Evaluation of glycosidic bond cleavage and formation ...

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Andrea Iurlaro a,b, Giuseppe Dalessandro a, Gabriella Piro a, Janice G. Miller b ..... a b c. Glc. Cellobiose. Man. All. Gal. Origin. Orange G ii). Orange G. Origin iii).

Food Research International 66 (2014) 115–122

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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 b

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento, via prov.le Lecce-Monteroni, 73100 Lecce, Italy The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, King's Buildings, Edinburgh EH9 3JH, UK

a r t i c l e

i n f o

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

a b s t r a c t This study investigated the formation of oxo groups in pure solutions of barley mixed-linkage (1→3),(1→4)-βD-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 NaB3H4-reducible functions into MLG chains. Driselase or lichenase digestion of NaB3H4-reduced MLGs released a complex mixture of 3H-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 3H-aldoses (mainly glucose, mannose, galactose and allose) deriving from random •OH attack at positions 2, 3 or 4 to form glycosulose residues which were NaB3H4-reducible to epimeric mixtures of 3H-aldose residues. Furthermore, the production of [3H]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, cardiovascular 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 homopolymers consisting predominantly of β-(1,4)-linked glucose oligomers (3–4 residues), separated by single β-(1,3) bonds (Fig. 1). Less frequent Abbreviations: A, dehydroascorbic acid; AH2, 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, thinlayer chromatography. ⁎ Corresponding author. Tel.: +39 0832 298612; fax: +39 0832 298858. E-mail addresses: [email protected] (A. Iurlaro), [email protected] (G. Dalessandro), [email protected] (G. Piro), [email protected] (J.G. Miller), [email protected] (S.C. Fry), [email protected] (M.S. Lenucci).

http://dx.doi.org/10.1016/j.foodres.2014.09.008 0963-9969/© 2014 Elsevier Ltd. All rights reserved.

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 speciesspecific 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

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Fig. 1. Chemical structure of mixed linkage (1→3),(1→4)-β-D-glucans.

been recently recommended by the Food and Drug Administration and the European Food Safety Authority to have beneficial health effects (EFSA, 2011; FDA, 2006; Motilva et al., 2014). The great potential of MLGs as an ingredient for functional food preparation is related to their ability to form thick viscous solutions within the stomach and small intestine (Wood, 2010), which in turn is strongly related to their water solubility and concentration, high molecular weight and conformation. When ingested with food, MLGs act as soluble dietary fibre; they swell with water during digestion, increasing bolus viscosity and thus diminishing the absorption rate of the digested nutrients (e.g. glucose and cholesterol) from the small intestine by resisting the convective effects of intestinal contractions (Dikeman & Fahey, 2006; Würsch & Pi-Sunyer, 1997). In the colon, MLGs are readily fermented into physiologically active by-products and thus act as a prebiotic on the gastrointestinal micro-flora (Mitsou, Panopoulou, Turunen, Spiliotis, & Kyriacou, 2010). It is well established that cell wall polysaccharides may undergo, either in vitro or in vivo, non-enzymic oxidative scission by the action of radical reactive oxygen species (ROS), especially the hydroxyl radical (•OH) generated by the Fenton reaction (Reaction 1) (Fry, 1998; Fry, Miller, & Dumville, 2002; Schopfer, 2001). Oxidative scission of MLGs has been reported following the addition of Fe2 + ions or ascorbic acid, or during thermal treatments and cold storage (Faure, Andersen, & Nyström, 2012; Faure, Werder, & Nyström, 2013; Fry, 1998; Kivelä, Gates, & Sontag-Strohm, 2009; Kivelä, Henniges, Sontag-Strohm, & Potthast, 2012; Kivelä, Nyström, Salovaara, & Sontag-Strohm, 2009; Kivelä, Sontag-Strohm, Loponen, Tuomainen, & Nyström, 2011; Mäkinen et al., 2012). Under these conditions, the viscosity of MLG solutions and their molecular weight were found to significantly decrease, with a much greater effect in the presence of endogenous co-extracted contaminants. MLG that has been exposed to •OH acquires mid-chain oxo groups, whose presence can be documented by reduction with NaB3H4 to form unusual 3H-aldose residues (Fry et al., 2002) or by the HPSEC/CCOA (carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide) labelling method (Kivelä et al., 2012). An alternative labelling method, recently developed for pectic and other polysaccharides, using N-isopropyl 2-aminoacridone (Vreeburg, Airianah, & Fry, in press), would also be applicable to MLG. Ascorbic acid is frequently added to food (including flour) to boost vitamin C content and improve product quality acting as antioxidant. However, its potential crucial role in lowering the physiological efficacy of MLG-fortified functional foods came to the attention of food technology

