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The influence of the food matrix onallergenicity*
Christal BowmanNational Center for Environmental Assessment
US Environmental Protection AgencyApril 11, 2012
*This information has been reviewed by the Environmental Protection Agency's Office of Research and Development and approved for presentation. Approval doesnot signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement orrecommendation for use.
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Food matrix issues can affect:
Detection and quantitation of proteins
Processing alterations
Digestibility assays in vitro
Digestion and absorption in vivo
Interactions with the immune system
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Matrix effects: Digestibility
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Enzymatic digestion
Complete digestion (labile)
Stable
Protein
. .. .
. ..
Not a good target for immune system
Available for processing by antigen-presenting cells
Dong et. al, TOXICOLOGICAL SCIENCES 73, 8
16 (2003)
immunologic ignorance
Semi-stable
(antigen)
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Protein content
High protein meals can overwhelm digestive
enzyme levels
Matrix may contain proteins with specific
enzyme inhibition activity
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Plant structures
Cell walls may not be broken down in the
upper GI tract
Nut protein bodiesdry, relatively insoluble
hydration status may be deciding factor in
allergenicity of some of these
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Lipids
In vitro pepsin digestion of-lactalbumin
slowed by phosphatidylcholine (abundant
surfactant in milk and produced by thestomach) Moreno et al. 2005 J Agric Food Chem
Phosphatidylcholine did not affect
pepsinolysis of-lactoglobulin, butprotected from duodenal enzymesMandalari2009 Mol. Nutr. Food Res.
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Polysaccharides
Gum arabic, pectin, or xylan at 50 wt%
enhanced peptic digestion of-
lactoglobulin. Interaction withpolysaccharides reduced protein
aggregates/increased solubility Mouecoucou et al.2004, BBA
Affects the size of peanut peptides obtained
by two-step hydrolysis Mouecoucou et al. 2004, Clin ExpAllergy
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Polysaccharides contd
Pectin forms a gel at low pH
Effects of 3% or 6% pectin on fruit proteindigestibility: -- Polovic et al. 2007 Clin. and Exp. Allergy.
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Crude extract (CE)
and extensivelydialyzed extract (DE)of kiwi fruit insimulated gastricfluid
Polovic et al. 2007 Clinical and Experimental Allergy
In vitro digestion of crude and dialyzed kiwi extracts
3 hr
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The presence of a foodmatrix significantly impedes
the transepithelial transportof allergens in vitro
Schulten et al. 2011, Mol. Nutr. FoodRes., 55: 14841491.
no hazelnut extract
with hazelnut extract
(-lactoglobulin)
(hazelnut nsLTP)
(apple allergen)
(aqueous extract)
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Proteins in blood of ratsafter feed with and withouthazelnut extract
Schulten et al. 2011, Mol. Nutr. FoodRes., 55: 14841491.
no hazelnut extract
with hazelnut extract
(-lactoglobulin)
(hazelnut nsLTP)
(apple allergen)
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Lipids: long chain fatty acids enhance
antigen absorption
Wang et al. 2009 PLoS One
chylomicron
Palmer et al. 2012 Brit Med J. epi study
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Low pH
High pH
SMALLINTESTINE
STOMACH
Enterocoated beads releasecontents only at pH > 5
Encapsulatingprotein protectsagainst digestion;
low hydration
Method used with OVA to sensitize mice (Michael et al., Vaccine 1996)
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Oral tolerance
Immunologic hyporesponsiveness to
specific antigen encountered via feeding
Inhibits allergic responses to food
Generally thought that soluble
protein/antigen is better for inducing
tolerance
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Oral tolerance
1 week
reduced IgETolerance generated
Anergy or deletion = lessT cell help for IgEresponseGeneration of Tregs =
active suppression of Thand B cells
Anti-ovalbumin IgE
Cont
rol
IPova
Oral
,IP
ova
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
*
Absorbance450nm
Bowman and Selgrade 2008 Tox Sci
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Oral tolerance is no longer induced when ovalbumin is encapsulated
Also demonstrated
by JG Michael etal.1996 Vaccine
Bowman and Selgrade 2008 Tox Sci
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Ovalbumin was aggregated and did not induce oral tolerance
Anti-OVA IgE
NAVE
OVAIP
5M
GOV
AOr
al,IP
0.0
0.1
0.2
0.3
in water, pH 3.0
OD450n
m
no tolerance
Chemical and heatdenaturation also abrogateoral tolerance (Peng1998).
Matrix pH may affect protein characteristics and
therefore tolerance induction
Bowman and Selgrade 2008 Tox Sci
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Peanut specific IgE
naive
iponly
1m
goral
2m
goral
1m
gor
al+
bic
arb
2m
gor
al+
bic
arb
0.0
0.1
0.2
0.3
0.40.5
0.6
0.7
0.8
*
**
Absorbance450nm
Peanut becomes orally tolerizing with
sodium bicarbonate treatment
untreatedsodium
bicarbonateadded
increased
solubility
Increased solubility?
or digestion inhibition?
Bowman and Selgrade 2008 Tox Sci
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Matrix effects: stimulation of the
immune system Saponin adjuvants; increased antigen uptake
Maharaj et al., 1986but also act at a distance
Plant sterols
Liposomes, LPS, other lipids
Lectins
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The influence of lipids on IgE production in an injection hazelnutallergy mouse model
Dearman et al. 2007; Clin and Exp Allergy
Mice exposed ip to partially purifiedhazelnut extract or recombinant (lipid-
free) Ber e 1
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The influence of lipid fractions on IgE production in an injectionhazelnut allergy mouse model
Dearman et al. 2007; Clin and Exp Allergy
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Whole peanut extract stimulates an increase in cell number afterfootpad injection in mice
van Wijk F et al. Toxicol. Sci. 2005;86:333-341
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Processing
Protein aggregates are generally more
immunogenic
Glycosylation via heating with matrix-supplied sugars
i
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Detection
Need to understand
exposure; relies on
accurate determination of
specific protein content
Compatibility with standardized reference
materials
Matrix quenching influence in immunoassays
varies with protein of interest and matrix tested --Van den Bulcke 2007 Eur Food Res Technol
Potential cross-reactivity between assay antibody
and other proteins
R f
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Recap of
food matrix considerations
Detection and quantitation of proteins
Processing alterations
Digestibility assays in vitro Digestion and absorption/handling in vivo
Interactions with the immune system
Discussion
pHsugars fats
structure other
proteins adjuvants