Understanding Amylase/Trypsin Inhibitors in Barley: A New Path Toward Detecting Barley in Foods

Celiac.com 09/10/2025 - Amylase/trypsin inhibitors, often shortened to ATIs, are proteins found in cereal grains like wheat and barley. These proteins naturally occur in the grain and help protect the plant against pests by blocking digestive enzymes in insects. However, for some people, ATIs can cause health problems. They are known to trigger allergic reactions and are believed to play a role in conditions like non-celiac wheat sensitivity, where individuals experience symptoms like bloating, abdominal pain, and fatigue after eating wheat or related grains, but do not have celiac disease.

Why Study Barley?

Most of the research so far has focused on ATIs in wheat. Barley, although less often eaten directly than wheat, is widely used in foods like soups, cereals, and beer. Given its close relationship to wheat, researchers suspected barley might also contain ATIs that could cause similar reactions. However, until now, there has been no method to measure ATI levels in barley accurately and completely.

What Did This Study Do?

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Researchers in this study developed a new, highly accurate lab technique to measure specific ATI proteins in barley. The method, which uses a tool called LC-MS/MS (a type of mass spectrometry), allowed them to detect and quantify ten different types of ATIs that are unique to barley. These included:

• Two amylase-inhibitors (monomeric and dimeric forms)
• Four chloroform/methanol-soluble inhibitors
• Three subtilisin/chymotrypsin-inhibitors
• One inhibitor that blocks both amylase and subtilisin enzymes

Using this new method, they analyzed 181 types of barley from around the world. This included both two-row and six-row varieties (referring to the structure of the grain head) from different genetic backgrounds. Their goal was to see how ATI levels varied and whether certain types of barley naturally contained more or less of these potentially problematic proteins.

Key Findings

The study found that ATI content in barley varies significantly, ranging from about 1.1 to 5.2 milligrams per gram of barley. This makes up roughly 0.7% to 3.6% of the total protein in the grain. Importantly, they found no clear pattern between the type of barley (two-row vs. six-row) and the amount of ATIs present.

The researchers also identified a few barley varieties that had especially low ATI levels. These included cultivars from Eritrea, Greece, and Ethiopia. These specific types of barley may be useful in developing new food products that are easier to tolerate for people who are sensitive to ATIs.

How the Method Was Developed

The researchers used a comprehensive approach to identify “marker peptides,” which are small pieces of protein that are unique to each ATI. They carefully selected ten of these markers based on factors like signal quality, size, and stability during testing. They then validated their method by ensuring it could accurately and consistently detect the ATIs across many samples.

One challenge was that many ATI proteins in barley are very similar in structure, so distinguishing between them required high precision. The method they developed is the first to allow for absolute quantification—meaning it doesn’t just show the presence of ATIs, but gives an exact measurement.

Why This Study Matters for People with Celiac Disease or Sensitivities

Although ATIs are not the same as gluten (the protein that causes harm in celiac disease), they can still activate the immune system and contribute to gut inflammation. This can worsen symptoms for people with celiac disease or trigger issues in those with non-celiac wheat sensitivity. By identifying barley varieties with lower levels of ATIs, food producers can potentially create barley-based foods that are safer and more comfortable for sensitive individuals. This includes not only food items but also beverages like beer, which is traditionally made from barley.

For those with celiac disease this new test can potentially be used to detect barley, including barley-derived gluten, in foods at concentrations of 20 parts per million (ppm) or less—a threshold that aligns with international gluten-free labeling standards. Traditional gluten detection methods often lack the sensitivity or specificity to identify barley proteins, particularly in processed foods or when barley is present in trace amounts. However, the use of mass spectrometry targeting specific amylase-trypsin inhibitors (ATI proteins), which are unique to barley, offers a more precise and sensitive approach. This makes it possible to identify and quantify barley even when present at very low levels, potentially filling a critical gap in current food testing protocols. For individuals with celiac disease who must avoid barley as strictly as wheat and rye, this advancement could provide a much-needed tool for ensuring the safety of gluten-free labeled products.

Looking Ahead

The new method developed in this study opens the door to further research and development. Scientists can now look at how growing conditions or processing methods affect ATI levels in barley. Breeders can begin working with low-ATI varieties to create safer grains. Eventually, consumers may have access to barley foods and drinks that are both nutritious and less likely to cause discomfort.

Conclusion

This study represents a significant step in understanding how specific proteins in barley may affect human health. With a validated method to measure ATIs, researchers and food producers are better equipped to explore new dietary solutions for people with sensitivities. For individuals living with celiac disease or related conditions, this could mean more dietary choices that are easier to digest and better support overall well-being.

Read more at: sciencedirect.com