Occurrence and Synthesis Pathways of (Suspected) Genotoxic α,ß-Unsaturated Carbonyls in Chocolate and Other Commercial Sweet Snacks.

α,ß-Unsaturated carbonyls are highly reactive and described as structural alerts for genotoxicity. Ten of them (either commercially available or synthesized here by combinatorial chemistry) were first investigated throughout the chocolate-making process by solvent-assisted flavor evaporation (SAFE) coupled to GC-MS/SIM. Monitored α,ß-unsaturated aldehydes were formed during chocolate production, primarily through aldol condensation of Strecker aldehydes triggered by bean roasting. Notably, levels of 2-phenylbut-2-enal (up to 399 µg·kg-1) and 5-methyl-2-phenylhex-2-enal (up to 216 µg·kg-1) increased up to 40-fold. Dry conching caused evaporation of α,ß-unsaturated carbonyls, while wet conching partially restored or increased their levels due to cocoa butter addition. Further analyses showed that α,ß-unsaturated aldehydes also occurred in most commercial sweet snacks (up to 16 µg·kg-1), although often at lower concentrations than in roasted cocoa or derived chocolates. In the end, none of the monitored α,ß-unsaturated aldehydes did raise a health concern compared to current maximum use levels (2-5 mg·kg-1). On the other hand, much higher levels of genotoxic furan-2(5H)-one were found in crepe and cake samples (up to 4.3 mg·kg-1).

Use of Botanical Ingredients: Nice Opportunities to Avoid Premature Oxidation of NABLABs by Increasing Their ORAC Values Strongly Impacted by Dealcoholization or Pasteurization.

Even when fresh, non-alcoholic, and low-alcoholic beers (NABLABs) exhibit significant staling defects due to premature oxidation. In this study, the antioxidant power of eleven fresh commercial NABLABs was assessed by means of three different assays: the oxygen radical absorbance capacity (ORAC), the linoleic acid-induced oxidation (TINH), and the indicator time test (ITT). Only the first two assays, both involving radicalar degradations initiated by AAPH, were found to correlate with each other. NABLABs displayed lower ORAC values than conventional beers (on average, 6127 µmol eq. Trolox/L), except for three samples made with special-colored malts or dry-hopped. Dealcoholization was the step with the greatest impact on the ORAC value (up to a 95% loss) and on flavan-3-ols, sotolon, and polyfunctional thiols, while pasteurization strongly affected color, TBA, and Strecker aldehydes. ORAC assays applied to hop, alternative cereals, and various botanical ingredients indicated that mashing with red sorghum, dry hopping/spicing, and wood maturation could bring the antioxidant power of a NABLAB close to those of conventional beers. With an ORAC value not reached by any other tested botanical ingredient (5234 µmol eq. Trolox/g), African Vernonia amygdalina leaves (traditionally used for Rwandan Ikigage beers) emerged here as the best candidate.

Occurrence of Perillaldehyde and Other (Suspected) Genotoxic Flavoring Substances in Water-Based Beverages Consumed by Belgian Children.

Despite the extensive use of flavoring substances in food, their monitoring for regulatory purposes is currently limited. This raises public health issues, especially as some compounds are prohibited due to (geno)toxicity. A solvent-assisted flavor evaporation (SAFE) method coupled with GC/MS (SIM) was validated here for diverse water-based beverages. Thirty flavoring substances out of the 38 targeted were validated, showing good analytical performances and confirming the versatility of the SAFE technique. The method was then applied to 94 samples, including fruit juices, iced teas, lemonades, colas, and sports beverages. Overall, seven different flavoring substances of interest were detected in the samples. Perillaldehyde and furan-2(5H)-one, two genotoxic flavoring substances, were found at concentrations up to 153 and 143 µg·kg-1, respectively. Perillaldehyde levels were significantly higher in commercial citrus juices than in freshly squeezed juices. Food control laboratories could use the developed method to face the current growing need to improve flavoring substance monitoring and conduct risk assessments.

Evidence of Enzymatic and Chemical Interconversions of Barley Malt 3-Sulfanylhexanol Conjugates during Mashing.

Recent studies have highlighted in malt the occurrence of the glutathionylated precursor of 3-sulfanylhexanol (G-3SHol) at concentrations reaching hundreds of µg/kg. Here, SIDA-LC-MS/MS was used to investigate the potential conversion of G-3SHol to its dipeptide and cysteinyl analogues during mashing. At 45 and 55 °C, malt γGT and carboxypeptidase activities quickly degrade G-3SHol (up to 90% loss), first to the cysteinylglycine conjugate and then to the cysteine conjugate (up to 205% increase). No γ-glutamylcysteine S-conjugate formation is observed. At 80 °C, despite enzyme inactivation, the G-3SHol level decreases steadily because of suspected imine formation with wort aldehydes at pH 5.5. More surprisingly, CysGly-3SHol is still generated at 80 °C. This indicates the presence in the wort of as yet unidentified precursors.

