105.0

121.9

172.8 190.7206.7

+MS2(208.0), 3.2-3.6min #(74-84)

0

1

4x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

83.2 101.1

204.7

+MS2(222.0), 9.8-10.9min #(220-245)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

69.4 79.0

121.9

161.7

172.7

+MS2(190.0), 10.9-11.3min #(243-253)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

87.3 99.7

119.8147.8

190.7

+MS2(208.0), 3.2-3.6min #(74-84)

0.0

0.5

1.0

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

69.4 79.0

121.9

161.7

172.7

+MS2(190.0), 10.9-11.3min #(243-253)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

76762014 ASBC Annual Meeting2014 ASBC Annual Meeting

75th ASBC Annual Meeting75th ASBC Annual MeetingJune 4–6, 2014June 4–6, 2014

Palmer House, a Hilton HotelPalmer House, a Hilton HotelChicago, ILChicago, IL

Polyfunctional thiol potential of new "dual" hop varieties for late and dry hopping. Flavor stability through beer ageing.

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

Confirmation of the instability of cysteine-S-conjugates through beer ageing

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.

Synthesis of cysteine-S-conjugates

As depicted in Figure 6, yeast X leads to much more thiols than yeast Y for the same hopping procedure.

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.

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.

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).

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.

c)b)

Conclusions

Free thiols and terpenols in "dual" hops

867 362

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).

OH

OH OH

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.

Citrus/floral-like

Cysteine-S-conjugates in "dual" hopsHydroalcoholic 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).

hophydro-

alcoholic extract

Cation exchange NH4

+

Apotryptophanase (β-lyase activity)

HPLC-MS/MS

Fractions 1,2-1,5M

RT: 0.00 - 25.07 SM: 7G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

7.88

8.12

9.16

10.24

9.81

17.82

5.43

10.9416.07

6.67

3.8414.48

11.82 17.47

15.2812.89

22.570.66 24.4323.811.64

21.602.7919.31 20.36

NL: 4.23E6

TIC F: + c ESI Full ms2 222.11@30.00 [ 60.00-500.00] MS amarillo 2012_3

Cysteine 3SHol

HO

O

NH3

S OH

Figure 5. Amounts of thiols released from hop by apotryptophanase.Figure 4. RP-HPLC-MS/MS (m/z 222) chromatogram of the Amarillo hop extract. Figure 3. Method for hop cysteine-S-congugates analysis. Sulfur/empyreumatic-like Citrus/grapefruit/blackcurrant-like

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).

O

HO

NH2

S

Cys-3S3MBol

OH

a)

d)c)

b) N Boc cysteine

O

H

O

HO

NHBoc

SCHO

O

HO

NHBoc

SH

trans-2-hexenal O

HO

NHBoc

SCH2OH

O

HO

NH3

SCH2OH

TFA

TFA salt of Cys-3SHol

+ACN

Cs2CO3 (0.5 eq) NaBH4 (1,5 eq)

MeOH

TFA (30 eq)

DCM

L-cysteine. HClO

HO

NH2

S

O

HO

NH3

SH +

MeOH:H2O (45:55)

NaOH (2eq)

Cl

isoprenyl chlorideCys-MBT

Cl

L-cysteine. HCl

HOBr

O

HO

NH2

SOH

O

HO

NH3

SH +

MeOH:H2O (60:40)

NaOH (2eq)

Cl

2-bromo ethanol Cys-2SEol

1) CH3COOH (1eq)2) CH3COCl (2eq)

O

HO

NH2

SO

Cys-2SEA

O

DCM

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).

Yeasts-impact on the derived-beers. Stability through ageing

AMA-X

AMA-Y

IST3SProl 3S3MBol

1S3Pol

EST

3S4MPol2SEol3SPrA

2SEA

CIT-Y

CIT-X

IST3SPrA 3S3MBol1S3Pol

EST2SEol

2SEA

3SHA

CIT-X

CIT-Y

Figure 6. GC-PFPD chromatograms of fresh beers. Figure 7. Concentrations of 2SEA (a) and 3SPrA (b) through ageing of CIT-X and CIT-Y.

"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,…).

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.

SH

HS

OO

OO

SH

HO

HS

OH

SH

O

O

SH SH

HO

HS

O

HO

HSHS

OO

OH

HS

HO

HS

HS

HOOH

HS

OH

HS

OH

HS

SH

OH

"Dual" Hops

Derived-BeersPolyfunctional 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.

