2014 ASBC Annual Meeting 76 75 th ASBC

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2014 ASBC Annual Meeting 76 75 th ASBC Annual Meeting Polyfunctional thiol potential of new "dual" hop varieties for late and dry hopping. Flavor stability through beer ageing. June 4– 6, 2014 Palmer House, a Hilton Hotel Chicago, IL Marie-Lucie Kankolongo Cibaka*, Thi Thu Hang Tran*, Jacques Gros*, Raphaël Robiette$ and Sonia Collin*, ** Earth and Life Institute, ELIM, Université catholique de Louvain. Croix du Sud, 2 box L 7. 05. 07, B-1348 Louvain-la-Neuve, Belgium $ Institute of Condensed Matter and Nanosciences, IMCN, Université catholique de Louvain. Place Louis Pasteur, 1 box L 4. 01. 02, B-1348 Louvain-la-Neuve, Belgium ** E-mail: sonia. collin@uclouvain. be (corresponding author) * Introduction Derived-Beers Polyfunctional thiols contribute to the hop varietal aroma of beers [1], especially when dry hopping [2] or bottle refermentation [3] are applied. Very recently, a cysteine-S-conjugate [S-3 -(hexan-1 -ol)-cysteine] has been identified by HPLC/MS/MS in the Cascade variety [4]. Other cysteine adducts have been evidenced in Saaz, Nelson Sauvin and Tomahawk hops by enzymatic release assays [5]. Both methods were here applied on new "dual" hop varieties including Citra, Amarillo, Mosaïc and Hallertau Blanc. Terpenols and free polyfunctional thiols were also quantified in these four selected "dual" cultivars. Their aromatic profiles were further compared to those previously obtained for Tomahawk, Nelson Sauvin (both "dual"), Cascade and Saaz. Yeasts-impact on the derived-beers. Stability through ageing "Dual" Hops Free thiols and terpenols in "dual" hops Beer productions were carried out in our 50 L microbrewery (Co. En. Co, Belgium). Four beers were obtained by late hopping (Citra (CIT) or Amarillo (AMA)) and top fermentation (yeasts INBR Bras 214 (X) or INBR Bras 212 (Y)). Thiols were analysed by GC-PFPD in fresh (Figure 6) and aged beers (Figure 7). AMA-X Thiol specific extraction using an organomercuric compound (p. HMB) [1] coupled to the sulfur-specific detection (GC-PFPD) allowed us to quantify various thiols (Figure 1). Terpenols were quantified by GC-MS after flavor extraction by the Likens Nickerson method (Figure 2). 867 2 SEA IST 362 2 SEol CIT-X EST 3 SProl 3 S 3 MBol 3 SPr. A 1 S 3 Pol 2 SEA IST 3 S 4 MPol 2 SEol EST 3 SPr. A 3 S 3 MBol 1 S 3 Pol CIT-X CIT-Y AMA-Y geraniol Tomahawk linalool Mosaic Citra Tomahawk 400 300 200 100 0 Amarillo Nelson Sauvin Cascade Saaz Hallertau Blanc Citrus/floral-like Figure 1. Amounts of free thiols in eight hop cultivars. CIT-Y Mosaic Citra 250 200 150 100 50 0 3 SHA Nelson Sauvin Figure 6. GC-PFPD chromatograms of fresh beers. Figure 7. Concentrations of 2 SEA (a) and 3 SPr. A (b) through ageing of CIT-X and CIT-Y. As depicted in Figure 6, yeast X leads to much more thiols than yeast Y for the same hopping procedure. Cascade Therefore, the concentration of 2 SE-A was very high in CIT-X fresh beer (22. 8 µg/L), but dropped until 7. 25 µg/L after 6 months due to oxidation. In contrast, small amounts of 2 SE-A were found in fresh CITR-Y (1. 1 µg/L). This level revealed to increase very fast to reach 11. 8 µg/L after only 1 month. Chemical hydrolysis of cysteine adducts most probably occurred in that case in the bottle. Amarillo Saaz Hallertau Blanc Figure 2. Amounts of 3 free terpenols in eight hop cultivars (concentrations in mg/kg). Synthesis of cysteine-S-conjugates a) S-3 -(3 -methyl-butan-1 -ol)-cysteine (Cys-3 S 3 MBol, felinine, Figure 8 a) was bought from Immunosource (Belgium). Three commercially unavailable cysteine-S-conjugates have been synthesized. S-3 -(hexan-1 -ol)-cysteine (Cys-3 SHol) (Figure 8 b) was obtained according to the procedure described by Thibon et al. , (2008) [7]. S-(3 -methyl-2 -buten-1 -yl)-cysteine (Cys-MBT) (Figure 8 c) was synthesized according to the method of Adsara et al. , (1981) [8]. S-2 -(ethylacetate)-cysteine (CYS-2 SEA) was here obtained for the first time, by acetylation of S-2 -(ethan-1 -ol)-cysteine (Figure 8 d). Figure 9 shows mass spectra of Cys-3 S 3 MBol, Cys-3 SHol, Cys-MBT and Cys-2 SEA. b) All "dual" hops contained citrus/grapefruit-like thiols. Yet some thiols revealed more specific of a variety : 2 SEA, 2 SEol and 3 SHA for Citra, 4 S 4 M 2 Pone and 1 S 