only in the past few years. Ascorbic acid (AH2) contributes to the Fenton reaction by (i) converting the metal catalysts to their reduced status (Reaction 2); (ii) non-enzymically reducing molecular oxygen to hydrogen peroxide (H2O2; Reaction 3); and (iii) stabilising H2O2 thus enabling and promoting •OH production (Wardman & Candeias, 1996). þ



Cu =Fe



þ H2 O2 →Cu





AH2 þ 2Cu =Fe



=Fe

þ

þ •OH þ OH



þ



→A þ H þ 2Cu =Fe

AH2 þ O2 →A þ H2 O2 :

ð1Þ

ð2Þ ð3Þ

The by-products of ascorbate in these reactions include dehydroascorbic acid (‘A’ in the equations above), which is itself subject to further non-enzymic reactions including oxidation to 3- and 4-O-oxalyl-L-threonates (Green & Fry, 2005; Parsons & Fry, 2012) and hydrolysis to diketogulonate and a compound tentatively identified as 2-carboxy-L-threo-pentonate (Parsons, Yasmin, & Fry, 2011), substances whose significance as dietary components has not yet been evaluated. Endogenous transition metals are commonly present in foods; in addition, MLGs are able to complex with metals (Platt & Clydesdale, 1984), allowing conditions to trigger Fenton reaction during MLG fortified food production and storage. The aim of this study was to investigate in vitro how Fenton reaction generated •OH contributes to 1) the chemical modification and degradation of barley MLGs using tritium labelling; 2) their molecular weight decrease. It also provides further documentation of a fingerprinting method, based on enzymic or acidic hydrolysis, opening the way to develop assays to detect radical-mediated MLG degradation during food processing and storage. 2. Materials and methods 2.1. Materials High-viscosity barley MLG and lichenase were purchased from Megazyme (Ltd., Bray, Ireland). Driselase, dextrans, D-glucose, D-allose, D-mannose, D-galactose, D-glucitol, D-cellobiose, Sepharose CL-6B, Whatman 20 and 3MM paper chromatography sheets were from Sigma-Aldrich Co. (St. Louis, Missouri, USA). TLC silica-gel 60 plates

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(plastic sheets 20 × 20 cm) were from Merck (Darmstadt, Germany). NaB3H4 (specific radioactivity 433 MBq/μmol) was from Perkin Elmer (NEN radiochemicals), catalogue ref NET023H100MC. Dialysis tubing (14000 MWCO) were purchased from Thermo Scientific (Rockford, IL, USA). OptiPhase “HiSafe” scintillation fluid was purchased from Wallac, Bucks, UK. Gold Star scintillant was supplied by Meridian (Epsom, Surrey, UK). Driselase (Sigma-Aldrich) was partially purified as described by Fry (2000). 2.2. Reduction of naturally occurring oxo groups and exposure of reduced MLGs to H2O2 or •OH MLG solution (0.5% w/v) was treated with 0.025 M NaOH for 2 h at 0 °C before being pre-treated with 0.062 M NaBH4 (in 0.025 M NaOH) (pH 12.0–13.0) at room temperature overnight, thus reducing any naturally occurring oxo groups (including reducing ends) in the MLG chains. Excess NaBH4 was destroyed with acetic acid until the pH reached 4.5. The sample was dialyzed overnight in constantly stirring cool water. Acetate buffer (50 mM final concentration, Na+, pH 4.5) was added to 1-ml aliquots of the dialyzed sample. The samples were incubated with respectively: (a) distilled water (control); (b) 10 mM H2O2; (c) 1 μM CuSO4/10 mM H2O2/10 mM ascorbic acid [to generate •OH by Fenton reaction (Tabbì, Fry, & Bonomo, 2001)]. In some experiments 4% dimethylsulphoxide (DMSO) was added to the Fenton reaction sample. The reactions were carried out at room temperature overnight (16 h). Reagents were removed by dialysis, and aliquots [containing approximately 0.2 mg of MLG, as estimated by the phenol–H2SO4 method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956)] were freeze-dried.

