Hop is an exceptional source of resveratrol for brewers

Dr. Vesna Jerkovic 9 September 2008

Unité de brasserie et des industries alimentaires, UCL Prof. Sonia Collin

Polyphenols

Phenolic acids Flavonoids Stilbenes� Benzoic acid

� Cinnamic acid � Flavonols� Flavanoids

� Anthocyanidins

� Prenylchalcones

� Flavanones� 3,4-Flavanediols

� Flavan-3-ols� Flavones� Isoflavones

OH

(OH)n

(OH)n

O

(OH)n

(OH)n

COOH

(OH)n

(OH)n

COOH

Antioxidant, aroma precursors

Antioxidant, astringency, color, health benefits (cardioprotector, estrogenic activities,…)

Health benefits (cardioprotector, anti-carcinogenic, anti-inflammatory,…)

Resveratrol and glycoside

� Implication in the « French paradox »In grapes : 0.5 to 39 ppm

In red wines : up to 20 ppm

� anti-viral, anti-oxidant, anti-inflammatory and estrogenic activities

OH

OH

OH OGlc

OH

OH

trans-Resveratrol trans-Piceid

Low compared to flavonoids but

Anti-carcinogenic,

OH

O

OH

OM e

OH

OH

� Flavonoids (10000 – 50000 ppm) :

- flavonols (300 – 2000 ppm) :

quercetin, kaempferol,…

- prenylchalcones

(up to 6000 ppm) :

xanthohumol,…

- flavanols (10000 � 50000 ppm) : catechin, epicatechin, dimers, trimers � oligomers

Hop polyphenolsFermentation BeerMalt, water Mashing Boiling Hop

+

Fermentation BeerMalt, water Mashing Boiling Hop

+

1:500

O

OH

OHOH

OH

R3

R2

R1

(+)-catéchine: R1=H R2=OH R3=H(-)-épicatéchine: R1=OH R2=H R3=H(+)-gallocatéchine: R1=H R2=OH R3=OH(-)-galloépicatéchine: R1=OH R2=H R3=OH

1

2

3

456

7

3'

4'

5'

� Phenolic acids (10 – 60 ppm) : syringic acid, cafeic acid,…

OOH

OHO

OH

OH

OH

OH

OH

OH

OH

OH

4

8

OH

OH

OH OGlc

OH

OH

� Stilbenes : resveratrol piceid

OH

OH

O

OH

OH

O

OH

OH

OH

O

OH

OH

O

OH

OH

COOH

OH

OMe

MeO COOH

(1-20 ppm)(1 - 5 ppm) (1 - 15 ppm)

Context

2003 : Discovery of trans-resveratrol in hop

Callemien, Jerkovic, Rozenberg & Collin, Journal of Agricultural and Food Chemistry , 53,

2005, 424-429

Best cultivars ?

Impact of the harvest year ?

Impact of conditioning (pelletisation, …) ?

Fate of stilbenes through the brewing process ?

Hop processing

Harvest

Drying

Hop vines Hop cones

Pelletisation

Hop pellets

CO2 extraction

Solvent extraction

Hop extract

1. New methodologies

1.1. Standard synthesisJerkovic, Nguyen, Nizet & Collin, Rapid Communication in Mass spectrometry, 21, 2007, 2456-2466

One-pot approach

OH

R

R

OMe

R

R

Diazomethane in diethyletherRoom T°, 45 minutes

OH

OH

OH

KOH/ EtOH

Room T°, 1 week

OH

OH

OGlc

Acetobromo-α,D-glucose

O

O

O O

O

O

O

O

OBr

OH

OH

OH

KOH/ EtOH

Room T°, 1 week

OH

OH

OGlc

Acetobromo-α,D-glucose

O

O

O O

O

O

O

O

OBr

Synthesis of methoxylated stilbenesSynthesis of glycosylated stilbenes

a

d

cb

a

d

c

b

δδδδ

ββββ

χχχχ

a’

b’

b”

d’

Reactants with similar chemical properties

attended products

non attended products

a a

d d

ccb b

a a

d d

cc

b b

δδδδδδδδ

ββββββββ

χχχχχχχχ

a’ a’

b’ b’

b”b”

d’ d’

