Barley is a member of the grass family, subfamily Pooideae, belonging to tribe Triticeae. The same is true for wheat and rye. Also in subfamily Pooideae are oats, but oats belong to tribe Aveneae. Rice is subfamily Oryzoideae and belongs to tribe Oryzeae. Corn, or maize, is subfamily Panicoideae and belongs to tribe Andropogoneae. What all of these cereal grains have in common is that they are used in making beer.
There are at least three different conditions that can affect people who experience an adverse reaction to beer: an allergic reaction; an autoimmune response; or a response that is non-allergic and non-autoimmune, but may have similar symptoms.
An allergic reaction to barley may or may not be associated with gluten. People can be allergic to barley just as they can to wheat, horses, eggs, and peanut butter. There are two dozen different allergens in wheat, for instance, of which gluten is only one. A typical allergic reaction, whether triggered by gluten or not, can manifest as watery eyes, runny nose, and respiratory problems.
However, people can also be specifically sensitive to gluten due to an autoimmune disorder. One example is “gluten rash,” which is a type of dermatitis caused by an autoimmune response. Celiac disease is a very serious autoimmune disease that damages the mucosal layer of the small intestine, thereby compromising its ability to absorb nutrients. The typical initial symptoms include gastrointestinal “distress,” including bloating, diarrhea, and abdominal pain. Sufferers of celiac disease are also at increased risk of cancer.
Gluten sensitivity (more properly called non-celiac gluten sensitivity) is a condition where sufferers are intolerant of dietary gluten via a mechanism that is not autoimmune in nature. Gluten sensitivity will cause very similar initial symptoms to celiac disease, but without the damage to the small intestine. Sufferers of celiac disease have an immunoreactive response to gliadin, which is a prolamin (a storage protein, as will be explained below) in wheat that combines with other wheat proteins (such as glutenin) to form gluten, which gives bread dough both elasticity and structure.
There are 20 different amino acids that serve as building blocks for construction of larger structures in all living organisms. A polypeptide is the result of several amino acids joined together via peptide bonds. Proteins are constructed of one or more long-chain polypeptides and have different properties and functionalities based on their three-dimensional physical structure. In plant seeds, several different classes of protein serve as amino acid reservoirs (what brewers would call FAN) for the embryo. These are called storage proteins, and include albumins, globulins, glutelins, and prolamins. Storage proteins are characterized based on their solubility in the laboratory: albumins are soluble in water, globulins are soluble in dilute salt solutions, glutelins are soluble in dilute acid or base solutions, and prolamins are soluble in alcohol solutions. The storage proteins important for cereals are prolamins, globulins, and glutelins. (Actually, glutelins are a type of globulin, but they are treated separately due to differences in solubility.)
Cereal chemists organize barley proteins into two main groups, storage and non-storage, based on their location and function within the kernel. The major storage proteins in barley are the hordeins (a class of prolamin) and glutelins. Non-storage proteins are the structural proteins and metabolic proteins (i.e., enzymes). During malting, the endosperm protein matrix is hydrolyzed into polypeptides, oligopeptides, and free amino acids. These endosperm proteins are primarily a mixture of hordeins and, to a lesser extent, glutelins. It is the breakdown of this endosperm matrix during germination that provides the vast majority of FAN to the wort. The non-storage proteins are the source of the enzymes that are present in barley before malting, such as beta-amylase; they are also a source of (non-storage) albumins, such as protein Z, which along with hordein is a primary foam-forming component in beer.
Prolamins are also the major storage protein for wheat and rye. In wheat, prolamins include the gliadins, which are structurally very similar to hordeins. The equivalent prolamin in rye is secalin. Gliadin is the most studied class of cereal prolamin, and a major component of wheat gluten.
The structural similarity between prolamins of the Triticeae is the reason that, although barley doesn’t technically contain gliadin, beer can still be a problem for people with gluten sensitivity. Celiac disease sufferers can be sensitive to any of these prolamins, but gliadin and hordein account for 90% of immunoreactive response in T cell testing. About 10% of celiacs exhibit an immunoreactive response to oats and corn (maize). Oats contain much less prolamin (called avenin) than wheat, barley or rye, and this may be why many people with celiac disease do not react to it; or it may be that some celiacs are less sensitive to prolamins in general and only react to particular types. There are in fact several hundred polypeptides within each prolamin group that are immunoreactive. To frame this discussion another way, it is like saying that all fish, mammals, and reptiles are dangerous man-eaters because they have large teeth, when in fact some are and some aren’t, for various reasons.