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or butanol/acetic acid/water (BAW, 12:3:5, by vol.) for 48 h (Piro, Congedo, Leucci, Lenucci, & Dalessandro, 2005). TLC was performed on silica-gel 60 plates in BAW (2:1:1 by vol.) for 7 h (with 2 ascents). For improved resolution of 3H-oligomers, TLC strips containing the 3H-spots were eluted with water as described by Eshdat and Mirelman (1972), vacuum-dried and developed in a different solvent system. PE was performed on Whatman 3MM paper, path length 45 cm, in 76 mM sodium molybdate (pH adjusted to 3.5 with acetic acid), 1.7 kV for 60 min (Franková & Fry, 2012) in the apparatus described by Fry (2011). The samples were loaded as spots or 2-cm streaks and cold-air dried. Glucose, allose, mannose, galactose, glucitol, cellobiose, [3H] laminaribiitol, [3H]cellobiitol and [3H]cellotriitol were loaded as authentic markers. Markers were detected by staining with aniline hydrogen-phthalate (reducing sugars), AgNO3 (non-reducing sugars), thymol or by fluorography (3H-labelled oligosaccharides) (Fry, 2000). 2.7. Detection of radioactivity Paper chromatograms, TLCs and electrophoretograms were dipped through 7% (w/v) 2,5-diphenyloxazole (PPO) in diethyl ether (Randerath, 1970) and exposed to pre-flashed Kodak Biomax MR films at − 80 °C for 3 or 4 weeks. Paper electrophoretograms were dried, cut into strips (30 × 10 mm), placed in 20-ml Packard vials containing 2 ml of Gold Star scintillant. Solutions were mixed with 10 vol. of OptiPhase “HiSafe” scintillation fluid. Radioactivity was assayed in an LS 6500 Beckman scintillation counter (Beckman Coulter Ltd, High Wycombe, UK). The counting efficiency was approximately 7% on paper and 33% in solution.

2.3. Radio-labelling of MLGs 2.8. Average molecular weight (Mw) determination 3

H-Labelling of MLGs was performed according to Fry, Dumville, and Miller (2001), and Fry et al. (2002). Briefly, freeze-dried MLGs (0.2 mg) were dissolved in 40 μl distilled water and 10 μl of 5 mM NaB3H4 (1 MBq) was added to each sample. The samples were incubated in sealed vials at 20 °C for 2 days. The remaining NaB3H4 was removed by addition of 2.5 μmol xylose and incubation for a further 24 h. The solution was acidified with 100 μl of 20% acetic acid, the volume was adjusted to 1 ml with water and the samples were freed of buffers, borate, xylose and [3H]xylitol by dialysis for 1–2 days in constantly stirring cool water. The samples were adjusted to 1.5 ml and 10 μl was assayed for total 3H-labelled polysaccharide. The remaining volume was divided into aliquots (containing ~50 μg MLGs), dried in a SpeedVac and re-dissolved in 30 μl water. 2.4. Enzymic digestion of MLGs A 50-μg aliquot of 3H-labelled MLG was digested by addition of Driselase (2 mg/ml) or 1 U/ml lichenase [both given as final concentrations; in pyridine/acetic acid/water 1:1:98 by vol., pH 4.7] at room temperature for 24 h. Digestion products were resolved by TLC or paper chromatography. 2.5. Acid hydrolysis of MLGs A 50-μg aliquot of 3H-labelled MLG was hydrolysed in 2 ml 2 M trifluoroacetic acid (TFA) at 120 °C for 1 h, SpeedVac-dried and re-dissolved in 20 μl H2O. Hydrolysis products were resolved by paper chromatography and paper electrophoresis. 2.6. Paper chromatography (PC), thin-layer chromatography (TLC) and paper electrophoresis (PE) PC was performed on Whatman No. 20 paper by the descending method in ethyl acetate/pyridine/water (8:2:1, by vol.; EPW) for 72 h

