Transcript Decrease of allergenicity after action of proteolytic enzymes and

Effects of heat treatment and
proteolytic enzymes on allergenicity
Dr. Montserrat Fernández Rivas
Bischenberg, 23rd September 2007
The GA2LEN/EAACI Allergy School
M. Fernández Rivas
Food allergens
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In contrast to inhalant allergens (most) food allergens undergo
considerable changes in structure and may form complex
structures with other food components before they interact
with the individual’s immune system.
Food processing and digestion modify the allergenicity of
foods, in terms of potential to sensitise and/or induce
symptoms.
Therefore it is of paramount importance to understand the
molecular basis of the effects of food processing and digestion
(and we have just begun).
Furthermore, the role of the food matrix in which an allergen
is processed must also be evaluated.
Epitopes:
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Linear or sequential: binding is not afected by the folding state of the
protein
Conformational: binding is disrupted by changes in the protein folding.
M. Fernández Rivas
Effects of heat treatment
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Thermolabile proteins: unfolding conformational
epitopes
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Thermostable proteins:
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Bet v 1 homologues
Albumins
Limited unfolding and aggregation: 11S, 7S globulins
Resisting unfolding and refolding on cooling: 2S albumins, nsLTP,
parvalbumin
Covalent modification of proteins: Maillard reactions
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Bet v 1 homologues
Peanut allergens, Ara h 1 and 2
nsLTP
M. Fernández Rivas
Thermolabile proteins:
Bet v 1 homologues
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Birch pollen-fruit-vegetable
syndrome
Clinical finding: patients react to the
fresh fruit but tolerate it cooked.
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Patients sensitised to intact native
Bet v 1 through the inhalant route
recognise conformational epitopes on
plant foods that are destroyed during
cooking (unfolding)
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Api g 1 and Gly m 4 are more
thermostable than their equivalents in
Rosaceae fruits: this is in line with
reactions induced by cooked celery
and soya-based food supplements
M. Fernández Rivas
Thermolabile proteins: Bet v 1 homologues
CD spectra of Pru av 1 at different temperatures
Scheurer et al. JACI 2004; 114: 900-7
1)
2)
3)
4)
Fresh sweet cherries from the market
cherry fruit juice, 35% fruit content
preserved cherries, sweetened
cherry jam, 45% fruit content
M. Fernández Rivas
Thermostable proteins: nsLTP
Clinical findings:
• Patients allergic to peach/apple LTP react
with the fresh fruit and with processed
products
(+) OFC with cooked
apple
• Patients allergic to hazelnut-Cor a 8 react
after
ingestion of roasted hazelnuts
• Patients allergic to maize LTP react with
cooked
maize derivatives (polenta, roasted corn,
popcorn)
1) fresh sweet cherries from the market
2) cherry fruit juice, 35% fruit content
3) preserved cherries, sweetened
4) cherry jam, 45% fruit content
CD spectra of Pru av 3 at different temperatures
Scheurer et al. JACI 2004; 114: 900-7
M. Fernández Rivas
Maillard reactions and nsLTP
Effect of heat treatment +/- glucose on Mal d 3 IgE binding potency
SPT Mal d 3 heated 60 min 100ºC: no change
20 min 90oC
2 hr 100oC
(+)
(-)
2 hr 100oC
Protection of Mal d 3 by the presence of glucose during heating
Sancho AI. Allergy 2005;60:1262-8
The Maillard reaction is achemical reaction between an
amino acid and a reducing sugar, usually requiring heat.
M. Fernández Rivas
Maillard reactions and peanut allergenicity
Roasting peanuts: increased IgE-binding
x90
Maleki SJ. JACI 2000;106:763-8
M. Fernández Rivas
Maillard reactions and peanut allergenicity
Roasting peanuts: increased IgE-binding + stability to digestion
Maleki SJ. JACI 2000;106:763-8
Ara h 2 trypsin inhibitor activity is 3.6 fold increased by roasting
and protects Ara h 1 against trypsin digestion.
Maleki SJ. JACI 2003; 112:190-5
M. Fernández Rivas
Effect of proteolytic enzymes
on Bet v 1 homologues
Cherry extract digested with pepsin:
(A) Silver stained
(B) IgE immunoblot
1) 2 hr without pepsin
2) 30 sec
3) 1 min
4) 15 min
5) 30 min
6) 1 hour
7) 2 hours
8) BSA without pepsin
9) BSA 2 hours
Scheurer et al. JACI 2004; 114: 900-7
M. Fernández Rivas
Pepsin digestion of purified cherry allergens
Bet v 1 homologue
nsLTP
Profilin
Scheurer S. JACI 2004; 114: 900-7
M. Fernández Rivas
Stability of profilin
Melon profilin is completely
digested in < 1 min
Melon profilin is not affected
After heating 100ºC 15 min
López-Torrejón G. CEA 2005; 35:1065-72.
M. Fernández Rivas
Stability of profilin
Histamine release
Untreated rCuc m 2
Positive 2/3
SGF digested rCuc m 2
Negative 3/3
López-Torrejón G. CEA 2005; 35:1065-72.
M. Fernández Rivas
In summary
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There are no clear rules regarding how
different allergens respond to heat treatment
and proteolytic enzymes, with some as the
Bet v 1 homologues having their allergenicity
destroyed, whereas for many others it is
unaltered.
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On the other hand, the allergenicity of some
foods may even increase following food
processing.