food matrix and processing on allergenic activity

Food Matrix and Processing

on the Allergenic Activity

TOPICs

• Overviews• Development of Food Processing• Processing induced Modification• Impact of Processing on Food Allergens • Clinical Implication

Overviews

Food processing:• Modulate allergic reaction(sensitization)• Processing affects by – altering their shape, molecular weight and structural– modulating immunoglobulin E-binding capacity (changing

IgE reactivity) lead to mediator releases.

• Sites on molecule recognized by IgE are called “epitopes”

Epitopes

• short regions(8 to 15 amino) residues,• tend to be mobile, • adopt a disordered secondary structure,• be stable in food processing.

• various segments of polypeptide chain and brought together by protein folding,• food processing affect in folded and destroy conformational epitopes • found in unprocessed or raw foods or reveal new epitopes previously hidden by protein folding.

Middleton’s Allergy: Principles and Practice 8th Edition

Effects of thermal processing on allergenicity is difficult;• the time-temperature combinations used in food processing, • the impact of water activity on the stability of individual

allergens to thermal, • the different methods employed to determine allergenic

activity.

Allergen Reactivity Safety

Cow’s milk raw, pasteurized, or UHT extensively heated e.g., baked products such as muffins or cakes

* Baking modifies the proteins much more than pasteurization.

Hen’s eggPeanut

raw, boiling extensively heated e.g., roasted and fried

* Cooking usually reduces but does not abolish the allergenic potency of egg.** Maillard reaction introduced by roasting.


Allergen Source Protein molecules ProductsCupins plant • Vicilin-like 7S seed

storage globulins • Legumin-like 11S seed storage globulins

legumes (e.g., soybean, peanut, lupin), tree nuts (e.g., hazelnut, walnut, cashew, pecan, almond), seeds (e.g., sesame, mustard).

Prolamin superfamily (three group of proteins)

skeleton of cysteine residues.

• 2S albumins• Lipid-transfer proteins (LTPs)• Cereal α-amylase inhibitors

fresh fruits (e.g., apple, kiwi, peach)seeds (e.g., peanut, tree nut, sesame)wheat (monocotyledonous plants) and cereals (e.g., barley)

Bet v 1 superfamily

fruit • Bet v 1 homolog• Gly m 4, Api g 1

apple-Birch pollen (oral allergy syndrome)soy bean, celery splice

Tropomyosin shellfish species

• Met e 1 crustacean and molluscan, greasy back shrimp

Parvalbumin muscle fish • apo and holo form white muscle fish

Caseins mammalian milk

• phosphoserine• phosphothreonine

milk

Others:-Lipocalins -β-lactoglobulin -ovomucoid and ovalbumin

• Bos d 5• Gal d 1 and Gal d 2

inhalant allergencow’s milkeggs

Classification of Allergens

Development of Food Processing

• The ability of humans to make and control fire, which began about 125,000 years ago • Variety to cook foods including using heated stones for boiling, burying food wrapped in leaves in the hot embers of fires, or baking fish in clay • Advent of urban living need to preserve foods after the harvest • In the twenty-first century, when most of the world’s population live in cities


Development of Food Processing

• Post-harvest treatments • Primary processing • Secondary processing • Complex foods

Post-harvest treatments


Nature of ProcessingModified atmosphere for storage of fruit and vegetablessuch as cooling, cleaning, sorting and packing

Source: Apple Impact of ProcessingBet v 1 homologs is upregulated during storage,levels are higher in fruit stored for 4-5 monthsLTP allergens is downregulated during storagelevels are higher in fresh fruit

Primary processing


Nature of ProcessingRemoval of outer layers by physical or chemical peeling

Source: PeachImpact of ProcessingLoss of allergens such as LTPs located in the outer layers

Secondary processingNature of Processing

Source Impact of Processing

Fruit purees and fresh juices

Peach,apple

Labile Bet v 1 homologs are modified. Prolamin superfamily fruit LTPs retain a native-like structure.

Preparation of pasteurized, UHT fruit juices and milk

Peach, apple,

milk

Labile Bet v 1 homologs are modified. Prolamin superfamily fruit LTPs retain a native-like structure. Milk allergens retain their native structure.

