*4.2. Digestion of Mealworm Allergenic Proteins by Pressurization Treatments*

Recent publications demonstrated cross-reactivity between edible insects and other Arthropoda (crustaceans, mite), identifying different proteins that are involved in muscle contraction (actin, myosin, tropomyosin, troponin T and C, tubulin), in enzymatic pathways (arginine kinase 1, alpha-amylase) or part of the hemolymphatic system (hexamerin 1 and 2) as pan-allergens [48]. Globally, the results that are presented in Table 1 showed that pepsin was more efficient than Alcalase® for hydrolysis of allergenic mealworm proteins, despite the fact that, on the whole, the DH that was obtained with Alcalase® was higher (Figure 2). Our results also demonstrated that HHP, applied as a pretreatment before in vitro digestion or simultaneously with enzymatic hydrolysis, improved the in vitro digestion of specific mealworm allergenic proteins. These results are consistent with previous publications evaluating the effects of HHP coupled to enzymatic hydrolysis on the potential allergenicity of major protein allergens from different food matrices [49–52]. Indeed, after HHP-assisted enzymatic hydrolysis, these protein hydrolysates exhibited non-antigenic properties that were superior to those of proteins only treated with enzymatic hydrolysis. The production of hydrolysates with lower immunoreactivity from pressure-treated native protein is induced by increasing the protein susceptibility to enzymatic action by exposing new cleavage sites that allow for the proteases to reach otherwise buried hydrolysis sites.

However, our work also showed that HHP-assisted enzymatic hydrolysis had a limited effect on tropomyosin, myosin heavy chain, troponin, and tubulin proteolysis, as measured by TSC and enzyme efficiency when compared to the control condition. In the literature, the efficiency of Alcalase® and pepsin for the hydrolysis of these specific insect allergenic proteins has been scarcely reported.

Tropomyosin from shrimp, being classed as arthropods, was sensitive to HHP, since pressurization at 500 MPa for 10 min combined with thermal treatment at 55 ◦C decreased the protein allergenicity by 73.59% as compared to a boiling treatment [52]. However, tropomyosin is usually reported as heat stable and resistant to gastrointestinal digestion [53]. More specifically, it was demonstrated that pepsin could only slightly hydrolyze oyster tropomyosin, which demonstrated that tropomyosin has relatively good resistance to this enzyme. Nevertheless, Mejrhit et al. [54] found that shrimp tropomyosin IgE binding was decreased after heat and pepsin treatments. Hall, Johnson, and Liceaga [19] demonstrated that Alcalase® hydrolysis of cricket protein changed the binding characteristics of cricket tropomyosin to IgE, which indicated the susceptibility of tropomyosin to Alcalase® proteolysis. Similar results were obtained using house cricket *Acheta domesticus*, desert locust *Schistocerca gregaria* and yellow mealworm *T. molitor* [4]. To the best of our knowledge, there are no previous reports on the impact of Alcalase® or pepsin hydrolysis, coupled or not to HHP, on the vitro digestion of myosin heavy chain, troponin and tubulin from edible insects. However, Deng et al. [55] reported that pepsin was efficient for the proteolysis of myosin heavy chain and troponin from shrimp, while Alcalase® was often used for the hydrolysis of muscle proteins from meat and fish-based products [56]. Consequently, the low number of TSC for muscular mealworm proteins after enzymatic hydrolysis or the similar number of TSC obtained for control and pressurization conditions can be explained by irreversible protein aggregation induced by HHP, which drastically decreased the proteolytic activity of both Alcalase® and pepsin used at atmospheric pressure or in combination with HHP.