Beer haze, including chill haze, is a combination of haze-active proteins and haze-active polyphenols that come together via hydrogen bonding to create large visible molecules. Hydrogen bonding is strongest at colder temperatures. At warm temperatures, the component molecules are vibrating too much for the weak hydrogen bonds to hold the complex together, and that’s why chill haze disappears as the beer warms. With time, these complexes can oxidize and polymerize into permanent haze, but it all starts with haze-active proteins and haze-active polyphenols.
Why do we say, “haze-active”? What does that mean? Basically, it refers to the types and size of these proteins and polyphenols—at a certain size these entities cause haze. Haze-active proteins are of the same size class as foam-active proteins, which typically includes hordeins. Prolamins are so called because they contain an unusually high proportion of the amino acids proline and glutamine. It appears that the hydrogen bonding between haze-active polyphenols and proteins occurs at the proline sites on the protein.1 The point is that the basis for haze, at least in part but perhaps a majority, is the hydrogen bonding at proline sites in hordeins. These haze-active proteins can be broken down by protease enzymes (e.g., the enzyme papain, used in papaya-based meat tenderizer). This creates smaller peptide chains that are not haze-active. Unfortunately, those protease enzymes will also break down the foam-active proteins, which is obviously a problem.
There are two main categories of enzyme that degrade barley endosperm proteins during malting. The first are endoproteases and endopeptidases, which act to break up the protein molecules from inside the protein structure. There are at least 40 such enzymes involved in this stage.2 The second group are exoenzymes, such as carboxypeptidase, that produce individual amino acids from the carboxyl ends of the peptide chains (i.e., only at the outside ends of the protein structure). To clarify, peptides are short chains of amino acids joined together, whereas polypeptides are longer chains of amino acids (usually >50) or protein segments. Peptides, polypeptides, and proteins can be affected by various protease-class enzymes.
Proline-specific endoproteases, such as Brewers Clarex® (from DSM) and Clarity Ferm (White Labs), act by specifically cleaving the haze-active proteins at the proline segments in the chain, reducing them to non-haze-active sizes (i.e., cleaving polypeptides into peptides). The proline sites of the peptides will still bond with haze-active polyphenols, but the size of the complex can’t grow to form a haze. Thus, most beer haze should be prevented by using these enzymes.
The proline-specific endoprotease in Brewers Clarex and Clarity Ferm has the bonus benefit of breaking up the hordein proteins that form barley gluten (it has a similar effect on gliadin and secalin). Industry studies have shown that beer treated with this enzyme measured less than 20 ppm gluten based on the R5 Mendez Competitive enzyme-linked immunosorbent assay (ELISA). Further analysis by mass spectrometry of the concomitant proteolytic residues also indicated that the residues would not be immunoreactive.3 However, even though the current benchmark for considering a food to be gluten-free is <20 ppm gluten, there is enough variation among people with gluten-related allergies that marketing a beer as “gluten-free” may be impossible from a legal liability standpoint. For example, in the case of one celiac patient, the consumption of 1 mg of gluten per day from a communion wafer was sufficient to prevent mucosal recovery of the small intestine (i.e., it caused chronic inflammation). Avoiding the wafer allowed recovery within six months.
Confounding the issue is the fact that the current antibody tests (i.e., ELISA) only target a handful of the hundreds of gluten proteins that can cause immunoreactions. Furthermore, these tests are calibrated on commercial wheat gliadin, which is not representative of barley hordein that has been malted, mashed, and boiled. To quote a paragraph from the conclusion of Tanner et al. (2014):
Definitive evidence of the safety of treated beer for celiacs ideally requires a double-blind crossover dietary challenge. In this experiment, the effect on circulating T-cells and mucosal appearance of a large number of celiacs, including sensitive subjects, who have been challenged with either PEP-treated beer or untreated beer followed by a crossover to the other treatment regime, would provide convincing evidence for the efficacy of A. niger PEP on eliminating gluten peptides for the whole celiac population. In order to achieve this, subjects would have to drink 10 L of an average beer (at 100 ppm) per day to consume sufficient hordein (1 g) for a useful short term challenge. Sourcing volunteers for such an experiment may not be a problem, but ethics approval would be unlikely. (p.46)
In other words, achieving FDA “Gluten-Free” status for barley, wheat, and rye based beers treated with proline specific endoproteases is probably not going to happen. Nonclinical experimental approval would require isolation and identification of hundreds and hundreds of prolamin polypeptides that could cause an immunoreactive response, and also require characterization of those polypeptides and their post-treatment residues by concomitant mass spectrometry, high performance liquid chromatography (HPLC), and other tests. Currently, beers treated by proline-specific endoproteases are being marketed as “gluten-reduced,” and that is probably the best we can hope for in the foreseeable future.
Portions of this work were originally published as a feature article, For Geeks Only, Gluten-free Beer, in Zymurgy, Volume 39, No. 2. March/April 2016.