Boiling- Maize, lentil, fish, milk, egg

Wheat, peanut

Prolamin superfamily LTPs and 2S albumin retain a native-like structure after boiling, although seeds are boiled, much of the protein is lost into cooking water. Alters, the structure of cupin allergens (7S and 11S seed storage proteins) and can render them insoluble.

Roasting and frying Peanut Thermostable, notably prolamin superfamily 2S albumin retain their native structure and solubility after roasting and frying. Some cupin allergens (7S and 11S seed storage proteins) are altered by roasting and become insoluble. Maillard modification

Secondary processingNature of Processing


Preparation of powdered ingredients

Celery spice, milk

Maillard modifications

Deamidation Gluten Acidic conditions

Hydrolyzed food ingredients

Milk, egg, gluten

Enzymatic (usually microbial proteases) and chemical treatments used to hydrolyze protein ingredients to change their functional properties

Oil refining Soybean, peanut

Proteins are removed during the refining process, and residual protein levels in highly refined oils are very low.


Complex foodsNature of Processing


Fining agents added to alcoholic beverages

Beer, wine, spirits

Certain beverages contain residual levels of fining agents, usually are highly modified compared with the raw

Fermented foods Milk, soybean

Lactic acid fermentations, to unfold protein and precipitate or form gelled networks due to reduced pH. Secrete proteases that can hydrolyze proteins.

Baked foods Milk, eggs Food ingredients may be included that are raw or have already been processed (e.g., skimmed milk powder, pasteurized egg white). Complex interactions between different food ingredients


Novel processing techniques

Pulsed electric field and ohmic heating processes• to preserve more of the natural flavor, texture, and

nutritional quality of foods and • to address issues of sustainable food production and

energy cost reduction.


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=7WsanTOw7RcoUM&tbnid=XFs1lbaAbaGz7M:&ved=0CAUQjRw&url=http%3A%2F%2Fwww2.hawaii.edu%2F~soojin%2Fresearch.htm&ei=ZKHOUrjSI4XoiAfOkYCwBQ&bvm=bv.59026428,d.aGc&psig=AFQjCNE2ae_yZo7SnTzGEDS5RV5e73vAaw&ust=1389359783123477

Processing-induced Modification

Guanhao Bu. Dairy Sci. & Technol. (2013) 93:211–223Middleton’s Allergy: Principles and Practice 8th Edition

Heating processes(thermal processing)Process of structural and chemical changes such as denaturation, aggregation and the Maillard reaction, impacts on allergenicity of protein

Glycation reactionConjugation with reducing sugars through Maillard reaction, effect improving functional properties and masking food protein allergenicity. New epitopes emerged as hydrophobic

High pressureProcessing structural changes, such as denaturation and aggregates, also influence in allergenic potential. But application of high pressure during enzymatic hydrolysis more effectively reduce antigenicity and serum IgE-binding properties

Enzymatic hydrolysisProteolytic enzyme in animal, plant and organism destroy allergenic epitopes, by hydrolysis broken down into small peptide molecules and amino acids

Lactic acid fermentationProteolytic enzyme during fermentation by lactic acid bacteria(probiotic bacteria) break some epitopes and partial degradation IgE-epitope

Denaturation and Aggregation

Food processing conditions;• high temperature• extremes of pH on structure• aggregation

Native, secondary structure elements such as • α-helices and β-sheets (blue)• disordered polypeptide segments (pink)• intermediate structures with residual (black)


Maillard modification

Chemical reaction of amino acid + reducing sugar• requiring heat

Carbonyl group + nucleophilic amino acid

Glucosylamine and water formation• BUT unstable

Form a complex mixture• NEW flavor + odors


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=UdsaqTppz4C_mM&tbnid=I-ofsKchxCdeXM:&ved=0CAUQjRw&url=http://www.privateselection.com/our-ingredients/gourmet-coffee/&ei=cm7JUqulFaWciAe-14HoDA&bvm=bv.58187178,d.dGI&psig=AFQjCNFyRMAUauUiOqFVedG25sOIfWq-yw&ust=1389018943236182

