Investigating the factors influencing hop aroma in beer Graham Eyres, Tobias Richter, Jamie Scrimgeour, Pat Silcock and Phil Bremer Department of Food Science University of Otago, Dunedin, New Zealand IBD Conference, Wellington, 19-23 March 218
Outline Introduction: Hop aroma in beer Research program at University of Otago Research objectives Study 1: Effect of yeast strain on hop-derived compounds in beer Study 2: Effect of yeast pitch rate Study 3: Online measures during fermentation Summary and next steps Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Introduction: Hop Aroma in Beer Consumers crave novel aroma and flavour characters and seeking greater diversity. Hop aroma and flavour in beer is a major driver for this diversity. Wide diversity of sensory attributes. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Introduction: Hop Aroma in Beer New aroma cultivars being released around the world. E.g. Hops with a difference breeding program Selected for novel aroma characters But the compounds responsible in beer are not well characterised. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Introduction: Hop Aroma in Beer Hop aroma in beer is not well understood Opportunity Current resurgence of interest in aroma hops and hop chemistry due to growth of the craft beer industry. Add value to the industry through understanding of hop aroma in beer. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Research Objectives Long term goals Understand the aroma compounds responsible for hop derived aroma in beer. Understand the factors that influence hop flavour in beer. Target Outcome: Control and optimise hop flavour in beer Current research objective Investigate the effect of yeast strain and fermentation parameters hop compounds in beer. Graham Eyres, Factors Influencing Hop Aroma in on Beer, IBD Conference, Wellington, 19-23 March 218
Study 1: Effect of yeast strain on hop flavour Research Question What is the effect of yeast strain on hop volatile compounds in beer? + + + = + Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Study 1: Effect of yeast strain on hop-derived compounds in beer Introduction Evidence that yeast strain and fermentation factors have a dramatic effect on hop flavour in beer. Limited information in the literature of these interactions under controlled conditions. Hypotheses: Adsorption and loss during fermentation Biotransformation of hop compounds Release of glycosidically bound compounds Sensory interactions with fermentation derived compounds The fundamental mechanisms and relative magnitude of these different effects are not well understood. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
First Step: Identification of target compounds Beer fermented in 1L bottles With aroma hops No aroma hops Aroma Hop: Motueka (5g/L) 9 C for 5 min GCMS Hot water aroma hop extract 9 C for 5 min Key parameters: 1 P wort 2 IBU (with Waimea) 12 day fermentation at 2 C 5 day maturation at 2 C Yeast: US-5 (1x17cell/mL)
Hop-derived positively influenced by fermentation Hop-derived negatively influenced by fermentation 13 Compounds Detected 41 Compounds Significantly Differed Yeast-derived negatively influenced by hops Hop-derived not influenced Hot Water Aroma Hop Extract Beer without Aroma Hops Beer with Aroma Hops Yeast-derived positively influenced by hops
Main Study Design Yeast Selection 15 Yeast strains Fermented @ 2 C Fermented @ 15 C Strain Details Strain Details US-5 Fermentis ale strain W-34/7 Fermentis lager strain S-33 Fermentis ale strain S-23 Fermentis lager strain BE-256 Fermentis ale strain S-189 Fermentis lager strain WB-6 Fermentis wheat beer OTA 19 European lager yeast OTA 79 Ale strain VIN 13 Anchor Wine yeast hybrid WLP45 Scotch whiskey yeast OTA 29 Champagne yeast, S. bayanus WLP73 Chardonnay wine yeast Exotics SPH Anchor Wine yeast hybrid OTA 48 Schizosaccharomyces pombe
Methods Wort (1 P, 2 IBU) Late Hopped with Motueka (5g/L). Each strain: 2 fermentation replicates x 2 yeast cultures. Headspace sampling in duplicate with Gerstel Twister (PDMS) and analysed using GC/MS.
