Shotgun proteomics, in-silico evaluation and immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates

Abstract

Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG-and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. the relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. igG-immunoblotting analysis with anti-tropomyosin i antibodies and ige-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. the immunoassays were carried out also on protein hydrolysates extracted by treating insects with protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While igG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, igeimmunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate Novel food protein sources are being studied in order to meet the future requirement for food, in connection to the perspectives of growing population 1 . Insects represent a good source of proteins, not only because of the high protein content in some species, but also for their nutritional quality in terms of essential amino acids profile. Not of a secondary importance, insect breeding, in comparison to common livestock, is characterized by many environmental advantages, such as less land use, feed and water requirement, fewer greenhouse gas emission and high feed conversion ratio 2 .From January 1 st of 2018, insects have been included in the category of novel foods and the European Food Safety Agency (EFSA) opinion is mandatory before their marketing 3 . In 2015, EFSA assessed for the first time the safety related to insect consumption. The potential hazards connected to the use of insects in food or feed were deemed to be related to exogenous and endogenous factors, which could be influenced also by harvesting and processing methods 4 . The presence of allergens represents one of the main endogenous risk related to the consumption of insects. Insect-based food ingredients could cause an allergenic response either due to a primary sensitization, or to a cross-reaction. Primary sensitization is related to the ability of insect proteins to elicit an allergic reaction not related to other food allergies. This risk is widespread mainly in regions where edible insects are commonly used (e.g. China) 5 . A cross reaction, more likely where insects are not commonly consume...

High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis Affect Mealworm Allergenic Proteins

“…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).…”

Antimicrobial Biomasses from Lactic Acid Fermentation of Black Soldier Fly Prepupae and Related By-Products

“…They grow and reproduce easily, have low feed conversion rate compared to conventional livestock animals (one kg of insect biomass can be produced from 1.7 Kg of feed biomass, depending on species) and can be reared on agro-industrial by-products [ 3 , 4 ]. In addition to serving as feed, insects provide a high nutritional profile for humans, although the presence of allergens represents a potential hazards that must be further investigated [ 5 , 6 ]. Overall, it is demonstrated that insects production involves low energy, land area utilization and environmental footprints [ 7 ].…”

Why for feed and not for human consumption? The black soldier fly larvae

“…Hall and colleagues have demonstrated that hydrolyzing cricket proteins with alcalase reduced tropomyosin Immunoglobulin E (IgE) reactivity in shrimp allergic patients and that hydrolyzing the proteins even further to 60% to 85% degree of hydrolysis completely reduced the IgE reactivity to tropomyosin. Hydrolyzing BSFL proteins showed a reduction in immune reactivity, but there was partial preservation of IgE reactivity (Leni et al, 2020b), although with further research this could be a technique that could be used to reduce allergic reactions caused by BSFL proteins completely.…”

“…Arginine kinase, found in many varieties of prawn species, has been found in the lesser mealworm (Alphitobius diaperinus) (Leni et al, 2020b), field cricket (Gryllus bimaculatus), silkworm larvae (Bombyx mori) (Liu et al, 2011), the German cockroach (Blattella germanica) (Chuang, Su, Chiang, Lee, & Chow, 2010), and the Indian meal moth (Plodia interpunctella). In a shotgun proteomics study, arginine kinase was not identified as a major pan allergen in BSFL (Leni et al, 2020b); however, Broekman et al (2017) identified the presence of arginine kinase in BSFL protein extract. Tropomyosin and arginine kinase are declared allergens for Arthropoda in the Allergen Nomenclature database (http://www.allergen.org/index.php) and have been found in both crustacean species and insect species.…”