Impact of Processing on Food Allergens

Proteins in raw foods and ingredients, • To transformed into carry new adducts(e.g.sugars)

and behave different in digestion • To affected the way in food proteins are presented to

immune system(antibody responses, food-specific IgE-binding affected by the conformational state of molecule )


A consequence of complexity and diversity of food composition and processing

Food Allergens and Clinical Implications

Classification of these allergens into protein families provides a framework for examining various aspects of food,

Common structural attributes such as; • CUPINS, • PROLAMIN SUPERFAMILY, • BET V 1 SUPERFAMILY,• TROPOMYOSINS,• PARVALBUMINS,• CASEINS,• and others: Lipocalins, β-lactoglobulin(Bos d 5),

Ovomucoid(Gal d 1) and Ovalbumin(Gal d 2)


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=OlPGDyt2dXOjWM&tbnid=pg6GZz1avRehJM:&ved=0CAUQjRw&url=http%3A%2F%2Fcafe.monkiezgrove.com%2F2009%2F10%2F01%2Fcharacter-design-master-chef%2F&ei=BYTOUpiMIIqhigehlIGIDQ&bvm=bv.59026428,d.aGc&psig=AFQjCNGan0jWZBaSkw2GRmOg4GQILTBQIw&ust=1389352285354473

CUPINS

• Share core β-barrel motif in cupin superfamily– vicilin-like 7S seed(e.g. Ara h 1) and legumin-like 11S seed

storage globulins

• Range of important allergenic foods including; – legumes(e.g. soybean, peanut, lupin), tree nuts(e.g.

hazelnut, walnut, cashew, pecan, almond), and other seeds (e.g., sesame, mustard)

• Prone to aggregate formation after heating or solutions of extreme pH and low ionic strength.


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=TCBEZUDSl6TeMM&tbnid=olAvD73XPQAMiM:&ved=0CAUQjRw&url=http%3A%2F%2Fmylittlenzhouse.com%2F2013%2F09%2F27%2Fmy-first-review-request%2F&ei=UYXOUr_WL8WdiAf07IDYCQ&bvm=bv.59026428,d.aGc&psig=AFQjCNGx_xfwz93aTMjjVdR1FTMmu1R_Iw&ust=1389352500891201

http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=0CLJJ85H814lHM&tbnid=lyS8DFhpNwVFRM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.naturalfoodbenefits.com%2Fdisplay.asp%3FCAT%3D5%26ID%3D102&ei=n4XOUsCwH4XoiAfOkYCwBQ&bvm=bv.59026428,d.aGc&psig=AFQjCNGx_xfwz93aTMjjVdR1FTMmu1R_Iw&ust=1389352500891201

Aim: To investigated the effect of thermal treatment on allergen structure and IgE-binding capacity, potency to histamine release and ability to induce T-cell proliferation and cytokine production from peanut allergic patients.Patient 35 pts: challenge with Ara h 1 purified from

either raw (N-Ara h 1) or roasted (R-Ara h 1)Intervention To observed structure, IgE immunoreactivity,

mediator release assay and cytokine productionPrimary outcome To determined factors affect the allergenic

properties of a major peanut allergen, Ara h 1.

Fany Blanc. Mol Nutr Food Res. 2011(55):1887–1894

Figure1. Characterization of heat-treated Ara h 1 by SDS-PAGE analysis

Boiling(H-Ara h 1) resulted in aggregation and hydrolysis of Ara h 1• Boiling, alone or with of glucose caused a partial loss of secondary structure• Roasted peanuts (R-Ara h 1) appeared to be highly denatured


Figure 4. IgE-binding capacity of native and heat-treated Ara h 1.


Boiled H- and G-Ara h 1 had greatly reduced IgE-binding capacities for all pts •Boiling resulted in either a very slight native-Ara h 1 or more marked heated Ara h 1 reduction in IgE-binding.• Increase in IC50 values for H-Ara h 1 and G-Ara h 1, significantly less IgE reactive.

