The OSU Barley Flavor Project Dustin Herb Ph.D. Candidate The OSU Barley Project Oregon State University January 17 th, 2016

Goals Determine if flavor differences within modern barley varieties exist. Identify factors that influence flavor difference. Develop methods to select for barley flavor. Genetically map gene(s) controlling the flavor expression within barley.

Willamette Flavor 3 varieties (Full Pint; Copeland; Klages) grown in Lebanon, OR in yield strips. Malted at the CMBTC. Brewed at New Glarus Brewing Co. and Sierra Nevada Brewing Co. In house sensory, BIC sensory, and OSU sensory. Difference test – revealed that flavor differences were present among the three varieties.

3 Varieties x 3 Locations 3 varieties (Full Pint; Metcalfe; Klages) grown in 3 locations (Oregon; Minnesota; Saskatchewan). Malted at Rahr Malting Co. Wort Sensory and analytics at Sierra Nevada Brewing Co. Genotype x Environment interaction flavor effect. FlavorVarietyEnvironmentG X ERep Total TasteX MaltXXX FruityXXX ChocolateXXX ToffeeXXXX VegetableXX DairyX WoodyX ChemicalX

FlavorAroma CompoundTop VarietyTop Environment Buttery2, 3-butanedioneFull PintOregon CheesyButyric acidMetcalfeMinnesota WaxyNonanoic acidFull PintOregon Nutty2-ethyl-3,5-dimethyl pyrazineFull PintMinnesota Nutty2,6-diethyl pyrazineMetcalfeOregon Nutty2-acetyl furanKlagesOregon BreadyFurfuryl alcoholFull PintMinnesota Roasted2-formyl-1-methyl pyrroleKlagesMinnesota Chocolate2-methyl butanalFull PintOregon Spicy2-methyl propanalMetcalfeMinnesota Greenphenyl acetaldehydeMetcalfeOregon Floralphenyl ethyl alcoholMetcalfeMinnesota Fruityethyl hexanoateMetcalfeMinnesota Etherealethyl acetateFull PintOregon Aldehyde3-methyl butanalFull PintOregon Chemical2-ethyl furanFull PintOregon Sulferousdimethyl sulfideFull PintMinnesota FlavorTop VarietyTop Environment TasteKlagesSaskatchewan DairyFull PintOregon MaltyFull PintOregon ToffeeFull PintOregon RoastedFull PintOregon ChocolateFull PintOregon FruityMetcalfeMinnesota VegetableMetcalfeOregon ChemicalKlagesOregon DirtyMetcalfeMinnesota AROMA COMPOUNDS SENSORY

Enzyme Brewing – Assessing the Malting Effect Determine what flavor compounds are present in barley. Determine a flavor baseline for barley. Evaluate the persistence of barley and malt flavor compounds throughout the brewing process. Same source barley, ½ malted, ½ brewed with Ondea Pro®. Identical brewing recipe. Brewing parameters for the barley grain and barley malt brews at Oregon State University pilot brewery in Corvallis, OR Target Gravity Mashing Regime Kettle Boil BitternessYeast/rate (%) ABV Ferment Length 15˚P Rest I: 30 min, 54C Rest II: 60 min, 64C Rest III: 15 min, 78C 60 min 32 g / hl Iso- Alpha-Acids 1056 Ale yeast days Finished beer measurements for barley grain and barley malt samples at the Oregon State University pilot brewery in Corvallis, OR Beers Color ( ˚ SRM) Turbidity (NTU) Apparent Extract (˚P) Real Extract (˚P) RDF (%) pH ABV (%) Bitterness (BU) Barley Malt

Oregon Promise Pop’n Golden Promise x Full Pint 202 double haploid population. Golden Promise is an iconic variety for malting, brewing, and distilling. Full Pint is a contributor to the craft brew Renaissance. The Oregon Promise will provide a valuable resource for extending current knowledge of malting and brewing genes to the frontiers of sensory assessment.

