Food Chemistry 141 (2013) 589–596

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Food Chemistry

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Reactive oxygen species responsible for beta-glucan degradation

0308-8146/$ - see front matter � 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).

Audrey M. Faure, Julia Werder, Laura Nyström ⇑ETH Zurich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 9, 8092 Zurich, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 October 2012Received in revised form 21 January 2013Accepted 25 February 2013Available online 7 March 2013

Keywords:Beta-glucanOxidationCatalaseSuperoxide dismutaseFenton reactionReactive oxygen species

The presence of iron(II) in beta-glucan in solution causes the formation of hydroxyl radical, which furtheroxidises the polysaccharide. This degradation can be enhanced by the presence of a reducing agent, suchas 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 thedegradation in the presence of a reducing agent (ascorbic acid), while in the mere presence of iron(II) itaccelerates the degradation. The addition of catalase and superoxide dismutase (SOD) prevented thehydroxyl 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 stimulatedthe 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.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Hydroxyl radicals (�OH) are highly reactive molecules and areone of the reactive oxygen species (ROS), along with species suchas superoxide radical (O2

��) and hydrogen peroxide (H2O2). �OHare 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 abstractinghydrogen atoms of C–H moieties, leading to the formation of car-bon-centered radicals (von Sonntag, 1980). The radicals formedmay 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 canbe broken down under �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 controlledby its molecular weight (Wood, 2007). �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 formationof �OH are still controversial (Burkitt, 2003; Koppenol, 2001). More-

over, previous studies dealing with �OH-induced beta-glucan deg-radation have not focused on studying the mechanism involvedin the formation of �OH. Therefore, investigating the identity ofthe major intermediate oxygen species leading to the formationof �OH, as well as the parameters controlling the radical generationwould give precious informations on the mechanism of the radicalmediated beta-glucan degradation and possible ways to control itwhen introducing beta-glucan to functional foods.

Fry (1998) has shown that in the presence of ascorbic acid (AH2)and catalytic metal, xyloglucan was degraded. This degradationwas found to be fully inhibited by the addition of catalase, indicat-ing that endogenous H2O2 occurred in the system and was involvedin the degradation process (Fry, 1998). In contrast, the addition ofsuperoxide dismutase (SOD) did not have any effect on xyloglucandegradation. The mechanism proposed was an oxidative cleavageof the polysaccharide by �OH (Fry, 1998), which was formedthrough a reaction between iron(II) and H2O2 (Eq. (1), Fenton reac-tion) (Fenton, 1894):

Cuþ=Fe2þ þH2O2 ! �OHþ OH� þ Cu2þ=Fe3þ ð1Þ

Ascorbic acid (AH2), which possesses reducing properties, canpromote the Fenton reaction by reducing iron(III) and dissolvedO2 Eqs. (2) and (3), thus supplying the substrates necessary to pro-duce �OH (Buettner & Jurkiewicz, 1996; Fry, 1998):

AH2 þ 2Cu2þ=Fe3þ ! Aþ 2Hþ þ 2Cuþ=Fe2þ ð2Þ

AH2 þ O2 ! AþH2O2 ð3Þ

590 A.M. Faure et al. / Food Chemistry 141 (2013) 589–596

This process, in which ascorbate recycles iron(III) to iron(II) andmaintains the Fenton reaction, is called ascorbate-driven Fentonreaction (Burkitt, 2003). Similar to xyloglucan, beta-glucan maysuffer from �OH-induced oxidative cleavage in an AH2/iron(II) sys-tem. The route to �OH production was presumed to be similar tothat described by Fry (1998). In a previous study, we showed thatcatalase could prevent the degradation process, which demon-strated the involvement of H2O2 in the AH2-promoted beta-glucandegradation (Faure, Münger, et al., 2012). We have also shown thatSOD was able to significantly decrease the formation of �OH in beta-glucan solution treated with an AH2/iron(II) system (Faure, Münger,et al., 2012). The inhibitory effect of SOD suggested that superoxideradicals were being generated and were involved in the formationof �OH. These findings provided new insight into the mechanismof radical-mediated beta-glucan degradation and implied the exis-tence of different reaction pathways leading to �OH generation.Therefore, in the present study we explored the role of O2

��, H2O2

and �OH in beta-glucan degradation in more detail.Oxidation of beta-glucan may also be initiated simply by the

presence of iron(II) (Faure, Andersen, et al., 2012). Many studieshave already reported the iron(II) induced oxidative damage onbiomolecules in aerobic conditions (Qian & Buettner, 1999; Ryan& Aust, 1992; Welch, Davis, Van Eden, & Aust, 2002). Gutteridge(1987) demonstrated that iron(II) could damage deoxyribose byinducing �OH formation. The most likely mechanism leading tothe �OH radical formation is prior generation of superoxide radicalthrough iron(II) auto-oxidation (Eq. 4). The self-dismutation ofsuperoxide would then lead to the formation of H2O2 (Eq. (5))and �OH would evolve from the resulting Fenton reaction (Eq. (1)):

