Reactive oxygen species responsible for beta-glucan degradation Audrey M. Faure, Julia Werder, Laura Nyström ⇑ ETH Zurich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 9, 8092 Zurich, Switzerland article info Article history: Received 24 October 2012 Received in revised form 21 January 2013 Accepted 25 February 2013 Available online 7 March 2013 Keywords: Beta-glucan Oxidation Catalase Superoxide dismutase Fenton reaction Reactive oxygen species abstract The presence of iron(II) in beta-glucan in solution causes the formation of hydroxyl radical, which further oxidises the polysaccharide. This degradation can be enhanced by the presence of a reducing agent, such as ascorbic acid. In this study we investigated the effect the iron(II) concentration on the hydroxyl rad- ical-mediated degradation of beta-glucan and identified the intermediate species involved in the forma- tion of hydroxyl radicals. An increase in the iron(II) concentration did not have a significant effect on the degradation in the presence of a reducing agent (ascorbic acid), while in the mere presence of iron(II) it accelerates the degradation. The addition of catalase and superoxide dismutase (SOD) prevented the hydroxyl radical driven-degradation of beta-glucan induced by iron(II) or ascorbic acid/iron(II), demon- strating the involvement of both superoxide and hydrogen peroxide in the hydroxyl radical formation. SOD, which catalyses the dismutation of superoxide into hydrogen peroxide, should have stimulated the formation of radicals, since these radicals are generated from the reaction between hydrogen perox- ide and iron(II). In the present study, we hypothesise the mechanism of the inhibition of beta-glucan deg- radation by superoxide dismutase. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Hydroxyl radicals ( Å OH) are highly reactive molecules and are one of the reactive oxygen species (ROS), along with species such as superoxide radical (O 2 Å ) and hydrogen peroxide (H 2 O 2 ). Å OH are the most powerful ROS and are involved in the oxidative dam- age of biological molecules, such as carbohydrates (Duan & Kasper, 2011). These free radicals oxidise carbohydrates by abstracting hydrogen atoms of C–H moieties, leading to the formation of car- bon-centered radicals (von Sonntag, 1980). The radicals formed may undergo diverse transformations such as rearrangement, cleavage of the glycosidic bond, and ring opening (Gilbert, King, & Thomas, 1984; von Sonntag, 1980). Beta-glucan in solution can be broken down under Å OH attack, when exposed to iron(II) or/ and ascorbic acid (Faure, Andersen, & Nyström, 2012; Faure, Mün- ger, & Nyström, 2012; Kivelä, Gates, & Sontag-Strohm, 2009; Ki- velä, Nyström, Salovaara, & Sontag-Strohm, 2009; Paquet, Turgeon, & Lemieux, 2010). The result of this degradation is a po- tential alteration of the viscosity-related health benefits of beta- glucan since the viscosity of a beta-glucan solution is controlled by its molecular weight (Wood, 2007). Å OH are known to be gener- ated via the iron catalysed Haber–Weiss cycle and the ascorbate- driven Fenton reaction (Burkitt & Gilbert, 1990; Haber & Weiss, 1932). However, the reaction pathways that lead to the formation of Å OH are still controversial (Burkitt, 2003; Koppenol, 2001). More- over, previous studies dealing with Å OH-induced beta-glucan deg- radation have not focused on studying the mechanism involved in the formation of Å OH. Therefore, investigating the identity of the major intermediate oxygen species leading to the formation of Å OH, as well as the parameters controlling the radical generation would give precious informations on the mechanism of the radical mediated beta-glucan degradation and possible ways to control it when introducing beta-glucan to functional foods. Fry (1998) has shown that in the presence of ascorbic acid (AH 2 ) and catalytic metal, xyloglucan was degraded. This degradation was found to be fully inhibited by the addition of catalase, indicat- ing that endogenous H 2 O 2 occurred in the system and was involved in the degradation process (Fry, 1998). In contrast, the addition of superoxide dismutase (SOD) did not have any effect on xyloglucan degradation. The mechanism proposed was an oxidative cleavage of the polysaccharide by Å OH (Fry, 1998), which was formed through a reaction between iron(II) and H 2 O 2 (Eq. (1), Fenton reac- tion) (Fenton, 1894): Cu þ =Fe 2þ þ H 2 O 2 ! Å OH þ OH  þ Cu 2þ =Fe 3þ ð1Þ Ascorbic acid (AH 2 ), which possesses reducing properties, can promote the Fenton reaction by reducing iron(III) and dissolved O 2 Eqs. (2) and (3), thus supplying the substrates necessary to pro- duce Å OH (Buettner & Jurkiewicz, 1996; Fry, 1998): AH 2 þ 2Cu 2þ =Fe 3þ ! A þ 2H þ þ 2Cu þ =Fe 2þ ð2Þ AH 2 þ O 2 ! A þ H 2 O 2 ð3Þ 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.02.096 ⇑ Corresponding author. Tel.: +41 44 632 91 65. E-mail addresses: laura.nystroem@hest.ethz.ch (L. Nyström). Food Chemistry 141 (2013) 589–596 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem