Novel foods: allergenicity assessment of insect proteins

Abstract

Insects represent a promising source of proteins and have been reported as a great potential for being used as novel food and feed proteins. This makes them a valuable source of nutrients to face the increasing demand of food necessitated by the growing global population. The current European food legislation on novel food (EU Reg. 2015/2283), which entered into force in 2018, provides the provisions that should be considered in the applications for the authorisation of novel foods in the European market. Insects, intended as an alternative source of food proteins for human consumption, are considered novel foods. Since food allergens are mostly proteins, the analysis and identification of the potential allergenicity of novel proteins should be a fundamental activity that enables the applicants to fulfil the requirements for the application and authorisation to bring a novel food into the European market and ensures a high level of food safety for the European consumers. The main aims of the work of the EU‐FORA fellow were to: (i) Review, assess and identify gaps in the current strategies for predicting allergenicity of novel foods and new alternative protein sources; and (ii) Familiarise, understand and perform an allergenicity assessment of a novel food protein source by: (a) Working on an allergenicity assessment case study of insect proteins from black soldier fly larva (Hermetia Illucens); and (b) Taking into consideration other risk assessment aspects of insects as novel food, including toxicological, nutritional and microbial risks. The project contributed to the continuous learning of the fellow on practical assays and methodologies for the in silico, in vitro and in vivo analysis principles and complemented personal skills related to the food risk assessment requirement for the preparation and submission of an application for authorisation of a novel food.

1. Introduction

The global population is growing, thus, the demand for food is expected to increase. To meet the future demand for food, alternative protein sources are being studied. Insects represent a promising source of proteins, and the potential of using insects as a novel source of food and feed proteins has been reported in several studies (FAO, 2009a; Rumpold and Schlüter, 2013; Van Huis, 2013; Akhtar and Isman, 2018; Kim et al., 2019; de Carvalho et al., 2020).

The possibility of using insects for food and feed has been investigated by some food authorities, including the Food and Agriculture Organization of the United Nations (FAO) and the European Food Safety Authority (EFSA), and the risk assessments related to insects as food or feed have been performed in some European Union (EU) Member States.

There are currently nine novel food applications for insects undergoing safety evaluation by EFSA and there have been already three insects for which EFSA has finished their safety evaluations (European Commission, 2022). These are: ‘Tenebrio molitor’ larva – mealworm in frozen, dried and powder form (EFSA NDA Panel, 2021a,b); ‘Locusta migratoria’ – migratory locust, grasshopper (EFSA NDA Panel, 2021); and ‘Acheta domesticus’ – house cricket (EFSA NDA Panel, 2021d). These three insects have all been authorised by the European Commission to be placed on the EU market as a novel food (EU, 2021/882, 2021a; EU, 2022/169, 2022a; EU, 2021/1975, 2021b; EU, 2022/188, 2022b).

Currently, the European food legislation on novel food (EU Reg. 2015/2283) demands, among other information, a comprehensive assessment of nutritional, microbial and toxicological risks and evidence that the novel food is unlikely to trigger allergic reactions, before authorising the introduction of the novel food/protein source on the EU market. Well establish methods and standards are available for the assessment of nutritional, microbial and toxicological risks. On the contrary, the risk assessment of allergenic potential of novel proteins is still challenging (Verhoeckx et al., 2020). As several pieces of information or conduction of experimental methods are needed for the allergenicity assessment to provide sufficient evidence to predict allergenicity, which are currently not available or not yet validated, such as, among other, potential threshold doses of food allergens, integration and standardisation for in vitro/in vivo tests and protocols, and modernisation of the in silico tools and databases, as well as clinical data, a careful allergenicity assessment is difficult (EFSA GMO Panel, 2022).

Bearing this in mind, in the current ‘learning by doing’ education EU‐FORA working programme, the fellow was engaged in analysing and discussing strategies for predicting allergenicity of a novel source of food protein using the insect black soldier fly larva as a case study.

2. Description of work programme

In the course of the work programme, the fellow, was hosted at the Research Group for Food Allergy at the National Food Institute, Technical University of Denmark (DTU Food) under the supervision of Head of Research Group. Here the fellow was introduced to various aspects of allergenicity assessment and involved in practical laboratory test and analysis steps, from extraction of proteins from a novel biomass to in silico, physicochemical, in vitro and in vivo assessment of the allergenic potential of the novel proteins, by carrying out a case study.

Through such supervised case study, the fellow experienced the challenges met in the whole process of performing an allergenicity risk assessment, which improved the fellow's understanding of the allergenicity risk assessment.

2.1. Aims

The main activities related to the project consisted of:

• Reviewing, assessing and identifying gaps in the current strategies for predicting allergenicity of novel foods and new alternative protein sources.

• Familiarise, understand and perform an allergenicity assessment of a novel food by:

  • ᵒ
    Working on an allergenicity assessment case study of the insect black soldier fly larva (Hermetia Illucens) as a potential future source for alternative food proteins
  • ᵒ
    Taking into consideration other risk assessment aspects of insects as a novel food, including toxicological, nutritional and microbial risks.

