Evaluation of glycosidic bond cleavage and formation of oxo groups in oxidized barley mixed-linkage β-glucans using tritium labelling Andrea Iurlaro a,b , Giuseppe Dalessandro a , Gabriella Piro a , Janice G. Miller b , Stephen C. Fry b , Marcello S. Lenucci a, ⁎ a Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento, via prov.le Lecce-Monteroni, 73100 Lecce, Italy b The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, King's Buildings, Edinburgh EH9 3JH, UK abstract article info Article history: Received 25 June 2014 Accepted 8 September 2014 Available online 16 September 2014 Keywords: Ascorbic acid β-Glucans Oxidative scission Reactive oxygen species Soluble dietary fibre This study investigated the formation of oxo groups in pure solutions of barley mixed-linkage (1→3),(1→4)-β- D-glucans (MLGs) incubated in the presence of hydrogen peroxide or Fenton reaction-generated hydroxyl radicals (•OH), and it gives a fingerprint of products obtained after enzymic and acidic hydrolysis of •OH-attacked MLG. Hydroxyl radical, but not hydrogen peroxide, introduced a range of NaB 3 H 4 -reducible functions into MLG chains. Driselase or lichenase digestion of NaB 3 H 4 -reduced MLGs released a complex mixture of 3 H-labelled products due to the presence of unusual and incompletely digestible residues in •OH-attacked polysaccharide chains. Complete acid hydrolysis of •OH-treated MLGs yielded a mixture of 3 H-aldoses (mainly glucose, mannose, galactose and allose) deriving from random •OH attack at positions 2, 3 or 4 to form glycosulose residues which were NaB 3 H 4 -reducible to epimeric mixtures of 3 H-aldose residues. Furthermore, the production of [ 3 H]glucitol demonstrated the radical-mediated cleavage of mid-chain glucose residues to create new reducing termini. Oxidative scission of MLGs by hydroxyl radical caused a decrease in molecular weight of about 96%, which was partially inhibited by the addition of DMSO, an •OH scavenger. The results can be a starting point for developing an assay to detect changes due to •OH attack of MLGs in vivo, or during food processing and storage, based on the chemical or enzymic release of unusual sugar residues such as allose as diagnostic products after reduction/labelling treatment and partial purification (or selective digestion by lichenase) of polymeric material. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Public interest in dietary fibre-enriched functional foods is steadily increasing because of their ability to prevent metabolic disorders and/ or reduce the risk of contracting pathologies such as diabetes, cardiovas- cular diseases and some cancers (e.g. colorectal adenoma, breast cancer and renal cell carcinoma) (Jenkins, Jenkins, Zdravkovic, Wursch, & Vuksan, 2002; nning, 2007; Poutanen, Laaksonen, Autio, Mykkanen, & Niskanen, 2007; Sier, Gelderman, Prins, & Gorter, 2004). Dietary fibres which are very interesting for functional food production include mixed-linkage (1 →3),(1 →4)-β-D-glucans (MLGs), linear homopoly- mers consisting predominantly of β-(1,4)-linked glucose oligomers (3–4 residues), separated by single β-(1,3) bonds (Fig. 1). Less frequent segments of consecutively β-(1 →4)-linked glucose residues (2 and 5–28) were also evidenced (Fry, Nesselrode, Miller, & Mewburn, 2008; Izydorczyk & Dexter, 2008; Wood, 2010). It should be noted, however, that some MLG oligosaccharides initially assumed on the basis of HPLC to be relatively long actually turned out on more detailed analysis to be shorter — e.g. an apparent nonasaccharide turned out to be a novel, lichenase-resistant hexasaccharide (Simmons et al., 2013). These hemicellulosic polysaccharides are prevalent constituents (with some differences in structure, molecular weight and amount) of the primary cell wall of Poales (including cereals and grasses of the Poaceae family), but are also abundant in the walls of the horsetails (genus Equisetum; Fry et al., 2008; Sørensen et al., 2008). MLGs are a minor component of durum wheat kernel (0.5–2.3%), mainly located in the cell wall of aleurone layer cells and, to a lesser extent, in the starchy endosperm. In other cereals they can be more abundant (2.0–20.0% in barley, 3.8–6.1% in oat) depending on the cultivar, and show a species- specific distribution, being mainly distributed in the sub-aleurone layer or in the starchy endosperm in oat and barley kernels, respectively (Collins et al., 2010; Dornez et al., 2011). A cause–effect relationship has been established between the regular consumption of MLGs and reduction of blood cholesterol concentration; in addition, a dietary intake of MLGs of ~3 g/day has Food Research International 66 (2014) 115–122 Abbreviations: A, dehydroascorbic acid; AH 2 , ascorbate; DP, degree of polymerisation; GPC, gel-permeation chromatography; MLGs, mixed-linkage (1→3),(1→4)-β-D-glucans; PC, paper chromatography; PE, paper electrophoresis; TFA, trifluoroacetic acid; TLC, thin- layer chromatography. ⁎ Corresponding author. Tel.: +39 0832 298612; fax: +39 0832 298858. E-mail addresses: iurlaro.andrea@libero.it (A. Iurlaro), giuseppe.dalessandro@unisalento.it (G. Dalessandro), gabriella.piro@unisalento.it (G. Piro), janice.miller@ed.ac.uk (J.G. Miller), s.fry@ed.ac.uk (S.C. Fry), marcello.lenucci@unisalento.it (M.S. Lenucci). http://dx.doi.org/10.1016/j.foodres.2014.09.008 0963-9969/© 2014 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres