After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. Because free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21) and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by nuclear magnetic resonance and high-resolution mass spectrometry. High-performance liquid chromatography−positive electrospray ionization−tandem mass spectrometry evidenced both for the first time in hop. Both S conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Similar to G-23, G-21 appeared ubiquitous to all varieties. Of all of the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg/kg of dry matter. Cys-21 was found in all samples at a much lower concentration (up to 0.2 mg/kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed for the confirmation that brewing yeast is able to release free compound 21 from them.

The occurrence of a substantial pool of cysteinylated and glutathionylated forms of polyfunctional thiols has been evidenced for several dual-purpose hop varieties, and so is the ability of Saccharomyces cerevisiae yeast to release free thiols from these forms through fermentation. The present work aimed to investigate the effect of temperature, wort density, maturation time, and strain on the efficiency of free thiol release by S. cerevisiae yeasts. Model media at 12, 15, or 17°P were spiked with three cysteinylated (Cys-) or three glutathionylated (G-) sulfanylalkyl alcohols (Cys-or G-3-sulfanylpentan-1-ol, 3-sulfanyl-4-methylpentan-1-ol, and 3sulfanylhexan-1-ol), fermented for 7 days at 18, 24, and 28 °C, and kept at 4 °C for varying number of days. The released sulfanylalkyl alcohols and their corresponding acetates were extracted with a Ag-ion SPE cartridge and analyzed by gas chromatography−pulsed-flame photometric detection. The wort density and yeast strain greatly affected the acetate/alcohol ratio. This ratio varied from 1 to 80% according to the yeast strain and was at its highest at 17°P and 24 °C. Maturation appeared as the crucial step for free thiol excretion from yeast cells (no thiol was recovered in the fermented worts without maturation). Among the five yeasts tested, the yeast strain SafAle K-97 released the highest level of sulfanylalkyl alcohols into the medium (up to 0.45% of the added cysteinylated adducts and 0.08% of the glutathionylated adducts), whereas S-33 or S-04 should be preferred when release of esters is sought out (release efficiencies up to 0.35% from cysteinylated adducts and 0.02% from glutathionylated adducts are observed if both the alcohol and its acetate are considered).

The contribution of polyfunctional thiols (PFTs) to the overall flavor of a lager beer has been well documented, but their origin remains unclear. In comparison with Saccharomyces cerevisiae strains previously investigated (same conditions: 7 days at 24 °C, 3 days at 4 °C), we examined how Saccharomyces pastorianus yeasts are able to produce these PFTs from cysteinylated (Cys-) and glutathionylated (G-) conjugates. Up to 0.35% release was observed from G-conjugates against less than 0.08% for all yeasts studied so far. Lowering the wort nitrogen level and the fermentation temperature (12 °C) strongly increased the release efficiency and the ester/alcohol ratio from Cys-conjugates. However, it did not improve the release from G-conjugates and even prevented the yeast from producing any acetate. Although poor in free PFTs and their Cys-precursors, both Saaz hop and pale malt (classical ingredients for premium lager beers) confirmed to be significant sources of G-conjugates (especially those of 3-sulfanylhexanol and 3-sulfanyl-4-methylpentanol).

The cysteinylated and glutathionylated precursors of 3‐sulfanylpentanol (Cys‐3SPol up to 197 μg/kg, G‐3SPol up to 14 mg/kg), recently reported in hops, were quantitated for the first time in the Mandarina Bavaria variety, along with the ubiquitous cysteinylated and glutathionylated forms of 3‐sulfanylhexanol (Cys‐3SHol up to 897 μg/kg, G‐3SHol up to 46 mg/kg). In contrast to findings with another new German cultivar, Polaris, no trace of 3‐sulfanyl‐4‐methylpentanol adducts (Cys‐ and G‐ 3S4MPol) was found. To assess the transfer rate of thiols from hops to finished beer, the same pilot Mandarina Bavaria hopped wort was fermented with two different dry yeasts, bottle refermented or not, and analysed. The data were compared with results obtained for a similarly produced commercial beer. In conclusion, despite significant variation between harvest years, Mandarina Bavaria appears not to contain outstanding amounts of free thiols or thiol S‐conjugates. Its S‐conjugate pool is sufficient, however, to bring 3SHol above its odour threshold. This work also suggests that since 3S4MPol was found near or above its threshold in most late‐hopped beers it does not originate from hops and that malt may be its main contributor. © 2021 The Institute of Brewing & Distilling

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