Native-Ara h 1

Heated Ara h 1

Figure5. Effect of thermal treatment on b-hexosaminidase and proliferating cells and cytokine induction capacity

• Boiling Ara h 1(100oc 15min) form complex aggregates resulted in partial loss secondary structure and reduced IgE-binding capacity. • The aggregates also reduced capacity to elicit histamine release and potential reduced allergenic potency.


b-hexosaminidase IL-5 IL-13

Aim: To establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut(Ara h 2/6)Patient 12 peanut-allergic pts: purified Ara h 1 and Ara h

2/6, observed after native and roasting ingestion Intervention To assess the structure, IgE binding fragments

and degranulation capacitiesPrimary outcome To observed roasting process(Ara h 1 and Ara h

2/6 peanut) changes to affect their allergenicity

Y.M. Vissers. Clinical&Experimental Allergy. 41:1631–1642


Fig. 1. SDS-PAGE and Western blot of the native and thermally processed Ara h 1 and Ara h 2/6

Ara h 1 Ara h 2/6

Both Residual soluble Ara h 1, Ara h 2/6 that heating had significant hydrolysis of protein

Fig.4 Analysis of IC50(ng/mL)values obtained using IgE capture inhibition assay

• Increase of the IC50 value corresponds to decrease in IgE-binding capacity.• IgE-binding capacity of R+g and R-g Ara h 1,decreased 9000- and 3.6-fold, compared with native Ara h 1


Y.M. Vissers. Clinical&Experimental Allergy. 41:1631–1642Percentage of b-hexosaminidase release

Histamine release (ng/mL)

Extensive heating reduced the degranulation capacity of Ara h 2/6but significantly increased the degranulation capacity of Ara h 1

PROLAMIN SUPERFAMILY

• Several groups of proteins, conserved skeleton of cysteine residues by presence of a-helical globular domain

• 3 major proteins as: 2S albumins, Lipid-transfer proteins (LTPs) and Cereal α-amylase inhibitors

• Others as hydrophobic protein(Gly m 1, soybean), Indolines(wheat) and alpha-globulins(cereal grains)

• Theirs stability to heat and gastrointestinal digestion



• 3 majors group of proteins – 2S albumins, a low-molecular-weight major of seed

storage proteins of dicotyledonous plants such as Sin a 1 from yellow mustard, Ara h2 and Ara h 6 from peanut

– Cereal α-amylase inhibitors, structurally similar to LTPs and 2S albumins, storage protein in seeds of wheat, barley, rye, corn and rice of inhibitors trypsin and α-amylase


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=THoPd2nU-lbRjM&tbnid=WK3AhLDr4-LKIM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.yumsugar.com%2FYellow-Mustard-Love-Hate-3419813&ei=wnTOUomCNe6XiAfCt4CIDQ&bvm=bv.59026428,d.aGc&psig=AFQjCNHAVlbZuKNgFaYlCycnEYvyIZcsDw&ust=1389348355372739

https://www.google.co.th/imgres?imgurl&imgrefurl=http%3A%2F%2Fsakthidaran.learningprofessor.info%2Fblog%2F%3Fp%3D1163&h=0&w=0&sz=1&tbnid=SMX1uYnewkf35M&tbnh=259&tbnw=194&zoom=1&docid=UqrX8sjzcFM8-M&ei=lnTOUuCaPMnriAffq4Fo&ved=0CAsQsCUoAw



Lipid-transfer proteins (LTPs), a disulfide bonds to mediate phospholipid between vesicles and membranes

– High concentration in epidermal tissue of Rosaceae family in fresh fruits and vegetables

– Apple(Mal d 3) is greatest soon after picking, but it tends to decrease during postharvest storage in a modified atmosphere. (Sancho AI, et al. J Agric Food Chem 2006;54:5098-104)

– Peach(rPru p 3) is removed of outer layers, such as peeling, significantly reduces the potential for an allergic reaction. (Fernandez-R.M., et al. Clin Exp Allergy 1999;29:1239-47)

http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=egmIce__iu2WaM&tbnid=ahvrX5Z1wSPXEM:&ved=0CAUQjRw&url=http%3A%2F%2Flinkrandom.blogspot.com%2F2012_09_01_archive.html&ei=iHvOUoq_N8W8iAfA-ICYDQ&bvm=bv.59026428,d.aGc&psig=AFQjCNF_NvP9jpBc7551wcUReV-Vf_bM2g&ust=1389350138725196

