Characterisation of New Zealand hop character and the impact of yeast strain on hop derived compounds in beer Graham Eyres, B. Gould, V. Ting, M. Leus, T. Richter, P. Silcock, and P.J. Bremer Department of Food Science University of Otago, Dunedin, New Zealand IBD Conference, Sydney, 14-18 March 2016
Outline Introduction: Hop aroma in beer Research program at University of Otago Research objectives Study 1: Volatile Analysis of NZ Hops Study 2: Effect of yeast strain on hop-derived compounds in beer Next steps: Ongoing research
Introduction: Hop Aroma in Beer Hop essential oil is a complex mixture of compounds More than 500 have been identified. Composition and aroma characters depend upon hop variety. Not all impact aroma compounds have been identified. Wide diversity of hop aroma characters in beer Relationship between chemical composition and hop aroma characters in beer is complex. Combination of varieties and hopping regimes Influence of yeast strain and fermentation parameters
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. Aim to add value to the industry through understanding of hop aroma in beer.
Hop Varieties Recently released hop varieties Plant and Food Research, NZ Hops with a Difference program Brooklyn, Wai-iti, Kohatu, Waimea New cultivars being released around the world. Selected for novel aroma characters But the compounds responsible are not well characterised. Greater knowledge of the aroma chemistry will provide information to enhance further breeding. Determine varietal molecular markers http://nzhops.co.nz/ variety/brooklyn
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. Current research objectives Identify the impact odorants in hop essential oil from NZ hop varieties. Investigate the effect of yeast strain and fermentation parameters on hop compounds in beer.
Overview Identification of hop aroma compounds Study 1 Study 2 Characterisation of NZ hop varieties Effect of yeast strain on hop compounds in beer GC/MS 7 varieties GC-olfactometry 3 Yeast strains 3 hopping regimes GC/MS
Study 1: Volatile analysis of NZ hops Objectives Characterise the volatile composition of NZ hop varieties. Select samples for further investigation. Samples 7 NZ hop varieties Whole hop cones from 2015 harvest Nelson Sauvin, Riwaka, Motueka, Rakau, Wai-iti, Kohatu and Waimea Analysis of volatile compounds using GC/MS
Study 1: Volatile Analysis of NZ hops Research Question What are the aroma compounds responsible for unique aroma compounds in New Zealand hops
Study 1: Volatile Analysis of NZ hops Methods Hop cones ground in liquid nitrogen Powder (1g) Aqueous extract (5g/L, 90 C, 5 min) Headspace SPME sampling PDMS/Car/DVB fibre Exposure: 60 C for 30 min (+5 min pre-incubation) GC/MS Analysis Desorption at 250 C, 5 min, Split 10:1 Separation on both polar (wax) and non-polar (HP-5) columns Analysis in triplicate; randomised order
Study 1: Volatile Analysis of NZ hops Data Analysis Peaks were identified by comparison of mass spectra to NIST 2014 database, supported by RI. Data was processed using a metabolomic processing approach. Data alignment and normalisation. Top 50 compounds extracted which explained the greatest variation between samples. Visualisation of data using Principal Components Analysis (PCA).
PCA of Aqueous Hop extract Polar column
PCA of Aqueous Hop extract Polar column
PCA of Aqueous Hop extract Polar column
Summary: Study 1 Characterising compounds identified Motueka, Riwaka: Methyl geranate Kohatu: 3-Methylbutyl isobutyrate Wai-iti, Rakau, Waimea: Methyl esters Nelson Sauvin: Geranyl isobutyrate Hop varieties could be discriminated based upon their volatile composition. Provides an excellent screening tool What are the compounds responsible for the aroma characters?
Next Steps In progress More data analysis! Confirmation of identified compounds Selection of four samples for GC/MS-olfactometry Identification of odour active compounds Trained panel of 6 assessors GC-O identifies the players but cannot be used alone to explain sensory perception Sensory Evaluation Descriptive Analysis Provide quantitative aroma profile of NZ hops Correlate to volatile composition analysis
Study 2: 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.
