C eliac disease (CD) is an au-toimmune condition mainly characterized by the de-struction of the small intes-tinal villi, reducing nutri-ent absorption and causing various symptoms including

diarrhea, headaches, fatigue, and neurologi-cal disorders. CD has also been associated with increased rates of intestinal cancer and other malignancies. It is believed that 1 per-cent of the Western world suffers from CD, although a large percentage of the popula-tion remains undiagnosed, what is referred to as the “celiac iceberg.”

Diagnosis is possible through blood tests targeting specific antibodies and biopsies looking for intestinal damage to the villi. The presence of the DQ2 and DQ8 genes indicates a susceptibility to the disease, but it may not develop in all subjects carrying those genes. Absence of those genes, how-ever, rules out development of the disease al-together. CD is often triggered by a psycho-logical or physical trauma. There is no cure for CD, so this means a lifelong gluten-free diet for people affected.

Non-celiac gluten sensitivity is in con-trast a non-autoimmune and non-allergic di-sorder. Symptoms are similar to CD but are not considered life-threatening. Non-celiac gluten sensitivity has been defined as one or more of a variety of immunological, morpho-logical, or symptomatic manifestations that may also be shared by celiac disease and ir-ritable bowel syndrome. Non-celiac gluten sensitivity affects reportedly 10 percent of the Western world, with likely just as many undiagnosed subjects.

GLUTEN PROTEINS IN GRAINS AND BEERGluten is a storage protein found in wheat, barley, and rye and is composed of two frac-tions: prolamins and glutenins. The prolamin fraction (termed gliadin in wheat, hordein in barley, and secalin in rye) is composed

BY SYLVIE VAN ZANDYCKE

GLUTEN-REDUCED

BEERS MADE WITH

BARLEY

80 The New Brewer N o v e m b e r / D e c e m b e r 2 0 1 3 B r e w e r s A s s o c i a t i o n . o r g

ply the level of prolamin by a factor of 2 to obtain the gluten concentration when measuring gliadin from wheat; however for barley products such as beer it is estimated that 97 percent of the allergenic epitopes are present in the hordein fraction of the malt. There-fore, doubling it is largely overestimating the con-tent of gluten, thus giving the test an extra margin of safety for barley products. The R5 Competitive ELISA has been tested in two suc-cessful collaboratives orga-nized by the AACC Inter-national and the American Society of Brewing Chem-ists (ASBC). Other methods such as mass spectrometry, high performance liquid chromatography (HPLC), and polymerase chain re-

action (PCR) have been attracting a lot of attention; however the practicality of using such methods on a routine basis has not yet been established.

A UNIQUE ENZYMEA prolyl (or proline-specific) endoprotease from Aspergillus niger is widely used for beer stabilization4. The enzyme, known by the commercial name Brewers Clarex,TM hydrolyzes haze-sensitive proteins at the proline site, making it impossible for those proteins to bind to polyphenols and create haze. With a small addition of the enzyme during fermentation (Figure 1), the beer can be stabilized for up to one year with no effect on foam (which has very little pro-

not suitable for all types of food, especially food con-taining hydrolyzed gluten such as beer, malt extract, sourdough, and starch.

The ELISA test has been further developed over the last few years to accurately measure gluten in beer where it is partially hydro-lyzed, and small fragments of hordeins should be de-tected if they contain al-lergenic epitopes. The rec-ommended ELISA test for beer is the R5 Competitive, which can recognize as lit-tle as 1 epitope. The second (and improved) generation of ELISA standards consists of a peptic/tryptic digest of wheat, rye, and barley; the limit of detection and quan-tification were of 1.36 and 5.0 ppm respectively3. It is standard practice to multi-

of mostly proline and glutamin amino acids and is responsible for causing unwanted reactions in gluten-intolerant people. Al-lergenic gluten peptides are insufficiently degraded by gastrointestinal enzymes (pro-teases), leaving intact epitopes; those immu-notoxic epitopes bind to celiac-specific cells (antigen) to stimulate T cells and create the symptoms described above.

Beer is mostly made of barley malt and therefore contains gluten, rendering it un-suitable for people with the intolerance; however it has been observed that a large majority of proteins are eliminated during the brewing process at mashing (especial-ly), boiling, fermentation, and maturation due to action of proteolytic enzymes and precipitation. Addition of process aids can also contribute to reducing the levels of gluten proteins even further to reach unde-tectable levels when measured using immu-nological tests1.