Aim: to study the effect of heat-induced structural changes on allergenicity of 2S albumins, N-Ara h 2/6 was then further heated for 15 minutes at 110uC in the presence or absence of glucose.Patient 34 peanut-allergic pts: observed after heating

and glycation treatments of 2S albumins(Ara h 2/6) peanut ingestion.

Methods To assessed cellular proliferative potency and IgE reactivity and functionality of allergens

Primary outcome To evaluated effect of processing on structure and IgE reactivity/functionality of peanut protein.

Yvonne M.V. PLoS ONE/journal.pone. 2011:6(8)

Figure1. Effect of heating on secondary structure and oligomeric state of Ara h 2/6.



Effect of thermal treatment on the IgE binding capacity of Ara h 2/6.

Effect of thermal treatment on activation of effector cells induced by Ara h 2/6

No effect on T-cell reactivity via heat induced denaturation, reduced IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein.

BET V 1 SUPERFAMILY

• Major birch pollen allergen, relevant sensitizing protein causing called ‘birch-fruit-vegetable syndrome’

• Belongs to related protein family 10(PR-10)such as – fruits of Rosaceae (Mal d 1 in apple, Pru a 1 in cherry, and Pyr c 1 in

pear)– vegetables of Apiaceae (Api g 1 in celery and Dau c 1 in carrot)– Hazelnut(Cor a 1.04), soybean(Gly m 4), mungbean(Vig r 1) and

peanut (Ara h 8)

• Reactions confined to the oropharynx in termed of ‘oral allergy syndrome(OAS)’ and occasionally systemic reactions.


Aim: To evaluate whether cooked Bet v 1–related food allergens induce IgE- and T cell–mediated reactions in vitro and in vivoPatient Patients birch pollen allergy who experienced

OAS and AD on ingestion of fresh apple, celery, or carrot

Intervention To determined protein structures, mediator release, Bet v 1–specific T-cell lines with epitope specificity compared native and cooked food allergens.

Primary outcome To evaluated T-cell epitopes of Bet v 1 were relevant for cross-reactivity with each food allergen. And conformational changes, resulting in diminished IgE-binding capacity not structure

Bohle et al. J Allergy Clin Immunol 2006;118:242-9

Bet v 1 and Api g 1 partly restored structure, but Mal d 1 and Dau c 1 did not

Bohle et al. J Allergy Clin Immunol 2006;118:242-9

In vitro, cooked food allergens lost the capacity to bind IgE and to induce mediator release but had the same potency to activate Bet v 1–specific T cells as native proteins. In vivo, ingestion of cooked birch pollen–related foods did not induce OAS but caused atopic eczema to worsen. Bohle et al. J Allergy Clin Immunol 2006;118:242-9

• Incubation at 40C hardly reduced basophil activation by all allergens• At 60C reduced the mediator-releasing capacity of Api g 1 completely and to a lesser extent the capacity of Bet v 1, Mal d 1, and Dau c 1.• at 80C and 100C abolished their mediator-releasing capacity.

Aim: To determined information about the allergenicity of processed celery in celery-allergic patientsPatient 12 pts with allergic reactions to raw or raw and

cooked celery, DBPCFCs with raw celery, cooked celery and celery spice

Intervention To performed thermal stability of celery allergen and mediator release assay to assessed allerginicity of processes

Primary outcome To assess the allergenicity of processed celery bycooked celery and celery spice

B.K. Ballmer-Weber. Allergy 2002:57:228–235

B.K. Ballmer-Weber. Allergy 2002:57:228–235

Thermal stability of celery by EAST inhibition

• Monosensitized to celery allergen, Api g 1, almost complete inhibition• shortest heating period decrease of inhibition potency of celery

RBL cell mediator release assay.