Discriminating Aroma Compounds in Five Cocoa Bean Genotypes from Two Brazilian States: White Kerosene-like Catongo, Red Whisky-like FL89 (Bahia), Forasteros IMC67, PA121 and P7 (Pará).

Fine-grade cocoa beans are characterized by their great organoleptic quality. Brazilian cocoa producers increasingly privilege organoleptic quality over yield. Catongo and FL89, fine Brazilian cocoa genotypes from Bahia, are characterized by particular flavors (respectively, kerosene and whisky). The beans IMC67, PA121, and P7 from the state of Pará are genotypes that have high resistance to diseases. Solvent-assisted flavor evaporation (SAFE) aroma extraction was used here to identify and quantify the volatile compounds discriminating these genotypes. The results show that the kerosene aroma of Catongo is likely due to the presence, at high levels, of ethyl acetate and isobutyl acetate. On the other hand, ethyl benzoate, heptanoate, and octanoate, trans-2-nonenal, 1-octen-3-ol, and 3-methylbutanol could play a key role in the whisky notes of FL89. Heptan-2-ol, heptan-2-one, nonan-2-one, linalool (although still more concentrated in IMC67 from Pará state), benzaldehyde, and phenylacetaldehyde also discriminate these beans. Other compounds, although not discriminating, appear important in determining their aromatic quality. The PCA showed that cocoa from Pará state formed a cluster due to similar aromas, while FL89 was the most distinct among the genotypes. Beans from Brazil show great potential and diversity for the fine cocoa market.

Malt and Hop as Sources of Thiol S-Conjugates: Thiol-Releasing Property of Lager Yeast during Fermentation.

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).

Modulation of the Sulfanylalkyl Acetate/Alcohol Ratio and Free Thiol Release from Cysteinylated and/or Glutathionylated Sulfanylalkyl Alcohols in Beer under Different Fermentation Conditions.

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 3-sulfanylhexan-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).

Occurrence of Ehrlich-Derived and Varietal Polyfunctional Thiols in Belgian White Wines Made from Chardonnay and Solaris Grapes.

Although almost disappeared at the end of the 20th century, Belgian wine production reached a million liters in 2017. The aim of the present work was to identify aroma markers for two white cultivars widespread in Belgium: Chardonnay (the most commercially grown cultivar worldwide) and Solaris (an interspecific fungus-resistant variety). Specific p-hydroxymercuribenzoic acid extraction followed by gas chromatography with a pulsed flame photometric detector was applied to single-varietal wine samples from 2015 to 2018 harvests. Among the Ehrlich-derived thiols, all found below their sensory thresholds, 3-sulfanylpropyl acetate usually outranged 3-sulfanylpropan-1-ol, while 2-sulfanylethan-1-ol concentrations always exceeded 2-sulfanylethyl acetate levels. The data confirm the occurrence, in both Chardonnay and Solaris wines, of 3-sulfanylhexan-1-ol and phenylmethanethiol (grapefruit and gunflint aroma, respectively), at concentrations far above their thresholds. This work also revealed, for the first time, the presence of 3-sulfanylheptan-1-ol, the branched alcohols 2-methyl-3-sulfanyl propan-1-ol, 2-methyl-3-sulfanylbutan-1-ol, and 3-sulfanyl-3-methylbutan-1-ol, and the carbonyls 3-sulfanylbutan-2-one (not in sparkling wines) and 3-sulfanylpentanal.

First Evidence of the Cysteine and Glutathione Conjugates of 3-Sulfanylpentan-1-ol in Hop ( Humulus lupulus L.).

After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (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.

Occurrence and Antioxidant Activity of C1 Degradation Products in Cocoa.

Procyanidin C1 is by far the main flavan-3-ol trimer in cocoa. Like other flavan-3-ols, however, it suffers a lot during heat treatments such as roasting. RP-HPLCHRMS/MS(ESI(-))analysis applied to an aqueous model medium containing commercial procyanidin C1 proved that epimerization is the main reaction involved in its degradation (accounting for 62% of degradation products). In addition to depolymerization, cocoa procyanidin C1 also proved sensitive to oxidation, yielding once- and twice-oxidized dimers. No chemical oligomer involving the native trimer was found in either model medium or cocoa, while two C1 isomers were retrieved. C1 degradation products exhibited antioxidant activity (monitored by RPHPLC-Online TEAC) close to that of C1 (when expressed in µM TE/mg·kg-1).