Reactants with similar chemical properties

attended products

non attended products

Synthesis and characterization of 22 analogs and 9 commercial standards

1.2. Optimization of analyses of trans-resveratrol and analogs in hopCallemien, Jerkovic, Rozenberg & Collin, Journal of Agricultural and Food Chemistry, 53, 2005, 424-429

1. Elimination of hydrophobic compounds by toluene and cyclohexane

2. Stilbene extraction by ethanol:water (80:20), 60°C

3. Concentration

4. Analysis by RP-HPLC-MS/MS-APCI(+)

RT: 12.95 - 24.84 SM: 9B

13 14 15 16 17 18 19 20 21 22 23 24Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

ela

tive

Abu

ndan

ce

NL:6.92E4

m/z= 134.5-135.5 MS Res-APCI36

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

45

0

10

20

30

40

59

0

10

20

30

40

50

Rel

ativ

e A

bund

anc

e

100

0

20

40

60

80

Res-APCI36#826-843 RT: 16.16-16.50 AV: 18 NL: 3.80E4 T: + c APCI Full ms2 229.20@37.00 [ 60.00-239.00]

Res-APCI36#903-918 RT: 17.65-17.94 AV: 16 NL: 2.25E4 T: + c APCI Full ms2 229.20@37.00 [ 60.00-239.00]

Res-APCI36#1082-1097 RT: 21.16-21.46 AV: 16 NL: 1.72E4 T: + c APCI Full ms2 229.20@37.00 [ 60.00-239.00]

Rel

ativ

e A

bund

anc

e

Rel

ativ

e A

bund

ance

100

0

0

100 135.0

211.0

135.0

135.0

211.0

211.0

107.0

119.1107.0

119.0107.0

119.0

228.7

228.2

229.1

100 200

17.78 (2)

21.38 (3)

16.38 (1)a. b.

(1)

(2)

(3)

Polar Apolar

trans-resveratrol

trans-piceid

cis-piceid

Prevail C18 (150 x 2.1 mm, 3 µm)Temp. 30°C, Inj. vol : 10 ml, Flow 0,2 ml/min.Linear gradient from 95 % water (0,1% ACF +1% ACN) / 5 %ACN to 100% ACNAPCI (+)

1.3. Quantification of trans-resveratrol and trans-piceidin beerJerkovic, Nguyen, Timmermans & Collin, submitted

Polyacrylate fiber

1. Extraction2. Derivatization

(BSTFA)3. GC-MS Liquid-liquid extraction

1. Extraction (Ethyl acetate)

2. Concentration

Solid-phase extraction

1. Conditioning

2. Loading of beer

3. Elution (Ethanol)4. Concentration

LOD : 5 ppb/ LOQ : 15 ppb

Elimination of hydrophobiccompounds(Toluene/Cyclohexane)

RP-HPLC-MS/MS-APCI(+)

+ trans-resveratrol+ trans-piceid- Bad recovery

+ trans-resveratrol+ trans-piceid+ High recovery (76%)- Long (8 h)

SPME - DIRECT ANALYSIS RP- HPLC ANALYSIS AFTER EXTRACT ION

Elimination of hydrophobiccompounds(Toluene/Cyclohexane)

RP-HPLC-MS/MS-APCI(+)

OH

OH

OH

NO

FF

F

Si Si+ 3

OSi(CH3)3

OSi(CH3)3

(CH3)3SiO F F F

NO

H

Si+ 3

+ Avoid pre-cleaning+ Easy and rapid (1.5 h)+ trans-resveratrol ok− trans-piceid N.D.− Bad reproducibility

Lager beer without adjunctPrevail C18 (150 x 2.1 mm, 3 µm)Temp. 30°C, Inj. vol : 10 ml, Flow 0,2 ml/minLinear gradient from 95 % water (0,1% ACF +1% ACN) / 5 %ACN to 100% ACNAPCI (+)

Polar Apolar

14 16 18 20 22 24Time (min)