• IgE raised against raw celery reacted with Api g 1, the major celery allergen• But, IgE-heated celery did not reactwith Api g 1

TROPOMYOSINS

• Allergen from crustacean and molluscan shellfish• α-helical structural protein in both muscle and

nonmuscle cells, highly conserved across kingdom. • Greasy back shrimp (Met e 1) is the major heat-

stable allergen in many crustacean species.


Aim: To detected shellfish-derived tropomyosin range of crustacean and mollusc species and to analysed impact of heat-processing on antibody recognition for improved allergen.Patient Fresh or frozen specimens of 11 different

crustacean and 7 mollusc speciesIntervention To determined protein profile of shellfish in raw

and heated extracts and effect of heat treatmentPrimary outcome

S.D.Kamath et al. Food Chemistry 141(2013);4031–4039


• The heated-shellfish displayed a more protein-banding pattern. • The heated-crustacean signified of heat-stable proteins.But the heated-mollusc have different intensities.


Fig. 4. Inhibition-ELISA for the quantitative analysis of cross-reactivity of the mAb

Cross-reactive tropomyosin was detected in crustacean species, with partial in detect in molluscs but none in oyster, octopus and squid.

Heating has a profound effect on major shellfish allergen tropomyosin, targetting N-terminal region of tropomyosin, must be developed to differentiate in crustaceans and molluscs

• Lobster and crab heated were able to inhibit reactivity in a dose-dependent• Molluscs heated were not able to achieve any inhibit, even concentration.

PARVALBUMINS

• major allergen found in the white muscle of fish.• apo form reduced IgE-binding capacity, lacks the

calcium ions found in the holo form• Thermal processing usually reduces but does not

abolish the allergenic activity of fish


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=YYKoOHuQwK1zhM&tbnid=6m7-VbZTZRXGfM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.fao.org%2Fwairdocs%2Ftan%2Fx5916e%2Fx5916e01.htm&ei=pMnOUoGLNaLyiAfFloGQBg&bvm=bv.59026428,d.aGc&psig=AFQjCNFfaiq73qyboscllkwtlC4x8CnSgw&ust=1389370128266158

http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=YYKoOHuQwK1zhM&tbnid=6m7-VbZTZRXGfM:&ved=0CAUQjRw&url=http%3A%2F%2Fmlmlblog.wordpress.com%2Fcategory%2Fauthors%2Ferin-loury-featured-authors%2Fpage%2F2%2F&ei=vMnOUp6VOYqhigehlIGIDQ&bvm=bv.59026428,d.aGc&psig=AFQjCNFfaiq73qyboscllkwtlC4x8CnSgw&ust=1389370128266158

Aim: To investigate the effect of glycosylation on the digestibility and IgE-binding of codfish parvalbuminPatient 21 pts: positive history of immediate-type

hypersensitivity reactions to fish, with specific IgE >5 kU/L

Intervention Native and glycosylated parvalbumins at various conditions were analysed for apparent molecular weight and IgE-binding

Primary outcome To investigated result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin

Harmen H.J. BioMed Research International. Volume 2013,10:756-789

Figure 3: Secondary structure content of different forms of parvalbumin


• The difference in intensity is slightly difference in protein concentration.• Native parvalbumin is structurally sensitive to low pH conditions, while glycosylation provides some protection against such sensitivity.

• Glycosylation of parvalbumin, formation of higher structures, are more potent IgE- binders than native


Fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

CASEINS

• A mammalian milk proteins with a loose tertiary, highly hydrated structure.

• To possess clusters of phosphoserine and phosphothreonine residues that bind amorphous calcium phosphate


Aim: To compare the allergenicity of caseins(CSN) and whey proteins(WP) of thermally processed cow and buffalo milkPatient 3-4 wks-old male mice were sensitised by CSN or

WP from cow or buffalo milk then obtained raw and different thermal processed milk

Intervention To assessed humoral response(IgG, IgE level) and splenocyte stimulation index.

Primary outcome To determined boiling and sterilisation of cow and buffalo milk may be affect the allergenicity

Umesh K.S. J SciFoodAgric 2013; 93:2287–2292

Relative percentage change in allergenicity of cow’s and buffalo’s milk compared to raw milk with respect to milk protein-specific IgE response.