MEASURING GLUTEN IN BEERAccurately measuring gluten is key to pro-ducing a safe product for gluten-intolerant individuals. Sensitivity to gluten varies con-siderably among affected people. For in-dividuals diagnosed with CD, damage can occur at levels as low as 10 mg per day of gluten; however, most damage occurs at 50 mg per day. An intake of less than 10 mg per day of gluten is recommended to avoid nega-tive effects. As such, the Codex Alimentarius states that if gluten is not present at more than 20 ppm, exposure in a product would remain below 10 mg per day2.

The Codex Alimentarius also refers to the Sandwich R5 Enzyme-Linked Immu-noassay (ELISA) to measure gluten in food; the monoclonal R5 antibody recognizes the QQPFP (glutamine-glutamine-proline-phenylalanine-proline) motif, among others. QQPFP is a common repetitive sequence present in all prolamins and allergenic epi-topes. However, the Sandwich R5 test is

“It has been observed that a large majority of proteins

are eliminated during the brewing

process at mashing (especially), boiling, fermentation, and maturation due to

action of proteolytic enzymes and precipitation.”

Wort coming in100+ ppm gluten

Green beer coming out<10 ppm gluten

(measured with R5 comp ELISA)

PSEP (Brewers Clarex™)

2-3 g/hl

FV

Figure 1: Practical example of Brewers ClarexTM usage in the brewing process to degrade gluten

82 The New Brewer N o v e m b e r / D e c e m b e r 2 0 1 3 B r e w e r s A s s o c i a t i o n . o r g

In 2012, the Craft Brew Alliance launched what is believed to be the first U.S. gluten-free barley malt-based beer on the market, Omission. The New Brewer talked with brewmaster Joe Casey to get his thoughts on the product and the process.

The New Brewer: How did the idea of creating Omission come about?

Joe Casey: We’re not sure if we were truly the very first, but we are the first doing it at our scale. Our launch was in Oregon in March 2012. The need for Omission came about when my wife was diagnosed as a celiac in 2005-06, and when I learned our company CEO was also a longtime celiac. The idea of using Brewers ClarexTM to potentially make malt-based gluten-free beer was something I learned of at a presentation at the CBC in Seattle in 2006.

TNB: What characteristics differentiate Omission from other glu-ten-reduced beers already on the market?

JC: Omission utilizes barley malt as a carbohydrate source. Because of that, it has all of those same malty flavor/aroma attributes that al-ternative grain brews simply can’t replicate. Omission is also the most transparent gluten-free food/beverage I know of. We test each batch multiple times, and developed a way for consumers to see the actual gluten analysis for the bottle of beer in their hand. Lastly, we only pack-age Omission in bottles, and not kegs. We can demonstrate chain of custody all the way from the brewery to the consumer with a bottle, and one cannot do that with a keg of beer. It’s not uncommon for people to get served the wrong pint of beer in a draft setting like that of a bar or restaurant. By keeping our beer in bottles only and creating a serving ritual in which the customer is always served in the bottle, we remove that risk for the consumer.

TNB: How can you make sure the beer is suitable for people who display intolerance to gluten?

JC: We have procedures and testing protocols in place that are ex-

traordinary and time and time again have shown that we can produce a GF beer that meets the FDA gluten concentration threshold as well as complies with the international Codex standard that has been accepted without issue in countries outside of the U.S. for years. We know we are pushing new boundaries of brewing science. We also know that celiacs and gluten-intolerant people who have for decades in some cases been told they cannot consume beer are not suddenly going to just trust us because we have a fancy website and a catchy name. We see our job to be fully transparent about what we’re doing: We brew using malted bar-ley and declare it clearly on the label; we use the most widely accepted and scientifically validated gluten test available, the R5 Competitive ELISA; we use that test at points A-Z throughout our process; and we share the results with our consumers. We also have mass specrometry-based data demonstrating that Omission beers lack the epitopes that trigger gluten sensitivity. Beyond that, it is the customer’s decision to try our product or not.

TNB: How long did the process development take? JC: At our pilot brewery, we worked on developing Omission beers

for about one year prior to moving the trials to our main production brewery. At the main brewery, we worked for about six months to fine-tune the brewing process and demonstrate to ourselves that we could reliably make GF Omission products in a facility that also makes beers that contain significant levels of gluten. Across that time we intentionally tried to make the process fail to simulate what could happen, and used those findings to make our Omission process even more robust before we actually ever sent beer to market.