Testing Environments 37 agronomic selection + parents grown in all three locations. 14 random selection grown only in Corvallis, OR. Corvallis, OR Lebanon, OR Madras, OR

Micro-malting & Malt analysis

Malt Quality Parents & Control SampleAABGColorDPExtPlumpMoistpHProtTurbViscos Full Pint AVG STDEV %CV Golden Promise AVG STDEV %CV Control AVG STDEV %CV

Malt Quality Malt quality of agronomic Oregon Promise selections from all environments (Corvallis, OR; Lebanon, OR; Madras, OR) analyzed by Rahr Malting Co. in Agronomic Selections Sample AABGColorDPEXTPlumpMoistpHProtTurbViscos COR AVG STDEV %CV LEB AVG STDEV CV% MAD AVG STDEV CV% Malt quality of random Oregon Promise selections from Corvallis, OR environment analyzed by Rahr Malting Co. in Random Selections Sample AABGColorDPEXTPlumpMoistpHProtTurbViscos COR AVG STDEV CV%

Nano-brewing 1.Malt samples were milled. 2.75g of malt grist and ~400 mL of water was placed in mash bath cans. 3.Mash cans are brought to standardized volume of 450 mL. 4.Congress mash is poured over Ahlstrom filters. 5.Wort was filtered three times for clarity. 6.Final wort was collected

Nano-brewing 7.IsoHop® is added to the congress wort and boiled for 35 min. 8.Residual sediment and trub filtered out with Ahlstrom filters. 9.WhiteLabs Czech yeast pitched and wort ferments in tube at 9°C. 10.Beer is centrifuged, bottled, and carbonated then conditioned

Sensory Comparison to reference Difference testing Descriptive analysis ~13 experimental beers 2 parental beers - FP & GP 1 internal standard - Rahr Pils 1 embedded reference - Miller High Life 17 beer sample/session X10 tasting sessions ~170 beer samples tasted

Full Pint Golden Promise

New Glarus Brewing Co.Rahr Malting Co Full, Malty, Smooth, Rich, Nice! - Best "Euro" Type Malty, Toasted/Bread, Roasted/Toffee Nice Foam, Clean, Smooth - Best "American" Type Honey, Nutty, Floral Best Foam, Sweet, Clean, MildHerbaceouse, Earthy, Green Apple Balanced Malty and Hoppy, Clean, Sweet Malty, Toffee, Caramel, Vanilla, Papery Sweet, full, very clean, bread, maltMalty, Nutty, Floral, Phenolic, DMS Malty, sweet / dry, morish, low colorMalty, Toffee, Caramel, DMS Light, clean, malty, sulfur, Euro, juicy, niceDiacetyl, DMS, Worty Sweet, full, biter after taste, malty, clean Acidic, Sour, Tart, Rubber Wow, malty, biscuit, sweet, candy, floral Fruity, Floral, Sweet Coffee! Roasted bitter, "White Stout"?, very unique Malty, Worty Malty, bread, European, very nice Nutty, Bananas, Floral Full PintFull, malty, EuropeanMalty, Sweet, Popcorn, Grain, Grass Golden PromiseHarsh bitternessFruity, Floral, Grass/Green, Astringent

Gas Chromatography-Mass Spectrometry Identify aroma compound peaks. Determine if significant compound(s) affect sample flavor. Track compound deviation from parental genotypes. Identify compounds that will be useful in flavor selection programs.

GC-MS

COMPOUNDFLAVOR 1-ButanolBananas, Sweet 1-HexanolFruity, Sweet, Oil, Green, Ethereal 1-OctanolFruity, Floral, Citrus 1-PropanolAlcoholic, Ethanol, Fruity, Apple, Nutty 2,3-ButanediolFruity, Buttery 2-HeptanoneFruity, Coconut, Sweet, Spicy, Green Acetic acidFruity, Sour, Vinegar BenzaldehydeSharp, Cherry, Sweet, Almond BenzeneacetaldehydeHoney, Sweet, Floral, Rose, Cocoa, Spicy, Green, Earthy BenzothiazoleSulfur, Vegetable, Nutty, Coffee, Meat, Rubbery Isobutyl acetateSweet, Fruity, Bananas, Tropic, Ethereal Isopentyl hexanoateFruity, Pineapple, Sour NonanalRose, Orange peel, Cucumber, Fatty