The average molecular weight of control and treated MLGs was estimated by gel-permeation chromatography (GPC) on a Sepharose CL-6B column (1.5 × 100 cm) in pyridine/acetic acid/water buffer (1:1:23 by vol., pH ~ 4.7). Molecular weight estimations were based on dextran standards of known molecular weight. The amount of carbohydrate in each fraction was estimated by the phenol–H2SO4 method (Dubois et al., 1956). 2.9. Statistical analysis Statistical analysis was based on a one-way ANOVA test. Tukey's post hoc method was applied for establishing significant differences between means (p b 0.05). All statistical comparisons were performed using SigmaStat version 11.0 software (Systat Software Inc., Chicago, IL). 3. Results 3.1. Non-enzymic oxidation of MLGs Fig. 2 reports the amount of radioactivity from NaB3H4 incorporated into MLGs pre-incubated in the presence of water (control), H2O2 or H2O2/ascorbate/CuSO4 (Fenton reaction) for 16 h. Although naturally occurring oxo groups had been preventatively pre-reduced with cold NaBH4, a low but detectable amount of radioactivity was incorporated into control MLGs indicating the de-novo formation of traces of new NaB3H4-reacting groups. An amount of NaB3H4-reacting groups considerably higher than expected only from the contribution of the reducing termini was reported for control and NaBH4-pre-treated xyloglucan and homogalacturonan (Fry et al., 2001), suggesting the formation of unknown, or nonspecific, interactions between polysaccharides and NaB3H4 or radioactive impurities present in the NaB3H4 solution. Although the presence of iron contaminants in the MLG powder (~ 12.3 ± 0.3 μg/g) has been reported (Faure, Andersen et al., 2012),

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Radioactivity (cpm x 106)

2.5

°

2.0 1.5 1.0 0.5 0.0

*

*

a

b

c

Fig. 2. Formation of new oxo groups in MLG by hydroxyl radicals. Pre-reduced non-radioactive MLG was incubated in (a) H2O, (b) 10 mM H2O2, or (c) •OH (generated by a Fenton reagent: 1 μM CuSO4, 10 mM H2O2 and 10 mM ascorbic acid), in each case acetate-buffered at pH 4.5, for 16 h. Newly formed oxo groups were reacted with NaB3H4 and radiolabel (counts per minute; cpm) incorporated into MLGs was measured by scintillation counting. Data, expressed as mean ± standard deviation, are representative of three independent replicates (n = 3). Data were submitted to one-way analysis of variance (ANOVA), bars marked with different symbols indicate significant differences among treatments (Holm–Sidak post-hoc test, p b 0.05).

H2O2 treatment did not induce a significant change in the number of NaB3H4-reacting groups, while H2O2/ascorbate/CuSO4 treatment increased the number of NaB3H4-reducible groups detectable in MLGs about 10-fold, supporting the idea that Fenton reaction-generated •OH are able to oxidise MLG chains. 3.2. Qualitative analysis of •OH-attacked MLGs To investigate the nature of radio-labelled residues, we digested the NaB3H4-reduced MLGs with Driselase (a mix of fungal glycanases and glycosidases, including cellulase, pectinase, β-xylanase and β-mannanase, able to convert hemicelluloses and pectic polysaccharides into mono- and disaccharides; Fry, 2000) or with lichenase [an endo-1,3(4)-β-glucanase from Bacillus sp. which specifically cleaves β-(1 → 4)-glucosidic linkages of the 3-O-substituted glucose residues in MLGs, resulting in oligosaccharides with degree of polymerisation (DP) typically 3 or 4 but with a proportion at DP 6 (Simmons et al., 2013)]. Among the Driselase-digestion products, no 3H-labelled monosaccharides were detected by fluorography of control (Fig. 3i, a) or H2O2treated (Fig. 3i, b) MLG. The absence of 3H-alditols (e.g. glucitol) confirmed that oxo groups normally occurring at the reducing terminus of a polysaccharide chain had been successfully reduced by the pretreatment with non-radioactive NaBH4, strengthening the hypothesis that the small amount of radioactivity detected in these two samples was the result of non-specific interactions between MLGs and NaB3H4. On paper chromatography in BAW, Driselase-digestion products of MLGs subjected to Fenton reaction (Fig. 3i, c) gave, on the contrary, three radioactive spots. The fastest (spot A) co-migrated with authentic markers of glucose and/or galactose and partially overlapped with glucitol, allose and mannose, which were not fully resolved; a faint, slower-migrating spot (B) overlapped with marker cellobiose and a chromatographically immobile third spot (C) probably containing large 3H-oligosaccharides. TLC (in BAW) of the Driselase-digestion products confirmed the presence of a 3H-disaccharide that comigrated with cellobiose and gave additional evidence of the presence of other 3H-oligosaccharides, but it did not definitively resolve monosaccharides (Fig. 3ii, c). These were better separated by paper chromatography in EPW, which showed that any free mannose and allose after extensive Driselase digestion of •OH-attacked MLGs were below the detection limit (Fig. 3iii, c). The presence of radio-labelled di- and oligosaccharides (Fig. 3ii, c) suggests that a consistent amount of 3H