The boiling and sterilisation of milk clearly affect the allergenicity bydecreasing humoral and cell-mediated responses. CSN and WP of sterilised milk are less allergenic than raw milk.

Others ALLERGEN FAMILIES

• Inhalant allergens: Lipocalins • Cow’s milk protein: β-lactoglobulin(Bos d 5)• Egg white proteins: ovomucoid(Gal d 1) and

ovalbumin(Gal d 2).


Clinical Implications

Post-harvest treatmentsIndividuals with birch pollen–related fruit allergies may tolerate freshly picked but not stored fruit. Individuals may experience reverse symptoms, although severity of LTP allergies may completely preclude consumption of problem fruitPrimary processingPeeling fruits can make them safe for consumption by certain individuals with LTP allergies. Secondary processing

Complex foods




Primary processing

Secondary processingFruit purees and fresh juicesIndividuals Bet v 1–related fruit allergies usually can consume pureed fruit products. These foods can still trigger reactions in individuals with LTP fruit allergies. Preparation of pasteurized, UHT fruit juices and milk Individuals Bet v 1–related fruit allergies usually can consume UHT-processed fruit products. Processing is insufficient to make UHT juices safe for individuals with LTP fruit allergies. The allergenic potential of UHT milk resembles that of pasteurized and raw milk. Boiling Boiling seeds and milk reduces their allergenicity but does not abolish it. For some foods (e.g., polenta), the extent of cooking affects the residual level of allergenic activity Complex foods


http://www.google.co.th/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&docid=8eAT2GyprOMjcM&tbnid=mOFgu5UnBvpqhM:&ved=0CAUQjRw&url=http://www.21food.com/products/pasteurised-fruits-70838.html&ei=H3_JUoPrCsWdiAfrooDADA&bvm=bv.58187178,d.dGI&psig=AFQjCNF8Q7aXZfmcTbTOQmO3LR2FbkFgFg&ust=1389023363132133



Primary processing

Secondary processingRoasting and frying The major peanut 2S albumin allergens retain their allergenic activity, explaining why roasted peanuts possess significant allergenic activity. Allergenic activity of the 7S and 11S globulins is retained to some extent after roasting. Maillard modifications may further contribute to the allergenic activity of roasted peanuts. Preparation of powdered ingredients Powdered food ingredients have allergenic activity similar to unprocessed foods and can promote trigger reactions when included in recipes or through cross-contact in other foods (e.g., residual milk powder in foods otherwise free from milk). Complex foods




Primary processing

Secondary processingDeamidationInduces formation of novel IgE epitopes through glutamine deamidation, which results in individuals reacting to foods prepared with deamidated gluten ingredients who can otherwise safely consume wheat flour–containing foods. Hydrolyzed food ingredientsHydrolysis reduces IgE reactivity but does not abolish it completelyOil refining In the EU, highly refined soybean oils are considered safe for consumptionHighly refined peanut oils do not appear to cause adverse reactions, but these do not have EU labeling derogation. Complex foods




Primary processing

Secondary processing

Complex foodsFining agents added to alcoholic beverages Individuals with allergies to fish triggered by collagen or egg can react to alcoholic beverages Reactions to fining agents are rare, and many individuals with egg, milk, or fish allergy can safely consume these beverages. Fermented foods Although fermented milk and yogurt may retain some allergenic activity, other highly modified foods, such as soy sauce, may have substantially reduced allergenic activity and may not present a hazard to certain allergic patients.


Aim: To determine traces of egg, milk, and fish-derived processing aids used in wine making might elicit clinical reactions in food-allergic patients.Patient 14 pts: allergy to egg (n=5), milk (n=5), or fish

(n=4)Methods Skin prick tests with fining agents, and

fined/unfined winesDouble-blind placebo-controlled food challenges with fined and unfined wines.

Primary outcome The allergenicity of fining agents: ovalbumin, lysozyme, casein and fish protein

S Kirschner. J InvestigAllergolClinImmunol 2009; Vol.19(3):210-217

fish protein

ovalbumin or lysozyme casein

Skin prick test with fined and unfined wines in patients allergic to egg, milk and fish.