Fe2þ þ O2 ! Fe3þ þ O��2 ð4Þ

2O��2 þ 2Hþ ! H2O2 þ O2 ð5Þ

The implications of superoxide and H2O2 on iron-promoted oxi-dation has been described by Qian and Buettner (1999). By meansof electron spin resonance (ESR), they have shown that the oxida-tion of dimethyl sulphoxide (DMSO), ethanol (EtOH) and glucose inaerobic solution treated with iron could be partially inhibited bythe addition of catalase and SOD. These results clearly demon-strated that H2O2 and superoxide were intermediate species inthe formation of oxidative species in aerobic systems in which ironis present. In the case of iron-induced beta-glucan degradation therole of superoxide and H2O2 in the formation of �OH has not yetbeen explored.

Parameters such as iron concentration may influence the �OHgeneration and further beta-glucan degradation. So far, no investi-gations about the effect of the iron(II) concentration on theAH2- and iron(II)-induced beta-glucan degradation have beenreported. However, studies on hyaluronic acid have shown thatthe iron(II)-induced degradation of the carbohydrate increaseswith increasing concentration of iron(II) (Wong, Halliwell,Richmond, & Skowroneck, 1981). Therefore, we have reasons tobelieve that the concentration of iron(II) might also influence theradical-mediated degradation of beta-glucan.

There is an obvious lack of information concerning the oxygenspecies involved in the formation of �OH that leads to beta-glucandegradation. Therefore, in the present study we have investigatedthe role of superoxide and H2O2 in the beta-glucan degradationpromoted by iron(II) and AH2/iron(II) in detail. In addition, we havestudied the influence of iron(II) concentration, which is potentiallyable to affect the radical-mediated degradation of beta-glucan. Theradical formation was monitored by the means of ESR spin trap-ping, and the beta-glucan degradation by rheology measurements.

2. Materials and methods

2.1. Materials

High viscosity barley beta-glucan (purity >97%) was purchasedfrom Megazyme (Ireland). The spin trap POBN (a-(4-Pyridyl N-oxide)-N-tert-butylnitrone; 99%) and the reference TEMPO (freeradical, sublimed, P99%) were from Sigma–Aldrich (St. Louis,MO, USA) and were stored at �20 �C. Type V�1000 IU/g, catalasefrom bovine liver (P10,000 units/mg protein); superoxide dismu-tase from bovine liver (P1500 units/mg protein), and iron(II)sulphate heptahydrate (FeSO4�7H2O) were bought from Sigma–Aldrich Chemie GmbH (Germany). Ascorbic acid (P99.5%) and eth-anol (puriss. p.a., ACS reagent, absolute alcohol, without additive,P99.8%), were purchased from Fluka (Germany). Hydrochloric acidand sodium hydroxide were obtained from Merck (Germany).

2.2. Preparation of the solutions

The solutions of high viscosity barley beta-glucan were pre-pared by adding 0.6 g of dry beta-glucan to Milli-Q water in a100 ml volumetric flask. The solution was heated in a water bathfor 3 h at 80 �C under continuous shaking. After complete dissolu-tion of beta-glucan, the pH of the solution was adjusted to 4.5 with1 mM HCl and the volume in the volumetric flask was adjusted to100 ml. Stock solutions of the spin trap POBN (4 M) and the re-agents AH2 (10 mM), iron(II) (2 and 40 mM) were prepared inMilli-Q water immediately before sample preparation.

2.3. Sample preparation

To study the effect of iron(II) concentration on the formation of�OH, the reagents (iron(II) with or without AH2) were applied to abeta-glucan solution at pH 4.5. This pH was selected since it allowsrapid formation of �OH in beta-glucan solutions treated withiron(II) with and without AH2 (Faure, Andersen, et al., 2012). Thefinal concentration of AH2 in the samples analysed was 250 lM.In the beta-glucan solution treated with both reagents (AH2 andiron(II)), the final concentrations of added AH2 and iron(II) were250 and 50 lM, respectively.

SOD and catalase were added as solutions and were alwaysmixed into the beta-glucan solution before addition of the iron(II)with and without AH2, to reach a final concentration of 300 and1000 U/ml, respectively. In all the samples with AH2 (final concen-tration 250 lM), AH2 was always mixed into the system last be-cause it initiates �OH formation. Each sample was analysed onthree different days in order to obtain three independent results.All samples were incubated in a dry incubator at 20 �C (Revolution-ary Science, Incufridge RS-IF-203).