was associated with unusual and not completely Driselase-digestible compounds in the polysaccharide chain. Lichenase digestion of NaB3H4-reduced •OH-attacked MLGs followed by TLC in BAW gave a complex pattern of radioactive spots, where the two most intense (spots A and B) approximately co-migrated respectively with cellobiitol and cellotriitol and probably comprised different di-, tri- and tetrasaccharides (Fig. 4i, c). In addition, other 3 H-oligosaccharides with DP 5–7 were detected. The presence of radioactive spots approximately co-migrating with cellobiitol and/or cellobiose, and cellotriitol and/or cellotriose, in lichenase-digested MLGs was also evidenced by paper chromatography in BAW of spot A + spot B (Fig. 4ii) eluted from the BAW TLC (Fig. 4i, c). After acid hydrolysis of NaB3 H4 -reduced MLGs, no radioactive spots were detectable in control (Fig. 5i, a) and H2 O 2-treated (Fig. 5i, b) samples by paper chromatography in BAW and EPW, while MLGs subjected to Fenton reaction gave a single spot co-migrating with monosaccharide standards on BAW (data not shown) and two spots on EPW (Fig. 5i, c): the slower of these (B) approximately co-migrated with authentic glucose, glucitol or galactose which were only partially resolved, the faster (A) with authentic allose or mannose. No radioactivity was associated with di- or oligomeric products, showing that TFA treatment gave a complete hydrolysis of •OH-attacked MLGs. Paper electrophoresis in molybdate buffer of the TFA hydrolysates (Fig. 5ii) confirmed the presence of radioactivity in corresponding with marker glucose, mannose and allose (the last two sugars overlapped) only in the •OH-treated MLGs and allowed to us identify the presence of a radioactive peak co-migrating with glucitol. The presence of [3H]glucitol was the evidence that •OH is able to cleave the polysaccharide chain, generating new reducing termini. A mechanism for this has been suggested (reaction d4 in Fig. 8 of Lindsay & Fry, 2007). The molar ratio of 3H-aldoses/[3H]glucitol was ~ 3:1, suggesting that for every 3 mid-chain oxo groups introduced, a new reducing terminus is created. 3.3. Molecular weight of •OH-attacked MLGs The approximate molecular weight of control and H2O2 and •OHtreated MLGs was estimated by gel-permeation chromatography. Control and H2O2-treated MLGs had an average molecular weight (Mw) between 2000 and 500 kDa (from a calibration graph prepared with dextran markers), while •OH-treated MLGs showed a Mw lower than 19 kDa (Fig. 6), symptomatic of very extensive intra-molecular cleavage of MLGs by ascorbate-induced oxygen radicals. The shift observed between the peak of control and H2O2-treated MLGs does not exclude the possibility of a slight degradation of the latter. To determine whether MLG degradation was due to Fenton reaction-generated •OH, we tested its susceptibility to inhibition by dimethylsulphoxide (DMSO). The k•OH (rate-constant for reaction with •OH) of DMSO is 6.6 × 109 M− 1 s− 1 (Buxton, Greenstock, Helman, & Ross, 1988), so it should rapidly react with •OH, acting as scavenger and inhibiting MLG cleavage. After treatment with Fenton reagents in the presence of ~ 0.5 M DMSO, the Mw of Fenton reactiontreated MLGs was ~ 42–282 kDa, significantly higher than in absence of the scavenger (b19 kDa) (Fig. 6), supporting the idea polysaccharide scission was due to •OH attack. The moderate effect of •OH radical even in the presence of DMSO may be partly due to the fraction of the radical formed in close proximity to the target hemicellulose chain that escapes scavenger action. The chance that Fenton reactions occur very close to polysaccharide chain is increased by the ability of MLGs to form complexes with metal ions (Platt & Clydesdale, 1984). 4. Discussion Fenton reaction-generated hydroxyl radicals introduce a range of NaB3H4-reducible functions into MLG chains. •OH attack at position 2, 3 or 4 will abstract a C-bonded H atom, leaving a carbon-centred

