First Identification of Two Potent Thiol Compounds in Ripened Cheeses

Different methods of sample preparation followed by extractions with phydroxymercuribenzoate (pHMB) were used to investigate the occurrence of thiol compounds in ripened cheeses. The analysis of cheese extracts by GC coupled with PFPD, MS and olfactometry detections enabled the identification of ethyl 2mercaptopropionate (ET2MP) and ethyl 3-mercaptopropionate (ET3MP) in Munster and Camembert cheeses. The average concentrations of ET3MP measured in Munster and Camembert cheeses were 3.88 and 1.67 μg/Kg respectively while ET2MP was only found in trace amounts in Munster cheese. Introduction It is nowadays well established that hundreds of compounds are involved in the overall cheese flavour. Among them, sulphur ones are of particular interest because of their very powerful odours and low perception thresholds. As reviewed by Molimard and Spinnler (1) there are lot of evidence that sulphur odorants like methanethiol, polysulphides and S-methyl thioesters play an important role in the flavour of cheese. Conversely, until now only little attention has been given to the possible occurrence of thiols in cheese. To date, apart from methanethiol, only one thiol compound was identified in Gouda cheese (5). The difficulty of carrying out thiols isolation in cheese is due to the complexity of its matrix and to the very low concentrations of such compounds which make them undetectable by the classic means. The objective of this study was to isolate new thiols in cheese using a method inspired from Tominaga et al. (2, 3). The method was considerably modified to overcome the problems related with the fat content and heterogeneity of cheese. Experimental Initial sample preparation. Several varieties of cheeses were used for this study due to their strong sulphur odour (6).The first step of preparation consisted in removing from the surface of each sample thin layers that were subsequently cut into small cubes. After addition of dichloromethane (DCM) the mixtures were stirred for 18 to 20 h. Then, the volatile thiols contained in the organic phases were extracted twice using a pHMB solution and purified on a strongly basic anion-exchanger column. Extraction of thiols from the columns (6). The volatile thiols were released from the thiol–pHMB complexes by eluting an L-cysteine solution. The eluates were successively extracted by 4 and 3 mL of DCM and dried on anhydrous Na2SO4. They were finally concentrated under a nitrogen flux and 3 μL was injected in GC. Expression of Multidisciplinary Flavour Science 440 Identification and quantification of volatile thiols. The apparatus and analytical methods were recently depicted in (6).The volatile thiols were identified by comparing their retention times and their mass spectra in SCAN mode with those of the commercial references. The calculation of linear retention indices (RIMS and RIPFPD) and the quantification of volatile thiols were performed as previously described (6). Determination of the olfactive perception threshold. Ascending forced-choice procedures were used to evaluate the odour threshold value of ET3MP in cream (6). The best estimated thresholds were calculated according to Meilgaard et al. (7). Results Thiols identification and quantification. Among the cheese samples analysed, thiols could only be identified in Munster brand “Ermitage” and Camembert brand “Jean Vernier” (6). The PFPD chromatogram of the Munster cheese extract shows numerous peaks. After a comparison of its PFPD retention time with that of a pure reference and GC-MS identification, a first peak (RIMS= 935, RIPFPD= 1012) was identified as ET2MP, a potent odorant found in cashew apple juice (8), strawberry oil (9) and wine (10). Following the same procedure, a second peak (RIMS = 1024, RIPFPD= 1101) was identified as ET3MP, a volatile previously reported in Concord grape (11) and wine (10). As presented in (Figure 1) its mass spectrum (Figure 1A) tightly matched up to that of the commercial reference (Figure 1B). Figure 1. Mass spectra (MS/EI) of ET3MP detected in a final L-cysteine extract of Munster “Ermitage” (A) compared to that of its commercial analogue (B). To our knowledge, this is the first report of the occurrence of these two thiol compounds in cheese. Moreover, an additional attribute for their identification was represented by their odour quality assessed by GC-O which was identical to those of the pure references at the same concentration. The average concentration for ET3MP in the surfaces of Munster and Camembert samples were 3.88 ± 0.22 μg/kg and 1.67 ± 0.19 μg/kg respectively while ET2MP was only found in trace amounts in Munster. As cheese aroma formation often begins by the surface before diffusion into the core, and given that thiols are more sensitive to oxidation on the surfaces, the concentrations of these thiols in the whole cheeses may be higher than those of the surfaces. 50 100 0% 25% 50% 75% 100%