2.4. Spin trapping

2.4.1. Samples with SOD, catalase or only iron(II)The initial �OH formation in beta-glucan solutions treated with

only iron(II), or containing SOD or catalase was very low. For thisreason a long incubation time of the spin trapping system was re-quired in order to reach an ESR-detectable concentration of spinadduct. In previous studies, we have reported that initial additionof POBN in combination with DMSO was suitable for measure-ments where formation of radicals was low, such as samples withonly iron(II), as the POBN–CH3 adduct formed is highly stable overtime (Faure, Andersen, et al., 2012). Thus POBN (80 mM) and DMSO(2%, v/v) were added to beta-glucan solution before the addition ofany reactants. The solution was then mixed for 1 min by vortex,followed by the addition of the reactants. Spectra of each sample

A.M. Faure et al. / Food Chemistry 141 (2013) 589–596 591

were recorded at different durations of incubation in order tocheck the evolution of the ESR signal.

2.4.2. Samples with ascorbic acid and iron(II)In samples with a combination of AH2 and iron(II), we expected

to see a higher rate of radical formation, and thus applied the peri-odic addition of spin trap POBN in conjunction with EtOH to studythe formation of radicals. Details for the method selection (initialaddition with DMSO vs. periodic addition with EtOH) are reportedin our previous study (Faure, Andersen, et al., 2012). In this proce-dure of periodic addition, a 200 ll aliquot sample of beta-glucansolution was drawn and transferred into 0.5 ml eppendorf tube,followed by 4 ll of POBN stock solution and 4 ll EtOH, in orderto reach final concentrations of 80 mM and 2% (v/v), respectively.The sample was mixed by vortex for 1 min, incubated for 1 h, afterwhich a spectrum was recorded.

2.5. Electron spin resonance measurements

The samples were loaded in 50 ll micropipettes (Brand GmbH,Wertheim, Germany) and the ESR spectra were recorded with aHigh Sensitive Benchtop EPR Spectrometer MiniScope MS300(Magnettech, Berlin, Germany) at room temperature. The settingsused were as follows: B0-field, 3350 G; sweep width, 100 G; sweeptime, 30 s; steps, 4096; number of passes, 4; modulation fre-quency, 1000 mG; microwave attenuation, 10 dB; receiver gain,900. The relative ESR signal corresponding to the content of spinadducts was obtained by calculating the ratio between the peak-to-peak-amplitude of the first doublet in the ESR signal of POBNadduct and the peak-to-peak-amplitude of the first singlet in theESR signal of a TEMPO solution (2 lM in H2O). TEMPO was usedas a standard and was measured in triplicate on each day ofmeasurements.

2.6. Rheology measurements

Viscosity properties of the samples were characterised with anAR-2000 rheometer (TA Instruments, New Castle, DE, USA) withcomputer control, using a cone and plate geometry (40 mm, 2�)over a shear rate range of 20–2000 s�1. All the viscosity measure-ments were performed at +20 �C.

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Blank10 µM50 µM 100 µM 200 µM500 µM1000 µM

(a)

Fig. 1. (a) Intensity of the ESR signal of POBN–CH3 adduct in 0.6% beta-glucan solution coof storage. (b) The effect of the different Fe2+ concentrations on the viscosity loss (%) of 0.6significant difference (p < 0.05).

2.7. Statistics

Statistical analyses were performed with SPSS Statistics 17.0.Significant differences between samples were determined by usingthe analysis of variance and the multiple range tests. A confidencelevel of 95% was required for differentiation of samples.

3. Results

3.1. Effect of iron(II) concentration, catalase and SOD on the beta-glucan degradation induced by iron(II)

The level of �OH (demonstrated as the formation of POBN–CH(CH3)OH spin adducts) was studied until 7 days of storage inbeta-glucan solutions treated with different added iron(II) concen-trations (0, 10, 50, 100, 200, 500 and 1000 lM) under aerobic con-ditions. The ESR data showed that �OH formation increased with anincreasing concentration of iron(II) in the beta-glucan solution(Fig. 1a). For instance, after 7 days of incubation approximatelythree time more spin adducts were produced in the sample contain-ing 500 lM iron(II) than in the 50 lM iron(II) sample. Furthermore,in most of the samples (except the blank), the formation of �OH wassignificantly higher after 7 days of storage compared to 1 day(Fig. 1a). In order to confirm that the formation of radicals waslinked to beta-glucan degradation, viscosity measurements wereperformed parallel to ESR on beta-glucan solutions containing 0,50, 200, 500 and 1000 lM iron(II). After 24 h of storage, the viscos-ity loss of the blank and the beta-glucan solutions treated with dif-ferent iron(II) concentrations were not significantly different fromeach other. Moreover, the decrease in viscosity in all samples wasbelow 15%, even though spin adducts indicating radical formationwere detected in these samples by ESR (Fig. 1a and b). The low levelof �OH formed after 24 h of storage was most likely not sufficient tolead to a significant loss of viscosity in the beta-glucan solutions.However, after 7 days of storage, the viscosity loss of beta-glucansolutions treated with 500 or 1000 lM of iron(II) was significantlyhigher compared to the blank and the beta-glucan with 50 or200 lM iron(II) (Fig. 1b). Moreover, the viscosity loss of the solutionwith 200 lM iron(II) was significantly higher than the ones of theblank and the 50 lM iron(II) beta-glucan solution (Fig. 1b). The par-allel increase in the viscosity loss with increasing iron(II) concen-tration was clearly related to the increase in �OH formation withthe increased iron(II) concentration (Fig. 1a and b).