Study 1: Preliminary Results PCA of all yeast strains selected compounds Spread of differences in volatile compounds between yeast strains No distinct groups according to Ale, Lager or wine yeast types Champagne Yeast 8 OTA29 6 OTA29 4 PC 2 (16 %) Chardonnay Yeast OTA48 WLP73 OTA48 2 WB-6 WLP45 WLP45 WLP73 S-23 VIN 13 S-189-6 -4-2 European Lager OTA19 OTA19 S-33 2 OTA79 W-34/7 Exotics S-33-2 VIN 13 OTA79 BE-256 S-189-4 US-5-6 4 US-5 W-34/7 S-23 Exotics BE-256 PC 1 (25 %) WB-6 6 8 1 Wheat beer yeast 12
Study 1: Preliminary Results PCA of all yeast strains selected compounds 8 6 citronellol acetate PC 2 (16 %) 4 geranyl acetate terpinen-4-ol methyl geranate linalool 2-6 -4-2 -2 neryl acetate -4 2 4 myrcene citronellol geraniol nerol -6 PC 1 (25 %) 6 8 1 12
Ale Yeasts Selected Compounds
Study 1: Conclusions Different yeast strains resulted in a variety of volatile compound profiles. Evidence in differences in biotransformation of hop derived compounds. Expected to have impacts on hop character and intensity in beer. Ongoing research to look at Sensory Evaluation.
Study 2: Yeast pitch rate on hop compounds in beer Research Question What is the effect of pitch rate on hop volatile compounds in beer? Three pitch rates with Late hopping with Fermentis SafAle US-5 Nelson Sauvin (5g/L) Late Hop No Late Hop Low Pitch 2 M cells / ml Standard Pitch 1 M cells / ml High Pitch 5 M cells / ml LH_LP LH_SP LH_HP NH_SP Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Sample preparation and analysis Wort (1 P, 2 IBU) were fermented at 2 C to constant gravity in EBC fermentation tubes. Conditioned at 3 C for 1 days. Fermentations performed in duplicate. Samples taken for volatile analysis using GC/MS using a headspace SPME method (n=3). Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Fermentation Profiles High Pitch 4 YEAST CELLS PER ML (MILLIONS) 35 NH_SP 1 NH_SP 2 LH_LP 1 LH_LP 2 LH_SP 1 LH_SP 2 LH_HP 1 LH_HP 2 3 25 2 Std Pitch 15 1 12 Std Pitch 1 YEAST CELLS PER ML (MILLIONS) 45 Low Pitch NH_SP 1 NH_SP 2 LH_LP 1 LH_LP 2 LH_SP 1 LH_SP 2 LH_HP 1 LH_HP 2 8 Low Pitch 6 4 2 5 High Pitch 5 1 15 TIME (HOURS) 2 25 5 1 15 TIME (HOURS) Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218 2 25
Results Fermentation esters decreased with increasing pitch rate. No Late Hop Low Std High No Late Low Hop Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218 Std High
Significant effects of pitch rate on hop-derived compounds
Study 2: Conclusions Concentrations of hop derived compounds varied with yeast pitch rate. Compounds followed different trends. Potential for different hop flavour balance. Biotransformation pathways require further investigation (not clear correlations). Large differences in fermentation compounds (e.g. esters) expected to have an impact on hop flavour through sensory interactions. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Study 3: Tracking aroma compounds during fermentation Beer prepared with different treatments Yeast\Hop No aroma hop Motueka Hops Nelson Sauvin Hops No yeast Control MT NS California Ale Yeast CA CA / MT CA / NS Scottish Ale Yeast SA SA / MT SA / NS Key parameters: 1 P wort 2 IBU (with Waimea) Flame-out hop addition: 5g/L Pitching: ~1x17cell/mL Volume: 6 replicates with 3mL 5 day fermentation at 2 C PTR-TOF-MS Proton transfer reactiontime of flight-mass spectrometer 3 ml fermentation in HS vials Richter et al. (218) Submitted to Food Research International
