pubs.acs.org/JAFC Published on Web 09/13/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 10761–10767 10761 DOI:10.1021/jf102575r The Kinetics of β-Elimination of Cystine and the Formation of Lanthionine in Gliadin BERT LAGRAIN,* KRISTEL DE VLEESCHOUWER,INE ROMBOUTS,KRISTOF BRIJS, MARC E. HENDRICKX, AND JAN A. DELCOUR Centre for Food and Microbial Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium When gliadin, a mixture of wheat storage proteins containing only intramolecular disulfide (SS) bonds, is heated at high temperatures and preferably at alkaline pH, the SS bonds are cleaved by β-elimination reactions leading to decreased cystine levels and the generation of dehydroalanine (DHA) and free sulfhydryl (SH) groups. DHA and the free SH group of cysteine can further react to form the irreversible cross-link lanthionine (LAN). The kinetics of this reaction were studied by heating model systems containing gliadin at different pH values (pH 6.0, 8.0 and 11.0) at temperatures up to 120 °C. Multiresponse modeling was applied to simultaneously describe the course of the reaction partners, intermediates and products. The estimated kinetic parameters indicate that the reaction rate constant for the elimination reaction increases with temperature and pH. Moreover, the predominant reaction consuming the intermediary DHA is the cross-link with cysteine to form LAN following second-order reaction kinetics. The corresponding reaction rate constant is less dependent on temperature and pH. Use of the proposed kinetic model to estimate reaction product concentrations in cereal-based foods allowed us to conclude that the β-elimination reaction may be less important during, e.g., bread making, but may well contribute to gluten network formation during the production of soft wheat products. It may also well be relevant in the production of bioplastics made from gluten. KEYWORDS: Gluten; cross-links; beta-elimination reaction; dehydroalanine; lanthionine INTRODUCTION The storage proteins of wheat consist of monomeric gliadin and polymeric glutenin. Together, they are referred to as gluten. Gluten plays a key role in structure and texture of different wheat- based food products and is, for instance, responsible for the bread making capabilities of wheat flour ( 1 ). Furthermore, wheat gluten and its fractions have a large potential for use in nonfood appli- cations, such as adhesives, coatings and thermoplastic materials ( 2 ). During the processing and setting of gluten containing products, temperature plays a crucial role. Heat treatment of gluten proteins results in large aggregates due to (further) polymeriza- tion of glutenin and formation of gliadin-glutenin bonds ( 3 ). Besides high temperatures, also alkaline conditions and/or mechan- ical energy input strongly contribute to protein cross-linking ( 4 , 5 ). Gluten network formation has mainly been attributed to the formation of intermolecular disulfide (SS) bonds by oxidation of sulfhydryl (SH) groups of cysteine and/or SH-SS-exchange reactions between cysteine and cystine. Such reactions lead to glutenin aggregation at moderate heating, and also involve the gliadin fraction at higher temperatures ( 6 -9 ). It has been demonstrated that gliadin becomes part of the glutenin network by SH-SS-exchange reactions, which are initiated by free SH groups in the glutenin fraction ( 3 ). However, not all reaction phenomena with respect to hydrothermal treatment of gluten or its fractions can be fully explained by this mechanism. Especially at alkaline pH, high temperatures and/or long exposure times, β-elimination of cystine in different proteins has been demonstrated to result in dehydroalanine (DHA) and cysteine formation ( 10 ). The impact of such β-elimination reactions on cross-linking of gliadin and gluten has been studied at pH 8.0 ( 11 ). Gliadin consists of proteins containing R-, γ-, and ω-gliadins. The R- and γ-gliadins contain 3 and 4 intramolecular disulfide (SS) bonds respectively, while ω-gliadins lack cysteine resi- dues ( 12 ). Due to β-elimination of intramolecular cystine, demon- strated at pH 8.0 and 130 °C, free SH groups are formed ( 11 ). On the other hand, under conditions of mild alkaline pH, DHA residues can react further with cysteine to form the DHA cross- link lanthionine (LAN). At higher pH values, lysinoalanine, a cross-link formed from DHA and lysine, can also be formed in gluten proteins ( 11 , 13 ). Such β-elimination reactions may well be relevant for several processes involving gluten as they form covalent nonreducible cross-links and new free SH groups which can participate in SH-SS-interchange reactions leading to for- mation of new intermolecular SS bonds. In order to predict and control β-elimination of cystine and subsequent formation of irreversible cross-links, and to determine its relevance for different processes, kinetic parameters, such as rate constants and activation energies, are very useful. By apply- ing multiresponse modeling, changes in the concentrations of the different reaction partners, intermediates and products are simul- taneously taken into account. Purified gliadin is a good model to *Corresponding author. Tel: (þ32)-16-321634. Fax: (þ32)-16-321997. E-mail: bert.lagrain@biw.kuleuven.be.