One of the traditional ewe´s milk production is bryndza cheese or Oštiepok cheese (Zajác et al., 2019; Šnirc et al., 2019). Slovak bryndza cheese is natural, white, spreadable cheese, manufactured according to the traditional method. It is recognized in the European Union by Protected Geographic Indication (PGI) status as cheese produced in specified mountainous regions of Slovakia (Commission Regulation (EC) No 676/2008; Šaková et al., 2015; Zeleňáková et al., 2016) where unpasteurized ewe´s milk is processed to obtain the Slovak bryndza cheese. The mountainous regions of Slovakia differ in altitude, climate, geological, and vegetation profiles and there exists some scientific evidence about the variability of bryndza which is lacking in connection to common characteristics of this Slovak cheese (Šaková et al., 2015). Ewe´s cheese represents a matrix with a specific composition which reflected ewe´s milk and also by different autochthonous lactic acid bacteria (LAB) produce typical aroma profile of ewe´s lump cheese, barreled ewe´s cheese as well as bryndza cheese (Šaková et al., 2015; Sádecká et al., 2016). Kačániová et al. (2019) identified 870 isolates from coliforms, enterococci, lactic acid bacteria, and yeasts in Slovak bryndza cheese by MALDITOF MS profiling. Hafnia alvei, Klebsiella oxytoca, Lactococcus lactis, and Lactobacillus paracasei were the most frequently identified species of bacteria. LAB group was represented by Lactobacillus, Lactococcus, and Pediococcus.

Several volatile organic compounds (VOC) of cheese, including raw milk-based ewe´s cheese, are formed by proteolysis and by the subsequent transformation of amino acids (Ozturkoglu-Budak et al., 2016) to α-keto acids (Čaplová et al., 2018). Two different major pathways of amino acid degradation have been identified in Lactococcus lactis (Yvon and Rijnen, 2001). The first pathway is initiated by an elimination reaction of methionine catalyzed by amino acid lyases and leads to major sulphur aroma compounds (Dias and Weimer, 1998a; Dias and Weimer, 1998b). The second pathway is initiated by a transamination reaction catalyzed by aminotransferases, and has been observed especially for aromatic amino acids, branched chain amino acids, and methionine (Rijnen et al., 1999; Bourdat-Deschamps et al., 2004). The resulting α-ketoacids are then degraded to aldehydes, alcohols, carboxylic acids, esters, methanethiol, and other sulphur compounds. Most of these compounds are produced by enzymatic degradation but a few ones result from chemical degradation in particular oxidation (Nierop-Groot and de Bont, 1998, Nierop-Groot and de Bont, 1999). VOC are usually analyzed by gas chromatography after the extraction or pre-concentration of the volatile fraction. The most exhaustive methods for this purpose are high vacuum distillation (HVT), solventassisted flavour evaporation (SAFE), or solid phase microextraction (SPME) (Sádecká et al., 2014) combined with headspace. Sádecká et al. (2014) used SPME with gas chromatography-olfactometry (GC-O) for the determination of volatile odorants in May bryndza cheese. Depending on the degree of cheese maturation, from a GCO point of view, 25 olfactometric responses from groups of alcohols, aldehydes, esters, ketones, fatty acids, and hydrocarbons were recorded.

The aim of this study was to obtain, for the first time, parallel information of principal volatile organic compounds in bryndza cheese determined by an electronic nose and a gas chromatography mass spectrometry with head-space solid phase microextraction sample pretreatment and to confirm the possibility of the use of enose for monitoring bryndza cheese quality.

In this study, the aromatic profiles of ten bryndza cheese samples by e-nose and HS-SPME GC-MS were evaluated. Bryndza cheese samples were collected from the Slovak dairies. Bryndza cheese is a soft spreadable cheese, made from unpasteurized ewe´s milk or a mixture of ewe´s and cow´s milk. Kačániová et al. (2019) previously described the microbiota studies of ewe´s bryndza cheese from the same mountainous regions of Slovakia. LAB, enterococci, and yeasts Galactomyces candidus play a key role in flavor development during cheese ripening (Kačániová et al., 2020; Pangallo et al., 2014; Sádecká et al., 2019). The VOC´s are generated by the enzymatic degradation of amino acids in cheese, especially in cheese containing the only LAB.

