What’s Behind the Rise in Gluten Toxicity? Hypothesis 2 – Food Glues

The second in a short series considers emerging ideas that attempt to explain the rising tide of gluten-related disorders. We start by considering the role of food enzymes (transglutaminases) — now used in many industrial food processes.

Previous post: Background Hypotheses and the Role of Antibiotics

Reasding time

Are rising rates of Coeliac Disease linked to the use of modern food ‘glues’?

The incidence of coeliac disease has risen dramatically in the last twenty years (see graph A below). In this article, we will consider the hypothesis that the rise in industrial microbial transglutaminases (mTgs) — also known as food glues — may be contributing to coeliac disease incidence (graph B).

(A) The change in celiac disease incidence over time in different countries, and (B) the direct correlation between the increase in celiac disease incidence and the increased annual expenditures on food-processing enzymes in the food and commercial baking industries in Western countries. Adapted from references7,8,15,16,73–76.
The increased use of microbial transglutaminase in food processing may promote celiac pathogenesis, possibly explaining the surge in incidence of celiac disease.
(PDF) Possible association between celiac disease and bacterial transglutaminase in food processing: a hypothesis. Available here: [accessed Dec 26 2018].

IN BRIEF (TL;DR)

● Human tissue transglutaminase (tTg) is considered a key player in the development of the autoimmune response that defines coeliac disease.
● However, a related class of enzymes, microbial transglutaminase (mTg) — extracted from bacteria — have become an important part of industrial food processing in the last 20 years. These are increasingly used as food ‘glues’ in bakery, dairy, seafood and meat products.
● Researchers suggest that gluten may become ‘activated’ by mTg in the gut, before absorption, or even outside the body in the case of baked wheat products using mTg.
● If future research substantiates this hypothesis, the findings will affect food product labelling, policies of the food industry, and consumer health education.

Read time 10 minutes (2000 words)

The Key Role of Tissue Transglutaminase in Coeliac Disease

Human tissue-transglutaminase (tTg) enzymes are found throughout the human body, where their primary function is to cross-link proteins creating protein polymers. Acting as a kind of glue, they are important in maintaining tissue strength and integrity, especially at surfaces such as the skin and mucosa.

Because of its ubiquity in the body it is not surprising that tTg plays a significant role in diseases of inflammatory, degenerative, age-related, neurodegenerative, malignant, metabolic, hormonal, genetic, and autoimmune nature. Examples include: type 1 diabetes, dermatitis herpetiformis, multiple sclerosis, SLE, bullous pemphigoid, Sjogren’s syndrome, and rheumatoid arthritis (Lerner et al. 2017).

As currently understood, tTg is a key player in the development of coeliac disease as it binds tightly to gluten peptides, that is, the fragments of gluten produced by the digestive process (see diagram below). This is thought to only happen inside the gut mucosa, i.e. after gluten peptides have passed through the gut wall where they can come into contact with th tissue transglutaminase enzyme (‘TG2’ in the diagram below).

Coeliac autoimmune sensitisation is understood to occur through the following steps: (1) Gluten is partially, but incompletely, digested in the lumen of the gut, producing polypeptides such as gliadin. Under certain circumstances, these protein fragments are able to travel across the gut wall (2) where they become bound to an enzyme called tissue transglutaminase 2 (TG2), by a process known as deamidation. It is these transglutaminase-gliadin complexes which produce a robust immune response in genetically susceptible people, leading to an immune response which includes the production of the antibodies known as anti-tissue transglutaminase antibodies (TG2 Abs)
Adapted from: Tye-Din et al, Celiac Disease: A Review of Current Concepts in Pathogenesis, Prevention, and Novel Therapies, Frontiers in Pediatrics, 2018

The immune system reacts strongly to these tTg-gluten complexes creating antibodies to destroy them. These anti-tissue transglutaminase (anti-tT) antibodies are a hallmark of classic coeliac disease and are now considered the most reliable blood markers for diagnosing the condition. Put simply: if your blood test shows elevated anti-tTg antibodies then you have coeliac disease.

What makes coeliac disease such a devestating autoimmune disease is that the anti-tTg antibodies also attack parts of the body which are rich in tTg linked proteins, such as those found in the gut lining. It is this auto-immune reaction that leads to the gut damage seen in coeliac disease.

The role of tTg as the primary mechanism for inducing coeliac auto-immunity has led to this being a major research area, but some scientists believe that transglutaminases of food and microbial origin may be active in the digestive tract and contributing to coealiac disease. They point out that many of these non-human transglutaminases are able to bind gluten peptides inside the gut, potentially priming them to be immune-triggers before passing across the gut wall.

Although research supporting their hypotheses is in its infancy, if shown to be true, it will have many implications for food manufacturing, labelling and our understanding of gluten-related disorders.