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119

i)

Man All Gal

Spot A

GlcH Orange G Glc Cellobiose

Spot B Spot C

Origin a

b

c

ii)

Orange G Man Glc Gal All Cellobiose

Origin a

b

c

iii) Man All GlcH Gal Glc

Orange G Origin a

b

c

Fig. 3. Radiochemical characterisation of •OH-attacked MLG: Driselase digestion products. MLG was treated with (a) H2O, (b) H2O2 or (c) •OH, and then radiolabelled, all as in Fig. 2. The radiolabelled polysaccharide was digested with Driselase, and products were analysed by (i) paper chromatography in BAW (12:3:5), (ii) TLC in BAW (2:1:1) and (iii) paper chromatography in EPW (8:2:1). Radioactivity was detected by fluorography (left); marker sugars were stained with AgNO3, thymol or aniline hydrogen-phthalate (right). All, allose; Gal, galactose; Glc, glucose; GlcH, glucitol; Man, mannose.

free radical which, in an aerated solution, will react with O2 to form a peroxyl radical (R–O2•). The latter may then eliminate a hydroperoxyl (equivalent to the superoxide anion) radical, leaving the sugar residue in the form of a glycosulose residue (i.e., carrying an oxo group on C-2, 3 or 4) (von Sonntag, 1980). Glycosulose residues can be reduced by NaB3H4 to yield [3H]glucose residues in addition to the corresponding 2-, 3- or 4-epimers, namely [3H]mannose, [3H]allose and [3H]galactose,

respectively, within the polysaccharide chain. Concurrently, •OH attack at C-5 would be expected to convert a glycosidic bond to a (more labile) ester bond (Fry et al., 2001), without generating any NaB3H4-reducible product. Attack at C-1 or at the carbon to which another glucose residue is linked (i.e. approximately 30% of C-3 positions and 70% of C-4 positions in barley MLG), may lead to polysaccharide cleavage producing in some cases a new reducing terminal glucose moiety

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i)

Orange G

Glc Laminaribiitol

Cellobiose G4G3G (dp3) G4G4G3G (dp4) dp5 dp6 dp7

Spot A

Cellobiitol Cellotriitol

Spot B

Origin a

b

c

d

ii) Orange G GlcH Glc

Laminaribiitol Cellobiose

Cellobiitol

G4G3G (dp3)

Cellotriitol Origin Spot A + Spot B

Fig. 4. Radiochemical characterisation of •OH-attacked MLG: lichenase digestion products. MLG was treated with (a) H2O, (b) H2O2 or (c) •OH, and then radiolabelled, all as in Fig. 2. The radiolabelled polysaccharide was digested with lichenase, and products were analysed by (i) TLC in BAW (2:1:1); radioactivity was detected by fluorography (left), and marker sugars were stained with thymol; lane (d) shows the lichenase-digestion products of non-radioactive MLG. Spots A and B were eluted from the TLC and separated by (ii) paper chromatography in BAW (12:3:5); markers were stained with AgNO3. Glc, glucose; GlcH, glucitol.