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osity

loss

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ntaining 2% DMSO, 80 mM POBN and different Fe2+ concentrations at different times% beta-glucan solution. Different letters within one time point denote a statistically

Table 1Effect of catalase and SOD on the remaining viscosity (%) of 0.6% beta-glucan solution treated with (a) 500 lM Fe2+ and (b) 250 lM ascorbic acid and 50 lM Fe2+ at different timesof storage. Different letters within one time point denote a statistically significant difference (p < 0.05).

(a) Remaining viscosity (%)

t Blank 500 lM Fe2+ 500 lM Fe2+ + SOD 500 lM Fe2+ + CAT

5 h 100 ± 1a 90 ± 5a 96 ± 2a 96 ± 5a

24 h 97 ± 4a 88 ± 1a 97 ± 4a 98 ± 5a

7 days 91 ± 3a 68 ± 2b 82 ± 9a 91 ± 1a

(b) Remaining viscosity (%)

t Blank 250 lM AH2 + 50 lM Fe2+ 250 lM AH2 + 50 lM Fe2+ + SOD 250 lM AH2 + 50 lM Fe2+ + CAT

2 h 99 ± 1a 45 ± 3b 96 ± 3a 97 ± 3a

24 h 100 ± 2a 14 ± 1b 83 ± 3c 100 ± 2a

7 days 94 ± 6a 8 ± 1b 42 ± 6c 82 ± 9a

AH2+50µMFe2+, 2h

AH2+50µMFe2+, 24h

AH2+50µMFe2+, 7 days

AH2+50µMFe2++SOD, 24h

AH2+50µMFe2++SOD, 7 days

592 A.M. Faure et al. / Food Chemistry 141 (2013) 589–596

SOD or catalase were added to a beta-glucan solution at pH 4.5treated with 500 lM iron(II), and the formation of spin adduct aswell as viscosity changes were monitored at 5 h, 24 h and 7 days.Catalase was able to stop the formation of �OH promoted by iron(II)for at least 7 days of incubation, since no ESR signal was detected.This was further confirmed by an absence of viscosity loss com-pared to the blank in the beta-glucan solution treated with cata-lase, and thereby to the inhibition of the beta-glucan degradation(Table 1a). SOD was observed to inhibit the formation of �OH forthe first 24 h, since no signal was observed on the ESR spectra at5 or 24 h (Fig. 2). However, after 7 days of incubation, the samplewith SOD exhibited an ESR signal, which indicated that �OH radi-cals were formed in this sample between 24 h and 7 days (Fig. 2).The height of this signal was significantly lower compared to thatof the control (beta-glucan with 500 lM iron(II)) (Fig. 2). Although,�OH radicals were formed in the beta-glucan solution with SOD, nosignificant differences between the remaining viscosity of the beta-glucan solution with SOD and the blank were observed (Table 1a).This is likely due to the fact that between 24 h and 7 days the levelof �OH formed in the solution with SOD was too low to cause beta-glucan degradation at a level that would lead to significant viscos-ity loss.

500µMFe2+, 5h

500µMFe2+, 24h

500µMFe2+, 7 days

500µMFe2++SOD, 7 days

Fig. 2. ESR spectra of 0.6% beta-glucan solution with 80 mM POBN and 2% DMSOunder different conditions. When ascorbic acid was present in the solution its finalconcentration was 250 lM.

3.2. Effect of added iron(II) concentration, catalase and SOD on thebeta-glucan degradation induced by an ascorbic acid/iron(II) system

We hypothesised that iron(II) concentration has an influence onthe level of �OH formed through the ascorbate-driven Fenton reac-tion. Therefore, we studied the effect of six different iron(II) con-centrations (0, 10, 50, 100, 200, 500 and 1000 lM) on the �OHformation in beta-glucan (pH 4.5) solutions treated with 250 lMAH2. In the solutions with added iron(II), the adduct formationreaches its maximal value during the first hour of incubation andthen either remains stable or decreases slightly up to 4 h (Fig. 3).In the sample treated only with AH2, the formation of adductswas slightly lower than in the other samples during the first hourof incubation, but increased progressively until 4 h (Fig. 3). For in-stance, at 4 h of storage the adduct concentration in the solutionwith AH2 was about two times higher than in the beta-glucan solu-tions with both reagents (AH2 and iron(II)). Thus, the addition of ir-on(II) together with AH2 in beta-glucan solution remarkablystimulated the formation of radicals.