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).

a)

Figure 9. Mass spectra of Cys-3S3MBol (a), Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (d).

a) c) d)b)

d) e)

6000

O

HO

NH3

SO

Cys-2SEA

O

O

HO

NH3

S

Cys-3S3MBol

OH

O

HO

NH3

SCH2OH

Cys-3SHolO

HO

NH3

S

Cys-MBT

Introduction

Free thiols and terpenols in "dual" hops

867 362

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).

OH

OH OH

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.

Citrus/floral-like

SH

HS

OO

OO

SH

HO

HS

OH

SH

O

O

SH SH

HO

HS

O

HO

HSHS

OO

OH

HS

HO

HS

HS

HOOH

HS

OH

HS

OH

HS

SH

OH

"Dual" Hops

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.

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).

Cysteine-S-conjugates in "dual" hopsHydroalcoholic 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).

hophydro-

alcoholic extract

Cation exchange NH4

+

Apotryptophanase (β-lyase activity)

HPLC-MS/MS

Fractions 1,2-1,5M

RT: 0.00 - 25.07 SM: 7G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

7.88

8.12

9.16

10.24

9.81

17.82

5.43

10.9416.07

6.67

3.8414.48

11.82 17.47

15.2812.89

22.570.66 24.4323.811.64

21.602.7919.31 20.36

NL: 4.23E6

TIC F: + c ESI Full ms2 222.11@30.00 [ 60.00-500.00] MS amarillo 2012_3Cysteine 3SHol

HO

O

NH3

S OH

Figure 5. Amounts of thiols released from hop by apotryptophanase.Figure 4. RP-HPLC-MS/MS (m/z 222) chromatogram of the Amarillo hop extract.

Figure 3. Method for hop cysteine-S-congugates analysis.

Sulfur/empyreumatic-likeCitrus/grapefruit/blackcurrant-

like

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).

6000

105.0

121.9

172.8 190.7206.7

+MS2(208.0), 3.2-3.6min #(74-84)

0

1

4x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

83.2 101.1

204.7

+MS2(222.0), 9.8-10.9min #(220-245)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

69.4 79.0

121.9

161.7

172.7

+MS2(190.0), 10.9-11.3min #(243-253)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

87.3 99.7

119.8147.8

190.7

+MS2(208.0), 3.2-3.6min #(74-84)

0.0

0.5

1.0

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

69.4 79.0

121.9

161.7

172.7

+MS2(190.0), 10.9-11.3min #(243-253)

0

1

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

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.

Synthesis of cysteine-S-conjugates

As depicted in Figure 6, yeast X leads to much more thiols than yeast Y for the same hopping procedure.

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.

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).

O

HO

NH2

S

Cys-3S3MBol

OH

a)

d)c)

b) N Boc cysteine

O

H

O

HO

NHBoc

SCHO

O

HO

NHBoc

SH

trans-2-hexenal O

HO

NHBoc

SCH2OH

O

HO

NH3

SCH2OH

TFA

TFA salt of Cys-3SHol

+ACN

Cs2CO3 (0.5 eq) NaBH4 (1,5 eq)

MeOH

TFA (30 eq)

DCM

L-cysteine. HClO

HO

NH2

S

O

HO

NH3

SH +

MeOH:H2O (45:55)

NaOH (2eq)

Cl

isoprenyl chlorideCys-MBT

Cl

L-cysteine. HCl

HOBr

O

HO

NH2

SOH

O

HO

NH3

SH +

MeOH:H2O (60:40)

NaOH (2eq)

Cl

2-bromo ethanol Cys-2SEol

1) CH3COOH (1eq)2) CH3COCl (2eq)

O

HO

NH2

SO

Cys-2SEA

O

DCM

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).

Yeasts-impact on the derived-beers. Stability through ageing

AMA-X

AMA-Y

IST3SProl3S3MBol

1S3Pol

EST

3S4MPol2SEol3SPrA

2SEA

CIT-Y

CIT-X

IST3SPrA3S3MBol1S3Pol

EST2SEol

2SEA

3SHA

CIT-X

CIT-Y

Figure 6. GC-PFPD chromatograms of fresh beers. Figure 7. Concentrations of 2SEA (a) and 3SPrA (b) through ageing of CIT-X and CIT-Y.

Derived-Beers

Figure 9. Mass spectra of Cys-3S3MBol (a), Cys-3SHol (b), Cys-MBT (c) and Cys-2SEA (d).

a) c) d)b)O

HO

NH3

SO

Cys-2SEA

O

O

HO

NH3

S

Cys-3S3MBol

OH

O

HO

NH3

SCH2OH

Cys-3SHolO

HO

NH3

S

Cys-MBT

Confirmation of the instability of cysteine-S-conjugates through beer ageing

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.

c)b)

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,…).

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)d) e)