This presentation was held at the 10th Protein Summit in Reims, France on September 27, 2017. Dr. Kitty Verhoeckx, lead scientist on allergenicity risk assessment at TNO, presented a cases study on mealworm allergenicity risks, associated with cross-reactivity in shrimp allergic people and de novo sensitization. This case study demonstrates the importance of allergenicity risk assessment in the R&D of novel and processed protein products.

1. Risk assessment of novel and
processed proteins in food
Kitty Verhoeckx

2. Reduce Food Loss and Wasteo
Create energy efficient productiono
systems (Sustainable food
production)
Climate/pest resistant cropso
Alternative protein sourceo
Background
Growing world population
And changing dietary habits

3. Alternative sustainable protein sources
Insects (May 2013: report FAO on edible insects)
Refining waste streams (e.g. beet leaves)
Algae, duckweed, seaweed (no agricultural land
required)
Engineered/ processed proteins in food industry
(e.g. GMO crops, hydrolysed wheat-proteins)

4. General food law
(EC regulation No 258/97 and EU recommendation 97/618)
The law requires that safety is assured for all food
ingredients placed on the market.
Responsibility of the producers

5. Novel food law
(EC regulation 2015/2283)
Regulation is in force but will be fully implemented January 2018.
Food additives, flavorings, enzymes, extraction solvents or
GMO's not in scope.
Novel food means any food that was not used for
human consumption to a significant degree within EU before 15
May 1997 and falls at least in one of listed categories.

6. Novel Food Categories
Food with a new or modified molecular structure
Food consisting of, isolated from or produced from:
microorganisms, fungi, algae
material from mineral origin
plants or their parts without history of use
animals or their parts without history of use
cell or tissue cultures
Food resulting from a production process not used for food production
within the Union before 15 May 1997, which significantly changes the
food’s composition or structure
Food consisting of engineered nanomaterials
Vitamins and minerals produced with a new method or which are in
nanoform

7. Safety assessment comprises:
Toxicological
Allergenic
Nutritional Microbiological

8. Food allergy
High prevalence 2-4 %
>20 million food allergic consumers in EU
Health impact (quality of life, lethality)
Social impact (allergy sufferers and surrounding
people)
>80 million people confronted with food allergy on daily basis
Economic impact (food incidents, recalls)
No cure avoidance of allergenic food products
Read ingredient labels & look for hidden allergens

9. Labelling
Regulation (EU) No 1169/2011
Added as ingredient : Legislation on declaration of 14 foods
Unintentionally present :Voluntary measures, such as
Precautionary labelling--> no guidance
may contain…. or produced in a factory that also produces….
Precautionary labelling should be used with prudence

10. May contain labelling Fruit Juice
TNO is working on science and
tools to improve the precautionary
labeling to warn consumers for
possible Unintended Allergen
Presence in food product.

11. Food allergy vs sensitisation
IgE
No clinical signs
Clinical signs
Sensitisation
Allergic reaction

12. • Is the novel (processed) protein able to elicit an
allergic reaction in a food allergic population
(cross reactivity, IgE binding)?
Allergy risk assessment strategy
Is the novel (processed) protein able to induce a•
new allergy (sensitization, IgE production)?
new allergycross reactivity

13. The mealworm is the larval stage of the Yellow mealworm✓
beetle
Originally produced as feed for animals such as fish, reptiles✓
and birds.
Commercially available for human consumption✓
Allergenicity assessment of a novel
food: Yellow Mealworm (Tenebrio Molitor)

14. New protein
(source)
History of human
exposure (work/food)
Relationship:
Taxonomy, Homology
Identification
proteins in source
Information on usage:
raw, matrix, processing
Research material:
Extract(s), GMP
Research protocol, METC
Cross reactivity
Sera known allergy
with new protein
Functional Cross
reactivity
Sera known allergy
with new protein
Sensitising potency
Immuno• -blot
ELISA•
CAP/ISAC•
Skin prick test
Provocation
History Sensitisation
Screening (interview and/or serology )
Working population•
Targeted people with symptoms•
No history
New allergy
Sera new allergy
with new protein
• In vitro
• In vivo
Cross reaction
Sera new allergy
with known allergen
In vitro•
In vivo•
Identification
proteins
Identification reactive proteins in source
(potential new allergens)
New allergiesCross reactivity
New allergies &
Cross reactivity
Basophil activation
test
Verhoeckx et al 2016, Regul Toxicol Pharmacol

15. History of Allergic reactions
Historyofhuman
exposure(work/food)
Allergic reactions to✓ insects bites
Allergic reactions caused by✓ inhalation (e.g.
dust with cockroach feces) and contact (e.g.
caterpillar hairs) and primarily occur with people
who regularly come into contact with insects
(e.g. entomologists, fish bait breeders)
A number of cases have also been documented✓
in which the consumption of insects has
caused an allergic reaction and even
anaphylactic shock in humans

16. Mealworms are closely related to shrimp
and house dust mite.
Relationship:
Taxonomy,Homology
Domain EukaryotaDomain bacteria Domain protozoa
Life
Kingdom Animalia
Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014)

