Summary
Enzymes have been of increasing interest in the field of beer and
spirits. Various enzymes are used in the
brewing and fermentation processes at nearly every step. There are some differences in the types of
enzymes used for the production of beer and alcohol, but the processes of
rendering fermentable sugars from a source and then fermenting them are
similar. There has been considerable
work on enzyme science and the production of alcohol. The creation of new processes to prevent
deactivation of enzymes in low calorie beer production, to genetically altered
enzymes that can split non-fermentable dextrins in to fermentable sugars, are
two examples that will be covered in better detail later.
In simple home brewing operations there are only two basic enzymes that
are of concern, alpha-amylase and beta-amylase.
These two enzymes can control flavor, body, alcohol content, and a
creamy feeling. An entire family of enzymes
known as proteolytic enzymes is capable of splitting complex protein chains in
to simpler proteins and amino acids which can affect rate of fermentation,
clarity, quality of the beer, and the head of the beer. Small scale home brewers would not have to
worry about enzyme concentration in most cases but as production moves to large
scale manufacturing enzyme controls can give a much better control of the end
product as well as speed up processes.
In other alcohol processes, wine liquor and even juice, enzymes are
added to increase the yield of fermentable juices. Most fruits and berries contain pectin which
can bind the fruit juices to the cellulose of the fruit. Enzymes are used to
dissolve the pectin in the fruit to improve fluidity. Enzymes are also used to de-bitter the fruit
juices before fermenting occurs.
In industrial scale operations there has been a lot of research and
development with respect to enzyme uses in all alcohol production processes. One recently filed patent, 8334118, uses gene
insertion to alter enzymes to remain active at higher temperatures. Anheuser Bush has patents dating back to the
1970’s for the use of enzyme manipulation to produce low calorie beer. These advancements in the use of enzymes for
beer, wine, and spirits have created a new problem with the marketing of beer
in certain global markets where the definition of what can be marketed as beer
are strictly controlled. Other people
have worked to create enzyme supplements derived from sources that do not
violate foreign market definitions for what can be marketed as beer.
Beer, wine and spirits have been
around for several millennium of human history but recent discoveries in
bio-processes have begun to change the process of their manufacture. By understanding the complex work of naturally
occurring enzymes in biological systems today’s beverages are made with a new
level of control that has never before been possible. The market for low calorie beer would not
exist without modern process designs that use enzymes. Wine makers can now use enzymes to boost
production from the same acreage of vineyards.
In every fermentation process enzyme activity works to improve the
growth of yeast cells.
This is a very exciting area of
bio-process engineering and life sciences to get in to. Enzymes and their uses are still being discovered
and have broad implications that go way beyond beer, wine and spirits. This particular field of interest, adult
beverages, is worth getting in to since these marketable consumer goods have
shown, through several recessions, to be relatively unaffected by economic
downturns. As an industry it is a very
stable business climate. Beer is also
something that not every person can successfully create and it is something
worth taking pride in when others, in mass, enjoy and celebrate your
product. For these reasons I find the
topic of using modern science and engineering to produce a better tasting
beverage very interesting.
From the beginning there was always
biological processes happening in the production of beer, wine and
spirits. Beer has always been made by
steeping grains in water and then fermenting the liquid. During the early stages of soaking the
grains, the grain seeds begin to germinate.
This process of germination does a few things that all vitally important
to successful beer and one unfortunate side effect. The germination of the grain seeds increases
the water content of the grain, causes the grain seed to produce its own
natural enzymes, and uses some of the starch stores in the grain to begin germination.
Of all the enzymes needed to make
good beer only one, beta-amylases is present before germination. The others: beta-glucanases, xylanases,
alpha-amylases, proteases, carboxypeptidases, and lipoxygenases are all produced
as a result of germination. The enzyme’s
produced are heat sensitive and the process of terminating germination must be
done carefully to prevent protein de-naturalization. The germination process is
so closely controlled because the process of germination consumes the starches
that are also required to produce fermentable sugars. According to most studies best results are
obtained by halting germination as soon as rootlets can be observed sprouting
from the grain.