Study 2: Effect of yeast strain on hop-derived compounds in beer Objective: Investigate the effect of yeast strain on hop derived volatile compounds in beer under constant conditions. Experimental Design 3 Ale Yeast strains 3 Hopping Regimes Late hop with Nelson Sauvin or Motueka No aroma hop Analysis by GC/MS
Experimental Design No Aroma Hops Nelson Sauvin Aroma Hops Motueka hops Aroma Hops Fermenter 1 Fermenter 2 Fermenter 3 Fermenter 4 Ale Yeast 1 Ale Yeast 2 Ale Yeast 3 Ale Yeast 1 Ale Yeast 2 Ale Yeast 3 Ale Yeast 1 Ale Yeast 2 Ale Yeast 3 Yeast 3 Replicate Yeast 2 Replicate Yeast 1 Replicate Three pilot scale (200L) worts were brewed at Emerson s Brewery (Dunedin, NZ). All malt Pilsner-type grain bill. Constant bittering to 25 IBU with Waimea hop pellets (60 min boil). Late hopping: Aroma hops (2.5g/L pellets) added at 5 min prior to flame-out. Wort separated into 4 Fermentors (40L) Pitched with three different yeast strains + 1 replicate
Methods Fermentation Conditions Yeast Culture Preparation: 2L culture of 10 P wort Stirred 24 h @ 20 C Pitch rate: 7.5 x 10 6 cells / ml Original gravity: 1045 (11.2 P) Fermentation: 20 C for 7 days Monitored Temperature, density & yeast counts Maturation: 4 C for 10 days GC/MS Analysis Beer samples (8mL) + 2g NaCl in 20 ml headspace vials Headspace SPME sampling PDMS/Carboxen fibre Exposure: 40 C for 30 min (+5 min pre-incubation) GC/MS Analysis Desorption at 230 C, 5 min, SL Separation on 60m ZB-Wax column Analysis in triplicate; randomised
Results Fermentation curves were similar; final gravities ranged between SG1008 to 1010. A total of 79 compounds were found using GC-MS across all beer samples 32 compounds derived from late hopping Statistical analysis with ANOVA determined: Significant effects of Yeast Significant effects of Hopping Significant Yeast*Hop interactions
Compounds derived from Aroma hop addition RI Hop Derived NS Motueka F (2,18) p value 1183 3-Methylbutyl 2-methylpropanoate 127 < 0.0001 1193 2-Methylbutyl 2-methylpropanoate 23 < 0.0001 1283 2-Methylbutyl 2-methylbutanoate 366 < 0.0001 1349 Methyl 3-methyl-2-hexenoate 1266 < 0.0001 1508 Ethyl Decanoate 43 < 0.0001 1601 Nonyl Acetate 74 < 0.0001 1711 Humulene 47 < 0.0001 1862 Geraniol 113 < 0.0001 1159 Myrcene 386 < 0.0001 1566 Linalool 136 < 0.0001 1687 2,6-Dimethyl 2,6-octadiene 344 < 0.0001 1775 Isobutyl decanoate 97 < 0.0001 2341 Unknown 65 < 0.0001
Compounds from Nelson Sauvin in Beer RI Hop Derived NS Motueka F (2,18) p value 1085 2-Methylpropyl 2-methylpropanoate 15 < 0.001 1292 2-Methylbutyl 3-methylbutanoate 55 < 0.0001 1319 Geranyl isobutyrate 4 < 0.05 1391 Hop Ether 62 < 0.0001 1627 (E)-p-Menth-2,8-dien-1-ol 33 < 0.0001 1752 Geranyl butanoate 101 < 0.0001 1757 Ethyl undecanoate 4 < 0.05 1796 Methyl benzeneacetate 50 < 0.0001 1907 2-Methylpropyl 2-methylpropanoate 4 < 0.05 1910 Ethyl benzenepropanoate 84 < 0.0001 1964 Heptanoic acid 79 < 0.0001 1987 beta-phenylethyl butyrate 4 < 0.05 2019 Unknown 1266 < 0.0001 2027 Unknown 8 < 0.01
