"dual" hop varieties for late and dry hopping. Flavor stability through
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2014 ASBC Annual Meeting 76 75th 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 L7.05.07, B-1348 Louvain-la-Neuve, Belgium $ Institute of Condensed Matter and Nanosciences, IMCN, Université catholique de Louvain. Place Louis Pasteur, 1 box L4.01.02, 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 Beer productions were carried out in our 50 L microbrewery (CoEnCo, 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). "Dual" Hops Free thiols and terpenols in "dual" hops AMA-X Thiol specific extraction using an organomercuric compound (pHMB) [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 IST OH OH CIT-X 2SEA 2SEol EST 3SProl 3S3MBol 3SPrA 1S3Pol 2SEA IST 3S4MPol 2SEol EST 3SPrA 3S3MBol1S3Pol CIT-X OH CIT-Y AMA-Y geraniol linalool Mosaic Citra Tomahawk 400 300 200 100 0 Amarillo CIT-Y Nelson Sauvin Cascade Saaz Hallertau Blanc Mosaic Citra Tomahawk 250 200 150 100 50 0 Amarillo 3SHA Figure 6. GC-PFPD chromatograms of fresh beers. Nelson Sauvin Figure 7. Concentrations of 2SEA (a) and 3SPrA (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 2SE-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 2SE-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. Saaz Hallertau Blanc Synthesis of cysteine-S-conjugates S-3-(3-methyl-butan-1-ol)-cysteine (Cys-3S3MBol, felinine, Figure 8a) was bought from Immunosource (Belgium). Three commercially unavailable cysteine-S-conjugates have been synthesized. S-3-(hexan-1-ol)cysteine (Cys-3SHol) (Figure 8b) was obtained according to the procedure described by Thibon et al., (2008) [7]. S-(3-methyl-2-buten-1-yl)-cysteine (Cys-MBT) (Figure 8c) was synthesized according to the method of Adsara et al., (1981) [8]. S-2-(ethylacetate)-cysteine (CYS-2SEA) was here obtained for the first time, by acetylation of S-2-(ethan-1-ol)-cysteine (Figure 8d). Figure 9 shows mass spectra of Cys-3S3MBol, Cys-3SHol, Cys-MBT and Cys-2SEA. 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). a) b) All "dual" hops contained citrus/grapefruit-like thiols. Yet some thiols revealed more specific of a variety : 2SEA, 2SEol and 3SHA for Citra, 4S4M2Pone and 1S3Pol for Mosaic, 3S4MPol for Hallertau Blanc, 3S2EPrA and 3SOal for Tomahawk, 3S4MPol (less than in Hallertau Blanc) and 3SOal for Nelson Sauvin. Although not referred as "dual" (only 4,5 – 8 % α-acids), the Cascade hop contained high amounts of the citrus-like 3SHol and 3SHptol. 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 d) Figure 8. Chemical structure of the commercially available Cys-3S3MBol (a) and synthesis scheme of Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (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). Intens. x104 +MS2(208.0), 3.2-3.6min #(74-84) a) 1 SM: 7G 95 Cysteine 90 hop hydroalcoholic extract NL: 4.23E6 7.88 100 3SHol 8.12 60 6000 85 100 140 160 180 Intens. x105 121.9 121.9 +MS2(208.0), 3.2-3.6min #(74-84) d) 147.8 119.8 1.0 172.7 172.7 11 190.7 206.7 120 +MS2(190.0), +MS2(190.0),10.9-11.3min 10.9-11.3min#(243-253) #(243-253) c) 200 83.2 0 220 240 m/z 60 80 101.1 100 69.4 69.4 00 120 140 160 180 200 220 240 m/z 60 60 79.0 79.0 80 80 161.7 161.7 100 100 120 120 140 140 160 160 87.3 0.0 180 180 200 200 220 220 240 240 60 m/z m/z 80 190.7 99.7 100 120 140 160 180 200 220 240 m/z Figure 9. Mass spectra of Cys-3S3MBol (a), Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (d). 80 Confirmation of the instability of cysteine-S-conjugates through beer ageing 75 70 65 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. NH4 + Relative Abundance 60 Cation exchange 80 Intens. Intens. x10 x1055 204.7 0.5 105.0 0 TIC F: + c ESI Full ms2 222.11@30.00 [ 60.00-500.00] MS am arillo 2012_3 +MS2(222.0), 9.8-10.9min #(220-245) b) 1 172.8 RT: 0.00 - 25.07 Intens. x105 121.9 9.16 55 50 10.24 9.81 45 17.82 40 5.43 35 30 10.94 25 Fractions 1,2-1,5M 16.07 6.67 20 3.84 14.48 11.82 15 17.47 15.28 12.89 HPLC-MS/MS Apotryptophanase (β-lyase activity) 10 22.57 0.66 23.81 24.43 1.64 2.79 5 19.31 21.60 20.36 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Tim e (m in) 14 15 16 17 18 19 20 Figure 3. Method for hop cysteine-S-congugates analysis. Figure 4. RP-HPLC-MS/MS (m/z 222) chromatogram of the Amarillo hop extract. 21 22 23 24 25 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 3SHol. 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. 