There are two types of barley grains
that are used for malting, 2 row and 6 row.
The row of the barley refers to the size of the grain kernel. Two row barley has are larger kernel than 6
row and can yield more weight per acre.
The 2 row barley kernel gets its larger bulk from more starch stored in
the grain. Six row barley will be able
to produce more enzymes, by weight, during the malting process but will yield less
starch per kernel. The 2 row barley will
have more starch but produce fewer enzymes, by weight.
The process of malting starts by
increasing the percent of moisture content in the grain. As germination proceeds the grains will begin
to sprout rootlets. The germination is
halted by reducing the moisture content by blow drying the grains with 120F
air. Once the grain has been dried it is
then roasted, malted, to the desired degree of darkening in an oven at 175F or
higher.
Because of the high temperatures
endured during the malting process some activity is lost in some of the
enzymes. A recent study published in
2006 found a variation of one key enzyme used to convert starch to fermentable
sugars. Two of the enzymes primarily
used to break starches in to fermentable sugars are highly susceptible to high
temperatures. Alpha-glucosidase has only
five percent functionality at high temperatures but a variant of the enzyme
with better temperature tolerances was found to exist in sugar beet
plants. This variant enzyme was then
used as the model to create a new barley enzyme by adding the specific amino
acid group responsible for the sugar beet enzyme stability to the barley enzyme. Subsequent rearrangements of the amino acid
chain for the enzyme have yielded three new barley enzymes that have improved
heat resistance.
Alpha-glucosidase is a member of a
family of enzymes known as glucosidases.
Four of the enzymes in this family of enzymes are responsible for all
the starch to sugar conversion that happens.
Alpha-amylase, beta-amylase, alpha-glucosidase, and beta-glucosidase each
play important roles in splitting different bonds in starch molecules. Each enzyme works by splitting the 1,4 bond
holding together single sugar molecules, alpha enzymes attack alpha 1,4 bonds
while the beta enzymes attack beta 1,4 bonds.
Alpha and beta amylase are the two
starch converting enzymes most commonly manipulated during the brewing
process. Alpha-amylase splits the 1.4
bond between chained glucose molecules at alpha 1,4 bond sites in the center of
long chain starch molecules, while the beta-amylase splits the beta 1,4 bond
sites on the edges of long chain starch molecules. This duel action between enzymes makes them
complimentary to each other. The every
splitting creates two ends to the starch molecule which can then be attacked by
the beta-amylase.
Since both of these enzymes need to work together for maximum efficiency,
during the mashing stage of beer brewing, the temperature range of each enzyme
must be considered. Alpha-amylase works
best at the temperature range 149F to 153F and the beta-amylase from 126F to
144F. Any temperature between 126F and
153F is acceptable to operate in.
“It is important that the all grain homebrewer realize that both enzymes
generally work well together at temperatures between 145F to 158F.” (Papazian
247)
However, within
this 27F range of operations there is a large variability in the end
product. At higher temperatures the
active period of the enzymes is shorter and will lead to beer that is “heavy”,
has a higher specific gravity caused my increased amounts of non-fermentable
sugar. Lower temperatures will have a
longer enzyme activation period which will lead to beer that is “lighter”, has
a lower specific gravity caused by fewer non-fermentable sugars and a higher
alcohol content.
In other alcoholic beverage
production this step may not be required, such as wine, when the sugars
required for fermentation are not stored as starch in the starting
material. In others this step is
complicated by the lack of needed enzymes to convert starch to sugar. An example of this is vodka, which can be
made from several different starting materials from potatoes to grapes. In the instances where liquor is made from
high starch materials enzymes are required to break the starch in to
fermentable sugars in the same way they are used for grain brewing of beer.
With some starting materials,
notably fruit based liquor and wine, different challenges arise with the
release of fermentable sugars. Naturally
occurring pectin binds the sugars of fruit juices to the inner membrane of the
fruit, as in the membranes of oranges.