Compounds from Motueka in Beer RI Hop Derived NS Motueka F (2,18) p value 1187 2-Pentyl propanoate 259 < 0.0001 1437 Perillene 2288 < 0.0001 1636 Caryophyllene 197 < 0.0001 2126 2-Phenylethyl methoxyacetate 266 < 0.0001
PCA : All Beer Samples Motueka Nelson Sauvin No Aroma Hop
PCA Nelson Sauvin Nonyl acetate 2,6-Octadiene, 2,6-dimethyl- Heptanoic acid Isobutyl decanoate Geranyl acetate Unknown RI 2341 Perillyl acetate
PCA of Motueka Ethyl undecanoate Caryophyllene 3-methylbutyl isobutyrate Isobutyl decanoate Nonyl acetate 2-Pentyl propanoate 2-methylbutyl 2-methylbutyrate
Influence of Yeast on Hop-derived compounds 44 compounds exhibited significant Yeast*Hop interactions. The top 10 hop-derived compounds were: RI Compound F p-value 1775 Isobutyl decanoate 116 < 0.0001 1601 Nonyl acetate 74 < 0.0001 1187 2-Pentyl propanoate 68 < 0.0001 2341 Unknown 65 < 0.0001 1159 Myrcene 63 < 0.0001 1687 2,6-dimethyl 2,6-octadiene 23 < 0.0001 1964 Heptanoic acid 20 < 0.0001 1183 3-Methylbutyl 2-methylpropanoate 13 < 0.0001 1779 Geranyl acetate 11 < 0.0001 1711 Humulene 10 < 0.001
Conclusions: Study 2 Significant differences in hop derived compounds in beer with three different yeast strains. Yeasts suppressed / enhanced levels of specific compounds in combination with each hop variety. A lot more work to be done in this area Repeat brewing experiments confirm results Extend range of hop varieties and yeast strains Correlate to sensory perception of hop aroma and flavour Descriptive sensory analysis with trained panel
Ongoing Research: Where to from here? GC/MS-olfactometry of NZ Hop varieties Identify the aroma compounds responsible for novel aroma characteristics Correlate to sensory evaluation Investigate thiol compounds in hops and beer Characterisation of yeast*hop interactions on hop aroma compounds in beer and hop flavour Evaluate impact of hopping regime Investigate effect of fermentation parameters Directly test hypotheses to elucidate the mechanisms responsible for these yeast*hop interactions. Evaluate impact on sensory perception.
Research Program at University of Otago Aroma compounds in hops Add value to hop breeding Understand hop flavour in beer Identify hop derived aroma compounds in beer Effect of yeast and fermentation on hop flavour in beer Target Outcome: Control and optimise hop flavour in beer
Acknowledgements University of Otago Research Grant Emerson s Brewery, Dunedin, NZ Richard Emerson, Chris O Leary and Brendan Bransgrove Brewing trials Emerson s Brewery JP Dufour scholarship Bridget Gould Ron Beatson (Plant and Food Research) 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 (03) 479 7661 THANK YOU
Brew 1: NS Brew 3: Mo Brew 2: NH Yeast 1 Yeast 2 Yeast 3
PCA No Aroma hops
GC/MS-Olfactometry Identification of aroma-active compounds Multifunctional Autosampler Sampling Liquid Headspace SPME Twister Thermal desorption MSD GC oven Odour Port
GC-MS/Olfactometry Identifies the players that drive flavour character and intensity. Limitation: odorants are evaluated in isolation. No effect of sensory interactions. No effect of the food matrix and release. Important to evaluate the importance of an identified odorant in the food matrix.