Effects of heat treatment and proteolytic enzymes on allergenicity

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Effects of heat treatment and proteolytic enzymes
on allergenicity

• Dr. Montserrat Fernández Rivas

2
Food allergens

• 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 individuals 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
• Linear or sequential binding is not afected by
  the folding state of the protein
• Conformational binding is disrupted by changes
  in the protein folding.

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Effects of heat treatment

• Thermolabile proteins unfolding conformational
  epitopes
• Bet v 1 homologues
• Albumins
• Thermostable proteins
• Limited unfolding and aggregation 11S, 7S
  globulins
• Resisting unfolding and refolding on cooling 2S
  albumins, nsLTP, parvalbumin
• Covalent modification of proteins Maillard
  reactions
• Bet v 1 homologues
• Peanut allergens, Ara h 1 and 2
• nsLTP

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Thermolabile proteins Bet v 1 homologues

• Birch pollen-fruit-vegetable syndrome
• Clinical finding patients react to the fresh
  fruit but tolerate it cooked.
• Patients sensitised to intact native Bet v 1
  through the inhalant route recognise
  conformational epitopes on plant foods that are
  destroyed during cooking (unfolding)
• 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

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Thermolabile proteins Bet v 1 homologues

• Fresh sweet cherries from the market
• cherry fruit juice, 35 fruit content
• preserved cherries, sweetened
• cherry jam, 45 fruit content

CD spectra of Pru av 1 at different temperatures
Scheurer et al. JACI 2004 114 900-7
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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
Scheurer et al. JACI 2004 114 900-7
CD spectra of Pru av 3 at different temperatures
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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 2005601262-8

• The Maillard reaction is achemical reaction
  between an amino acid and a reducing sugar,
  usually requiring heat.

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Maillard reactions and peanut allergenicity Roasti
ng peanuts increased IgE-binding
x90
Maleki SJ. JACI 2000106763-8
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Maillard reactions and peanut allergenicity Roasti
ng peanuts increased IgE-binding stability to
digestion
Maleki SJ. JACI 2000106763-8
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 112190-5
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Effect of proteolytic enzymes on Bet v 1
homologues

• Cherry extract digested with pepsin
• (A) Silver stained
• (B) IgE immunoblot
• 2 hr without pepsin
• 30 sec
• 1 min
• 15 min
• 30 min
• 1 hour
• 2 hours
• BSA without pepsin
• BSA 2 hours

Scheurer et al. JACI 2004 114 900-7
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Pepsin digestion of purified cherry allergens
Bet v 1 homologue
nsLTP
Profilin
Scheurer S. JACI 2004 114 900-7
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Stability of profilin
Melon profilin is completely digested in lt 1
min Melon profilin is not affected After
heating 100ºC 15 min
López-Torrejón G. CEA 2005 351065-72.
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Stability of profilin
López-Torrejón G. CEA 2005 351065-72.
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In summary

• 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.
• On the other hand, the allergenicity of some
  foods may even increase following food processing.