(Lindsay & Fry, 2007), which we would pick up as [3H]glucitol. Recently, two different scission pathways of the β-(1 → 3) glycosidic bond of barley MLGs, initiated by the presence of a radical at the anomeric carbon C-1, have been proposed, which lead to the release of a lactone in C-1 (Faure, Sánchez-Ferrer, Zabara, Andersen, & Nyström, 2014). Driselase digestion of NaB3H4-reduced •OH-attacked MLGs released a complex mixture of 3H-labelled mono-, di- and oligosaccharides. [3H] Glucose was the most abundant identified monosaccharide residue, followed by [3H]galactose, while free [3H]mannose and [3H]allose were not detected. The presence of significant amounts of [3H]mannose, in addition to [3H]glucose, [3H]galactose and several larger 3H-saccharides, among Driselase digestion products of MLGs subjected to Fenton reaction, was previously detected by scintillation counting (Fry et al., 2002). The undetectability of [3H]mannose in the present work (Fig. 3i–iii, c) may be due to the lower sensitivity of fluorography compared with scintillation counting. With regard to allose, Driselase probably lacks the activity that would be required to release it as a free monosaccharide. Driselase digestion products would therefore include Driselase-resistant 3H-disaccharides such as [3H]allosyl-glucose and other 3H-labelled low-Mw products. The labelling pattern was supported confirmed by acid hydrolysis of NaB3H4-reduced •OH-treated MLG. As expected, [3H]galactose and/or [3H]glucose, and [3H]mannose and/or [3H]allose residues were detected. Furthermore, [3H]glucitol was detected by electrophoresis, demonstrating that a proportion of the oxidative scission events involves the formation of new reducing termini, not only aldonic acid termini. Concurring conclusions were reached by Kivelä, Gates et al. (2009), Kivelä, Nyström et al. (2009)

and Mäkinen et al. (2012) to explain the viscosity loss and molecular degradation of pure MLG solutions, crude MLG extracts or oat MLGs in modelled beverage conditions. Transition metals (Fe, Cu, Zn) and ascorbic acid are typically present in plant-derived foods (Kivelä, Gates et al., 2009; Kivelä, Nyström et al., 2009; Slavin, Martini, Jacobs, & Marquart, 1999). Ascorbic acid is also commonly added during food processing as an anti-oxidant. In the presence of food-related ascorbic acid concentrations (10 mM), traces of Cu2+ (1 μM) and H2O2, MLG average molecular weight was reduced from N 500 kDa to less than 19 kDa, a molecular weight loss of N 96%. MLGs can create complexes with metal ions (Platt & Clydesdale, 1984) and the precise location of Cu2 + will tend to target •OH production, and thus also •OH action, at nearby sugar residues of the polysaccharide chain. Such targeting would explain why scavengers, like DMSO, even at concentrations of up to 0.5 M, are only partially able to inhibit •OHinduced scission of MLG. Again, it means that MLG degradation may occur also in food processing even in the presence of naturally occurring radical scavengers. In recent years several molecules with radical scavenging potential (including sugars, amino acids, organic acids and phenols), metal chelators or H2O2 or O2 removal agents have been tested to inhibit •OH-induced degradation of β-glucans (Faure, Münger, & Nyström, 2012; Kivelä, Nyström et al., 2009; Kivelä et al., 2012; Paquet, Turgeon, & Lemieux, 2010). Depending on the kind and, in most cases, on concentration of such agents added to β-glucan solutions subjected to Fenton reaction, different levels of protection were achieved. Catalase, 4-hydroxybenzoic acid, sucrose and phenylalanine, for example, strongly inhibited the net formation of •OH in the

A. Iurlaro et al. / Food Research International 66 (2014) 115–122

i)

All Spot A Man GlcH

Spot B

Gal Glc

Orange G Origin a

ii)

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Distance from origin (cm) Fig. 5. Radiochemical characterisation of •OH-attacked MLG: acid hydrolysis products. MLG was treated with (a) H2O, (b) H2O2 or (c) •OH, and then radiolabelled, all as in Fig. 2. The radiolabelled polysaccharide was hydrolysed with TFA, and products were analysed by (i) paper chromatography in EPW (8:2:1); radioactivity was detected by fluorography (left), and marker sugars were stained with AgNO3 (right). In (ii) an identical aliquot of the total products was analysed by paper electrophoresis in molybdate buffer at pH 3.5; radioactivity was quantified by scintillation counting. Marker sugars were stained with aniline hydrogen-phthalate or AgNO3. All, allose; BPb, bromophenol blue; DNP-Lys, dinitrophenol-lysine; Gal, galactose; Glc, glucose; GlcH, glucitol; Man, mannose; OG, orange G.