Increasing the iron(II) concentration in the AH2 treated beta-glucan solution demonstrated that no significant increase in theradical formation was observed, meaning that AH2 was the limitingreactant in the formation of the radicals (Fig. 3). In all samples, therate of radicals formed decreased significantly between 4 and 24 h,and remained relatively stable over subsequent days (data notshown).

To confirm that the radical formation was related to beta-glu-can degradation, we studied the viscosity of beta-glucan solutionstreated with AH2 without and with added iron(II) (50, 200, 500 and1000 lM) over time. After 2 h of storage, the remaining viscosity ofthe beta-glucan solutions treated with AH2 and different iron(II)

A.M. Faure et al. / Food Chemistry 141 (2013) 589–596 593

concentrations were significantly lower (between 38% and 49%)compared to the beta-glucan solution with only AH2 (85%) (Ta-ble 2). These results correlated with the ESR data, where the lowest�OH formation was observed in the beta-glucan solution with onlyAH2 during the first hour of storage. Thus, the addition of iron(II)with AH2 accelerates the process of radical-mediated beta-glucandegradation. Moreover, increasing the concentration of iron(II)added into the beta-glucan solution treated with AH2 did not leadto an observable significant decrease in the viscosity (Table 2). Thiscorresponds with the ESR data showing that in the AH2 containingsamples, the formation of radicals was not dependent on the addediron(II) concentration (Fig. 3). After 24 h storage, the remaining vis-cosity of the samples containing AH2 was between 14% and 16%and did not significantly differ between samples (Table 2). Further-more, after 7 days of storage, less than 11% of the original viscositywas observed without significant differences between samplescontaining different iron(II) additions (Table 2).

In the present study, we also investigated whether the ascor-bate-induced degradation of beta-glucan in solution can be pre-vented by SOD and catalase. Thus, SOD or catalase were added toa beta-glucan solution at pH 4.5 treated with AH2 and iron(II),and the formation of radicals and the viscosity changes of the solu-tion were investigated. The present results show that catalaseinhibited the formation of radicals and beta-glucan viscosity loss(such as in samples where iron was added alone). In fact, thebeta-glucan solution treated with the AH2, iron(II) and catalasenever exhibited an ESR signal, and its remaining viscosity neversignificantly deviated from the one of the blank (Table 1b). Onthe other hand, the presence of SOD in beta-glucan solution treatedwith AH2 and iron(II) inhibited formation of �OH and the viscosityloss of the beta-glucan solution until 2 h. For instance after 2 h, thepositive control (beta-glucan treated with AH2 and iron(II)) had aremaining viscosity of 45%, while the remaining viscosity of thesame solution with SOD was 96% (Table 1b). After 24 h, the beta-glucan solution treated with the AH2/iron(II) system and SODexhibited an ESR signal of low intensity, which indicates formationof �OH (Fig. 2). This production of �OH was associated with a signif-icantly lower remaining viscosity of the sample with SOD (83%)compared to that of the blank (100%) after one day of storage(Table 1b). After seven days, the ESR signal had increased in inten-sity, and the rate of remaining viscosity of the beta-glucan solutionhad significantly decreased to 42% of the original (Fig. 2; Table 1b).Even though SOD could not completely stop the formation of �OH,and thereby beta-glucan degradation, the fact that this enzymewas capable of significantly inhibiting this process shows thatsuperoxide is an intermediate species in the AH2 driven degrada-tion of beta-glucan.

4. Discussion

4.1. Beta-glucan degradation promoted by iron(II)

The rate of radical formation was found to be greater with in-creased iron(II) concentration, and this corresponded well withbeta-glucan degradation (monitored as decreased viscosity)(Fig. 1). These results supported our hypothesis, as �OH radicalsare believed to rise from iron(II) autoxidation, with ascendingiron(II) concentration the supply of �OH increases. At the same time,these findings were in good agreement with those reported byWong et al. (1981), who showed that an increase of iron(II) concen-tration (between 10 and 500 lM) led to an increase in hyaluronicacid degradation. At all iron(II) concentrations tested, the formationof �OH in beta-glucan was sustained for one week of storage.Regarding the fact that the autoxidation of iron(II) promotes forma-tion of �OH, we assume that iron(II) was present at least in a low

concentration in the system for at least one week. However, iron(II)is known to oxidise rapidly at mildly acidic and physiological pH.According to Morgan et al. the oxidation rate of iron(II) is lower atpH 4.5 than at higher pH such as 6.5 and 7.5 (Morgan & Lahav,2007). Nevertheless, with the low iron(II) concentration used here(14 mg/l) compared to the one used by Morgan and Lahav(100 mg/l), it is likely that within 5 h all the iron(II) was oxidisedin the beta-glucan solution with pH 4.5. Moreover, this pH condi-tion is close to the optimal pH of superoxide self-dismutation;4.8, and thus we suppose that the fast dismutation of superoxidecompensates for the lower rate of iron(II) autoxidation.

