Beer has existed for centuries and
has been enjoyed by countless individuals. There are even some who argue that
without beer, civilizations would not have developed as they did.
Unfortunately, beer contains wheat, and therefore gluten, which means that those
with celiac disease or gluten intolerance cannot consume most beers without
experiencing some sort of adverse reaction. Though there are a few gluten free
beers on the market, their taste is often sub-par and leaves something to be
desired. There are few words which can describe the enjoyment one can derive
from a glass of a perfectly crafted beer, and it is the purpose of this
research to bring this joy back to individuals with celiac disease and gluten
intolerance.
When
gluten is digested by the body, it breaks down into a few different molecules,
one of which is a polypeptide known as gliadin. Due to the structure of
gliadin, it cannot be digested by normal gastrointestinal enzymes, so it passes
to the intestines intact. Once in the intestines, it begins to aggregate and
clump due to the fact that it has not been broken down. In a normal individual,
there are no adverse reactions to the gliadin molecules. However, in an
individual with celiac disease, this aggregation of gliadin and hordein (the
barley homolog) causes an autoimmune response which damages or villi, meaning
that nutrients which would usually be absorbed by the body cannot be absorbed.
Therefore, those with celiac disease who consume gluten containing products are
at severe risk for malnourishment, even if it seems as if they are eating a
balanced diet. The number of gluten free products is certainly on the rise at
the moment, though their taste is not always on par with their gluten
containing products. This fact is truly unfortunate for those with celiac
disease and is the subject of much research at present.
As
mentioned above, gliadin is one of the major components of gluten and the part
which those with celiac disease have problems digesting. Gliadin is very rich
in proline residues, which makes the peptide bonds more difficult to break down
due to their cyclic nature. A class of enzymes, known as prolyl endopeptidases,
cleaves the C-terminus of proline residues. They belong to the class of serine
proteases and consist of a catalytic domain analogous to chymotrypsin
(Histidine, Aspartate, Serine) and possess a beta-propeller domain.
Prolyl endopeptidases could be used
in treatment of celiac disease as they would help to break down the proline
rich gliadin thereby eliminating the autoimmune response to the aggregated
gliadin. Currently, there is no pharmaceutical prolyl endopeptidase available,
but there is a great amount of research underway to produce one. White Labs
produces a commercial prolyl endopeptidase called Clarity-Ferm which is used to
a large extent in research of celiac disease and gliadin breakdown.
Beer has four main ingredients:
water, hops, yeast, and grain. There are also other adjuncts used, but those
vary greatly between styles. The first three present no problems to those with
celiac disease, but, due to the presence of gliadin or hordein, the grains
(barley and wheat) cause concern. Without the grains, the enzymes that cause
fermentation are not present and there is no sugar to ferment. Essentially,
without grain, beer cannot truly be produced. This poses an issue as it means
that almost all beer is unsafe for those with celiac disease to consume.
However, if all the excess gliadins were to be broken down during or after
fermentation, the beer could be safe for consumption.
In order to brew beer, one of the
necessary steps is the mash. This process involves the mixing of the grains
with warm (or hot) water to activate the enzymes contained within the grains. A
major class of enzymes which becomes activated in this process are the
amylases. In beer, α-amylase
and β-amylase play the major
roles in the breakdown of the starch to form the fermentation products and,
eventually, the alcohol. Most enzymes have an optimum temperature at which they
become most active, which is usually not problematic. α-amylase and β-amylase,
however, ideally operate at different temperatures. This presents a problem to
breweries as they would like both enzymes to be operating as efficiently as
possible. A few different methods have been devised over the years to confront
this issue.
Traditionally, brewers tend to
split the difference of the ideal operating temperatures for α-amylase and β-amylase resulting in a mash temperature of roughly 66-67°C. This method, referred to as a
single-step mash, is by far the most common and produces beer which is very
good. Another process, referred to as a
step mash, operates under the premise that the enzymes need to spend time at
their optimal temperatures. The mash is started at approximately 49 °C to
activate the proteases. The mash is raised to about 62°C to properly activate β
-amylase, and is increased to around 67°C for α-amylase.
Though this small temperature change may not seem like much, it is enough to
change certain flavor characteristics of the beer. There is also a possibility that the step
mash could make a prolyl endopeptidase more effective at degrading gliadin,
hence its focus in this research.