GC-MS

12_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ SL_LOD:4.0_a:0.3 1HA, 1HB 11_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ SL_LOD:8.5_a:0.4 SL_LOD:30.3_a:-0.9 H_LOD:4.5_a:-2.8 TKW_LOD:7.6_a:-1.4 P_LOD:3.8_a:-1.1 BC_LOD:9.2_a:3.1 ME_LOD:15.6_a:0.8 WC_LOD:4.3_a:0.1 BP_LOD:6.9_a:-0.5 S/T_LOD:6.0_a:1.3 S/T_LOD:3.9_a:0.8 DP_LOD:3.3_a:-7.7 DP_LOD:3.6_a:8.5 BG_LOD:6.6_a:-60.7 HD_LOD:10.1_a:2.4 2H 11_ _ _ _ _ _ _ _ _ H_LOD:9.9_a:3.8 TKW_LOD:9.4_a:1.5 P_LOD:6.9_a:1.5 HD_LOD:4.1_a:-1.5 3H 12_ _ _ _ _ _ _ _ _ _ _ H 11_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ H_LOD:20.3_a:-6.3 TKW_LOD:28.4_a:-3.2 P_LOD:10.0_a:-2.1 BC_LOD:9.2_a:-3.2 BC_LOD:5.5_a:-2.5 ME_LOD:5.9_a:-0.5 ME_LOD:23.9_a:-1.0 WC_LOD:20.9_a:-0.3 WP_LOD:34.5_a:-0.6 S/T_LOD:5.3_a:-4.9 DP_LOD:3.1_a:6.8 AA_LOD:56.5_a:-18.6 BG_LOD:6.8_a:64.2 FAN_LOD:46.3_a:-39.4 BSR_LOD:4.3_a:4.1 HD_LOD:4.6_a:-2.3 5H 11_ _ _ _ HA, 6HB 12_ _ _ _ _ _ _ _ _ AA_LOD:9.0_a:-6.5 BG_LOD:3.5_a:-41.8 HD_LOD:9.4_a:1.2 11_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ SL_LOD:4.8_a:0.3 H_LOD:5.0_a:-2.6 TKW_LOD:6.6_a:-1.3 S/T_LOD:52.1_a:-0.9 BG_LOD:2.8_a:37.4 7H AA = Alpha Amylase BC = Barley Color BG = Beta-glucan BP = Barley Protein BSR = Barley Stripe Rust DP = Diastatic Power GPD = ari-e (dwarf gene) LR = Leaf Rust FAN = Free Amino Nitrogen H = Height HD = Heading Date ME = Malt Extract P = Plump SL = Spike Length FPD = sdw1 (Dwarf) HD14 = 2014 Heading Date S/T = Soluble/Total Nitrogen TKW = Thousand Kernel Weight WC = Wort Color WP = Wort Protein BD = Braird BSR14 = 2014 Barley Stripe Rust Flavor QTL ? FLAVOR

QTL Mapping Pop’n Full Oregon Promise Population Nano-brewing sensory Pilot brewing trials with OP agronomic and flavor selections Maris Beaver population (Full Pint x Maris Otter) Winters & Facultative Full Stein population (Full Pint x Violetta) Winters & Facultative

Exotic Barley Flavor Screening 174 genetically diverse lines from the USDA Core Collection. Micro-malted at Rahr Malting Co. Wort analysis at Sierra Nevada Brewing Co. Nano-brewed at New Glarus Brewing Co. and OSU. Sensory at OSU.

Modification Flavor Degree of modification Under-, Well-, Over- modification Beta-glucan structure High- & low-molecular weights Environmental factors Barley spec profile Structural: Soluble carbohydrates ratio Lipid and fat levels

Barley Brand Projects Nitrogen – Protein Level and Amino Acid Flavor Barley “Terroir” Flavor Organic vs Conventional Flavor Effects

Summary Significant flavor differences detected between genotypes. Random selections were highest in fruit, grass, cereal, malt, bread, and toffee flavors in Corvallis environment. Significant flavor differences detected between environments. Genotype x Environment effects detected. Barley Flavor ≠ Malt Flavor Distribution of sensory data indicates that exploratory sensory methods are effective in nano-brew samples. Identification of aroma compounds segregation indicates genotypic differences for select compounds. GC-MS is effective for high-throughput screening and characterization of “potential” flavor contributions. Flavor QTLs discovered!!!

Impact Breeders More tools on the tool belt (nano-brewing, sensory, GC-MS). Marker Assisted Selection for flavor QTLs Growers Regional taste or “terroir” production. Maltsters Identification of production regions and/or farms with best agronomics and flavor. Barley flavor specific malting (Custom malting). Value added product and new markets. Brewers Product diversification. Increased single malt brews. Proprietary or exclusive varieties contracts. Consumers More choices.

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