A Improved extraction of prolamins for gluten detection in processed foods (1)
Gluten intolerance is one of the most common food intolerances in the Western countries. The gluten-free products may contain a maximum of 20 mg of gluten per kg of food. The prolamin group consists of monomeric and polymeric proteins. Monomeric prolamins may contain disulfide bonds in their in- ner structure, whereas polymeric prolamins are joined together by the disulfide bonds, forming large protein complexes. The ratio between monomeric and polymeric prolamins in wheat is between 1.5 and 3.1 and in barley between 1.4 and 5.0. The ratio in rye is between 6.2 and 8.2. The recommended extraction method with sandwich R5 ELISA analysis for processed food samples is the so-called cocktail extraction. The cocktail extraction solution contains 2-mercaptoethanol as a reducing agent and guanidine hy- drochloride as a disaggregating reagent to improve protein solubility. Proteins may also be hydrolyzed into smaller peptides during the process (e.g. in fermentation processes and brewing), which usually increases their solubility.
The details of the samples are listed.
The addition of DTT to the 50% 1-propanol extraction yielded 31% and 34% higher protein concentra- tions for wheat and barley, and 23% higher con- centration for rye compared with the unreduced extracts.
A 40% concentration of 1-propanol was the most efficient in extraction with all three cereals, extracting about 54% ± 2% of prolamins of the total protein of wheat meal, 46% ± 4% of the total protein content of the barley meal and 56% ± 2% of the total protein content of rye meal.
The prolamins from the bread samples were most efficiently extracted with 40% 1-propanol and 1% DTT at 50C for 60 min at an extraction ratio of 1:10.
Wheat prolamins clearly differed from barley and rye prolamins. Wheat contained a large number of prolamins that required reduction. In the wheat extracts, antibody R5 reacted mainly with α-, β-, and γ-gliadins and low-molecular weight (LMW) glutenins. It also recognized ω-gliadins and HMW glutenin subunits, but less intensively.
Heating during extraction increased especially the yield of the polymeric and higher molecular weight prolamins. The yield is also improved by selecting more hydrophobic solvents, since prolamins are hydrophobic due to their high content of hydrophobic amino acids. The hydrophobicity of 1-propanol is slightly higher than that of ethanol, which increases its efficiency in prolamin extraction. The gluten network is formed when wheat flour is mixed with water. Mixing induces changes, such as new disulfide bonds between prolamins, that influence their extractability in different solvents. Heating also causes such strong bonds between the prolamins that even reduction cannot result in the same solubility of the prolamins that they had before the heat treatment. Low regulatory gluten limits demand high accuracy from the analysis method that is used for gluten quantification. The relative error of the gluten assay with antibody R5 is 30%.
1. P. Kanerva, T. Sontag-Strohm, O. Brinck, H. Salovaara, A Improved extraction of prolamins for gluten detection in processed foods. Agricultural and Food Science. 20 (2011), doi:10.2137/145960611797471525.