Roasting conditions for preserving cocoa flavan-3-ol monomers and oligomers: interesting behaviour of Criollo clones.

BACKGROUND: Cocoa bean roasting is important for creating the typical chocolate aroma through Maillard reactions, but it is also a key step deleterious to the polyphenol content and profile. RESULTS: Compared with usual roasting at 150 °C, keeping the beans for 30 min at 120 °C or for 1 h at 90 °C proved much better for preventing strong degradation of native P1, P2 and P3 flavan-3-ols in cocoa (shown for Forastero, Trinitatio and Criollo cultivars). Surprisingly, Cuban, Mexican and Malagasy white-seeded beans behaved atypically when roasted for 30 min at 150 °C, releasing a pool of catechin. Enantiomeric chromatographic separation proved that this pool contained mainly (-)-catechin issued from (-)-epicatechin by epimerisation. As the (-)-epicatechin content remained relatively constant through Criollo bean roasting, flavan-3-ol monomers must have been regenerated from oligomers. This emergence of (-)-catechin in Criollo beans only, reported here for the first time, could be due to increased flavan-3-ol monomer stability in the absence of anthocyanidin-derived products. CONCLUSION: The degradation rate of flavan-3-ols through roasting is higher in cocoa beans containing anthocyani(di)ns. The liberation of a pool of (-)-catechin when submitted to roasting at 150 °C allows to distinguish white-seeded cultivars. © 2017 Society of Chemical Industry.

Procyanidin A2 and Its Degradation Products in Raw, Fermented, and Roasted Cocoa.

Cocoa is known as an important source of flavan-3-ols, but their fate "from the bean to the bar" is not yet clear. Here, procyanidin A2 found in native cocoa beans (9-13 mg/kg) appeared partially epimerized into A2E1 through fermentation, whereas a second epimer (A2E2) emerged after roasting. At m/z 575, dehydrodiepicatechin A was revealed to be the major HPLC peak before fermentation, whereas F1, a marker of well-conducted fermentations, becomes the most intense after roasting. RP-HPLC-ESI(-)-HRMS/MS analysis performed on a procyanidin A2 model medium after 12 h at 90 °C revealed many more degradation products than those identified in fermented cocoa, including the last epimer of A2, A2 open structure intermediates (m/z 577), and oxidized A-type dimers (m/z 573).

Fate of Anthocyanins through Cocoa Fermentation. Emergence of New Polyphenolic Dimers.

Fresh, ripe cocoa beans from Cameroon (German cocoa/Amelonado group and ICS 40/Trinitario group) were subjected to fermentation-like incubations in acetic acid, lactic acid, or both and to natural fermentation. Two naturally fermented samples from Cuba (UF 654/Trinitario group and C 411/Criollo group) were also investigated. Both cyanidin-3-galactoside and cyanidin-3-arabinoside (found as major anthocyanins in colored beans only) were drastically degraded through fermentation, especially in small beans and in the presence of acetic acid. On the other hand, emergence of a cyanidin-rhamnose isomer was evidenced, even in Criollo beans. In addition to the recently described structures F1 and F2 [m/z = 575 in ESI(-)], three additional polyphenolic structures [F3, F4, and F5; m/z = 557 in ESI(+)] were found after fermentation, the two former ones resulting from epicatechin oxidation. Synthesis of F5 requires an interclass reaction between cyani(di)n and epicatechin, which explains its absence in fermented Criollo beans.

3-Sulfanyl-4-methylpentan-1-ol in Dry-Hopped Beers: First Evidence of Glutathione S-Conjugates in Hop (Humulus lupulus L.).