3.0

a b c d

Blue dextran 2000 kDa

2.5 2.0

A490

77 kDa

19 kDa

This research was supported by the following projects: PRIN-MIUR 2010–2011, Prot. 2010Z77XAX_002 and PON, progetto Pro.Ali.Fun. — PON02_00186_2937475, and the UK BBSRC project 15/D19626.

Cobalt chloride

1.0 0.5

generating system and significantly slowed down the viscosity decrease of β-glucan solutions during up to two weeks of storage; while phytic acid, citric acid and sulphite, despite being efficient in preventing •OH formation, were not able to avoid the dramatic decrease of β-glucan solution viscosity (Faure, Münger et al., 2012; Kivelä, Nyström et al., 2009). Furthermore, the addition of xanthan gum significantly reduced the viscosity loss but did not provide complete protection for β-glucan over time (Paquet et al., 2010). These findings corroborate the results obtained in the presence of DMSO. •OH-induced molecular weight decrease may affect the health properties of MLGs by interfering with their capacity to form a viscous solution. Low-Mw MLGs have a higher tendency for gel formation than molecules with high molecular weight (Lazaridou, Biliaderis, & Izydorczyk, 2003; Tosh, Wood, Wang, & Weisz, 2004). This has been explained by the higher mobility of the shorter chains (Doublier & Wood, 1995). Oat MLG with a molecular weight of approximately 50 kDa gelled in under 2 h in a 10% solution, while the gelation time was over 40 h for MLGs with molecular weight of approximately 150 kDa (Lazaridou et al., 2003). Furthermore, gels of low-Mw MLGs were weaker than those of high-Mw MLGs when determined by dynamic oscillation, but stronger and less brittle when determined by static compression (Kivelä et al., 2011). Normally MLGs in dilute aqueous solution occur as fringed micelletype aggregates, which grow side-to-side via hydrogen bonding of the cellotriose units (Böhm & Kulicke, 1999; Grimm, Krüger, & Burchard, 1995; Tosh et al., 2004). Formation of glycosulose (osone) residues due to •OH attack will induce changes in cellotriose units and this may affect the MLG's conformation and tendency to aggregate in water solution, altering water solubility. From a biological point of view, the capacity of •OH to cleave polysaccharides may allow it to play useful, highly localised, physiological roles if produced in the cell wall. The half-life of •OH in a biological milieu is ~ 1 ns, and the functional range is likely to be less than 1 nm before an •OH molecule reacts with a neighbouring organic molecule (Griffiths & Lunec, 1996; Halliwell & Gutteridge, 1999). •OH would be a site-specific agent of polysaccharide scission if generated in the cell wall, potentially playing an important role in wall-loosening during cell expansion and fruit ripening (Fry, 1998; Schopfer, 2001). The methodology discussed in the present manuscript is the starting point to develop an assay to detect chemical changes in MLG chains due to •OH radical attack in vivo or during food processing and storage based on the release of diagnostic sugars such as allose by chemical hydrolysis of partially purified reductively labelled MLGs or by selective digestion of the MLGs within the complex food matrix by the use of specific hydrolases. Acknowledgements

282 kDa

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References 42 kDa

500 kDa

60

50

55

45

40

35

30

25

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Fraction number Fig. 6. Size profiles of •OH-attacked MLG. MLG was treated with (a) H2O, (b) H2O2, or (c) •OH and then radiolabelled, all as in Fig. 2, then subjected to gel-permeation chromatography on Sepharose CL-6B. Profile (d): as in (c) but the Fenton reagent was supplemented with 4% dimethylsulphoxide (DMSO), a scavenger of hydroxyl radicals. Dextrans of 19–2000 kDa were run as markers. Carbohydrate in the eluate was monitored as A490 in the phenol–H2SO4 assay.

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