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.