3SHol and 3SNol were released from all hops here investigated. The S-conjugate of 3S4MPol was only detected in Hallertau Blanc (free 3S4MPol was also in higher concentration in this cultivar). a) b) c) d) e) Figure 10. Concentrations through ageing of a) released 3SHol and b) residual Cys-3SHol in beers spiked with 7 mg/L of synthetic Cys-3SHol. 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-2SEA in beers spiked with 15 mg/L of synthetic Cys-2SEA. Concentrations through ageing of e) residual Cys-3S3MBol in beers spiked with 5 mg/L of commercial Cys-3S3MBol. 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 (pHMB) [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 OH OH OH Tomahawk geraniol 250 linalool Mosaic Tomahawk 350 250 100 Nelson Sauvin 200 50 150 100 Citra 50 Citra Cascade 0 Cascade 0 Amarillo Nelson Sauvin 150 300 Mosaic 200 Saaz Amarillo Saaz Hallertau Blanc 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 : 2SEA, 2SEol and 3SHA for Citra, 4S4M2Pone and 1S3Pol for Mosaic, 3S4MPol for Hallertau Blanc, 3S2EPrA and 3SOal for Tomahawk, 3S4MPol (less than in Hallertau Blanc) and 3SOal for Nelson Sauvin. Although not referred as "dual" (only 4,5 – 8 % α-acids), the Cascade hop contained high amounts of the citrus-like 3SHol and 3SHptol. 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). RT: 0.00 - 25.07 SM: 7G 95 TIC F: + c ESI Full ms2 222.11@30.00 [ 60.00-500.00] MS am arillo 2012_3 8.12 6000 85 80 75 70 65 60 + NH4 Relative Abundance Cation exchange Cysteine 3SHol 90 hop hydroalcoholic extract NL: 4.23E6 7.88 100 9.16 55 50 10.24 9.81 45 17.82 40 5.43 35 30 10.94 25 Fractions 1,2-1,5M 20 3.84 14.48 11.82 15 HPLC-MS/MS Apotryptophanase (β-lyase activity) 16.07 6.67 17.47 15.28 12.89 10 22.57 0.66 23.81 24.43 1.64 2.79 5 19.31 21.60 20.36 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Tim e (m in) 14 15 16 17 18 19 20 21 22 23 24 25 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 3SHol. 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. 3SHol and 3SNol were released from all hops here investigated. The S-conjugate of 3S4MPol was only detected in Hallertau Blanc (free 3S4MPol 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 (CoEnCo, 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 2SEA IST CIT-X EST 2SEol 3SProl3S3MBol 3SPrA 1S3Pol 2SEA IST 3S4MPol EST 2SEol 1S3Pol 3SPrA3S3MBol CIT-X CIT-Y AMA-Y CIT-Y 3SHA 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 2SEA (a) and 3SPrA (b) through ageing of CIT-X and CIT-Y. Therefore, the concentration of 2SE-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 2SE-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) S-3-(3-methyl-butan-1-ol)-cysteine (Cys-3S3MBol, felinine, Figure 8a) was bought from Immunosource (Belgium). Three commercially unavailable cysteine-S-conjugates have been synthesized. S-3-(hexan-1-ol)-cysteine (Cys3SHol) (Figure 8b) was obtained according to the procedure described by Thibon et al., (2008) [7]. S-(3-methyl-2-buten-1-yl)-cysteine (Cys-MBT) (Figure 8c) was synthesized according to the method of Adsara et al., (1981) [8]. S-2-(ethylacetate)-cysteine (CYS-2SEA) was here obtained for the first time, by acetylation of S-2-(ethan-1-ol)-cysteine (Figure 8d). Figure 9 shows mass spectra of Cys-3S3MBol, Cys-3SHol, Cys-MBT and Cys-2SEA. b) c) d) Figure 8. Chemical structure of the commercially available Cys-3S3MBol (a) and synthesis scheme of Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (d). b) c) a) Intens. x104 +MS2(208.0), 3.2-3.6min #(74-84) Intens. x105 +MS2(222.0), 9.8-10.9min #(220-245) 121.9 172.8 100 140 160 180 d) 147.8 119.8 0.5 206.7 120 172.7 172.7 11 +MS2(208.0), 3.2-3.6min #(74-84) 1.0 190.7 105.0 80 Intens. x105 121.9 121.9 1 60 +MS2(190.0), +MS2(190.0),10.9-11.3min 10.9-11.3min#(243-253) #(243-253) 204.7 1 0 Intens. Intens. x10 x1055 200 83.2 0 220 240 m/z 60 80 101.1 100 69.4 69.4 00 120 140 160 180 200 Figure 9. Mass spectra of Cys-3S3MBol (a), Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (d). 220 240 m/z 60 60 79.0 79.0 80 80 161.7 161.7 100 100 120 120 140 140 160 160 87.3 0.0 180 180 200 200 220 220 240 240 m/z m/z 60 80 190.7 99.7 100 120 140 160 180 200 220 240 m/z 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 3SHol and b) residual Cys-3SHol in beers spiked with 7 mg/L of synthetic Cys-3SHol. 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-2SEA in beers spiked with 15 mg/L of synthetic Cys-2SEA. Concentrations through ageing of e) residual Cys-3S3MBol in beers spiked with 5 mg/L of commercial Cys-3S3MBol. 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,…).
