Workflow to Screen for Potential Allergens in Black Soldier Fly Insect Protein

Alternative protein is an emerging topic due to the demand for sustainable food sources. However, the safety of such novel foods and associated ingredients, and the possible health risks posed by allergic reactions associated with eating them, remain a vital concern. In this application brief, we demonstrate a simple workflow using LC-MS (ACQUITY UPLC M-Class and SYNAPT XS Mass Spectrometer) coupled with Progenesis QI Informatics, to screen for potential allergens in black soldier fly insect protein. Out of the 47 proteins detected, 21 were found to have strong evidence, 9 corresponding to weak evidence, whilst 17 were highlighted as having no evidence for potential allergenicity. The workflow is intuitive and can be applied to other types of novel foods that require allergenicity risk assessment. 

Benefits

• Easy-to-follow workflow to screen for allergens in insect protein
• ProteinWorks provides a simplified kit-based approach for protein digestion
• Guided menu in Progenesis QI for Proteomics to help you seamlessly move through the multiple stages in data-processing

With an increasing world population and demand for sustainable food sources, insects are one of a promising array of alternative sources of protein. The global production levels of animal-based foods place severe pressures on the environment through the emissions to air, water and soil, and the use of natural resources to produce them. It is predicted that primary protein production needs will increase by 50% in 2050, yet, at the current state, 85% of arable land is already in use. Land required for insect farming is substantially reduced compared to mainstream animal-sourced food. Moreover, insects can recover nutrients from biomass that humans cannot or do not want to eat, and bring them back to the food value chain, thereby contributing to a circular economy.¹ Insect species, such as the black soldier fly (BSF), are well-suited for growth on a large scale, and are also one of the best options for waste valorization. Furthermore, it is known that the protein from BSF contains high levels of essential amino acids, and the bioavailability of micronutrients such as iron, calcium, and zinc, which are comparable to those in beef.² Nonetheless, the safety of this novel food, and the possible health risks, including allergic reactions associated with eating them, remain a vital concern. Food allergens are mostly proteins. Cross-reactivity between insects and other invertebrates, such as crustaceans and mollusks, may occur since they are known to be closely related. Tropomyosin, myosin, and arginine kinase have been found to be the major allergens for cross-reactivity between crustaceans and insects.³ Thus, it is highly likely that those consumers which are allergic to eating shellfish, are also allergic to eating insects. It is therefore important to address these concerns to build consumer confidence in the consumption of alternative proteins.⁴ The default assumption when assessing novel foods containing proteins is that they have allergenic potential. The allergenic potential of the novel food should be explored by considering its composition, particularly its protein(s), its source (including taxonomic relationships), the production process, and available experimental and human data that includes information on cross-reactivity. This comprises a comprehensive literature review to retrieve available information on sensitization, case reports of allergic reactions, that requires support by analytical chemistry to characterize the allergenic content of novel proteins.

In this application brief, we demonstrate how an analytical workflow can be used to screen for potential allergens in novel proteins, in a non-targeted manner, using a sample of BSF protein as a case study. 

Triplicates of the two different batches of BSF protein were analyzed using the summarized schematic workflow as shown in Figure 1.

The extracted BSF protein solution followed a 5-step protocol of the ProteinWorks Auto-eXpress Low Digest Kit (p/n: 176004078), whereby the trypsin digestion was carried out at 37 °C overnight instead of 45 °C for 2 hours as suggested in ProteinWorks. The supernatant was then diluted 10x with 97/3/0.1 H₂O/ACN/FA for UDMS^E acquisition.

The analysis was carried out using an ACQUITY UPLC M-Class equipped with the nanoEase M/Z HSS T3 Column, 100 Å, 1.8 µm, 75 µm x 250 mm (p/n: 186008818) and the SYNAPT XS Mass Spectrometer. Data were acquired in positive ion mode, utilizing the UDMS^E acquisition mode. A representative extracted chromatogram is illustrated in Figure 2. 

The raw data was imported into Progenesis QI for Proteomics, which performed chromatographic alignment, data normalization, and peak picking automatically. A total of 2,473 peptides were identified using the Ion Accounting identification workflow with the settings: FDR less than 1%, fixed modification (carbamidomethylation of cysteines), and variable modifications (oxidation of methionines), using the reviewed Insecta and Hermetia illucens UniProt databases.

In Progenesis QI for Proteomics, the reviewing of selected proteins is intuitive and easy, based on the identified peptides. For example, the properties of the individual peptide ions of troponin are shown in Figure 3, whereby you can further refine the identification by tagging any outliers. 47 proteins were shortlisted after reviewing. 

The AllerCatPro⁵ is a comprehensive model, available freely online, to predict the protein allergenicity potential, based on the union of five major databases: IUIS, UniProtKB, Allergome, COMPARE, and FARRP. The shortlisted 47 proteins were submitted in their FASTA format, which provided an output table ranking the result as strong, weak, or no evidence of allergenicity per protein. Out of the 47 proteins detected, 21 were found to have strong evidence, 9 corresponding to weak evidence, whilst 17 were highlighted as having no evidence for potential allergenicity.

Table 1 summarizes the list of proteins that gave strong, weak, or no evidence of allergenicity. Tropomyosin, myosin, and arginine kinase, all detected in the BSF protein, are known to be the major allergens for cross-reactivity between crustaceans and insects.⁵ Where potential allergenic hazards have been identified, they should be investigated further by in vitro allergenicity studies of the novel food and/or its source(s). 

In conclusion, we have demonstrated how a workflow can be used to screen potential allergens found in BSF protein. Food safety risk assessment of novel foods and their ingredients is not only a regulatory requirement in many regions, but is also an important step to address consumers’ concerns, in order to build confidence in the consumption of alternative protein. This workflow is intuitive and can be applied to other types of novel alternative protein. 

Acknowledgements

This is part of a collaborative project between Waters International Food and Water Research Centre (IFWRC, Singapore) and AgriProtein Singapore. The authors would like to thank Insect Technology Group for the BSF samples and expertise in the area of insect protein.

1. Parodi A, Leip A, De Boer I J M, Slegers P M, Ziegler F, Temme E H M, Herrero M, Tuomisto H, Valin H, Van Middelaar C E, Van Loon J J A, Van Zanten H H E. The Potential of Future Foods for Sustainable and Healthy Diets. Nat Sustain, 2018, 1, 782–789.
2. Wang Y-S, Shelomi M. Review of Black Soldier Fly (Hermetia Illucens) As Animal Feed and Human Food. Foods, 2017, 6, 91.
3. Romero M R, Claydon A J, Fitches E C, Wakefield M E, Charlton A J. Sequence Homology of the Fly Proteins Tropomyosin, Arginine Kinase and Myosin Light Chain with Known Allergens in Invertebrates. J Insects as Food Feed, 2016, 2, 69–81.
4. Pali-Schöell I, Verhoeckx K, Mafra I, Bavaro S L Mills E N C, Monaci L. Allergenic and Novel Food Proteins: State of the Art and Challenges in the Allergenicity Assessment. Trends Food Sci Technol, 2019, 84, 45–48.
5.  Maurer-Stroh S, Krutz N L, Kern P S, Gunalan V, Nguyen M N, Limviphuvadh V, Eisenhaber F, Gerberick G F. Allercatpro-Prediction of Protein Allergenicity Potential from the Protein Sequence. Bioinformatics, 2019, 35, 3020–3027.