As we assumed that in the beta-glucan solution pH 4.5 theadded iron(II) autoxidised rapidly, the generation of �OH shouldhave been interrupted relatively early on. However, the presenceof a reducing agent (such AH2) in the system may have regeneratediron(II) from iron(III) and sustained formation of �OH over time.Reducing sugar such as glucose can reduce weak oxidising agentslike iron(III). Further polysaccharides of D-glucose monomer, suchas cellulose or beta-glucan, possess a reducing end due to the car-bon 1 of the last glucose unit, which is not involved in a glycosidicbond. As the formation of �OH was maintained in our system up to1 week, we hypothesise that, due to its reducing ends, beta-glucanwas able to reduce iron(III) to iron(II). As a result of the radical-mediated degradation of beta-glucan, smaller fragments of thepolysaccharide were released over time, which consequently, in-creased the number of reducing ends in the solution and thereforethe reducing potential. Thus at an adequate pH (around 4.5), thereaction of beta-glucan degradation induced by iron(II) continued,due to the regeneration of iron(II).

The implications of the results reported here are highly relevantfor the use of beta-glucan as a functional ingredient in aqueousfood systems. In fact, because iron is present in most foods andthe pH of beverages such as milk or juice fruit is above or closeto 4.5, the conditions to form �OH and promote beta-glucan degra-dation are present. Consequently, the oxidative degradation ofbeta-glucan must be controlled when added into aqueous food sys-tems. However, one can control the oxidative degradation by themeans of radical scavengers and antioxidative enzymes (Faure,Münger, et al., 2012).

The effects of antioxidant enzymes, namely SOD and catalase,were studied in beta-glucan treated with 500 lM iron(II). Theaddition of superoxide dismutase and catalase to remove superox-ide and H2O2, respectively, succeeded to inhibit formation of spinadducts indicating that no or low amounts of �OH were produced.SOD was able to prevent the formation of �OH until 24 h, but afterthis time radicals were formed in the beta-glucan solutions(Table 1a). We assume that this formation of radicals after 24 his due to a loss of enzyme activity starting at 6 h of incubation atroom temperature. Although radicals were formed in the beta-glu-can solution with SOD after 24 h, no significant viscosity loss of thesolution compared to the blank was observed after 7 days of incu-bation. Consequently, the amount of �OH generated between 24 hand 7 days was insufficient to cause significant damage to thepolysaccharide. As the addition of catalase inhibited the formationof �OH and subsequently beta-glucan degradation (no viscosity lossof the beta-glucan solution), it is evident that H2O2 participates ingeneration of the �OH responsible for the polysaccharide degrada-tion. These results indicate that formation of both superoxideand H2O2 are essential for the formation of �OH in beta-glucan solu-tions containing iron(II). This is consistent with the suggestion thatiron(II) autoxidation promotes �OH formation, and further beta-glucan degradation (Eqs. (4), (5) and (1)).

Our observation that SOD prevents radical formation and beta-glucan degradation was inconsistent with some previous studiesdealing with the degradation of carbohydrates promoted by iro-n(II) salt. For instance, Halliwell and Gutteridge (1981) showed

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Day 1, 24h

Fig. 3. Intensity of the ESR signal of POBN–CH(CH3)OH adduct (periodic addition) in 0.6% beta-glucan solutions containing 2% EtOH, 80 mM POBN, 250 lM ascorbic acid anddifferent Fe2+ concentrations and at different time of storage. In periodic addition the time indicated is the time of sampling. After the addition of POBN and EtOH, the samplewas incubated for 1 h to allow the formation of the spin adduct.

Table 2Effect of iron(II) concentration on the remaining viscosity (%) of 0.6% beta-glucansolution treated with 250 lM ascorbic acid. Different letters within one time pointdenote a statistically significant difference (p < 0.05).