Monovarietal dry-hopped beers were produced with the dual-purpose hop cultivars Amarillo, Hallertau Blanc, and Mosaic. The grapefruit-like 3-sulfanyl-4-methylpentan-1-ol was found in all three beers at concentrations much higher than expected on the basis of the free thiol content in hop. Even cysteinylated precursors proved unable to explain our results. As observed in wine, the occurrence of S-glutathione precursors was therefore suspected in hop. The analytical standards of S-3-(4-methyl-1-hydroxypentyl)glutathione, never described before, and of S-3-(1-hydroxyhexyl)glutathione, previously evidenced in grapes, were chemically synthesized. An optimized extraction of glutathionylated precursors was then applied to Amarillo, Hallertau Blanc, and Mosaic hop samples. HPLC-ESI(+)MS/MS revealed, for the first time, the occurrence of S-3-(1-hydroxyhexyl)glutathione and S-3-(4-methyl-1-hydroxypentyl)glutathione in hop, at levels well above those reported for their cysteinylated counterparts. S-3-(1-Hydroxyhexyl)glutathione emerged in all cases as the major adduct in hop. Yet, although 3-sulfanylhexan-1-ol seems relatively ubiquitous in free, cysteinylated, and glutathionylated forms, the glutathione adduct of 3-sulfanyl-4-methylpentan-1-ol, never evidenced in other plants up to now, was found only in the Hallertau Blanc variety.

Occurrence of Theaspirane and its Odorant Degradation Products in Hop and Beer.

In model oxidized media, six theaspirane-derived compounds were identified by gas chromatography-high resolution mass spectrometry: 4-hydroxy-7,8-dihydro-ß-ionone, 6-hydroxy-7,8-dihydro-α-ionone, dihydrodehydro-ß-ionone, two monoepoxides, and a derived alcohol. Only 4-hydroxy-7,8-dihydro-ß-ionone and dihydrodehydro-ß-ionone have been described previously in the literature. Investigation of hop revealed five of these compounds in free form together with theaspirane (especially in the Mosaic variety), while the Citra and Amarillo hop varieties emerged as very interesting for the release of theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, and dihydrodehydro-ß-ionone from glucoside precursors. For the first time, theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, 6-hydroxy-7,8-dihydro-α-ionone, and both monoepoxides were found in a fresh commercial top fermentation beer (only theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, and dihydrodehydro-ß-ionone have recently been mentioned as Gueuze constituents).

How sotolon can impart a Madeira off-flavor to aged beers.

4,5-Dimethyl-3-hydroxy-2(5H)-furanone or sotolon is known to impart powerful Madeira-oxidized-curry-walnut notes to various alcoholic beverages. It has been much studied in oxidized Jura flor-sherry wines, aged Roussillon sweet wines, and old Port wines, in which it contributes to the characteristic "Madeira-oxidized" aroma of these beverages. No scientific paper describes how sotolon might be involved in the Madeira off-flavor found in aged beers. The specific extraction procedure applied here allowed us to quantify this lactone in 7 special beers, at levels sometimes well above its threshold (from 5 to 42 µg/L after 6, 12, 18, and 24 months of natural aging, while unquantifiable in fresh beer). Investigation of spiked beers led us to highlight the key role of pro-oxidants and acetaldehyde. Addition of ascorbic acid without sulfites should be avoided by brewers, as the former would intensify sotolon synthesis. Acetoin, a beer fermentation byproduct, also emerged as possible precursor in beer when combined with serine.

Quantitation of selected terpenoids and mercaptans in the dual-purpose hop varieties Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace.

Free terpenoids and both free and bound polyfunctional thiols were investigated in five selected dual-purpose hop cultivars. Surprisingly, the dual-purpose Sorachi Ace variety was found to contain higher amounts of farnesene (2101 mg/kg) than aromatic hops such as Saaz but only traces of 3-methylbutylisobutyrate, a compound that usually distinguishes all bitter varieties. All five cultivars investigated here showed an exceptional citrus-like potential explained by either monoterpenic alcohols or polyfunctional thiols. Among the monoterpenic alcohols, ß-citronellol at concentrations above 7 mg/kg distinguished Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace from Nelson Sauvin and Tomahawk, two previously investigated dual-purpose hops, while linalool (312 mg/kg) and geraniol (211 mg/kg) remained good discriminating compounds for Nelson Sauvin and Tomahawk, respectively. Regarding polyfunctional thiols, higher amounts of 3-sulfanylhexyl acetate (27 µg/kg) characterized the Citra variety. Free 4-sulfanyl-4-methylpentan-2-one proved discriminant for Sorachi Ace, while the bound form is predominant in Nelson Sauvin. On the other hand, an S-conjugate of 3-sulfanylhexan-1-ol was found in Sorachi Ace at levels not far from those previously reported for Cascade, although the free form was undetected here. Both free and bound grapefruit-like 3-sulfanyl-4-methylpentan-1-ol (never evidenced before the present work) emerged as discriminating compounds for the Hallertau Blanc variety. The apotryptophanase assay also allowed us to evidence for the first time an S-conjugate of 2-sulfanylethan-1-ol.

Guaiacol and 4-methylphenol as specific markers of torrefied malts. Fate of volatile phenols in special beers through aging.