17. New protein
(source)
Relationship:
Taxonomy, Homology
Identification
proteins in source
Information on usage:
raw, matrix, processing
Research material:
Extract(s), GMP
Research protocol, METC
Cross reactivity
Sera known allergy
with new protein
Functional Cross
reactivity
Sera known allergy
with new protein
Immuno• -blot
ELISA•
CAP/ISAC•
Identification
proteins
Cross reactivity
Basophil activation
test
History of human
exposure (work/food)
Cross reactivity
Skin prick test
Provocation
Broekman et al 2016, J Allergy Clin Immunol

18. Conclusions cross reactivity
All shrimp allergic patients were sensitized to mealworm based on
SPT, BAT, Immunoblot and CAP mealworm
87% of the Shrimp allergic patients had a positive DBPCFC to
mealworm. (Muller 0-3, Subjective symptoms at 216 mg dose).
Allergens involved: Tropomyosin and Arginine kinase
Processing did not effect allergenicity (SPT, BAT)
Shrimp allergic patients are at risk when eating mealworm

19. Sensitisation
Broekman et al 2017, J Allergy Clin Immunol

20. Conclusions de novo sensitisation
All subjects (n=4) were sensitized to mealworm according to all
tests.
Two subjects had a positive DBPCFC to mealworm, but were
not allergic to shrimp or any other food.
Responsible allergen: Larval Cuticle protein.
De novo sensitization to mealworm is possible.
The risk may be larger than for the shrimp allergic population
alone

21. New protein
(source)
History of human
exposure (work/food)
Relationship:
Taxonomy, Homology
Identification
proteins in
source
Information on usage:
raw, matrix,
processing
Research material:
Extract(s), GMP
Research protocol, METC
Sensitising potency
No history
New allergies
New allergies &
Cross reactivity
No History of sensitisation?

22. Processing affects biological processes
Protein Change in functionality/nutritional value
Treatments
- Enzymatic
- chemical
- physical (e.g. heat)
Physiological
Solubility-
Water binding-
Gelation-
Viscosity-
Emulsification-
Texture-
Flavor-
color-
Foaming-
Structure-
stability-
Biological
Digestibility-
Uptake-
Immunological
- Allergenicity
- Health effects

23. Processing affects digestion kinetics
glycinin
Β-conglycinin
Acid treatment (pH < 2.5)
Enzyme
Thermal treatment (>70 °C)
denaturation
Enzyme
hydrolysis
Kazunobu Tsumura 2009
Β-conglycinin hydrolysed glycinin hydrolysed

24. Does the processing technique influences the allergic response of the
already allergic population  most studies investigate the recognition of
processed protein by antibodies
Does processing have an impact on the ability of food proteins to induce
a new allergy (sensitization: capacity stimulate the IgE production)
Allergenicity assessment of processing
techniques
(Review on food processing and allergenicity Verhoeckx, 2015)

25. Pasteurization increased allergenicity of milk (IgE binding)
No effects of UHT processing, vacuum condensing and spray drying
on allergenicity of milk were found
Fermentation of milk strongly reduced allergenicity, (IgE-binding)
extensively hydrolyzed milk formulas
Extensive heating diminishes the allergenicity of egg white proteins
(egg allergic patients)
Acid hydrolyzed wheat gluten induced allergenicity (wheat tolerant
people
Heat induced glycation (Maillard reaction) enhanced allergenicity of
raw peanut (IgE binding)
Allergenicity assessment of processing
techniques - examples
(Review on food processing and allergenicity Verhoeckx, 2015)

26. Allergenicity assessment of a new
processing techniques-conclusions
processing can induce and reduce the allergenic potential of allergens.
Microbial fermentation and enzymatic or acid hydrolysis may have the
potential to reduce allergenicity.
Other processing methods such as pressure treatment show promising
results (lowering allergenicity), though further studies are needed to
clarify the effects
Information on the effect of processing on sensitization is scarce.
(Review on food processing and allergenicity Verhoeckx, 2015)

27. 2 food allergy initiatives
Shared Research Program TNO www.srpfoodallergy.com
COST Action ImpARAS www.imparas.eu

28. GOALS
Shared Research Program Food Allergy
Food
allergy
free
world
Protect the
allergic
consumer
Prevent
introduction
strong allergenic
foods
Preventive &
curative
strategies
TNO is looking for industrial and
academic partners to join us!
Interested?
www.srpfoodallergy.com

29. Aim: To build an interdisciplinary European network of scientists with a
broad range of expertise to discuss, with an out-of-the-box view, new
ideas and more predictive models and approaches to improve the
current allergenicity risk assessment strategy.
30 EU countries involved & Supported by Industry & Governments
COST Action ImpARAS

30. Take home messages
Food safety assessment of novel foods and new processing
techniques is required by law.
Allergenicity can be influenced by processing and should therefore be
investigated to protect the consumers.
Pitfall is the assessment of the onset of new allergies methods
need to be developed.
Start early in development pipeline with safety assessment, this will
safe a lot of time and strengthen your market position!

31. Thank you for your attention
Dr. Kitty Verhoeckx
Scientist, TNO
T: +31 (0)88 8665136
@: Kitty.verhoeckx@tno.nl
SRP TNO www.srpfoodallergy.com
COST Action ImpARAS www.imparas.eu