Comparative digestion of thermally treated vertebrates and invertebrates allergen pairs in real food matrix

Highlights

• Invertebrates tropomyosin (abalone, oyster, shrimp) revealed pepsin resistance.

• Vertebrates tropomyosin (chicken, pork, beef) were less stable to digestion.

• Raw shrimp myosin light chain (MLC) showed pepsin resistance among invertebrates.

• Vertebrate (chicken) MLC was thermally stable.

• A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork.

Abstract

The digestion stability of allergen pairs, tropomyosin, TM (fish and seafood allergen), and myosin light chain, MLC (chicken meat allergen) is compared among vertebrates and invertebrates in raw and cooked food matrix under standardized simulated in vitro gastrointestinal (GI) digestion. SDS-PAGE followed by multiple TM and MLC-specific antibodies in semidry WB revealed pepsin resistance of invertebrate TMs (abalone, oyster, shrimp) under diet-relevant conditions (raw, cooked). Vertebrate TMs (chicken, pork, beef) were less stable to digestion except that the raw chicken TM was observed pepsin resistant (not diet-relevant). Vertebrate (chicken) MLC was thermally stable. A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork; could be the aggregates of MLC. Raw shrimp MLC showed pepsin resistance among invertebrates. A good correlation was observed between combined resistance of TM and MLC to GI digestion following the diet-relevant thermal treatment and reported protein allergenicity among vertebrates and invertebrates.

Introduction

Shellfish is listed as one of the 14 most common allergenic foods and food groups in Europe (milk, egg, fish, crustaceans, molluscs, tree nuts, peanuts, soy, wheat, lupine, sesame, mustard, celery, and sulfite (for hypersensitivity reasons)); labelling of these foods is mandatory when used as ingredients (Mazzucchelli et al., 2018). Allergies to crustacean shellfish are very common, but molluscan shellfish allergies do not occur that frequently. However, abalone, a marine gastropod commonly consumed in Asia, is one of the 25 allergenic foods in Japan. Tropomyosin (TM) is a common and major allergen in the disc abalone Haliotis discus (Emoto et al., 2009). The Korean Ministry of Food and Drug Safety (MFDS) has established food labels for 21 foods, including shrimp, pork, chicken, beef, and mollusc shellfish (clams, mussels, abalone, and oyster) (Suh et al., 2019). Although many shellfish are consumed raw, they are also frequently consumed in cooked form. Therefore, studying the effects of heating on food protein allergenicity is important for identifying reliable diagnostic markers and appropriate measures for specific immunotherapy. Heating has been shown to induce marked effects on the protein profiles and IgE reactivity of shellfish extracts (Abramovitch et al., 2017, Guang-Ming et al., 2010, Mills et al., 2009, Nowak-Wegrzyn and Fiocchi, 2009).

In addition to TM, the most prevalent and thoroughly studied pan-allergen in shellfish allergy, arginine kinase (AK, Lit v 2), myosin light chain kinase (MLC, Lit v 3), and sarcoplasmic calcium-binding protein (SCP, Lit v 4) have been identified as clinically relevant major shellfish allergens. MLC 1, a skeletal muscle isoform, has been identified and listed as an allergen, Gal d 7, in chicken (allergome.org) (Ayuso et al., 2008, Shiomi et al., 2008). The subunit structure of this vertebrate allergen is identical to that of invertebrate allergens, that is, a hexamer of two heavy chains and four light chains (UniProtKB P02604, allergome.org). This calcium ion-binding muscle protein has been documented as a sensitising agent for shrimp and/or chicken allergies in children and adults. Recombinant Gal d 7 has been produced and used for identifying patients with primary sensitisation to poultry meat (not limited to chicken meat) (Klug et al., 2020). No complete confirmatory assessment has been reported for high-risk allergens or pan-allergens. However, the Food and Agriculture Organization (FAO) and Codex Alimentarius 2013 has proposed resistance to pepsin digestion as a predictor of allergenicity, claiming that this characteristic is shared by most allergens (Commission Codex Alimentarius, 2003, FAO/WHO, 2001). Therefore, simulated digestion has gained significant attention in recent years. Numerous comparative studies have revealed the practicality and pitfalls of simulated in vitro digestion in assessing allergens (Akkerdaas et al., 2018, Bøgh and Madsen, 2016, Fu et al., 2002, Gámez et al., 2015, Naegeli et al., 2021). The major pitfall is the exclusion of the real food matrix effect on allergenic protein digestion and the lack of research on digestion-resistant allergen peptide fragments. Ofori-Anti et al. (2008) has suggested that the results could be misleading if the possible effect of the food matrices are not considered; digestibility tests are performed using only the purified protein (Ofori-Anti et al., 2008).

Previously, the allergenicity of invertebrate TM in shrimp and clams was compared with that of vertebrate TM in fish. The invertebrate TM triggered stronger elicitation of anaphylaxis in a mouse cell model compared with vertebrate TM, regardless of TM thermal processing (Xu et al., 2020, Xu et al., 2020). In a case report on fish-sensitised patients, the authors demonstrated that studying TM on a wider scale is clinically relevant because not only invertebrate TM but also vertebrate TM could elicit clinically relevant reactions in patients with allergy (González-Fernández et al., 2018). These studies further emphasise the need for comprehensive research on structurally and functionally similar proteins of vertebrates and invertebrates in terms of potential allergenicity and cross-reactivity. The effect of thermal treatment on invertebrate TM has been studied. For example, in the case of boiled, fried, and roasted oysters, TM IgE binding with patient sera was the lowest in roasted and fried samples, higher in boiled samples, and the highest in raw extracts, according to immunoblotting studies (Yadzir et al., 2015). Previous research has also focused on the comparison of structure and biophysical features between allergenic (invertebrate, i.e., shrimp) and non-allergenic (vertebrate, i.e., pig) TM. The allergenicity depended not only on sequence but also on contributions of protein structure and dynamics (James et al., 2018). Additionally, considering the high structural similarity between vertebrate and invertebrate TM, it is enigmatic why invertebrate TMs are allergenic, whereas vertebrate TMs are not (https://www.allergen.org) (Jenkins et al., 2007, Klueber et al., 2020).

Numerous studies on in vitro digestion of allergenic proteins followed by appropriate IgE measurement assays do not clarify whether a correlation exists between digestion stability and protein allergenicity. Comprehensive reviews on this topic indicate that more than half in vitro digested allergenic proteins are not digestion-resistant; the predictability of distinguishing allergenic from non-allergenic protein pairs is not enhanced by sub-optimal pH, low pepsin-to protein ratio, and resistance to peptic and pancreatic digestion (Akkerdaas et al., 2018, Bøgh and Madsen, 2016). The in vitro digestion stability test alone is not a definitive assessment. For improving digestibility testing strategies, physiologically relevant conditions need to be used, taking the food matrix into consideration (Brodkorb et al., 2019). Therefore, the objective of our study was to compare the digestion stability and patterns of homologous allergen pairs (TM, a major allergen of invertebrates, and MLC, a newly established chicken meat allergen) among vertebrates and invertebrates in raw and cooked food matrices under standardised simulated in vitro gastrointestinal (GI) digestion. As a novel approach in our work, we considered the food matrix and used diet-relevant treatments (either raw or cooked) to observe the digestibility of these TM and MLC pairs via specific antibody-based western blotting (WB).