Microbial transglutaminase

Many bacteria produce microbial transglutaminases (mTgs), which bind a range of proteins. In the last two decades, the food industry has found many uses for these mTg ‘glues’. They can, for example, be used to bind mechanically recovered meat slurry to create firm textured processed meats. Their ability to glue meats together means that lower quality meat cuts can be utilised in processed meat and fish products giving manufacturers control over texture, water retention and cutting quality of their products.

Its use in food production has been increasing by more than 20% year after year, aided by genetic engineering techniques. According to Aaron & Torsten, Clinical Immunology, Dec 2018 the derived enzymes are “used in a variety of nutritional industries, including dairy, bakery, meat, sausage, fish, tofu, confection, oil, coffee, beverage, confection and convenience food production”

In dairy products such as creams and yoghurts, mTg can help manufacturers tightly control the thickness and setting of deserts. In baking, mTgs have widespread use in pasta, bread and baked goods, enabling low-quality flours to be used, whilst improving texture and performance.

Application of MTGase on food processing (Kieliszek & Misiewicz, 2013)

Not on the label

Modern microbial genetic engineering techniques have enabled mTgs to be developed and produced on industrial scales. So popular and versatile are they in food processing that their use is increasing at a rate of 20% year-on–year [ref]. Despite their ubiquity in modern processed foods, most people will never have heard of them because in most countries there is no requirement to declare them on the product label.

Marketing video showing how microbial transglutaminase can turn beef offcuts into whole ‘steaks’ BDF Ingredients “Leading transglutaminase Solutions”

Manufacturers claim that mTg is an entirely safe food processing additive so has no need to be labelled. They claim that even if it were harmful, it is denatured during manufacture, cooking and digestion, so, labelling is unnecessary. However, it is now recognised that workers using mTg can become seriously sensitised and suffer allergic reactions. Some countries have shown more caution. Switzerland, for example, requires mTg use to appear on food labels. For the rest of us, however, even “gluten-free” foods may contain mTg from their manufacture and, as consumers, we have no way of telling.

mTg interactions with gluten

As we have already seen, human tissue transglutaminase binds strongly to gluten once it has passed through the gut wall, prior to triggering an immune reaction in some genetically-susceptible people.

Worryingly, microbial transglutaminase is also able to deamidate gluten and form transglutaminase-gluten complexes. The molecular structure of mTg and human tTg-bound gliadin, whilst superficially similar, have sufficient differences to suggest that they are unlikely to trigger similar immune reactions (see below). It was something of a surprise, therefore, when recent tests demonstrated that coeliac blood produced immune reactions of similar potency to both kinds of molecule, raising questions over mTg safety.

3d-models of microbial transglutaminase (left) and tissue transglutaminase (right) docked with gliadin (a gluten peptide)
Modified from Matthias et al, 2016

An additional problem with food-derived microbial transglutaminase may well come from the fact that it is able to bind to gluten peptides in the digestive system before traversing the gut lining, which suggests an alternative route to autoimmune initiation. Even more alarming is the possibility, that these immunogenic complexes may be created in the bakery when mTg is mixed with the dough, suggesting a possible route by which modern bread may be contributing to the initiation of disease. If this is demonstrated to be true it may well explain (at least in part) the recent increase in gluten-related disorders.

However, the hypothesis is far from proven. For instance, it is not yet known if mTg or mTg-gluten complexes are able to reach the gut. It is possible that baking or other industrial processes neutralise their ability to bind to gluten in the gut. Even then, before they could affect the gut, they would have to survive digestion. Whether any significant amounts of active mTg or mTg-gluten complexes make it into the digestive tract is a moot point: the research has not yet been done.

As a microbial TG is included in many food technological processes, its safe use should be checked. This assessment must cover not only the safety of the TG itself but also that of the deamidated/cross-linked proteins generated by this enzyme.

Malandain H (2005)

What we know so far

In-Vitro Study, 2012 (Francisco Cabrera-Chávez et al)

  • mTg treated gluten-containing (wheat) and mTg treated gluten-free (maize and rice) flour is found to provoke strong immune responses in the blood of coeliac patients.
  • Reactions were notably higher in the blood of a subset of patients tested, suggesting that mTgs may affect individuals differently, calling into question blanket safety claims

In-Vitro Study, 2012 (Denery-Papini et al)

  • Microbial transglutaminase deamidates and binds to gluten in the same way as does human tissue transglutaminase.
  • Deamidated gluten can provoke allergic reactions in people otherwise tolerant of wheat.

In-Vitr Study, 2012 (Elli et al)

  • Gluten and transglutaminase-treated gluten provoked equally strong reactions in duodenal tissue taken from coeliac patients.
  • Addition of lysine to the transglutaminase-treated gluten neutralised the immunological reactivity by sufficiently altering the structure of gluten.

