Use of beta-glucosidaseactivity for flavour enhancement in specialty beers

Luk DaenenChair De Clerck 2012

Flavour complexity

Malt, hops, spices, fruit

flavour

Yeast

Flavour of the

final beer

Flavour precursors

Specific enzymes

flavour

addition

Process

Flavour modification

Malt, hops, spices, fruit

flavour

Yeast

Flavour of the

final beer

Flavour precursors

Specific enzymes

flavour

addition

Process

Here, focus on biological approach (no flavour addition): � Use of plant parts/extracts, enzymes, microorganisms

Preferred by consumers: Natural product, traditional process, authenticity, health, environment

Advantage for producer: “clean label”, often cheaper, development of complex and subtle flavours

Interest for wine-making

Some grape varieties have a significant pool of glycosidically bound flavour compounds � hidden flavour potentialHydrolysis by yeast, enzymes,...

Gun

ata

et a

l 198

4

Interest for brewing industry

HopsFlavour precursors in “green” part of hops (flowers, leaves, ...)Enhance “hoppy” aroma by precursor hydrolysisInterest for lager, ale, geuze, specialty beers, ...

CherryFlavour precursors in juice, kernel, skinEnhance cherry flavour by precursor hydrolysisInterest for fruit beers

Release of hidden flavour potential

Glycosidically bound flavour compounds = glycosides

O OHO

OHOH

OH OH

OH OHO

OHOH

OH

+

Linalool-glucoside� non-volatile� odourless

Linaloollavender, citrus, floral

glucose� Yeast� enzymes (beta-glucosidases)� acid

Treatment Comment

Acid hydrolysis Low pH (3,0 -3,5) Separate proces; Sour beers

Enzymatic hydrolysis Eg. Enzymes from Aspergillus niger

Side activities possible:� HCD activity � 4-VG� Anthocyanidin breakdown (loss of colour)� Esterase activity � isoamylacetate ↓

Yeast biotransformation Saccharomyces sp. Mostly low or moderate activity

Brettanomyces sp. High activity; but also other Brett flavours (!)

���� Different treatments lead to different flavour profi les

Selection of yeast strains

Saccharomyces sp.Mostly no β-glucosidaseAll have exo-1,3- β-glucanse activity� Low to moderate activity

Some commercial wine strains with inducible β-glucosidase

Brettanomyces sp.Some with high β-glucosidaseMainly cell associated(low extracellular)Also minor glycosidase activities

0

10

20

30

40

50

60

70

80

enz

yme

act

ivity

(m

U/m

l)pNPG pNPR pNPX pNPA

p-nitrophe nyl glycoside

■ extracellular

■ intracellular

■ cell insoluble fraction

Isolation of hop glycosides

Hop pelletsSupercritical CO2 extraction Essential oils

Soft resins

spent hops / hop residue / hop solids1) ethanol / water2) ethanol evaporation

watery extractSolid Phase Extraction (XAD2)1) wash with water2) elution with ethanol3) evaporation4) PVPP � remove polyphenols

hop glycoside extract

extraction

purification

(Saaz)

�%

�%

1-octen-3-ol

benzyl alcohol

linalool methyl salicylate

theaspirane A

theaspirane B

4-terpineol

α-terpineol

nerol

dihydroedulan I

�A

�B

Aglycones from hop glycosides

OH O

OH

OMe

O

OH

OH

control pH 5.0

AR2000 enzyme prep.

4-hydroxy-7,8-dihydro-β-ionol

SPME GC/MS

OH

Lab strain wild type S. cerevisiae BY4742Lab strain deletion transformant S. cerevisiae BY4742 exg1∆S. cerevisiae brewing strain LD25 ���� low exo-1,3- β-glucanase activityS. cerevisiae brewing strain LD40 ���� high exo-1,3- β-glucanase activityCommercial S. cerevisiae wine strain ���� inducible β-glucosidase activityBrettanomyces custersii LD72 ���� β-glucosidase activityMixed culture S. cerevisiae LD25 and B. custersii LD72