An enzyme also occurs in these fruit that breaks down the pectin binding
these sugars but substitute enzymes maybe desired to increase the rate of
pectin decomposition.
Pectin refers to a mixture of
polysaccharides that bind fruit juices to the cellulose fibers of fruit. Polygalacturonic acid, polygalactose, and
polyarabinose are the primary polysaccharides.
The enzymes that degrade these pectin compounds, pectinases, occur
naturally in the fruit and are the cause of softening during ripening. The natural pectinase enzymes partially
degrade the pectin and create a soluble end to the pectin compounds. The insoluble end remains attached to the cellulose
of the fruit and increases the viscosity of the juice. These pectinase enzymes do not act quickly
enough during extraction. During the
process of extraction additional pectinase enzymes are added to increase juice
yield. Adding cellulases to the
extraction process helps to break the bond between the cellulose and the pectin
while the pectinase breaks down the pectin molecules resulting in better juice
production and fluidity of juice. The
most common enzymes used in this process are pectin esterase and
polygalacturonase. A secondary effect of
pectinase enzymes is the clarification of the juices by hydrolyzing the pectin
chains and destabilizing the suspensions.
The common source of pectinase enzymes in both industrial and home scale
production of wine is the fungus Aspergillus Niger
Another important family of enzymes
during the beginning mash of almost all alcoholic beverages is proteolytic
enzymes. These enzymes split up
naturally occurring protein molecules that exist in the mash for the sake of
creating free amino acids. These free
amino acids remain in the beer, called wort at this stage, until fermentation. These amino acids are used by the yeast
during fermentation as yeast nutrients to increase the rate of fermentation. At
113F to 122F certain proteolytic split some nitrogen based proteins in to amino
acid groups, these amino acid groups are most useful a yeast nutrients. From 122F to 140F still other proteolytic
enzymes break down other protein molecules which effects character and quality
of the resulting beer. This can change
the foam, texture, and clarity of a beer.
One of the more disconcerting uses for
modern enzyme development in beer, wine and spirits production is as a way to
make these products from cheaper starting materials. By identifying the enzymes required to
produce quality reproducible beverages commercial enzymes can be created and
used in place of naturally occurring enzymes which gives brewers the ability to
use other starch sources that would be cheaper than standard grain malt. These enzyme supplements, exogenous enzymes,
are also used to speed up the brewing process, produce lower calorie beer,
increase the percentage of cheap adjuncts, or reduce variability in per batch
production.
BioZoom, #2 2008
There are three steps in industrial
beer manufacturing where enzymes are added to the system. Most of the enzyme supplements are added
during the initial mash to convert as many starches as possible to fermentable
sugars. The additional alpha-amylase
added to the decoction vessel is an added step that is required for the use of
cheap adjunct starch. This step
hydrolyses the starch molecules of the cheaper “cereals” and aids in
liquefaction and viscosity. The enzymes
added to the mash tun perform the functions of splitting starches to
fermentable sugars. The beta-glucanase
and xylanase are used to alter secondary characteristics of the liquid malt to
make filtering in the lauter tun easier.
Filtering is required to prevent grain husks from entering the copper
kettle where temperatures will allow harmful chemicals, tannins, to leach from
the grain husk in to the beer.
The copper cook pot will exceed the
temperatures most of these enzymes will be able to survive as vigorous boiling
typically happens at this stage. The
additional alpha-amylase added during fermentation is only done if low calorie
light beer is desired as it will increase the maltose and glucose content in
the fermenter. The second addition of
beta-glucanase will hydrolyze glucans to reduce the viscosity and aid
filtration of the beer. The lager tank
is a special step only required for lager beers, ales will skip this step.
The temperature control at the
beginning mash stage and decoction step is the most sensitive area of
control. During fermentation the
temperature ranges required for yeast survival are well below those that would
inactivate the enzymes added in that stage.
BioZoom, #2 2008
The alpha-amylase
added to the decoction vessel, indicated by this temperature control graph, is
a specially designed enzyme similar to what was mentioned earlier. The particular enzyme being used here is an
enzyme with alpha-amylase functionality that is sold under the name,
Termamy®BrewQ and is designed for its ability to remain active at higher
temperatures.
