Beer Is a Beverage That Has Been Brewed by Many Civilizations for Thousands of Years. For

ABSTRACT

Beer is a beverage that has been brewed by many civilizations for thousands of years. For the past four hundred years, hops have been utilized in the brewing process to impart bitterness to the brew – and are now considered essential for creating a balanced and palatable beverage. In this poster we present a study of the extraction and isomerization of the alpha acids from hops – the chemical components of the hop extracts responsible for bittering beer. Alpha acids extracted from hops are isomerized to iso-alpha acids during a protracted boil of the wort. In this study, samples were collected from the boiling wort at ten minute intervals and analyzed for their iso-alpha acid content using solid phase extraction and HPLC. By measuring the concentration of iso-alpha acids, we can determine the bitterness of the brew, and the rate of isomerization. Our initial results indicate that for the first twenty minutes of the boil the isomerization of alpha acids remains low, but rapidly increases after this time, leveling out after about 50 minutes. The analysis of iso-alpha-acids in beer will provide procedural information for further research on the extraction and isomerization of these acids in homebrewed beer. The concentration of isomerized alpha acids was determined in four different types of beer. The compounds were extracted from a can of Miller High Life, a bottle of Miller High Life, Heineken, and a homebrewed Imperial Stout. Approved standards were obtained from the European Brewery Convention (EBC) and concentration was measured using high performance liquid chromatography (HPLC). Results from HPLC indicate that iso-alpha-acids can be determined in simple brews with few ingredients. However, the homebrewed Imperial Stout contained other ingredients that interfered with concentration determination. Further research can be performed on the extraction process of alpha acids and iso-alpha-acids from hops during the brewing process, but the affects of other beer flavorings will require additional procedures. Hops (Humulus lupulus) are added to beer during the brewing process to preserve the beer, complement the sweetness of the malt, and provide a citrus aroma. Bittering hops are adding at the beginning of wort boiling and give the beer the bitter taste. The hops contain the insoluble alpha acids humulone, cohumulone, and adhumulone that isomerize during the boiling process to form the more soluble iso-alpha-acids isohumulone, iso- iso-cohumulone, and iso-adhumulone (Keukeleire, 2003). The iso-alph-acids are the main compounds causing the bittering of beer (Malowicki, 2005). Bitterness is given in international bitterness units (IBU) or one part per million isohumulone. Measuring the concentration of iso-alpha-acids will provide information on the IBU and the effects of other flavoring compounds on HPLC analysis. By applying this information, homebrewed beers can be analyzed during the brewing process to determine the rate at which alpha acids add to the wort and the rate the compounds isomerize. An understanding of alpha acids and iso-alpha-acids will increase the ability of brewers to alter hop flavors.

Iso-alpha-acids were extracted from a can and bottle of Miller High Life® beer, Heineken®, and a homebrewed Imperial Stout. Miller High Life is one of the oldest brands and is considered an American lager. Miller does not boil hops in the wort, rather the company uses hop extracts to bitter the beer, not to flavor it. Miller beer contains approximately 5-6 IBU. Heineken is a pale lager that contains 18-19 IBU and is marketed as a premium beer. Imperial Stout has several variations but all brews contain several malts and hops. The malts used to make Imperial Stout are dark and contain several minerals and ingredients. The stout is usually made to have about 50 IBU.

Solvent for Standard (acidic methanol)  0.175 mL phosphoric acid combined with 250 mL HPLC grade methanol

Reagents for Extraction  Desorbing Solvent A . 0.40 mL phosphoric acid dissolved in 100 mL deionized water  Desorbing Solvent B . 0.20 mL phosphoric acid dissolved in 50 mL deionized water and 50 mL methanol  Desorbing Solvent C . 0.10 mL phosphoric acid in 100 mL methanol

Mobile Phase  77.5% (613.025 g) HPLC Grade Methanol  22.5% (224.602 g) HPLC Grade Water  1% (16.850 g) Phosphoric Acid Standard Solution Preparation 1. Warm standard to room temperature in a desiccantor and weigh 20.10 mg 2. Quantitatively transfer standard to a 100 mL volumetric flask and dilute to mark with acidified methanol 3. Protect standard from light with aluminum foil and store in freezer at -10ºC (stable for one month)

Solid Phase Extraction of Iso-Alpha-Acids 1. Degas approximately 200 mL of beer sample by transferring of beer from beaker to beaker 2. Add one drop octanol and make additional transfers until all foam is gone 3. Adjust pH to 2.5 using phosphoric acid 4. Condition the SPE C8 column a. 2 mL methanol, discard liquid b. 2 mL water, discard liquid c. Pipet 40 mL of beer sample into column using a volumetric pipet and discard flow through d. 6 mL solvent A, discard flow through e. 2 mL solvent B, discard flow through f.3 aliquots of 0.6 mL of desorbing solvent C, collect eluants in volumetric flask and calculate volume using mass and density of methanol g. Filter and store in sealed container

HPLC Analysis 1. Run HPLC a. Hops specific column b. 20 μL injection loop c. Flow rate: 1.0 mL/min d. Wavelength: 274 nm RESULTS

CONCLUSION-1 CONCLUSION-2 FUTURE DIRECTIONS REFERENCES Keukeleire, J., Ooms, G., and Heyerick, A. “Formation and Accumulation of alpha-acids, beta-acids, desmethylxanthohumol, and Xanthohumol during Flowering of Hops.” J. Agri. Food Chem. 2003 Jun 18;51, 4436-41. Malowicki, M. and Shellhammer, T. “Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system.” J. Agric. Food Chem. 2005 Jun 1;53(11):4434-9.