0

10

20

30

40

50

60

70

80

90

1000 NL: 3.50E4

m/z= 134.5-135.5 F: + c APCI Full ms2 229.20@37.00 [ 60.00-239.00] MS gv biere 090504 09

OG lc

OH

OH

OH

OH

OH

R T : 1 0 .0 0 - 4 0 .0 0

1 0 1 5 2 0 2 5 3 0 3 50

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

3 0 0 0 0

3 5 0 0 0

4 0 0 0 0

4 5 0 0 0

5 0 0 0 0

5 5 0 0 0

6 0 0 0 0

6 5 0 0 0

7 0 0 0 0

7 5 0 0 0

8 0 0 0 0

8 5 0 0 0

9 0 0 0 0

9 5 0 0 0

1 0 0 0 0 0

Inte

nsity

1 8 .1 8

1 6 .4 5

3 3 .3 9

3 4 .1 52 6 .9 2 2 9 .7 7

3 0 .9 02 2 .8 1 2 4 .6 41 9 .7 3 2 8 .5 5 3 7 .9 8

3 4 .5 3

1 3 .0 7

3 2 .9 3

3 5 .4 21 2 .5 3

1 5 .1 7

1 2 .2 3

SPME-DIRECT ANALYSIS RP-HPLC ANALYSIS

Lager beer without adjunctPolyacrylate fiberCP-Sil 5 CB columnDesorption 7 min at 280°C100 � 250°C at 10°C/min, 250°C for 30 minSIM m/z = 444Helium = 1 ml/min

trans-resveratrol

2. Stilbenes in hop and other raw materials

2.1. Secondary metabolite� relationship stilbenes/ α-acids (hop bitterness)Jerkovic, Callemien & Collin, Journal of Agricultural and Food Chemistry, 53, 2005, 4202 – 4206

AmarilloVanguard

R2 = 0,9793

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 18

α-acid (%)

Glo

bal

stilb

ene

cont

ent

(m

g/kg

)

American pellets

0

2

4

6

8

10

12

14

Ha

llertau M

ittelfruh

er (G) 2

00

5

He

rsbrucker S

pat (G

) 20

05

Sp

alter (G) 2

00

5

Ha

llertau M

agnum

(G) 2

00

5

Sm

aragd (G

) 20

05

Ha

llertau T

aurus (G) 2

00

5

Ha

llertau T

radition

(G) 2

00

5

Sap

hir (G

) 20

05

Wye T

arget (G) 2

00

5

Nugget (G

) 20

05

Wye T

arget (G) 2

00

6

Prem

ian

t (CZ

) 20

05

Ha

llertau M

ittlefrüher (G

) 20

06

Ha

llertau M

agnum

(G) 2

00

6

Sm

aragd (G

) 20

06

Ha

llertau T

radition

(G) 2

00

6

He

rsbrucker S

pat (G

) 20

06

To

mah

aw

k (USA

) 20

05

Ha

llertau T

aurus (G) 2

00

6

Sim

coe

(US

A) 2

00

5

Nugget (G

) 20

06

Sap

hir (G

) 20

06

Sp

alter (G) 2

00

6

Saa

z (CZ

) 20

05

Warrio

r (US

A) 2

00

5

Warrio

r (US

A) 2

00

6

Willa

me

tte (USA

) 20

05

Cascade (U

SA) 2

00

5

To

mah

aw

k (USA

) 20

06

Nugget (U

SA) 2

00

5

Sla

deck (C

Z) 2

00

6

Sim

coe

(US

A) 2

00

4

To

mah

aw

k (USA

) 20

04

Saa

z (CZ

) 20

06

Cascade (U

SA) 2

00

6

Nugget (U

SA) 2

00

4

Warrio

r (US

A) 2

00

4

Cascade (U

SA) 2

00

4

Willa

me

tte (USA

) 20

06

Willa

me

tte (USA

) 20

04

Stil

bene

con

cent

ratio

n (m

g/kg

)

trans-Piceid trans-Resveratrol

ALL GERMAN VARIETIES

4.9

2.75.4

� American varieties

� Low α-acids < 5.5 %

� ! Harvest-dependant

Necessary but not sufficient

Jerkovic & Collin, Journal of Agricultural and Food Chemistry, 55, 2007, 8754-8758