One of the goals of the proposed
research is to study whether a single-step mash or a multi-step mash is more
effective at producing a gluten free beer after adding the prolyl
endopeptidase. Due to the different temperatures found in the multi-step mash,
it is known that activity of the amylases increases, but the effect the
increased amylase activity will have on the prolyl endopeptidase is unknown.
Theoretically, since the amylases are more effective at hydrolysis of sugars,
the prolyl endopeptidases are expected to be more efficient. For both beers
that will be brewed, the single-step mash will be held at 66 °C for the entire
process (about 60 minutes). The multi-step mash will start at 49 °C for about
20 minutes. The temperature will be increased to 62 °C then to 67 °C with the
two steps taking 30 minutes each. In both cases, the mashout will take place at
77 °C.
The first beer to be brewed will be
a clone of a Sierra Nevada Stout. This
beer will contain 4.1 kg of American pale malt, 1.4 kg of Munich malt, 0.45 kg
of American Black Patent malt, and 0.30 kg of American crystal malt (60 °L)
mashed for a total of 60 minutes. The presence of the Black Patent and crystal
malt contributes largely to the darkness of this beer. After the mash, the wort
will be drained and boiled. 14 AAU
(alpha acid units) of Magnum hops will be added at the beginning of the boil,
5.8 AAU of Cascade hops will be added with 10 minutes left in the boil, and 10
AAU of Willamette hops will be added at the end of the boil. Wyeast 1056 yeast
will be used for fermentation. Primary fermentation will occur at 20 °C for
approximately three weeks. After the three week period, some of the beer will
be bottled with corn sugar added to facilitate carbonation while some will be
set aside and left uncarbonated.
The second beer to be brewed while
the first is carbonating is a clone of Bell’s Oberon, an American Wheat Ale.
The mash for this beer will proceed via the same method as the first, but will
contain 5.4 kg of 2-row brewer’s malt, 3.6 kg of white malt, and 0.50 kg of
Carapils grain. The mash will occur for
a total of 60 minutes. The boil of the wort will occur for 60 minutes and 8 AAU
of Saaz hops will be added with 30 minutes remaining and again with 15 minutes
remaining. Fermentation will occur at 20 °C for three weeks and use an American
Wheat Ale yeast, likely Wyeast 1010. The beer will be dry-hopped for five days
post-fermentation with 11 AAU of Cascade hops 4 AAU of Saaz hops. After dry-hopping, some beer will be bottled
and some will be set aside.
This
research will mainly use White Labs commercial prolyl endopeptidase,
Clarity-Ferm, to degrade the gliadin in beer while it is fermenting. Alongside
this batch, a control will also be run to ensure that it is indeed the
Clarity-Ferm breaking down the gliadin. Brittany Hulett, a former research
student of Dr. Hamilton, determined that 2 mL of enzyme added to one gallon of
beer was sufficient, so that amount will be used in this project. Due to the
high levels of wheat used in the Oberon clone, another batch with 4 mL of
enzyme added will also be created. All batches will be done in triplicate
to ensure consistency of results.
From mash
to bottling, the brew process usually takes about three weeks. During this period of fermentation, every
batch will be tested daily for gliadin levels. Testing this often will allow
for a more accurate determination of the activity of the prolyl endopeptidase
and whether or not it is performing as expected over time. The wheat ale
contains much more gliadin than a normal beer and will therefore be a good marker
for whether or not the addition of prolyl endopeptidase to a beer is
commercially viable across styles. Once the beer is finished carbonating in the
bottles, a blind tasting panel will be selected to determine the difference in
taste, if any, between the different samples and brew methods .
In order to test the effectiveness
of the prolyl endopeptidases, a RIDASCREEN
Gliadin Competitive test kit will be used to detect the presence, or absence,
of gliadin. This test is an ELISA (enzyme-linked immunosorbent assay) which
utilizes an R5 antibody. The test looks for gliadin fragments which
can be harmful to those with celiac disease. Ideally, this test will show that
the control contains moderate to high levels of gliadin, while the beer with
added prolyl endopeptidase will have little to no gliadin present.
The goal of this research is to
find the best method for producing a gluten-free American stout and American
wheat ale through adjustment of a few variables. The single-step mash verses
the multi-step mash will be the largest of the variables manipulated. The
results of the research will be obtained through usage of a RIDASCREEN test kit
and blind taste tests by a randomly chosen panel. If successful, the process
could be implemented commercially and provide enjoyment to those with celiac
disease and gluten intolerance.