Review, 2015 (Lerner & Matthius, 2015)

  • mTg crosslinks proteins, thereby changing their antigenicity.
  • These novel proteins are more resistant to digestion
  • Crosslinked mTg and gluten are likely to increase the permeability of the intestines.
  • This could result in increrased intestinal permeability (leaky gut) allowing more immunogenic foreign molecules to induce celiac disease.

In-Vitro Study, 2016 (Matthius et al)

  • Blood sera from children with coeliac disease showed immune reactions to mTg alone and stronger reactions to mTg-gluten complexes.
  • The level of reaction correlated to the amount of intestinal damage.

In-Vitro Study, 2018 (Stricker et al)

  • Duodenal biopsies from coeliac patients showed marked uptake of mTg and gliadin.
  • mTg localised in the basolateral membrane which may indicate a potential antigenic interaction .

Comments from the researchers:

…increased consumption of industrial food mTg can explain the recently described epidemiological changes in the phenotype and incidence of CD worldwide…

…it is suggested that until more knowledge is available and further studies have been performed, there should be a full transparency of mTg use and clear labeling of the mTg-using products for the benefit of the consumers and the public health.

T. Matthias et al. / Autoimmunity Reviews 15 (2016)

The latest thoughts on mTg

The mTg hypothesis is certainly interesting if not compelling and certainly far from proven: There are simply too many unknown factors. For example, we do not know whether mTg-modified proteins can survive cooking and digestion. If not, then it becomes rather a moot point. That said, I think this is an area of research to keep an eye on.

In their most recent paper summarising the current hypothesis on microbial transglutaminase and coeliac disease, authors Aaron and Torsten (who publish prolifically on this topic) point to a complex picture of potential mTg-gut interactions, which I have summarised in the infographic below:

1 The use of food glues (microbial transglutaminases) is increasing at 20% per year and entering the human food chain via a wide variety of products including dairy, baked goods, meat and fish products.

2 Residues of mTg in food, along with mTg-bound proteins (such as gliadin) and other novel protein complexes may affect the gut directly, potentially affecting the tight-junctions — thereby increasing leaky gut — and/or triggering immune reactions

3 Some species of normal gut bacteria produce mTg which appears necessary for their survival in the gut. Processed foods can disrupt the normal balance of gut flora, leading to dysbiosis. This has the potential to increase gut mTg exposure from mTg-producing species. Antibiotic use can cause dysbiosis likewise contributing to the potential.

4 Some commercial probiotics include species which produce mTgs. Probiotic consumption has risen around the world in the last two decades and it is possible that this has lead to increased mTg exposure, potentially higher up in the digestive tract than where these microbes are commonly found. The potential for horizontal gene-transfer between gut species may lead to mTg production occuring in different species.

Counterarguments

Few researchers have invested research in the mTg hypothesis, which in and of itself says nothing about its potential veracity, but does mean there is little solid evidence to go on. Over the coming years, I hope we see studies identifying whether active mTg or mTg-proteins actually make it through digestion into the gut.

In what might seem like a perverse move, however, there is growing interest in the use of mTgs as a possible route to creating wheat-based products that are coeliac-safe. The idea is that treating gluten-containing flours with mTg may make actually them less immunogenic with the possibility of making gluten-based foods that are suitable for coelaics. You can appreciate the commercial and medical interest in perfecting such a process.

In one such study in 2017 (Lin Zhou et al) researchers demonstrated that gluten can be modified with mTg in a way that does indeed reduce its immune-reactivity, suggesting that mTg could actually make gluten-containing products safer for coeliacs. However, to achieve the reduction in immune stimulation was produced under very specific conditions where the intentions were to reduce the immunogenicity of just one of the gluten-peptides (33-mer), which is in no way representative of typical mTg industrial food uses.

Furthermore, when tested on coeliac patients previously, such “mTg-deactivated flour” was not as safe as expected: After 15 days of using this ‘coeliac safe’ flour, only 37% of the coeliac patients remained symptom-free, while 63% saw a relapse in their condition. Then, in a more recent small-scale test, bread modified with mTg to make it less immunogenic for coeliac patients demonstrated reduced immunogenicity, but again a subset of individuals had unacceptable reactions including small intestine damage. (Lerner & Matthius, 2015)

Gluten has been shown to exert multiple toxic effects even in people without coeliac disease. In the words of one researcher: (Lerner, 2017) “Gluten affects the microbiome and increases intestinal permeability. It boosts oxidative stress and affects epigenetic behavior. It is also immunogenic, cytotoxic, and pro-inflammatory. Gluten intake increases apoptosis and decreases cell viability and differentiation.”

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