The amino acid transamination is catalyzed by lactococci aminotransferases and it is the first step in the degradation of aromatic and branches-chain amino acids which are precursors of aroma compounds (Yvon et al., 1997; Tanous et al., 2005). The resulting α-ketoacids are then degraded to aldehydes, alcohols, fatty acids, esters, methanethiol, and other sulphur compounds (Savijoki, Ingmer and Varmanen, 2006).

The identification of the compounds determined by enose was performed by matching the measured peaks with Kovats ́retention indices with the NIST library. Aroma compounds identified in bryndza cheeses by e-nose are shown in Table 1. Total, 25 compounds with a discriminant >0.900 from class alcohols, esters, fatty acids, terpenes ketones, and aldehydes were identified. Ten compounds – 3-methyl butanal, 2-methyl-1-propanol, 2- butanone, 2-pentanone, ethyl acetate, isopentyl acetate, ethyl butyrate, acetic acid, butanoic acid, and butane-2,3- dione were identified in this study by e-nose and by gas chromatography-olfactometry previously described in bryndza cheese from Slovakia by Sádecká et al. (2014, 2016, 2019) and Šaková et al. (2015).

Identified compounds by e-nose with a discriminant >0.900 were selected, based on which the semi-qualitative evaluation was performed by the principal component analysis (PCA). Figure 1 displays result processed by the PCA technique of the aroma profile of bryndza cheese samples. The first dimension (PC1 86.927%) allows the separation of July2 and August2 (positive score) from the other samples of cheese (negative score). Among samples May1, July2 and September2 (negative score of the second dimension PC2; 9.402%) and other samples of cheese (positive score PC2) statistically significant differences (p <0.05) were evident in their aroma profiles.

We confirmed differences in aroma profiles between May bryndza and summer or winter bryndza produced from ewe´s milk by the first dairy. On the contrary, more aroma profile differences were in bryndza cheese produced from the mixture of ewe´s and cow´s milk by the second dairy.

The samples collected in May and June showed no significant differences in aroma profiles when compared to each other but the aroma profiles were significantly different in comparison with July, August, and September samples and at the same time, the samples collected from July, August, and September showed statistically different (p <0.05) aroma profiles compared to each other.

On the contrary, the position of May2, June2, June1 – September1 samples on the negative score of axis 1 and a positive score of axis 2 could be explained by its higher proportions of acetic acid, benzyl alcohol, butyl acetate, butanal, benzaldehyde, n-butanol, 2-pentanone, and hexanol. The August2 sample is positioned at the opposite on axis 1 due to its higher proportions in butanoic acid, 2- propanol, 2-methyl propanal, 3-methyl butanal, α-pinene, and ethyl acetate. The sample of July2, positioned on the positive score of axis 1 and the negative score of axis 2, contains higher proportions of isopentyl acetate, 2- butanone, 2-methyl propanol, furfural, and propanoic acid and the position of May1 and September2 samples on the negative scores of axes 1 and 2 could be explained by their higher proportions of ethyl butyrate, ethyl propanoate, propanal, 2,3-butandione, and acetaldehyde.

Semi-quantitative content of identified volatile organic compounds in bryndza cheese samples determined by GCMS is shown in Table 2. In total, 6 higher alcohols, 3 esters, 5 fatty acids, 3 ketones, and methoxy-phenyloxime were determined in the samples. Findings showed that principal odorants detected in all samples were acetoin (3.88% – 27.1 %), acetic acid (1.77% – 18.7%), dimethylsilanediol (1.07% – 6.93%), methoxy-phenyl-oxime (1.78% – 7.01%), and isopentanol (1.72% – 7.44%).

The bryndza cheese from the first dairy contains about 15% of acetoin collected from May and June, and, on the contrary, the next months its content decreased (10.6%, 7.04%, and 10.5% respectively). The content of acetoin in samples from the second dairy was more variable, its amount was decreased (3.88%) in samples collected from July production and increased in samples collected from August production (27.1%).