3 Pol for Mosaic, 3 S 4 MPol for Hallertau Blanc, 3 S 2 EPr. A and 3 SOal for Tomahawk, 3 S 4 MPol (less than in Hallertau Blanc) and 3 SOal for Nelson Sauvin. Although not referred as "dual" (only 4, 5 – 8 % α-acids), the Cascade hop contained high amounts of the citrus-like 3 SHol and 3 SHptol. Only the "dual" hop cultivars revealed to be exceptional sources of citrus-like terpenols : β-citronellol in Citra and Hallertau Blanc, β-citronellol and geraniol in Mosaic, geraniol and linalool in Tomahawk, and linalool in Nelson Sauvin and Amarillo. c) Cysteine-S-conjugates in "dual" hops Figure 8. Chemical structure of the commercially available Cys-3 S 3 MBol (a) and synthesis scheme of Cys-3 SHol (b), Cys-MBT (c) and Cys-2 SEA (d). Hydroalcoholic extracts were purified on a cation exchanger resin according to Starkenmann et al. (2011) [6] (Figure 3). The resulting fractions were analyzed by RP-HPLC-MS/MS (Figure 4) or subjected to apotryptophanase beta-lyase activity before free thiols were extracted and quantified by GC-PFPD (Figure 5). Cysteine hop hydroalcoholic extract Cation exchange d) 3 SHol 6000 b) a) c) d) Figure 9. Mass spectra of Cys-3 S 3 MBol (a), Cys-3 SHol (b), Cys-MBT (c) and Cys-2 SEA (d). Confirmation of the instability of cysteine-S-conjugates through beer ageing Felinine and synthesized cysteine-S-conjugates were spiked into a commercial beer in order to asses their instability through beer natural or accelerated ageing. Even in absence of yeast, S-cysteine conjugates can be chemically degraded to release their corresponding thiol (Figure 10). Temperature has a great influence on this mechanism. NH 4+ Fractions 1, 2 -1, 5 M HPLC-MS/MS Apotryptophanase (β-lyase activity) Figure 3. Method for hop cysteine-S-congugates analysis. Figure 4. RP-HPLC-MS/MS (m/z 222) chromatogram of the Amarillo hop extract. Sulfur/empyreumatic-like Citrus/grapefruit/blackcurrant-like Figure 5. Amounts of thiols released from hop by apotryptophanase. Recently, RP-HPLC-HRMS/MS analyses enabled us to evidence, for the first time, S-3 -(hexan-1 -ol)-cysteine in Cascade hop [4]. This S-cysteine conjugate was here also identified in the "dual" Amarillo hop (Figure 4) which contained, however, very few free 3 SHol. As previously shown by Gros et al. , (2013) [5], this confirms that the polyfunctional thiol potential of a cultivar can not be directly deduced from the amount of free thiols. The β-lyase activity of apotryptophanase revealed able to release 8 odorant polyfunctional thiols from our "dual" hop extracts. 3 SHol and 3 SNol were released from all hops here investigated. The S-conjugate of 3 S 4 MPol was only detected in Hallertau Blanc (free 3 S 4 MPol was also in higher concentration in this cultivar). a) b) c) d) e) Figure 10. Concentrations through ageing of a) released 3 SHol and b) residual Cys-3 SHol in beers spiked with 7 mg/L of synthetic Cys-3 SHol. Concentrations through ageing of c) residual Cys-MBT in beers spiked with 15 mg/L of synthetic Cys-MBT. Concentrations through ageing of d) residual Cys-2 SEA in beers spiked with 15 mg/L of synthetic Cys-2 SEA. Concentrations through ageing of e) residual Cys-3 S 3 MBol in beers spiked with 5 mg/L of commercial Cys-3 S 3 MBol. Conclusions "Dual" hops are characterized by high contents of both terpenols (+ glucosides) and polyfuntional thiols (+ cysteine adducts). By synergy, they will impart a typical citrus aroma to the derived beers. Free thiols are very instable through beer ageing. Fortunately, part of them can be regenerated from cysteine-S-conjugates. Terpenols offer the great advantage to be much more stable through beer ageing. Dry hopping and refermentation procedures can take advantage of the exceptional thiol and terpenol profile of "dual" hops (good choice of yeast strain, optimized contact time and temperature, …). References [1] Gros et al. , J. Agric. Food Chem. , 2011, 59, 8853 -8865; [2] Collin et al. , Cerevisia, 2012, 36, 119 -124; [3] Nizet et al. , J. Am. Soc. Brew. Chem. , 2013, 71, 15 -22; [4] Gros et al. , J. Agric. Food Chem. , 2012, 60, 7805 -7816; [5] Gros et al. , J. Inst. Brew. , 2013, 119, 221 -227; [6] Starkenmann et al. , J. Agric. Food Chem. , 2011, 26, 378 -384; [7] Thibon et al. , J. Chromatogr. A, 2008, 1183, 150 -157; [8] Adsara et al. , US Patent, 1981, 4 243 679.

