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

2. 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.

3. 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.

4. 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

5. 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

6. 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 5.514 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) Barley3.910.0383.25.366.8 4.85 5.2216.4 Malt4.430.0421.74.572.2 5.13 5.9615.6

8. 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.

9. 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

10. Micro-malting & Malt analysis

11. Malt Quality Parents & Control SampleAABGColorDPExtPlumpMoistpHProtTurbViscos Full Pint AVG83.50381.331.46156.0078.4110.304.955.9940.785.271.12 STDEV7.9917.930.2512.170.932.171.160.071.650.910.97 %CV9.574.7016.967.801.1921.0323.481.114.0417.2386.65 Golden Promise AVG42.67461.001.50119.0076.6533.476.205.8331.8019.031.09 STDEV4.38155.190.2638.201.7011.271.800.023.617.600.94 %CV10.2533.6617.1732.102.2233.6829.060.3411.3539.9486.61 Control AVG63.5397.331.35139.0079.1516.604.705.9240.294.301.01 STDEV11.46120.560.3317.061.249.370.960.075.381.230.87 %CV18.0361.0924.3914.341.5756.4620.361.1213.3628.5886.79

12. Malt Quality Malt quality of agronomic Oregon Promise selections from all environments (Corvallis, OR; Lebanon, OR; Madras, OR) analyzed by Rahr Malting Co. in 2015. Agronomic Selections Sample AABGColorDPEXTPlumpMoistpHProtTurbViscos COR AVG57.52281.761.52109.0577.4021.314.955.8835.019.391.63 STDEV15.80198.930.2326.471.498.071.780.078.338.110.12 %CV27.4770.6015.4724.271.9237.8835.991.1223.7886.387.63 LEB AVG50.02383.951.47114.0577.9214.155.415.9534.0512.261.31 STDEV12.57227.900.2227.061.766.382.040.067.1210.020.71 CV%25.1359.3615.1923.722.2645.0637.671.0520.9181.7054.31 MAD AVG47.49524.051.31136.1975.1725.484.815.8733.5415.431.67 STDEV12.35279.690.1725.571.5115.511.320.053.9916.740.47 CV%26.0153.3713.2618.772.0160.8927.440.8111.88108.4927.91 Malt quality of random Oregon Promise selections from Corvallis, OR environment analyzed by Rahr Malting Co. in 2015. Random Selections Sample AABGColorDPEXTPlumpMoistpHProtTurbViscos COR AVG 70.83127.571.61118.9376.7032.985.225.8439.845.541.55 STDEV 19.6295.640.2325.481.338.401.490.068.051.770.08 CV%27.7074.9714.4021.431.7425.4828.520.9520.2232.015.06

13. 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. 2 3 5 4 6

14. 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. 7 8 9 10

15. 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

16. Full Pint Golden Promise

20. New Glarus Brewing Co.Rahr Malting Co. 120031Full, Malty, Smooth, Rich, Nice! - Best "Euro" Type Malty, Toasted/Bread, Roasted/Toffee 120058Nice Foam, Clean, Smooth - Best "American" Type Honey, Nutty, Floral 120090Best Foam, Sweet, Clean, MildHerbaceouse, Earthy, Green Apple 120145Balanced Malty and Hoppy, Clean, Sweet Malty, Toffee, Caramel, Vanilla, Papery 120285Sweet, full, very clean, bread, maltMalty, Nutty, Floral, Phenolic, DMS 120322Malty, sweet / dry, morish, low colorMalty, Toffee, Caramel, DMS 120331Light, clean, malty, sulfur, Euro, juicy, niceDiacetyl, DMS, Worty 120341Sweet, full, biter after taste, malty, clean Acidic, Sour, Tart, Rubber 120359Wow, malty, biscuit, sweet, candy, floral Fruity, Floral, Sweet 120373Coffee! Roasted bitter, "White Stout"?, very unique Malty, Worty 120744Malty, bread, European, very nice Nutty, Bananas, Floral Full PintFull, malty, EuropeanMalty, Sweet, Popcorn, Grain, Grass Golden PromiseHarsh bitternessFruity, Floral, Grass/Green, Astringent

21. 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.

22. GC-MS

23. 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

24. GC-MS

25. 12_30918 0.0 12_30877 16.0 11_20712 23.9 11_10744 27.7 12_30336 37.4 11_10764 38.4 11_10438 41.2 12_31208 41.8 11_10552 47.1 11_20095 47.5 11_10516 49.6 11_10890 56.3 11_20990 58.9 11_20434 69.1 12_31160 71.1 11_21373 71.8 11_10522 78.6 11_20169 79.0 SL_LOD:4.0_a:0.3 1HA, 1HB 11_10911 0.0 11_20908 1.6 11_10586 3.6 11_10722 3.7 11_20383 17.1 11_10443 23.5 11_20772 25.1 11_21187 0.0 11_21261 4.7 11_11073 23.2 11_10525 23.7 11_10399 24.2 11_10648 24.7 11_10342 25.2 11_10297 30.4 11_11054 31.3 11_21166 36.3 11_10265 39.3 11_11402 42.8 11_20667 43.6 11_20419 45.8 12_10859 51.3 11_21242 55.0 11_11100 57.3 11_11533 64.4 11_10876 77.8 11_10900 78.8 11_21340 82.3 11_11323 85.7 11_11480 86.8 12_20183 109.4 11_20590 124.8 11_10791 146.6 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_10710 0.0 12_30953 0.5 11_10335 8.5 12_30754 14.6 11_20778 15.4 12_31346 18.3 12_30663 30.6 12_31367 32.2 11_21212 51.8 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_31324 0.0 11_20557 18.7 11_20411 31.0 11_11180 38.2 11_10262 11_10946 43.8 11_10668 44.0 11_20939 44.3 12_30994 45.9 11_10467 48.2 11_21504 52.3 4H 11_21324 0.0 12_30214 12.5 11_10671 14.4 11_11221 14.9 11_20441 16.4 11_20645 30.3 11_11355 35.4 12_31427 36.5 11_20526 42.0 11_10518 43.4 11_21168 57.6 11_21061 60.5 11_11273 63.0 12_30524 90.8 11_21422 92.6 11_10805 98.5 12_30929 106.1 11_10783 106.2 12_30930 106.3 11_10095 106.4 12_30869 107.4 12_30635 111.2 11_10819 118.2 11_21355 132.0 11_20829 146.3 11_10869 148.5 12_30382 186.9 11_20132 187.5 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_20232 0.0 11_11479 11.0 11_20415 12.0 12_30843 18.2 6HA, 6HB 12_30430 12_30856 0.0 11_10817 11_10462 0.2 11_21339 3.1 12_31289 6.0 11_20468 6.1 11_21025 19.6 11_10202 27.8 AA_LOD:9.0_a:-6.5 BG_LOD:3.5_a:-41.8 HD_LOD:9.4_a:1.2 11_20722 0.0 12_30530 2.9 11_10451 12.9 12_31305 26.7 12_30880 47.0 11_10431 55.3 11_10153 55.7 11_11098 56.0 11_20975 56.2 11_11219 61.2 11_20771 11_10143 64.9 11_21448 67.5 12_31395 68.2 11_20092 83.6 12_20832 109.5 11_21160 117.3 11_20139 122.3 12_31325 123.9 11_20504 130.9 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

26. 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

27. 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.

28. 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

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

30. 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!!!

31. 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.

33. THINK BARLEY DRINK BARLEY