The content of acetic acid was in a range of 8.55% – 13.3% in samples produced in May-July, it was decreased in August (1.77% and 6.92%, respectively) and increased in September (18.0% and 18.7%, respectively). While acetoin and acetic acid were the most representative compounds in samples produced in May and June, samples produced in July and August had different profiles. While the samples from the first dairy had a higher content of acetoin and acetic acid (July) and 2-phenethyl acetate and 2-phenyl ethanol (August), the samples from the second dairy had a higher content of 2-butanone and acetic acid (July) and 2-butanone and acetoin (August). Bryndza cheese produced in September had a weaker aroma, but acetic acid and acetoin were identified in the higher amount than in the May and June samples. Statistical analysis by the Test of Tukey´s Multiple Comparison Test confirmed that the amount of eight aromatic compounds (Table 2) was influenced by the month of production. On the other hand, only two aroma substances were notably influenced by the kind of milk.

The compounds identified by the electronic nose with a discriminant >0.900 with ethyl acetate, isopentyl acetate, 2-butanone, acetic and butanoic acids, and butane-2,3- dione were confirmed by GC-MS. Several of the identified compounds (ethyl acetate, 2-phenylethyl acetate, ethyl propanoate, 2-methyl-propanol, 2-phenyl ethanol, 2,3- butanediol, 3-hydroxy-2-butanone (acetoin), 3-methyl butanol, 2,3-butanedione, acetic, butanoic, pentanoic, and octanoic acids) are known to be components of different foreign ewe´s cheese such as the Oscypek, Canestrato Pugliese, Fiore Sardo, Torta del Casar, Terrincho, Roncal, Manchego, and Pecorino Romano (Barron et al., 2005; Massouras, Pappa and Mallatou, 2006; Majcher et al., 2011; Sádecká et al., 2014; Delgado-Martínez et al., 2019).

Other identified compounds, benzaldehyde (Bourdat- Deschamps et al., 2004), 2-pentanone (Gallegos et al., 2017), hexanal (Gómez-Torres et al., 2016), 2,7- dimethyl-4,5-octanediol (Nájera-Domínguez et al., 2015) were previously described as secondary metabolites by LAB. Passerini et al. (2013) confirmed that strains of L. lactis with the citP gene and the citM-G cluster produced a larger amount of aroma-active compounds than the strains without this genetic information. Bozoudi et al. (2018) and Iussig et al. (2015) reported the quality differences of milk and dairy products from different grazing areas. The terpenoid composition (limonene, myrcene, carvone) of milk and cheese are directly transferred from ingested botanical species and free fatty acids (from acetic to dodecanoic acid) can also be effective to trace animal management and feeding systems (Moran et al., 2019). The free fatty acids are also precursors of methyl ketones, alcohols, lactones, and esters, so they may play an important role in the global aroma development of cheese (Delgado-Martínez et al., 2019). Fatty acids were the most abundant VOCs in the barrelled ewes' types of cheese (intermediate product in the production of winter bryndza) from Slovakia (Sádecká et al., 2016) and in the raw ewe´s milk cheese, Feta cheese and Torta del Casar (Bozoudi et al., 2018; Delgado et al., 2010; Delgado- Martínez et al., 2019). The identified 2-methyl propanal (Griffiths, 2010) was previously described as milk aromas. The other compounds, n-butanol, dimethylsilanediol, and methoxy-phenyl-oxime were identified in cheeses produced from pasteurization milk fermented with mixed cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) and with Dregea sinensis Hemsl. protease (Wang, Wang and Huang, 2017).

The e-nose technology in this study can detect the fingerprint of volatile organic compounds present in the headspace of the bryndza sample and parenica cheese previously described (Štefániková et al., 2019) by the means of a gas chromatography principle. Several studies used an e-nose method containing 10 metal-oxide semiconductors for characterization of the aroma profile of French cheese types, Danish blue cheese, or Pecorino cheese (Ghasemi-Varnamkhasti et al., 2019; Trihaas, Vognsen and Nielsen, 2005; Cevoli et al., 2011). There was used the e-nose with sensors in the above-mentioned studies, which could not determine or identify concrete volatile organic compounds and therefore there was a need to confirm the results by GC methods.

This study has proved for the first time the possibility of bryndza cheese quality evaluation using an e-nose with GC columns and FID detectors. The results were compared with gas chromatography with mass spectrometry. The e-nose method can determine the aroma profile of samples in a short time and the results may be supplemented by aensory evaluation by the assessors. The e-nose may take great advantages over GC-MS in distinguishing the integral aroma profiles, although it cannot identify the explicit volatile compounds of different samples.