TNB: CBA is very open about disclosing its process to remove glu-ten from the beer. What is the rationale behind sharing this knowl-edge?

JC: What we learned early on with Omission is that its consumers would accept nothing less than complete transparency. For us disclo-sure was really about building trust and demonstrating reliability, as well as showcasing the extraordinary efforts we go through to maintain a GF status in a large scale production facility that also makes beers that contain gluten. Secondarily, we want to show the industry as well as government regulators what we think is the gold standard of a produc-tion, quality, and testing program built specifically for the production of barley malt-based GF beers. We believe anyone trying to do what we do should be held to the same standards that we’re holding ourselves to.

TNB: How are you currently alleviating the issues associated with the labeling of gluten-reduced beers made with raw materials con-taining gluten?

JC: Currently we have two different beer labels within each Omission product. The first claims gluten free, and is legal only in the state of Oregon as well as various countries outside the U.S. such as Canada and the EU. The other labels for use outside of Oregon are not legally allowed to use the term “gluten free,” and thus have language indicating the gluten has been removed. In order to use the latter term, the TTB requires very specific language and the posting of a disclaimer indicat-ing their belief that there is no valid test to detect gluten in beer. (There are in fact two validation studies recently published that used beer as one of the tested products. The first was done by the American Associa-tion of Cereal Chemists International, and it was followed by a study by the American Society of Brewing Chemists.) In addition, we continue to work with the FDA and the TTB, as well as several international labora-tories and allergen detection experts, to ensure our analytical techniques are current with the best that science has to offer.

CBA’S OMISSION BREAKS GROUND

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83B r e w e r s A s s o c i a t i o n . o r g The New Brewer N o v e m b e r / D e c e m b e r 2 0 1 3

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free. However, TTB Rul-ing 2012‐2, Interim Policy on Gluten Content State-ments in the Labeling and Advertising of Wines, Distilled Spirits, and Malt Beverages, is still in force despite the FDA ruling. Currently the only beers that are allowed to be labeled gluten-free are made with grains that do not contain gluten, such as millet, sorghum, buck-wheat, and rice. Those beers display a very dif-ferent flavor profile than traditional malt beers.

The labeling situa-tion is exacerbated by the fact that some beers scoring less than 20 ppm gluten with the R5 Com-petitive ELISA test have been shown to contain some hordein fragments, although their toxicity

and exact concentration has not been deter-mined7. This can be easily explained by the fact that the ELISA test targets specific repeti-tive sequences within allergenic epitopes and therefore represents a measure of reac-tive gluten only, not total gluten. Additionally, it is not clear if those beers have been enzy-matically treated or simply measure under 20 ppm as the result of protein loss during the brewing process. With an adequate dosage of enzyme, no epitope should be left intact in the beer.

Sylvie Van Zandycke, Ph.D. is account manager at DSM Food Specialties. She would like to thank DSM for its autho-rization to publish the data; Chantal Christis and Michael Akeroyd for their expertise with the ELISA testing and mass spectrometry; and Joe Casey from Craft Brew Alliance. n

line) or flavor. The haze-sensitive proteins are contained in the hordein fraction of the barley grain, so essentially the enzyme has a double application and cleaves the allergenic epitopes of the gluten (Figure 2), rendering them non-reactive to T cells5 and making the beer safe for consumption. Guerdrum and Bamforth1 studied the ac-tion of this enzyme when used in the brew-ing process (in fermentation or maturation) and measured the gluten levels in the re-sulting beers, which were well below the level of detection as measured with the R5 Competitive ELISA assay.

LEVELS OF GLUTEN IN COMMER-CIAL BEERSLevels of gluten were measured in 37 com-mercially available beers (Figure 3). Fifteen of those revealed levels that would qualify them as gluten-free products, containing less than 20 ppm of gluten. Among those, seven were treated with Brewers ClarexTM during the brewing process either for beer stabiliza-tion or for the purpose of detoxifying gluten. All scored undetectable gluten (under 10 ppm). The dosage of the enzyme is propri-etary to each product and brewery, and has been optimized to either reach a specific shelf-life and/or undetectable gluten levels. Two light American lagers (A,B) also tested under 10 ppm gluten; it is well documented that light beers contain up to 40 percent ad-juncts so the malt (and gluten) concentration is reduced and additionally the process may be such that most proteins are eliminated.

