cheeses--particularly fresh white varieties--this region reflects residual lactose or other sugars, as well as C-O and O-H vibrations associated with organic acids such as lactic acid [37]. The 1160-1186 cm\({}^{-1}\) region may represent a combination of ester and glycosidic C-O stretching, reflecting both milk fat and carbohydrate contributions. High VIP scores were consistently found in the 1200-1300 cm\({}^{-1}\) window, where Amide III, phosphate, and fatty ester vibrations overlapped. Further discriminatory features were observed between 1427 and 1727 cm\({}^{-1}\), including Amide I, aromatic ring modes, and ester carbonyls. The absorption band at ~1745 cm\({}^{-1}\) corresponds to the C=O stretching vibration of ester functional groups in lipids, particularly triglycerides, as documented in studies on cheese and food matrices [13]. The high-wavenumber range 2952-2964 cm\({}^{-1}\) corresponds to CH\({}_{3}\) and CH\({}_{2}\) asymmetric stretching--a reliable indicator of fatty acid chains in triglycerides. In general, comparable electrochemical markers have been reported in studies of PDO mozzarella, where regions around ~1745, 2925-2850, and 1500-900 cm\({}^{-1}\) were effective in discriminating milk origin [13; 17; 38]. Unlike prior FTIR-based studies which focused on general compositional profiling or microbiota analysis, the present work offers several novel contributions to the authentication of PDO Feta cheese.