YPD fermentation with added hop glycosides

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

0,016

Con

trol

pH

5

Sc

BY

4742

w

ild ty

pe

Sc

BY

4742

ex

g1∆

Sc

LD25

Sc

LD40

Sc

UV

A22

8

Bc

LD72

S

c LD

25 +

B

c LD

72

Co

ncen

trat

ion

(mg/

l)

linalool

Linalool:- citrus, floral, aniseed- important contributor to hop aroma

OH

0,000

0,001

0,002

0,003

0,004

0,005

0,006

0,007

0,008

0,009

0,010

Con

trol

pH

5

Sc

BY

4742

w

ild ty

pe

Sc

BY

4742

ex

g1∆

Sc

LD25

Sc

LD40

Sc

UV

A22

8

Bc

LD72

S

c LD

25 +

B

c LD

72

Co

ncen

trat

ion

(mg/

l)

methyl salicylate

O

OH

O Methyl salicylate:- wintergreen, minty, spicy

Control at pH 5.0 without yeast

Wort fermentation with S. cerevisiae brewing strains

Theaspirane B

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

begin end begin end begin end begin end +EXT

glycosides ~hopping rate

0 g/L

glycosides ~hopping rate

3 g/L

glycosides ~hopping rate

30 g/L

post-fermentationextract addition at

30 g/L

Rel

ativ

e ra

tio

LD25

LD40

0

1

2

3

4

5

floral

non-citrus fruity

citrus fruity

spicyhops

vegetable

overall appreciation

0 g/L 3 g/L 30 g/L extract

Wort + Saaz hop glycosides, or post-fermentation addition of a pre-hydrolyzed extractFermentation with S. cerevisiae LD25 and LD40 (low and high exo-1,3-β-glucanase)

Results : Linalool: significant ↑ in extract; similar for 2 yeastsDihydroedulan & theaspiranes: clear ≠ for 2 yeastsSensory: significant increase; other compounds seem to be involved

LD40

Linalool

0.0

5.0

10.0

15.0

20.0

25.0

30.0

begin end begin end begin end begin end +EXT

glycosides ~hopping rate

0 g/L

glycosides ~hopping rate

3 g/L

glycosides ~hopping rate

30 g/L

post-fermentationextract addition at

30 g/L

Con

cent

ratio

n (µ

g/l)

LD25

LD40

Refermentation of a dry-hopped beer by different ye asts

0

5

10

15

20

25

30

35

0 10 20 30 40

Time (days)

Rel

ativ

e ra

tio

.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 10 20 30 40

Time (days)

Co

nce

ntr

ati

on

(m

g/l

) .

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0 10 20 30 40

Time (days)

Co

nce

ntr

ati

on

(m

g/l)

.

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0 10 20 30 40

Time (days)

Co

nce

ntr

atio

n (

µg

/l)

.

Theaspirane B

Isoamyl acetate

4-ethyl guaiacol

β-ionone

S. cerevisiae + B. custersiiS. cerevisiae + B. bruxellensisS. cerevisiae

β-glucosidase activity vinylphenol reductase activity

esterase activityunknown activity

‘Kriek’-beer

Traditionally: intact sour cherries are added to wooden casks filled with young lambicleaving it for 5 to 6 monthsthe sugar from the sour cherries triggers a second fermentation

Currently:also cherry pulp, cherry juice and cherry stonesfruit extracts and essencesflavours

benzaldehydebenzyl alcohol

linalool

phenylethyl alcohol

benzyl acetategeraniol

eugenol

α-ionolcis-isoeugenol

α-terpineol

trans-isoeugenol

Aglycones from sour cherry glycosides

Control pH 5.0

AR2000

OHOHOH

OH

OOH

Schmid and Grosch (1986): benzaldehyde, linalool an d eugenol are some of the important contributors to cherry ar oma

SP

ME

GC

/MS

Cherry stones contain the cyanogenic glycoside amygdalin

O

OH OH

O

CH2O

OH

O

HOCH2

OH

OH

OH

C NH

OH2

C NH

OH

OH

O

HOCH2

OH

OH

OH

C

H

O

OH

O

O

O

O

HOCH2

OH

OH

OHOH2

+ 2

beta-glucosidase

Amygdalin

+

hydroxynitrile lyaseor spontaneousHCN +

Benzaldehyde

(R)-Mandelonitrile

1 or 2 steps

x 2

(stable at low pH)

Benzyl alcoholBenzyl acetate

Benzyl-beta-D-glucoside

beta-glucosidase

+

chemical or enzymatic esterification

Development of cherry flavour� Refermentation of a base beer, with addition of

200g sour cherries per liter

� Yeast: Brettanomyces custersii strain, selected for its high beta-glucosidase activity