0

2

4

6

8

10

12

14

2004 2005 2006 2004 2005 2006 2004 2005 2006 2004 2005 2006

Stil

bene

con

cent

ratio

n (p

pm)

trans-Picied trans-Resveratrol TP TRWillamette Tomahawk

(a)

Cascade Warrior

2.2. Phytoalexin � strong influence of the harvest yearJerkovic & Collin, Journal of Agricultural and Food Chemistry, 55, 2007, 8754-8758

trans-Piceid

trans-Resveratrol

2.3. Influence of hop processing and storageStilbene � light sensitive � strong influence of hop processing Jerkovic & Collin, Journal of Agricultural and Food Chemistry, 2007, accepted

trans-Piceid in cones trans-Piceid in pellets trans-Resveratrol

Willamette 4.9% Cascade 5.4 % Warrior 13.2% Tomahawk 12.1%

0

2

4

6

8

10

12

14

cones pellets cones pellets cones pellets cones pellets cones pellets cones pellets

Stil

bene

con

cent

ratio

n (p

pm)

trans-Piceid trans-Resveratrol

Nugget 13.8% Simcoe 12.8%

Harvest 2004 : influence of pelletisation

0

2

4

6

8

10

12

cones pellets cones pellets cones pellets cones pellets

Stil

bene

con

cent

ratio

n (p

pm)

trans-Resveratrol

trans-Piceid in pellets

trans-Resveratrol

trans-Piceid in cones

Willamette (4.0%) Cascade (5.0%) Warrior (13.1%) Tomahawk (14.2%)

trans-Piceid in cones trans-Piceid in pellets trans-Resveratrol

Harvest 2006 : influence of pelletisation

No rules for cultivars sensibility

0

2

4

6

8

10

12

Willam

ette 0 month of storage (4.9%

)

4 months

8 months

12 months

Cascade 0 month of storage (5.4%

)

4 months

8 months

12 months

Tomahawk 1 0 m

onth of storage (14.4%)

4 months

8 months

12 months

Warrior 0 m

onth of storage (13.2%)

4 months

8 months

12 months

Nugget 0 month of storage (13.8%

)

4 months

8 months

12 months

Tomahawk 2 0 m

onth (12.1%)

4 months

8 months

12 months

Simcoe 0 m

onth of storage (12.8%)

4 months

8 months

12 months

Stil

bene

con

cent

ratio

n (m

g/kg

hop

)

trans-Piceid trans-Resveratrol

One-year storage at 4°C leads to a huge loss of resveratrol and its glucoside

Hop cones : influence of storage

0

2

4

6

8

10

12

14

Willamette 0 month of storage (4.9%

)

4 months 8 months 12 months

Cascade 0 month of storage (5.4%)

4 months 8 months 12 months

Warrior 0 month of storage (13.2%

)

4 months 8 months 12 months

Nugget 0 month of storage (13.8%)

4 months 8 months 12 months

Tomahawk 0 month (12.1%)

4 months8 months 12 months

Simcoe 0 month of storage (12.8%)

4 months8 months 12 months

Stil

bene

con

cent

ratio

n (m

g/kg

)trans-Resveratrol in pellets

trans-Piceid in pellets

Série5

Total trans-stilbene in cones at 0 and 12 months

0

2

4

6

8

10

12

14

Willamette 0 month of storage (4.9%

)

4 months 8 months 12 months

Cascade 0 month of storage (5.4%)

4 months 8 months 12 months

Warrior 0 month of storage (13.2%

)

4 months 8 months 12 months

Nugget 0 month of storage (13.8%)

4 months 8 months 12 months

Tomahawk 0 month (12.1%)

4 months8 months 12 months

Simcoe 0 month of storage (12.8%)

4 months8 months 12 months

Stil

bene

con

cent

ratio

n (m

g/kg

)trans-Resveratrol in pellets

trans-Piceid in pellets

Série5

Total trans-stilbene in cones at 0 and 12 months

Hop pellets : influence of storage

OGlc

OH

OH

OH

OH

OH

trans-Piceid

trans-Resveratrol cis-Resveratrol 15 16 17 18 19 2 0 21 22 23Tim e (m in )