Another purpose to for specialty
enzyme additions is to produce beers that have lower levels of gluten in the
beer. By replacing traditional, gluten
source, ingredients with gluten free starch sources the concentration of gluten
in a beer can be reduced to levels required by market regulators to be called a
gluten free beer; in the EU this limit is no more than 20 parts per million
gluten. One currently existing patent
for gluten free beer lists: buckwheat,
sorghum, and millet as recommended gluten free adjuncts. Gluten free starting materials are comprised
almost entirely of starch and protein and lack the enzymes required to convert
starches to ferment-able sugars. In the
patent for gluten free beer buck wheat is the sole grain used to produce the
beer. Enzyme and coloration additives
are required to produce the final result, but the patent claims the end result
is a totally gluten free beer.
A final use of enzymes in beer is
the effect of enzymes found in bottled beer on human health. Recent study has shown one chemical imparted
to beer by hops during the copper kettle is useful for stimulating anticancer
enzymes in humans while inhibiting other enzymes that activate the cancer
process.
Beer, wine, and spirits have been
produced for several millennium but recent advances in biochemical processes
have been revolutionizing the industry.
In some cases enzymes are being used to make a more reproducible
product. In some cases enzymes are being
used to make a product that meets specific dietary needs. Enzymes are used to increase process
yields. The use of enzymes has even made
it possible to produce adult beverages from cheaper starting materials that
just 100 years ago would not have been possible to use. The current climate for beer and alcohol has
been trending towards more complex flavors and with what we now know of the
impact of enzymes on flavor, character and strength of alcoholic drinks
scientifically crafted beer, wine and spirits will slowly become the norm.
US patent US
20020012718
This patent was published on
January,31,2002.
This patent is for the process and
materials used in the production of gluten free beer. The process uses at least one gluten free
cereal, named in the patent as being, buck wheat, sorghum, or millet. The patent process examples all claim buck
wheat to be the best of these options as it has natural flavors that closely
resemble those of typical gluten malts used in beer. The patent also lists as required enzymes for
the saccharification of the gluten free starches, amylolytic and glucanases.
While buck wheat has properties of
color and taste that make it an acceptable replacement for barley it lacks the
enzymes required to hydrolyse starch to maltose. For this reason alpha and beta forms of both
amylase and glucanase must be added to split the starch in to fermentable
sugars. It is also recommended to add 20
to 60 percent by weight of a gluten free syrup to the mixture, which adds
additional fermentable sugars to the mixture.
The syrup is obtained by the hydrolysis of gluten free starches such as corn
starch, rice, or potatoes. By skipping
the buck wheat and using a 100 percent syrup the enzyme saccharification step
can be skipped. The patent states that
excellent results are typically obtained for 50/50 ratios of buck wheat to
syrup.
The starting mixture may also
require a protease enzyme addition.
Since beer color is also an important part of beer quality caramel color
may also be required.
The resulting product is completely
gluten free. The beer produced is
comparable to beer made from barley malt color, flavor, appearance and head texture.
The procedure for the production of
the beer is as follows.
·
Mix
buck wheat, calcium chloride, calcium sulfate, caramel color, and water in
copper kettle at 50C for 30 minutes
·
Add
alpha-amylase raise to 78C over 15 minutes then hold at 78c for 15 minutes more
·
Raise
temperature to boil, let boil for 30 minutes
·
Adjust
pH of mash with orthophosphoric acid to about 5.6 pH
·
Transfer
to mash tun and add 1 to 1 to 1 glucanase and alpha-amylase, and protease,
saccharification begins at this stage.
·
Bring
temperature to 72C over 15 minutes, hold for 25 minutes then raise to 76C for
10 minutes.
·
Sprague grains with water to remove spent grains and
extract remaining sugars.
·
Transfer
to a cooking copper with gluten free syrup, boil over an hour with batch hop
additions
·
Chill
ferment and bottle by the conventional means.