Phenol-specific extracts of 12 Belgian special beers were analyzed by gas chromatography hyphenated to olfactometry (AEDA procedure) and mass spectrometry (single ion monitoring mode). As guaiacol and 4-methylphenol were revealed to be more concentrated in brown beers (>3.5 and >1.1 µg/L, respectively), they are proposed as specific markers of the utilization of dark malts. Analysis of five differently colored malts (5, 50, 500, 900, and 1500 °EBC) allowed confirmation of high levels of guaiacol (>180 µg/L; values given in wort, for 100% specialty malt) and 4-methylphenol (>7 µg/L) for chocolate and black malts only (versus respectively <3 µg/L and undetected in all other worts). Monitoring of beer aging highlighted major differences between phenols. Guaiacol and 4-methylphenol appeared even more concentrated in dark beers after 14 months of aging, reaching levels not far from their sensory thresholds. 4-Vinylphenols and 4-ethylphenols, on the contrary, proved to be gradually degraded in POF(+)-yeast-derived beers. Vanillin exhibited an interesting pattern: in beers initially containing <25 µg/L, the vanillin concentration increased over a 14 month aging period to levels exceeding its sensory threshold (up to 160 µg/L). Beers initially showing an above-threshold level of vanillin displayed a decrease during aging.

Degradation of (-)-epicatechin and procyanidin B2 in aqueous and lipidic model systems. first evidence of "chemical" flavan-3-ol oligomers in processed cocoa.

Despite the key role of flavan-3-ols in many foods, very little is yet known concerning the modification of their chemical structures through food processes. Degradation of model media containing (-)-epicatechin and procyanidin B2, either separately or together, was monitored by RP-HPLC-DAD-ESI(-)-MS/MS. Medium composition (aqueous or lipidic) and temperature (60 and 90 °C) were studied. In aqueous medium at 60 °C, (-)-epicatechin was mainly epimerized to (-)-catechin, but it was also oxidized to "chemical" dimers, a "chemical" trimer, and dehydrodi(epi)catechin A. Unlike oxidation, epimerization was enhanced at 90 °C. In lipidic medium, epimerization proved slow but degradation was faster. Procyanidin B2 likewise proved able to epimerize, especially at 90 °C and in aqueous medium. At high temperature only, the interflavan linkage was cleaved, yielding the same compounds as those found in the monomer-containing model medium. Oxidation to procyanidin A2 was also evidenced. With little epimerization and slow oxidation even at 90 °C, procyanidin B2 proved more stable in lipidic medium. Synergy was also observed: in the presence of the monomer, the dimer degradation rate increased 2-fold at 60 °C. This work states for the first time the presence of newly formed flavan-3-ol oligomers in processed cocoa.

Occurrence of odorant polyfunctional thiols in beers hopped with different cultivars. First evidence of an S-cysteine conjugate in hop (Humulus lupulus L.).

Forty-one thiols, mainly ß-sulfanylalkyl acetates, ß-sulfanylalkyl alcohols, and ß-sulfanylalkyl carbonyls, were recently evidenced in hop. In a beer hopped with the Tomahawk cultivar, most of them were found at higher levels than expected. The aim of the present work was to investigate the polyfunctional thiols in beers hopped with different varieties. A few thiols proved not to come only from hop (mainly 2-sulfanylethyl acetate, µg/L levels, and 1-sulfanylpentan-3-one and 1-sulfanylpentan-3-ol, ng/L levels, internal standard (IST) equivalents). The thiol profile of Saaz-hopped beer proved similar to that of the reference beer produced without hop. A high level of 3-sulfanyloctan-1-ol emerged as an indicator of the use of Tomahawk hop (140 ng/L, IST equivalents; FD (flavor dilution) = 65536). In both Cascade- and Tomahawk-hopped beers, 3-sulfanylhexan-1-ol and 3-sulfanylheptan-1-ol were smelled at high flavor dilutions, although only for the latter, significant amounts of the unreduced 3-sulfanylheptanal were found in hop. As already claimed for hop authentication, 3-sulfanyl-4-methylpentan-1-ol remains a good marker of Nelson Sauvin-hopped beers (548 ng/L, IST equivalents; FD = 65536), together with 4-sulfanyl-4-methylpentan-2-one (128 ng/L, FD = 4096). As illustrated by the huge production occurring during fermentation, accurate prediction of hop varietal impact requires quantitating thiol adducts in hop. S-3-(1-Hydroxyhexyl)cysteine was evidenced here for the first time in Cascade hop.