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nda

nce

1 5 16 1 7 18 1 9 20 21 2 2 23Tim e (m in )

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

10 0

Re

lativ

e A

bun

da

nce

16.05 (1)

17.33 (2)

21.50 (3)

15.87 (1)

17.19 (2)

21.15 (3) 22.22

(4)

trans-piceid

trans-resveratrol

cis-piceid

cis-resveratrol

Fresh hop

Hop + 8 months

cis-Piceid

Glc

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11 12Times (months)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12Time (months)

2.3. Influence of hop processing and storage Jerkovic & Collin, Journal of Agricultural and Food Chemistry, 2007, accepted.

Close to hop pellets storage

4°C, nitrogen, dark

25°C, light

25°C, dark

trans-piceid recovery (%)

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11 12Time (months)

trans-resveratrol recovery (%)

4°C, dark

25°C, light

25°C, non protection, light

25°C, nitrogen, light

cis-piceid recovery (%)

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11 12Times (months)

25°C, non protection, light

25°C, nitrogen, light

cis-resveratrol recovery (%)

OGlc

OH

OHtrans-piceid

OH

HO

O

OHHO

OH

ESS

δ-Viniferin

2.4. Search for resveratrol analogs in hop Jerkovic & Collin, EBC, 2007, in press

(a)

(b) (c)

(d)

(d)

(e)

A

B

C

(d) in B and C

(e) in B

OMe

MeO

OH

107.0

Detection of a trans-pterostilbene analog (same M+1 = 257) : concentratio n close to 1 ppm in pterostilbene equivalents

m/z = 229

m/z = 257

m/z = 257

Standard

Unknown

trans-pterostilbenestandard

2.5. Other raw materials for brewers Jerkovic & Collin, EBC, 2007, in press

Adaptation of hop extraction : cyclohexane + ethanol:water

R T: 0.0 0 - 30 .10 SM : 15G

0 5 10 15 2 0 2 5 3 0Time (mi n)

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

NL: 2 .0 0E5m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 60 .0 0-3 29 .00 ] M S jf 0 20 40 7 st ilbe ne0 4

NL: 2 .0 0E5m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj cer eales

NL: 2 .0 0E5

m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj ce rea les0 2

NL : 1. 00 E 6

m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj ce rea les0 3

R T: 0 .0 0 - 30 .0 9 SM : 15G

0 5 10 15 2 0 25Time (mi n)

0

2 0

4 0

6 0

8 0

10 0R

ela

tive

Abu

nda

nce

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

15.4

20.4

15.320.4

OG lc

OH

O H

OH

O H

O HR T: 0.0 0 - 30 .10 SM : 15G

0 5 10 15 2 0 2 5 3 0Time (mi n)

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

NL: 2 .0 0E5m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 60 .0 0-3 29 .00 ] M S jf 0 20 40 7 st ilbe ne0 4

NL: 2 .0 0E5m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj cer eales

NL: 2 .0 0E5

m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj ce rea les0 2

NL : 1. 00 E 6

m/ z= 13 4.5 -13 5 .5 F : + c AP CI F ull ms 2 2 2 9.0 0@3 7.00 [ 6 0. 00 - 32 9 .00 ] M S vj ce rea les0 3

R T: 0 .0 0 - 30 .0 9 SM : 15G

0 5 10 15 2 0 25Time (mi n)

0

2 0

4 0

6 0

8 0

10 0R

ela

tive

Abu

nda

nce

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

0

2 0

4 0

6 0

8 0

10 0

Re

lativ

e A

bun

danc

e

15.4

20.4

15.320.4

OG lc

OH

O H

OH

O H

O H

malt + standard

pale malt

dark malt

barley

corn

white sorghum

redsorghum

wheat

For the first time, detection of traces of stilbenes in red sorghum : 0.5 – 1 ppm !

