Use of beta-glucosidase activity for flavour enhancement in specialty beers. Luk Daenen Chair De Clerck 2012

Transcription

1 Use of beta-glucosidase activity for flavour enhancement in specialty beers Luk Daenen Chair De Clerck 2012

2 Flavour complexity Malt, hops, spices, fruit Flavour precursors flavour Yeast Flavour of the final beer flavour Specific enzymes Process addition

3 Flavour modification 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 Malt, hops, spices, fruit Flavour precursors flavour Yeast Flavour of the final beer flavour Specific enzymes Process addition

4 Interest for wine-making Some grape varieties have a significant pool of glycosidically bound flavour compounds hidden flavour potential Hydrolysis by yeast, enzymes,... Gunata et al 1984

5 Interest for brewing industry Hops Flavour precursors in green part of hops (flowers, leaves,...) Enhance hoppy aroma by precursor hydrolysis Interest for lager, ale, geuze, specialty beers,... Cherry Flavour precursors in juice, kernel, skin Enhance cherry flavour by precursor hydrolysis Interest for fruit beers

6 Release of hidden flavour potential Glycosidically bound flavour compounds = glycosides H + H H Linalool-glucoside non-volatile odourless Yeast enzymes (beta-glucosidases) acid Linalool lavender, citrus, floral glucose 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 profiles

7 Selection of yeast strains Saccharomyces sp. Mostly no β-glucosidase All have exo-1,3- β-glucanse activity Low to moderate activity Some commercial wine strains with inducible β-glucosidase 80 Brettanomyces sp. Some with high β-glucosidase Mainly cell associated (low extracellular) Also minor glycosidase activities enzyme activity (mu/ml) extracellular intracellular cell insoluble fraction 0 pnpg pnpr pnpx pnpa p-nitrophe nyl glycoside

8 Isolation of hop glycosides Hop pellets (Saaz) Supercritical C 2 extraction Essential oils Soft resins 1) ethanol / water 2) ethanol evaporation Solid Phase Extraction (XAD2) 1) wash with water 2) elution with ethanol 3) evaporation 4) PVPP remove polyphenols spent hops / hop residue / hop solids extraction watery extract purification hop glycoside extract

9 Aglycones from hop glycosides SPME GC/MS Me 4-hydroxy-7,8-dihydro-β-ionol ❿A control ph 5.0 ❿% 1-octen-3-ol benzyl alcohol linalool methyl salicylate α-terpineol theaspirane A ❿B AR2000 enzyme prep. ❿% 4-terpineol nerol theaspirane B dihydroedulan I

10 YPD fermentation with added hop glycosides Linalool: - citrus, floral, aniseed - important contributor to hop aroma Methyl salicylate: - wintergreen, minty, spicy Commercial S. cerevisiae Lab Brettanomyces Mixed strain S. brewing Lab cerevisiae culture deletion strain Control strain S. custersii wild wine cerevisiae transformant at LD40 LD25 type ph strain 5.0 LD72 S. LD25 without cerevisiae high low S. inducible β-glucosidase cerevisiae and exo-1,3-β-glucanase yeast B. BY4742 custersii β-glucosidase BY4742 activity LD72 exg1 activity 0,016 0,014 linalool 0,010 0,009 methyl salicylate Concentration (mg/l) 0,012 0,010 0,008 0,006 0,004 0,008 0,007 0,006 0,005 0,004 0,003 0,002 0,002 0,001 0,000 0,000 Control ph 5 Sc BY4742 wild type Sc BY4742 exg1 Sc LD25 Sc LD40 Sc UVA228 Bc LD72 Sc LD25 + Bc LD72 Control ph 5 Sc BY4742 wild type Sc BY4742 exg1 Sc LD25 Sc LD40 Sc UVA228 Bc LD72 Sc LD25 + Bc LD72 Concentration (mg/l)

11 Wort fermentation with S. cerevisiae brewing strains Wort + Saaz hop glycosides, or post-fermentation addition of a pre-hydrolyzed extract Fermentation with S. cerevisiae LD25 and LD40 (low and high exo-1,3-β-glucanase) Results: Linalool: significant in extract; similar for 2 yeasts Dihydroedulan & theaspiranes: clear for 2 yeasts Sensory: significant increase; other compounds seem to be involved overall appreciation vegetable hops floral spicy LD40 non-citrus fruity 0 g/l 3 g/l 30 g/l extract citrus fruity Linalool Theaspirane B Concentration (µg/l) LD25 LD40 Relative ratio LD25 LD40 begin end begin end begin end begin end +EXT 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-fermentation extract addition at 30 g/l glycosides ~ hopping rate 0 g/l glycosides ~ hopping rate 3 g/l glycosides ~ hopping rate 30 g/l post-fermentation extract addition at 30 g/l