Two American lagers (D, E) also mea-sured under 10 ppm gluten; they were both from global brewing companies whereas the craft American lager (F) tested above 20 ppm gluten, possibly indicating the use of adjuncts in the global brands. Interest-ingly for those beers that scored undetect-able gluten (less than 10 ppm) with the ELISA test but were not treated with Brew-ers ClarexTM, the presence of allergenic epitopes6 may be detected using mass spec-trometry; this has not been observed to be the case with a Brewers ClarexTM treated beer where every single allergenic epitope should be degraded by the enzyme.

All the beers that test-ed above 20 ppm gluten originated from U.S. craft breweries; the hefewei-zen displayed the highest gluten content due to the large amount of wheat present in the recipe. The Triple also contained a sizable percentage of wheat and therefore higher levels of gluten. The Brown Ales (M, O, N) also seem to be on the high end, possibly due to the type of malt used; however one Brown Ale (L) scored much lower in gluten content, indicat-ing that more proteins were eliminated during the brewing process in that particular brewery. So apart from beers that contain wheat, in general it can be observed that the gluten content in beer is probably more dependent on the brew-ing process in place in a particular brewery than to the style of beer (see Table 1).

GLUTEN-FREE LABELINGThe FDA recently set a limit of 20 ppm for gluten-free food and defined it as not con-taining any of the following ingredients: • An ingredient that is any type of wheat,

barley, rye, or any crossbreeds of these grains

• An ingredient derived from these grains that has not been processed to remove gluten

• An ingredient derived from these grains that has been processed to remove gluten, if it results in the food containing more than 20 ppm of glutenThis clearly defines beer made with

barley malt and enzymatically treated to degrade gluten (under 20 ppm) as gluten

“Currently the only beers that are allowed to be labeled gluten-free are made with grains that do not

contain gluten, such as millet, sorghum,

buckwheat, and rice. Those beers display a very different flavor

profile than traditional malt beers.”

Figure 2: Cleavage site of T cell stimulatory gluten epitope Glia 31-43 with prolyl endopep-tidase. QQPFP is the repetitive motif in toxic gluten peptides targeted by the R5 antibody. All toxic epitopes tested had at least one cleavage site5.

Brewing Parameters Gluten Levels

Percentage of malt in recipe Increase

Type of malt Vary

Original gravity of the beer Increase

Use of stabilizers Decrease

Use of wheat Increase

Use of specific process equipment (centrifuge, whirlpool, filters) Decrease

Table 1: Factors influencing gluten levels during the brewing process

84 The New Brewer N o v e m b e r / D e c e m b e r 2 0 1 3 B r e w e r s A s s o c i a t i o n . o r g

REFERENCES1. Guerdrum, L.J. and C.W. Bamforth. “Prola-

min levels through brewing and the im-pact of prolyl endopeptidase.” Journal of the American Society of Brewing Chem-ists, 70, 35-38 (2012).

2. Codex Alimentarius Commission 2008. www.codexalimentarius.org

3. Haas-Lauterbach, Sigrid, et al. “Gluten Frag-ment Detection with a Competitive ELISA. Journal of AOAC International 95, Num-ber 2, March/April 2012, 377-381.

4. Craig, Harry and Jeroen van Roon. “Clarity assured – using a proline specific protease to prevent chill haze.” Brewing and Dis-tilling International 3, 35-38, 2007.

5. Stepniak, Dariusz et al. “Highly efficient gluten degradation with a newly identi-fied prolyl endoprotease: implications for celiac disease.” American Journal of Physiology – Gastrointestinal and Liver Physiology 291: 621-629, 2006.

6. Sollid, Ludvig, et al. “Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ mol-ecules.” Immunogentics, June 2012, Vol-ume 64, Issue 6, 455-460.

7. Colgrave, Michelle, et al. “What is in a Beer? Proteomic Characterization and Relative Quantification of Hordein (Gluten) in Beer.” Journal of Proteome Research, 2012, 11 (1), 386–396.

Figure 3: Gluten levels expressed in mg per L (ppm) and measured with the R5 Competitive ELISA assay (R7021). Each value represents an average of 2 gluten extractions and 3 readings for each extraction; the standard deviation is indicated. Gluten content in the beer = 2x gliadin levels. Beers considered as gluten free (under 20 ppm of gluten) are indicated above the blue arrow. Beers treated with the prolyl endopeptidase are indicated above the orange arrow.

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