2.2. Activities/methods

2.2.1. Current strategies for predicting allergenicity

A literature search and review of the state of the art of current available methodology and approaches for performing allergenicity risk assessment of novel food or food proteins were performed by the fellow. Outcomes showed that at present there are no validated approaches for allergenicity risk assessment of novel protein (EFSA NDA Panel, 2014; Verhoeckx et al., 2020; EFSA GMO Panel, 2021, 2022). The findings were discussed and analysed in ad hoc monthly meetings with Senior Researchers in the Research Group for Food Allergy.

The aspects discussed included questions regarding:

• The existing knowledge about (high) allergenic vs. (low) non‐allergenic foods and food proteins;

• The approaches and the tools currently available to predict allergenicity of new foods and food proteins;

• The need for efficient prediction of allergenicity and the purpose of the allergenicity risk assessment; and

• Which test and parameters could be included and what are the gaps that need to be filled in order to provide an improved allergenicity risk assessment procedure for novel food and food proteins.

Among the aspects that came to light, notably to consider were the limited options to assess the hazard and potential risks of new proteins causing de novo sensitisation (EFSA GMO Panel, 2017, 2022; Mazzucchelli et al., 2018; Remington et al., 2018), as there is currently no single test or parameter available that may provide sufficient evidence to predict de novo sensitisation. On the other hand, for cross‐reactivity aspect, meaning for the assessment of the risk of inducing reactions in already allergic patients, tools and methods are available and these may well predict the potential cross‐reactivity with a reasonable level of confidence (EFSA GMO Panel, 2010, 2022; Verhoeckx et al., 2020). Therefore, there is not a huge and urgent need for improving tools and methods, for assessing the risk of inducing reactions in already allergic patients due to cross‐reactivity with known allergens. The large gap, and where there is a great need for improving and developing new predictive tools, is for the evaluation of de novo sensitisation for assessing the potential risk of inducing a new allergy of similar severity as peanut allergy.

Key elements of knowledge and methods are moreover lacking, which includes among other: integration and standardisation for in vitro and in vivo tests and protocols, as well as modernisation of the in silico tools and databases; data regarding the allergenic potency of other allergenic foods than those recently published FAO/WHO for which a reference doses has been proposed; the consensus on the clinically relevant allergens and clinical data on threshold doses for individual allergenic protein molecules (EFSA GMO Panel, 2022; FAO and WHO, 2021).

Therefore, the ‘weight‐of‐evidence’ approach, currently in use, remains the most suitable method applicable for allergenicity assessment. Still, a better clarity on the use of the ‘weight‐of‐evidence’ approach is needed, since the results might differ depending on whether a food product is derived from genetically modified organisms, from new biotechnological food or true novel food is being assessed.

For this task, a draft document on reviewing and discussing strategies for predicting allergenicity has been initiated. The document is an ongoing work aiming at a potential manuscript for publication in a peer‐reviewed journal.

2.2.2. Allergenicity assessment of novel food – a case study

At present, the allergenicity assessment, as part of the risk assessment of novel food, evaluates the allergenic risk that a new protein or whole food pose to the existing allergic population identified by the immunoglobulin E (IgE) cross‐reactivity (EFSA Guidance, 2021; EFSA GMO Panel, 2022). Current methodology is suitable mainly for assessing the allergenic potential of new proteins for the cross‐reactivity, whereas methodologies for assessing allergenic potential of new proteins due to de novo sensitisation are limited. Food allergens are mostly protein; therefore, the analysis of the potential allergenic proteins becomes a fundamental aspect to evaluate the potential allergenic risk in the context of the allergenicity assessment of novel food (EFSA Guidance, 2021).

Taking into account that black soldier fly larva is considered a potential alternative protein source (EFSA Scientific Opinion, 2015) it is, therefore, important to investigate and characterise the allergenic potential, for both cross‐reaction and de novo sensitisation, of black soldier fly larva proteins.

As a preliminary step, a literature search was performed. This was carried out to identify studies on the risk assessment and findings of insects as a future source for alternative proteins and on the allergenicity assessment of black soldier fly larva. Further, a literature search with a focus on gathering information and reviewing studies related to the black soldier fly larva proteins was performed, by searching whether there were any proteins already identified and characterised and which method was applied for their characterisation (Figure 1 – I and V).

Figure 1.

Analytical approaches carried out within the case study, including literature review, material preparation (protein defatting and protein extraction), laboratory analysis (protein characterisation ‐ Electrophoresis, Mass Spectrometry, ELISA; in vitro analysis; in vivo/animal studies), and in silico analysis. Figures in I, II, III, V were created in BioRender.com

The next step of the allergenicity assessment consisted of the extraction and preparation of the material for characterisation of the composition and for use in further analyses. The original material, of the black soldier fly larva, was kindly provided by ENORM BioFactory A/S –Denmark in powder form and was afterwards treated by a defatting process. The defatted material was further processed for protein extraction to provide the material for use in further analyses. The protein concentration of the extract was determined by the bicinchoninic acid protein (BCA) protein assay and amino acid analysis. The amino acid analysis also provided the amino acid composition of the extracted proteins. Further, the extract was characterised by gel electrophoresis (sodium dodecyl sulfate–polyacrylamide gel electrophoresis – SDS–PAGE) for protein separation and protein size profiling as well as by tandem mass spectrometry (MS/MS) analysis in order to determine the presence of specific proteins (Figure 1 – II).