US patent # 4272552
This patent was
published on June, 9, 1981.
This patent is held by Anheuser
Busch and is for the process of making a low calorie beer.
The process of creating a low
calorie beer removes the carbohydrates from the beer.
By carrying out the mash of the malt
in a complete separate step at lower temperatures that inactivate microorganisms
without deactivating enzymes in the malt the wort from the copper can be moved
to the primary and secondary fermentors of a previous batch giving the
fermenting process increased enzyme activity to help convert longer chain
carbohydrates to fermentable sugars consumed by yeast giving the end product
decreased carbohydrate and calorie content.
By controlling the starting gravity
of the unfermented wort it is possible to reduce the final calorie content of
the beer. This watered down beer is then
added to separately mashed malt just before or during the fermentation to the
desired concentration to produce a beer with the desired calorie content. In this process each step is done by the
usual conventional means.
In a second way of controlling with
ending calorie content of the beer is by simply using a concentrated
fermentation process and then adding carbonated water to the ending beer. The carbonated water is deaerated water that
had the exit gases from fermenting bubbled through it.
The patent claims that by treating
malt mash at 55C for between 30 and 150 minutes the microorganisms present in
the mash will be deactivated while the retaining enzyme activity in the
mash. By combining this active enzyme
mash with wort headed to fermentation it is similar to direct enzyme additions
in the fermentation process. The treated
mash must have all the microorganisms deactivated before addition to the
fermenting wort to prevent contamination.
The patent defines low calorie beer
as being a beer between 100 and 95 calories for a 12 oz erving.
This patent references other patents
for low calorie beer for background information on the invention this patent
covers. The previous patents for low
calorie beer include 3379534 which adds the enzyme amyloglucosidase to split
dextrins that would otherwise not be fermented and make it in to the final
product. The patent 2223444 produces low
calorie beer by boiling the wort in a vacuum at lower temperature to prevent
lose of enzyme activity. Patent 3852495
produces low calorie beer that is also low in alcohol by boiling off some of
the alcohol and then introducing new yeast cultures to ferment the remaining
sugars. A final patent being referenced,
2782147 uses special hops, yeast, malt, and water with a diastase extract to
produce a beer that is low in calories but high in alcohol content.
US patent #8334118
This patent was
published on December, 18, 2012
This patent is held
by the Verenium Coorperation and is for a specially designed form of the
alpha-amylase enzyme.
The patent enzyme alters the
polynucleotide and polypeptides that encode for alpha-amylase enzymes to create
a new enzyme with alpha-amylase activity but is capable of remaining active at
larger pH ranges, higher temperatures, and oxidative states.
A comparison of the different amino
acid sequences being patented and the effects of each on the activity of the
alpha-amylase enzyme created illustrate the usefulness of the patent.
Below these figures
for the activity rates of the enzymes are shown the all the sequences being
patented in the new alpha-amylase enzyme. The genetic sequences given also show
the corresponding amino acid sequences below them.
The patent claims that the new
alpha-amylase enzyme is most useful in applications where the splitting of
starches in to glucose, maltose, and other base sugars is required under
conditions of high heat or extreme pH.
It is recommended in the use of beer making where large amounts of
adjuncts are being used and require high temperatures. The patent also suggests use of the new
enzyme in bio-fuel production applications.
The enzyme is also capable of removing starch based stains and remains
active in conditions typically found in detergent applications.
Drinking Beer: Hop
To It, Medical Post, V42 Issue 2, Pg 19, 1/17/2006
The Complete Joy of
Homebrewing, 3rd Edition, Charlie Papazian, Harper Collins 1996
Enzymes in Industry
and Medicine, Gordon F. Bickerstaff, Edward Arnold, 1987
Enzymes in Brewing,
Steen Aastrup and Hans Sejr Olsen, Biozoom, The Danish Society for
Biochemistry and Molecular Biology, 2008, #2
Making a Better Barley for Brewing, Erin Peabody,
Agricultural Research, V54 Issue 9, 2006
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