� Results : � Increase in benzyl compounds (especially

benzaldehyde reduced to benzylalcohol)� also other compounds important for cherry

flavour: geraniol, eugenol, linalool

0,0

0,1

0,2

0,3

0,4

0,5

2 d 13 d 43 d 2 d 13 d 43 d 2 d 13 d 43 d 2 d 13 d 43 d

Whole Cherries Cherry Pulp Cherry Juice Cherry Stones

Co

nce

ntr

atio

n [m

g/L

]

0,000

0,005

0,010

0,015

0,020

0,025

0,030

2 d 13 d 43 d 2 d 13 d 43 d 2 d 13 d 43 d 2 d 13 d 43 d

Entire Cherries Cherry Pulp Cherry Juice Cherry Stones

Con

cent

ratio

n [m

g/L]

geraniol

eugenol- clove, spicy, medicinal flavour- released from eugenyl-glycoside- flavour threshold: 0.040 mg/l

geraniol- floral, rose-like- released from geranyl-glycoside- flavour threshold: 0.010 mg/l

0

1

2

3

4almond

fruity

spicy

Brett-flavour

sour

overall impression

■ cherry stones

■ cherry juice

■ cherry pulp

■ whole cherries

SummaryTreatment Comment

Acid hydrolysis Low pH (3,0 -3,5) Separate proces; sour beers

Enzymatic hydrolysis Eg. Enzymes from Aspergillus niger

Some have side activities:HCD activity � 4-VGAnthocyanidin breakdown (risk of color loss)Esterase activity � isoamylacetate ↓

Yeast biotransformation Saccharomyces sp. Mostly low or moderate activity

Brettanomyces sp. High activity; but also other Brett flavours

���� Different treatments lead to different flavour profi les

Glycoside containing substrates:� Hop flowers, pellets or hop solids� Cherries (whole, juice, pulp and/ or kernels)� Other fruits, spices, ...

~ Thank you for your attention ~

Back-up slides

♦ acid hydrolysis at pH 3.0

♦ enzyme hydrolysis (almond β-gluc.)

♦ enzyme hydrolysis (AR2000)

Hop glycoside flavour potential

♦ control at pH 5.0

0

2

4

6

8 noncitrus fruity

citrus

floral

spicy

green

minty

concentrated hop glycosideextract in buffered medium

- Release of sensory active aglycones

- Possible impact on the overall beer flavor ?

Brettanomyces LD72 identification

1st identification � physiological tests2nd � PCR fingerprinting (primers M13 and OPA09)

Brettanomyces custersii CMBS LD72 Dekkera anomala CBS 8139

Experimental set-up of dry-hopping trial

lagering tank

ale

dry hopping

normal

(3.0 g l-1)

dry hopping

concentrated

(15.0 g l-1)

dry hopping

spent hops (15.0 g l-1) no dry hopping

Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

*Sc+Bc+D

lagering tank

ale

dry hopping

normal

(3.0 g l-1)

dry hopping

concentrated

(15.0 g l-1)

dry hopping

spent hops (15.0 g l-1) no dry hopping

Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

Sc+Bb

Sc+Bc

Sc+AR Sc

*Sc+Bc+D

S. cerevisiaeS. cerevisiae + AR2000 enzyme preparationS. cerevisiae + B. bruxellensisS. cerevisiae + B. custersii S. cerevisiae + B. custersii + glucose

� samples withdrawn after 1, 8, 18 and 32 days

� glucoside hydrolase activity against model substrat e pNPG

� analysis of volatile compounds by GC-MS

HCN potential in sour cherry stones

200 g cherries / litre beer

stones = 5-7% of total weight (Chaovanalikit & Wrolstad 2004)

10 - 14 g stones / litre beer

22.0 – 30.8 mg amygdalin / litre beer

amygdalin in stones: 0.22 (w/w)% (Chandra et al. 1993)

1.30 – 1.82 mg HCN / litre beer < 4 mg HCN / litre !

MWHCN = 27 g/mol MWamyg = 457 g/mol

Regulation of HCN in foodstuffs � EU-directive 88/388/EEC (1988)

1 mg HCN per (v/v)% alcohol

Example: cherry beer � 4 (v/v)% alcohol� maximum 4 mg HCN / litre

Considering:- maximum release- no evaporation

Released benzaldehyde:5.1 – 7.1 mg / litrethreshold: 2 mg / litre

Lethale dosis � 42 mg HCN / 70 kg body weight