Then a computer/database‐based analysis was performed where the amino acid sequences of black soldier fly larva were retrieved and grouped according to the allergenic protein super‐families (NCBI‐Protein, 1988; Radauer et al., 2014; Finn et al., 2016; Goodman et al., 2016; The UniProt Consortium, 2021). The degree of protein sequence homology between the studied proteins and known allergens was then determined by bioinformatic tools to evaluate the potential allergenicity risk due to cross‐reactivity as recommended in the Codex Alimentarius Commission (FAO, 2009b) (Figure 1 – IV).

To identify the sensitising capacity of black soldier fly larva an animal study was carried out with Brown Norway rats. The immunogenicity and sensitising capacity of black soldier fly larva proteins were evaluated in comparison to known (high) allergenic and known (low) non‐allergenic food proteins by means of several enzyme‐linked immunosorbent assays (ELISA) analyses. All the tests were carried out by developing and applying in‐house protocols generated at Research Group for Food Allergy in the National Food Institute.

To evaluate the digestibility, an in vitro simulated gastro and gastroduodenal digestion of black soldier fly larva was performed in simulated gastric fluid and simulated duodenal fluid solutions, respectively. Aliquots were taken for further analysis by SDS–PAGE and immunoblotting (Figure 1 – III).

The fellow was introduced to the above‐mentioned practical laboratory assays and methodologies. Nevertheless, most laboratory work was supported and carried out with the help of the lab technicians.

A manuscript, currently under preparation, will present a full description of the allergenicity risk assessment of the black soldier fly larva, including protein chemical analysis, in silico analysis, the in vitro analysis and the in vivo analysis (animal studies). This manuscript will be submitted for publication in a peer‐reviewed journal.

An overview of the activities carried out during the EU‐FORA fellowship are presented in the Figure 1 below.

2.3. Secondary activities

Besides being introduced to the aspects of the allergenicity risk assessment approach, the fellow was introduced to food enzyme allergenicity assessments conducted by the Research Group for Food Allergy to authorities, particularly on in silico allergenicity assessment cases.

In addition to the regular weekly meetings with the main supervisor and the research group meetings, the fellow took part of other activities:

• Attended the 9th BfR‐Summer Academy‐Lecture Series on food safety and appropriate risk assessment, organised by the German Federal Institute for Risk Assessment (BfR‐ Bundesinstitut für Risikobewertung).

• Attended the Fundamentals of Immunology course on Innate Immunity and B‐Cell Function. Offered by Rice University. Rice University in Houston, Texas, via Coursera online courses.

• Participated in the online Stakeholder workshop on small particles and nanoparticles in food. Organised by EFSA Working Group on Nanotechnologies (WG Nano).

• Participated in the ONE – Health, Environment, Society – Conference 2022. Organised by EFSA in partnership with the European Centre for Disease Prevention and Control (ECDC), the European Chemicals Agency (ECHA), the European Environment Agency (EEA), the European Medicines Agency (EMA), and the European Commission's Joint Research Centre. 21–24 June 2022, Brussels.

3. Conclusions

In the course of the EU‐FORA fellowship programme, the fellow, hosted at the DTU Food, was introduced to the food allergenicity assessment and familiarised with the role of the allergenicity assessment on the risk assessment of novel foods.

In this process, the fellow learned practical assays and methodologies for the in silico, in vitro and in vivo analysis principles and complemented personal skills related to the allergenicity risk assessment requirement for the preparation and submission of an application for authorisation of a novel food.

The EU‐FORA fellowship project at DTU Food contributed to the process of learning new skills and knowledge of the fellow on the food risk assessment methodologies, on the understanding of laboratory assays and tools, and on the use of in silico analysis and bioinformatics tools. The EU‐FORA programme also provided a rich social experience both during the training modules and the placement at the DTU Food.

Abbreviations

BCA

bicinchoninic acid

EFSA GMO Panel

EFSA Panel on Genetically Modified Organisms

EFSA NDA Panel

EFSA Panel on Dietetic Products, Nutrition and Allergies

ELISA

enzyme‐linked immunosorbent assay

FAO

Food and Agriculture Organization of the United Nations

GMO

genetically modified organisms

IgE

immunoglobulin type E

MS/MS

tandem Mass Spectrometry

SDF

simulated duodenal fluid

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SGF

simulated gastric fluid

Suggested citation: Liguori B, Sancho AI, Poulsen M and Lindholm Bøgh K, 2022. Novel foods:Allergenicity assessment of insect proteins. EFSA Journal 2022;20(S2):e200910, 9 pp. 10.2903/j.efsa.2022.e200910