Tracking Fermentation with PTR-TOF-MS California Ale Yeast m/z 145.121 concentration [pptv] Scottish Ale Yeast concentration [pptv] Scottish Ale with Nelson Sauvin California Ale with Nelson Sauvin California Ale with Motueka 18 Scottish Ale with Motueka 18 Scottish Ale without hop California Ale without hop 16 no yeast/no aroma hops Motueka without yeast 16 NS 14 Nelson Sauvin without yeast NS 14 Motueka without yeast Nelson Sauvin without yeast no yeast/no aroma hops Octanoic acid / Methyl heptanoate 12 12 Ethyl hexanoate 1 1 No yeast Control 8 6 6 4 8 MT MT 4 2 2 6 12 18 24 3 36 42 48 54 6 66 72 78 fermentation time [hours] 6 12 18 24 3 36 42 48 54 6 66 72 78 fermentation time [hours] Note the changes in hop related, yeast related and hop / yeast related compounds over time
Tracking Fermentation with PTR-TOF-MS Volatile profile differentiation: At the start of fermentation is due to the hop cultivar s characteristics At end of fermentation is due to differences in the metabolism of the yeast strains. CO2 and ethanol curves show rates of metabolism and changes in metabolism Concentration CO2 [au] h 42 h 6h 48 h 18 h 6 h 24 h 66 h 3 h 72 h 36 h 78 h Concentration EtOH [ppbv] 3 25 25 2 2 15 15 1 1 Control CO2 Fermentation CO2 Control EtOH Fermentation EtOH 5 5 12 h 54 h 6 12 18 24 3 36 42 48 54 6 66 72 78 Fermentation time [h] 631 m/z total 168 m/z Sign Diff
Tracking Fermentation with PTR-TOF-MS Possible to differentiate the beers based on the last 4 time points (time points 6-78h) California Ale with Motueka ms75.77 California with Nelson Sauvin8 Scottish Ale with Motueka Scottish Ale with Nelson Sauvin 6 Hop effects and Yeast / Hop interaction effects are evident. SA Loadings ms138.135 ms137.132 ms95.84 ms81.68 ms9.61 ms89.57 ms62.3 ms61.27 ms43.17 ms231.974 4 CA PC3 (9%) 2-1 ms75.42 ms13.73 Esters and Terpenes are mainly associated with the differences between the beers -5 5 1-2 ms131.15-4 ms117.9 ms118.93 ms146.125 ms145.122-6 ms87.78 ms11.94 ms73.63 ms129.126-8 PC1 (4%) ms173.153 15
Study 3: Conclusions Volatile generation during fermentation is complex and a dynamic process. PTR-ToF-MS can successfully differentiate and monitor the change in compounds during fermentation. Including hop derived compounds. Real-time monitoring provides temporal information that will enhance understanding of the biochemical pathways and chemical processes occurring during fermentation. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Ongoing Research Continuing to mine data for hop-yeast interactions. Explore biotransformation pathways and mechanisms for the differences. Link to sensory evaluation to determine the impact on perceived hop flavour and aroma. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Summary Apply advanced analytical tools to investigate hop flavour in beer. Effect of yeast strain and fermentation. Understand changes over time. Gain a mechanistic understanding of the hop-yeast related chemical changes. Enable brewers to select and manage yeasts cultivars to enhance hop aroma in beer. Achieve target flavour profile with consistency. Graham Eyres, Factors Influencing Hop Aroma in Beer, IBD Conference, Wellington, 19-23 March 218
Acknowledgements Dr Franco Biasioli, FEMS, San Michele all Adige Tobias Richter acknowledges Emerson s JP Scholarship to support his studies. Jo Pitt (Fermentis) for providing yeast. Ron Beatson (Plant and Food Research) and Doug Donelan (NZ Hops Ltd) for provision of hop samples.
Dr Graham Eyres Sensory Science Research Centre Department of Food Science University of Otago Dunedin, New Zealand graham.eyres@otago.ac.nz (3) 479 7661 THANK YOU