Matrix rich in flavonoids = potential source of stilbenes

2.6. Search for stilbene synthase in hop Jerkovic & Collin, EBC, 2007, in press

Method : PCR and DNA sequencingComparison with Stilbene synthase extracted from grapes

GRAPES SAMPLES

Grapes STS reference

Hop CHS references

H

O

P

S

A

M

P

L

E

S

H.lupulus Chs3 H.lupulus CHS-like 2 H.lupulus CHS-like 1 Hallertauer Merkur clone 7 Wye Target clone 1 Wye Target clone 2 Wye Target clone 5 Wye Target clone 3 Wye target clone 9 H.lupulus Chs4 Spalter select clone 6 Willamette clone 8 Wye Target clone 4 H.lupulus vps H.lupulus CHS-like 3 H.lupulus chs2 H.lupulus CHS-like 4 Pinus strobus STS Pinus strobus CHS Vitis sp.clone 10 Vitis sp.clone 13 Vitis STS2 A.hypogaea STS1 A.hypogaea CHS1 Cannabis CHS H.lupulus chs H1 V. vinifera CHS E Coli FA elongase

10099

99

98

94

96

32100

50

52

56

3152

100

4628

100

40

91

36

6979

91

9732

Hop CHS references

Target clone 9

No stilbene synthase was found in hop � chalcone synthase might be involved in

stilbene biosynthesis (Yamaguchi et al, 1999 )

3. Stilbenes in beer

Resveratrol in commercial beers ~ 5 µg/l

3.1 Stilbenes commercial beers Jerkovic, Nguyen, Timmermans & Collin, Journal of the Institute of brewing, 2008, in press.

<5<5TFL

<5<5TFP

>5*>5*TFA

>5*>5*TFW

<5<5LG4

>5*<5LG3

>5*>5*LG2

>5*>5*LG1

trans-Piceid(µg.L-1)

trans-Resveratrol (µg.L-1)

Beers

* Under the quantification limit (15 µg.L-1) of the SPE procedure.

How to increase resveratrol content ????���� Preparation of stilbene-rich hop extracts? Use of spent hops

0

20

40

60

80

100

120

0 minutes 7 minutes 75 minutes

Time of ebullition

Con

cent

ratio

n (

%)

trans-piceid trans-resveratrol

Ebullition = critical step

0

20

40

60

80

100

120

t=0 End of fermentation, without yeast End of fermentation, with yeast

tra

ns-s

tilbe

ne r

eco

very

(%)

- ini

tial c

once

ntra

tion

10

mg/

l

trans-Piceid trans-Resveratrol

Fermentation = release from the glucoside

Jerkovic, Callemien & Collin, Journal of Agricultural and Food Chemistry, 53, 2005, 4202 – 4206

0

1

2

3

4

5

6

7

8

9

Tomahawk Pellets Tomahawk Spent

trans-Piceid trans-Resveratrol

Pellets Spent

3.2 Enriched-hop extract CO2 extracts

CONCLUSIONS

Methods have been optimized for stilbenes analysis i n hop and beer

A new stilbene library is now available

� chalcone synthase could be involved in stilbene synthe sis.

E. Red sorghum is another source of stilbenes for br ewers

D. No stilbene synthase in hop

B. For hop stilbenes : � the American low- α-acid cultivars emerge as the most concentrated

before pelletization;

� strong influence of the harvest year;

� pelletization induces strong stilbene degradation;

� one-year storage at 4°C leads to a huge loss of res veratrol and its glucoside, especially in the case of hop cones.

A. Hop = exceptional source of polyphenols

C. Beer :

� resveratrol in commercial beers ~ 5 µg/l;

� spent hop extracts could be very interesting to inc rease and standardize resveratrol level in beer.

Thanks to…

� La fondation Inbev-Baillet Latour pour le soutien financier

� Prof. Sonia Collin

� L’équipe INBR : Fanny, Etienne,… � Les mémorants qui ont participé au projet

� Monsieur Stéphane Meulemans de chez Yakima Chief pour son aide précieuse dans l’obtention d’échantillons

� Ma famille

� Mes amis : Béné, Alexis, Aurore, Sabine, Delphine, …� Michaël

� Toutes les personnes qui de près ou de loin m’ont permises de mener à bien ce travail