Although concentrated fining agents containing ovalbumin, lysozyme, and casein were allergenic in the skin prick test, no patient reacted adversely in the provocation test to fined wine

S Kirschner. J InvestigAllergolClinImmunol 2009; Vol.19(3):210-217



Primary processing

Secondary processing

Complex foodsBaked foodsEvidence from oral food challenge studies indicates that baking reduces the allergenic activity of milk, and foods with baked milk may be given to children whose infantile cow’s milk allergy is beginning to resolve.


Aim: To evaluate the natural history of tolerance development who incorporated baked-milk products into their diets.Patient 88 egg-allergic children: underwent sequential

food challenges to baked-cheese (pizza) followed by unheated-milk.

Methods comparison group, Immunologic parameters were measured at challenge visits.

Primary outcome To evaluated tolerance to baked-milk underwent sequential food challenges followed by unheated-milk.

Jennifer S. Kim. J AllergyClinImmunol. 2011;128(1):125–131

Jennifer S. Kim. J AllergyClinImmunol. 2011;128(1):125–131

Initially baked-milk-tolerant are 7.6 times more likely to develop unheated-milk tolerance than subjects who were initially baked-milk-reactive over the follow-up period; HR=7.62 [1.75, 33.14] P=.007

Aim: to determine whether SPT wheal size and/or serum sIgE levels to milk proteins could be used to predict successfully passing a baked milk challengePatient 35 children: history of allergic reactions to milk

and food challenges to baked milkMethods retrospective chart review, determined by a

positive SPT or elevated serum sIgEPrimary outcome To determine serum sIgE and SPT that may be

useful in predicting outcomes to baked milk challenges.

Lisa M.B. Ann AllergyAsthmaImmunol. 2012;109(5):309–313


Milk SPT wheal size was a better marker for food challenge outcome to baked milk, compared to casein SPT and milk protein sIgE levels. Identified >90% NPVs

Immunologic responses

Symptoms at first reported


Aim: to investigate the mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergensPatient 39 C3H/HeJ mices: orally sensitized with

ovalbumin (OVA) or ovomucoid (OM) andchallenged native or heated proteins

Methods Evaluate allergenicity by Immunoreactivity and digestibility by mediator release assay and basophil activation assay

Primary outcome To investigate factors behind reduced allergenicity of two major egg white allergens, ovalbumin (OVA) and ovomucoid, when to heat-treatment.

Gustav BS. J Allergy ClinImmunol. 2011;127(4):990–7


Reduced allergenicity of heated egg white proteins partially resultingfrom altered digestion and absorption in the gastrointestinal tract may explain the clinical tolerance of extensively heated egg in the majority of egg-allergic children

Aim: To characterize immunologic changes associated with ingestion of baked egg and evaluate the role that baked egg diets plays in the development of tolerance to regular egg.Patient 79 baked egg-tolerated children: incorporated

baked egg into their diet.Methods comparison group, Immunologic parameters

were measured at follow-up visits.Primary outcome To evaluate predictors of baked and regular egg

tolerance and assess the time to development of regular egg tolerance.

Stephanie AL. J AllergyClinImmunol. 2012;130(2):473–480

Recommendations to perform baked egg challengebased on clinical status and testing

Dietary baked egg is safe, convenient, and well accepted by patients.Introducing baked egg to egg-allergic children presents an important shift in the treatment paradigm for egg allergy


TAKE HOME MESSAGE

• Food processing and food matrix can affect the structure change and allergenic activity(IgE-binding capacity and mediator release)

• Processing triggers chemical changes including unfolding, aggregation, addition of glucose(Maillard modification)

• Heat resistant allergen, retained structure and allergenic activity after severe thermal processing(e.g. canning, roasting)

• Thermal treatments such as baking can reduce the allergenicity of certain foods (e.g. milk, eggs) and induced early tolerance.

THANK YOU FOR YOUR ATTENTION

food matrix and processing on allergenic activity

Health & Medicine