Introduction Polyfunctional thiols contribute to the hop varietal aroma of beers [1], especially when dry hopping [2] or bottle refermentation [3] are applied. Very recently, a cysteine-S-conjugate [S-3 -(hexan-1 -ol)-cysteine] has been identified by HPLC/MS/MS in the Cascade variety [4]. Other cysteine adducts have been evidenced in Saaz, Nelson Sauvin and Tomahawk hops by enzymatic release assays [5]. Both methods were here applied on new "dual" hop varieties including Citra, Amarillo, Mosaïc and Hallertau Blanc. Terpenols and free polyfunctional thiols were also quantified in these four selected "dual" cultivars. Their aromatic profiles were further compared to those previously obtained for Tomahawk, Nelson Sauvin (both "dual"), Cascade and Saaz. Free thiols and terpenols in "dual" hops "Dual" Hops Thiol specific extraction using an organomercuric compound (p. HMB) [1] coupled to the sulfur-specific detection (GC-PFPD) allowed us to quantify various thiols (Figure 1). Terpenols were quantified by GC-MS after flavor extraction by the Likens Nickerson method (Figure 2). 867 362 Tomahawk geraniol 250 linalool Mosaic Tomahawk 350 250 100 Nelson Sauvin 200 50 100 Citra 50 Citra 0 Amarillo Nelson Sauvin 150 300 Mosaic 200 0 Cascade Saaz Amarillo Saaz Hallertau Blanc Citrus/floral-like Figure 1. Amounts of free thiols in eight hop cultivars. Figure 2. Amounts of 3 free terpenols in eight hop cultivars (concentrations in mg/kg). All "dual" hops contained citrus/grapefruit-like thiols. Yet some thiols revealed more specific of a variety : 2 SEA, 2 SEol and 3 SHA for Citra, 4 S 4 M 2 Pone and 1 S 3 Pol for Mosaic, 3 S 4 MPol for Hallertau Blanc, 3 S 2 EPr. A and 3 SOal for Tomahawk, 3 S 4 MPol (less than in Hallertau Blanc) and 3 SOal for Nelson Sauvin. Although not referred as "dual" (only 4, 5 – 8 % α-acids), the Cascade hop contained high amounts of the citrus-like 3 SHol and 3 SHptol. Only the "dual" hop cultivars revealed to be exceptional sources of citrus-like terpenols : β-citronellol in Citra and Hallertau Blanc, β-citronellol and geraniol in Mosaic, geraniol and linalool in Tomahawk, and linalool in Nelson Sauvin and Amarillo.

Cysteine-S-conjugates in "dual" hops Hydroalcoholic extracts were purified on a cation exchanger resin according to Starkenmann et al. (2011) [6] (Figure 3). The resulting fractions were analyzed by RP-HPLC-MS/MS (Figure 4) or subjected to apotryptophanase beta-lyase activity before free thiols were extracted and quantified by GC-PFPD (Figure 5). Cysteine 3 SHol hop hydroalcoholic extract Cation exchange 6000 NH 4+ Fractions 1, 2 -1, 5 M HPLC-MS/MS Apotryptophanase (β-lyase activity) Sulfur/empyreumatic-like Citrus/grapefruit/blackcurrant-like Figure 3. Method for hop cysteine-S-congugates Figure 4. RP-HPLC-MS/MS (m/z 222) chromatogram of the Amarillo Figure 5. Amounts of thiols released from hop by apotryptophanase. analysis. hop extract. Recently, RP-HPLC-HRMS/MS analyses enabled us to evidence, for the first time, S-3 -(hexan-1 -ol)-cysteine in Cascade hop [4]. This S-cysteine conjugate was here also identified in the "dual" Amarillo hop (Figure 4) which contained, however, very few free 3 SHol. As previously shown by Gros et al. , (2013) [5], this confirms that the polyfunctional thiol potential of a cultivar can not be directly deduced from the amount of free thiols. The β-lyase activity of apotryptophanase revealed able to release 8 odorant polyfunctional thiols from our "dual" hop extracts. 3 SHol and 3 SNol were released from all hops here investigated. The S-conjugate of 3 S 4 MPol was only detected in Hallertau Blanc (free 3 S 4 MPol was also in higher concentration in this cultivar).