t Blank 250 lM AH2

Fe2+ concentration

– 50 lM 200 lM 500 lM 1 mM

Remaining viscosity (%)2 h 99 ± 1a 85 ± 2b 49 ± 11c 45 ± 3c 40 ± 2.5c 38 ± 14c

24 h 100 ± 2a 14 ± 1b 16 ± 1b 16 ± 1b 16 ± 0.5b 16 ± 6b

7 days 94 ± 6a 10 ± 0.5b 9 ± 0.5b 8 ± 1b 9 ± 1b 10 ± 3b

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that SOD accelerated the degradation of deoxyribose promoted byiron(II) salt. Moreover, it was reported that the addition of SODstimulated the degradation of hyaluronic acid when treated withiron(II) (Wong et al., 1981). Indeed, SOD catalyses the dismutationof superoxide (Eq. (6)) and thereby increases the supply of H2O2,

which in turn boosts the generation of �OH. However, our resultswere consistent with the ones reported by Qian and Buettner(1999), who showed that SOD was able give some protectionagainst the oxidation of DMSO, EtOH, and glucose promoted by ir-on(II) salt. In the present study, it appears that H2O2 produced bythe action of SOD did not participate with iron(II) in the Fentonreaction to produce �OH. Here, we propose a sequence of reactionsby which SOD inhibits the �OH formation promoted by iron(II) salt.First, SOD would catalyse the dismutation of the superoxide result-ing from iron(II) autoxidation, which in turn leads to the formationof H2O2 (Eq. (6)). The iron(III) present in the solution (iron(II)autoxidation + possible contaminants from beta-glucan) may thenreact with H2O2 to give iron(II) and superoxide (Eq. (7)):

2O��2 þ 2Hþ �!SODH2O2 þ O2 ð6Þ

Fe3þ þH2O2 ! O��2 þ 2Hþ þ Fe2þ ð7Þ

According to Gutteridge (1985), the iron(II) resulting from reac-tion 7 will then autoxidise rapidly, before it can take part in theFenton reaction with H2O2, explaining the decrease in �OH forma-tion. The superoxide produced by reaction 7 would then again dis-

mute by the action of SOD (Eq. 6). Therefore, the describedsequence of events will proceed into a cycle of formation and dis-mutation of superoxide.

In the mechanism that we hypothesised, H2O2 reacts preferablywith iron(III) instead of iron(II). The reason for this may be due to aFe(III):Fe(II) concentration ratio favoring reaction 7. For example, aprevious study showed that SOD could reduce the formation of �OHpromoted by iron(II) salt, and the authors suggested that thisinhibitory effect from SOD was related to iron(III):iron(II) concen-tration ratio and/or metal redox cycle when the media is contam-inated with iron(III) (Urbanski & Beresewicz, 2000). Thebeta-glucan powder used in this study contains residual metalslike iron (12.27 ± 0.28 lg/g), and therefore we speculate that thepresence of iron(III) contaminant in our solution may have modi-fied the balance Fe(III):Fe(II) in a way that favored reaction (7). Thishypothesis accounts for our findings, but further work is needed tosupport this theory. Overall these results confirm that iron(II)autoxidation promotes beta-glucan degradation induced by iron(II)salt, and that both superoxide and H2O2 are involved in the forma-tion of �OH responsible for the beta-glucan degradation.

4.2. Beta-glucan degradation promoted by ascorbic acid and iron(II)

In the present study we also evaluated different iron(II) concen-trations to check whether iron(II) concentration influences theAH2/iron(II) induced beta-glucan degradation. The increase in theadded iron(II) concentration in beta-glucan solutions containingAH2 affected neither the radical formation nor the viscosity lossof the beta-glucan solution (Table 1). Even low level of residual ir-on(II) in the beta-glucan solution (sample blank, 1.32 ± 0.03 lM ofiron) was sufficient to promote �OH formation and thereby beta-glucan degradation. However, under this condition the degradationprocess was delayed compared to the sample with AH2 and addediron(II). A similar observation was reported in our earlier paper,where it was assumed that the iron contaminant is complexed tobeta-glucan and thereby more difficult to access by AH2, thusdelaying the radical formation (Faure, Andersen, et al., 2012). Here,we have demonstrated that AH2 is the limiting factor in the ascor-

A.M. Faure et al. / Food Chemistry 141 (2013) 589–596 595

bate-driven Fenton reaction since the iron(II) concentration has aminor effect on the ascorbate-induced degradation of beta-glucan.It appears that even residual iron is sufficient to initiate �OH forma-tion in the presence of AH2. These results are highly relevant forthe purpose of designing beta-glucan enriched aqueous food prod-ucts. In fact, because the coexistence of AH2 and iron(II) is verylikely in liquid food systems, incorporation of beta-glucan into thistype of food system may lead to its degradation at an early stage ofstorage. This effect of ascorbic acid on the degradation of polysac-charides was also demonstrated by both Kivelä et al. (2009) andWong et al. (1981), who showed that hyaluronic acid and beta-glu-can, respectively, were degraded in the presence of ascorbic acidand iron(II).

Results of the present paper show that �OH formation declinedsignificantly after 6 h in all the samples and remained constantover time (data not shown). This indicates that with time AH2 losespart of its pro-oxidative potential. Oxidation of AH2 leads to forma-tion of dehydroascobic acid, which is much less effective as areducing agent than AH2 (Fry, 1998).

