Enzymes used in beer, wine and spirits.

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, 3^rd 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

Wikipeadia.com