Derived-Beers Yeasts-impact on the derived-beers. Stability through ageing Beer productions were carried out in our 50 L microbrewery (Co. En. Co, Belgium). Four beers were obtained by late hopping (Citra (CIT) or Amarillo (AMA)) and top fermentation (yeasts INBR Bras 214 (X) or INBR Bras 212 (Y)). Thiols were analysed by GC-PFPD in fresh (Figure 6) and aged beers (Figure 7). AMA-X IST 2 SEA CIT-X EST 2 SEol 3 SProl 3 S 3 MBol 3 SPr. A 1 S 3 Pol 2 SEA IST 3 S 4 MPol EST 2 SEol 1 S 3 Pol 3 SPr. A 3 S 3 MBol CIT-X CIT-Y AMA-Y CIT-Y 3 SHA Figure 6. GC-PFPD chromatograms of fresh beers. As depicted in Figure 6, yeast X leads to much more thiols than yeast Y for the same hopping procedure. Figure 7. Concentrations of 2 SEA (a) and 3 SPr. A (b) through ageing of CIT-X and CIT-Y. Therefore, the concentration of 2 SE-A was very high in CIT-X fresh beer (22. 8 µg/L), but dropped until 7. 25 µg/L after 6 months due to oxidation. In contrast, small amounts of 2 SE-A were found in fresh CITR-Y (1. 1 µg/L). This level revealed to increase very fast to reach 11. 8 µg/L after only 1 month. Chemical hydrolysis of cysteine adducts most probably occurred in that case in the bottle. Synthesis of cysteine-S-conjugates a) c) b) d) Figure 8. Chemical structure of the commercially available Cys-3 S 3 MBol (a) and synthesis scheme of Cys-3 SHol (b), Cys-MBT (c) and Cys-2 SEA (d). b) c) a) Figure 9. Mass spectra of Cys-3 S 3 MBol (a), Cys-3 SHol (b), Cys-MBT (c) and Cys-2 SEA (d). S-3 -(3 -methyl-butan-1 -ol)-cysteine (Cys-3 S 3 MBol, felinine, Figure 8 a) was bought from Immunosource (Belgium). Three commercially unavailable cysteine-S-conjugates have been synthesized. S-3 -(hexan-1 -ol)-cysteine (Cys 3 SHol) (Figure 8 b) was obtained according to the procedure described by Thibon et al. , (2008) [7]. S-(3 -methyl-2 -buten-1 -yl)-cysteine (Cys-MBT) (Figure 8 c) was synthesized according to the method of Adsara et al. , (1981) [8]. S-2 -(ethylacetate)-cysteine (CYS-2 SEA) was here obtained for the first time, by acetylation of S-2 -(ethan-1 -ol)-cysteine (Figure 8 d). Figure 9 shows mass spectra of Cys-3 S 3 MBol, Cys-3 SHol, Cys-MBT and Cys-2 SEA. d)

Confirmation of the instability of cysteine-S-conjugates through beer ageing Felinine and synthesized cysteine-S-conjugates were spiked into a commercial beer in order to asses their instability through beer natural or accelerated ageing. Even in absence of yeast, S-cysteine conjugates can be chemically degraded to release their corresponding thiol (Figure 10). Temperature has a great influence on this mechanism. a) b) c) d) e) Figure 10. Concentrations through ageing of a) released 3 SHol and b) residual Cys-3 SHol in beers spiked with 7 mg/L of synthetic Cys-3 SHol. Concentrations through ageing of c) residual Cys-MBT in beers spiked with 15 mg/L of synthetic Cys-MBT. Concentrations through ageing of d) residual Cys-2 SEA in beers spiked with 15 mg/L of synthetic Cys-2 SEA. Concentrations through ageing of e) residual Cys-3 S 3 MBol in beers spiked with 5 mg/L of commercial Cys-3 S 3 MBol. Conclusions "Dual" hops are characterized by high contents of both terpenols (+ glucosides) and polyfuntional thiols (+ cysteine adducts). By synergy, they will impart a typical citrus aroma to the derived beers. Free thiols are very instable through beer ageing. Fortunately, part of them can be regenerated from cysteine-S-conjugates. Terpenols offer the great advantage to be much more stable through beer ageing. Dry hopping and refermentation procedures can take advantage of the exceptional thiol and terpenol profile of "dual" hops (good choice of yeast strain, optimized contact time and temperature, …).

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