Based on our previous study, catalase is able to stop generationof �OH (induced by a AH2/iron(II) system), which in turn inhibitsdegradation of beta-glucan (Faure, Münger, et al., 2012). In thesame study, we also demonstrated that SOD was capable of pre-venting formation of �OH in a beta-glucan solution treated withAH2 and iron(II) for up to 100 min of incubation. This suggests thatsuperoxide is involved in the formation of �OH promoted by anAH2/iron(II) system. To better understand the mechanism of thehydroxyl formation through the ascorbate-driven Fenton reaction,in the present study, we further investigated the activities of cata-lase and SOD in beta-glucan solution treated with AH2 and iron(II).The addition of SOD prevented the formation of �OH and therebybeta-glucan degradation up to 2 h, and reduced it significantlyfor up to 7 days. Catalase inhibited the �OH induced beta-glucandegradation until 7 days, which was in agreement with the resultsreported in our previous work. Thus, because SOD and catalasewere capable of protecting beta-glucan, it can be deduced thatsuperoxide and H2O2 occurred in the system and play a key rolein the ascorbate-driven generation of �OH responsible for thebeta-glucan degradation. Moreover, regarding the fact that SODprevents formation of �OH, it can also be deduced that superoxideis involved in the generation of H2O2 in the system as the �OH risefrom the reaction between H2O2 and iron(II). These results areinconsistent with the mechanism of ascorbate-induced �OH sug-gested by Fry (1998), who attributed the formation of H2O2 tothe reduction of dissolved O2 by AH2 (Eq. (3)). Moreover, he didnot observe any protection from SOD in the xyloglucan degrada-tion promoted by an ascorbate/iron(II) system. The discrepancy be-tween these two studies maybe due to the differences in thereaction system, such as different AH2 concentrations (250 lMvs. 1 mM) or different pH (4.5 vs. 5.5), which may affect the enzymeactivity (Fry, 1998).

Based on these results, we propose a chain of reactions for theascorbate-induced formation of �OH, in which superoxide areformed prior to H2O2. First, iron(II) undergoes a fast autoxidationto produce superoxide radicals and iron(III) (Eq. (4)). The resultingsuperoxide radicals will dismute into H2O2, which in turn reactwith iron(II) to produce �OH, which, finally, oxidise beta-glucan.AH2 in the mono-ionised ascorbate anion form reduces the iron(III)present in the media, thus providing the iron(II) necessary for theFenton reaction. The sequence of reactions may be more complexthan that described above, since it is likely that other reactionsare also involved in the formation of �OH. Nevertheless, the hypoth-esis accounts for all the findings made in this study.

An important finding of this study is that that SOD suppressedthe formation of �OH instead of enhancing it. Indeed, as mentioned,SOD should boost the generation of �OH by catalysing the dismuta-

tion of superoxide into H2O2, which would produce more �OH. Inthe present study, it appears that the Fenton reaction (Eq. (1))did not take place. We assume that the mechanism of inhibitionby SOD of the ascorbate-driven hydroxyl formation was the sameas described above in the presence of only iron(II). Therefore, theH2O2 formed in our system reacted preferably with iron(III) ratherthan iron(II) (Eq. (7). Gutteridge (1985) reported that SOD could in-hibit the degradation of deoxyribose in a H2O2/iron(III) system. Themechanism suggested was that first iron(III) would be reduced byH2O2, and the resulting iron(II) would auto-oxidise so rapidly thatit could not take part in the Fenton reaction with H2O2 to form �OH(Gutteridge, 1985). As explained previously, the reaction betweeniron(III) and H2O2 would be a favored by a specific iron(III):iron(II)concentration ratio and/or metal redox cycle when the media iscontaminated with iron(III).

In summary, our results demonstrate that the presence of iro-n(II) alone in beta-glucan solution is able to promote �OH formationand thereby beta-glucan degradation. The addition of AH2 withiron(II) in beta-glucan solution clearly accelerated these processes,and it was demonstrated that even a low amount of iron(II) wassufficient to initiate the oxidative cleavage of the polysaccharide.The most significant finding to emerge from this study is thatSOD and catalase were capable of preventing both types of degra-dation (iron(II)- or ascorbate-induced), which demonstrates theinvolvement of H2O2 and superoxide in the hydroxyl-radical med-iated degradation of beta-glucan. Moreover, it seems that the keyreaction of the mechanism leading to the formation of �OH in thetwo kinds of degradation is iron(II) autoxidation, which inducesthe formation of superoxide. Therefore, in order to stabilise beta-glucan in aqueous food systems, the oxidation of iron(II) must becontrolled.

Acknowledgement

We would to thank Prof. Raffaele Mezzenga (Laboratory of Foodand Soft Materials, ETH Zurich) for kindly allowing us to use therheometer.

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