how reliable are the measurements of residual gluten in gluten-free foods?

www.helsinki.fi/yliopisto

How reliable are the measurements of residual gluten in

gluten-free foods?

Päivi Kanerva

Department of Food and Environmental Sciences

University of Helsinki, Finland

11th International Gluten Workshop Beijing, China

August 12-15, 2012


Introduction

Conditions that affect reliability of gluten measurements:

• Variety of gluten-detecting antibodies

• Different reference materials

• Protein hydrolysis

• Protein deamidation

Conclusions

Content

11th International Gluten Workshop, Beijing, 12-15 Aug 2012

Päivi Kanerva


• Coeliac disease is one of the most common food intolerances in the world, with prevalence of 1-2 %

• Gluten proteins are known to be an environmental factor causing coeliac disease

• Gluten proteins of wheat and similar proteins of barley and rye are all harmful for coeliacs

• Harmful proteins trigger an adverse immunoreaction in people with coeliac disease leading to destruction of small intestine villi

• Current treatment for coeliac disease is a gluten-free diet

Introduction Coeliac disease


Päivi Kanerva


Introduction Measuring gluten content

• Gluten content is measured by immunological ELISA methods.

• Methods are based on prolamin-recognizing antibodies.

• The method currently recommended by Codex is known by names R5-ELISA or the method of Mendez


Päivi Kanerva

Sandwich Competitive


Variety of gluten-detecting antibodies

R5 antibody raised against rye secalin (Sorell et al. 1998)

– currently in use

Recognizes prolamins of wheat, barley and rye; reacts mainly with QQPFP

ω-gliadin antibody raised against wheat gliadin (Skerritt&Hill 1990)

– predecessor of R5

Recognizes heat-stable omega-type prolamins

α-gliadin antibody raised against wheat gliadin (Spaenij-Dekking et al. 2004)

Recognizes T-cell stimulatory epitopes of wheat, barley and rye

G12 and A1 antibodies raised against 33mer (Moron et al. 2008)

Recognizes prolamins of wheat, barley and rye, and weak reaction to oat prolamins; G12 reacts mainly with QPQLPY, A1 with QLPYPQP


Päivi Kanerva

Kanerva et al 2011 Agric Food Sci 20:206-216.


• Reference material has a major influence on the quantification.

• Reference material should be similar to the analysed protein; however, complexicity of prolamins makes it difficult

• Different standards are used today in gluten analysis:

PWG gliadin other gliadin standards (e.g. Sigma) Peptide standards (synthetic or hydrolyzed)

Different reference materials


Päivi Kanerva

Measurement of gluten with R5 ELISA using different reference materials

Amount of barley flour (%) Amount of barley flour (%)

Glu

ten

co

nte

nt

(pp

m)

Ho

rde

in c

on

ten

t (p

pm

)

Gliadin standard Hordein standard


• Hydrolysis of proteins occurs during many food manufacturing processes, e.g. brewing

• Hydrolysis can be enhanced by enzymes: either added or endogenous

• High amounts of prolamin degrading enzymes is developed during grain germination

Hydrolysis of proteins


Päivi Kanerva

•Rye protein hydrolysis during sourdough

process

Sourdough from germinated rye contained

only 240-480 ppm of prolamins, which is

about 0,5% from original amount

Both sandwich and competitive assay gave

similar results


Protein deamidation

In addition to hydrolysis, solubility of gluten can be improved by deamidation

Deamidation improves also foam and emulsion forming cababilities of gluten proteins

Deamidation can be done by acid or base treatment or enzymatically


Päivi Kanerva

Deamidation is a conversion of glutamine or asparagine to glutamic acid or aspartic acid


Päivi Kanerva

Chemical deamidation of vital gluten by acid treatment

Deamidation of gluten significantly

reduced its immunological detection

by the prolamin specific antibodies

R5 and omega-gliadin.

R5 sandwich ELISA

x 600

R5 competitive ELISA

x 125

Omega-gliadin sandwich

ELISA

Kanerva et al. 2011. J Cereal Sci


Reliability of the measurements of gluten content is questioned if the complexity of prolamins and changes in their structures are not carefully considered.

reliability can be substantially improved by selecting right reference material

reactivity of different prolamins with different antibodies should be taken into account and results interpreted accordingly

consequences of modification to protein reactivity with antibodies should be evaluated

Conclusions


Päivi Kanerva

Thank you Cereal Technology Group at the University of Helsinki

Professor

Hannu Salovaara

University lecturer

Tuula Sontag-Strohm

Researchers

Päivi Kanerva

Xin Huang

Technician

Outi Brinck

how reliable are the measurements of residual gluten in gluten-free foods?

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