12 Refermentation of a dry-hopped beer by different yeasts S. cerevisiae S. cerevisiae + B. bruxellensis S. cerevisiae + B. custersii Relativ e ratio Theaspirane B β-glucosidase activity Concentration (mg/l). 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 4-ethyl guaiacol vinylphenol reductase activity 0 0, Time (days) Time (days) Concentration (mg/l). 3,0 2,5 2,0 1,5 1,0 0,5 Isoamyl acetate esterase activity Co n cen tratio n (µ g/l). 0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 β-ionone unknown activity 0, Time (days) 0, Time (days)

13 Kriek -beer Traditionally: intact sour cherries are added to wooden casks filled with young lambic leaving it for 5 to 6 months the sugar from the sour cherries triggers a second fermentation Currently: also cherry pulp, cherry juice and cherry stones fruit extracts and essences flavours

14 Aglycones from sour cherry glycosides SPME GC/MS H Control ph 5.0 Schmid and Grosch (1986): benzaldehyde, linalool and eugenol are some of the important contributors to cherry aroma benzaldehyde eugenol AR2000 benzyl alcohol α-terpineol linalool benzyl acetate geraniol α-ionol trans-isoeugenol phenylethyl alcohol cis-isoeugenol

15 Cherry stones contain the cyanogenic glycoside amygdalin HCH 2 HCH 2 H H H CH 2 H C + 2 N H 2 beta-glucosidase 1 or 2 steps H H + H H x 2 C N H Amygdalin HCN + C H hydroxynitrile lyase or spontaneous (R)-Mandelonitrile (stable at low ph) Benzaldehyde H chemical or enzymatic esterification Benzyl alcohol Benzyl acetate HCH 2 beta-glucosidase H H 2 + H Benzyl-beta-D-glucoside

16 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 cherry stones cherry juice cherry pulp whole cherries overall impression sour almond Brett-flavour fruity spicy Concentration [mg/l] Concentration [mg/l] 0,5 0,4 0,3 0,2 0,1 0,0 0,030 0,025 0,020 0,015 0,010 0,005 0,000 eugenol - clove, spicy, medicinal flavour - released from eugenyl-glycoside - flavour threshold: mg/l 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 geraniol - floral, rose-like geraniol - released from geranyl-glycoside - flavour threshold: mg/l 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

17 Summary Treatment 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-VG Anthocyanidin 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 profiles Glycoside containing substrates: Hop flowers, pellets or hop solids Cherries (whole, juice, pulp and/ or kernels) ther fruits, spices,...

18 ~ Thank you for your attention ~

19 Back-up slides

20 Hop glycoside flavour potential concentrated hop glycoside extract in buffered medium noncitrus fruity 8 6 control at ph 5.0 acid hydrolysis at ph 3.0 enzyme hydrolysis (almond β-gluc.) enzyme hydrolysis (AR2000) minty green citrus floral - Release of sensory active aglycones - Possible impact on the overall beer flavor? spicy

21 Brettanomyces LD72 identification 1st identification physiological tests 2nd PCR fingerprinting (primers M13 and PA09) Brettanomyces custersii CMBS LD72 Dekkera anomala CBS 8139

22 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 S. cerevisiae + AR2000 B. bruxellensis custersii enzyme + glucose preparation samples withdrawn after 1, 8, 18 and 32 days Sc Sc+Bb glucoside hydrolase activity against model substrate pnpg Sc+Bc Sc+AR Sc Sc+Bb Sc+Bc *Sc+Bc+D Sc+AR Sc analysis of volatile compounds by GC-MS Sc+Bb Sc+Bc Sc+AR Sc

23 HCN potential in sour cherry stones Regulation of HCN in foodstuffs EU-directive 88/388/EEC (1988) Example: cherry beer Released benzaldehyde: mg / litre threshold: 2 mg / litre Considering: - maximum release - no evaporation 1 mg HCN per (v/v)% alcohol 4 (v/v)% alcohol maximum 4 mg HCN / litre 200 g cherries / litre beer stones = 5-7% of total weight (Chaovanalikit & Wrolstad 2004) g stones / litre beer amygdalin in stones: 0.22 (w/w)% (Chandra et al. 1993) mg amygdalin / litre beer MW HCN = 27 g/mol MW amyg = 457 g/mol mg HCN / litre beer < 4 mg HCN / litre! Lethale dosis 42 mg HCN / 70 kg body weight