Arabinoxylan

Sodium acetate, hydrogen peroxide (H₂O₂), sodium hydroxide (NaOH), sodium borohydride (NaBH₄), iron(III) sulfate pentahydrate (Fe₂(SO₄)₃), and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Amylopectin was obtained from Carbosynth (Compton, UK). Amylose, xyloglucan, arabinoxylan, xylan, glucomannan, galactomannan, mannan, curdlan, galactan, β-glucan, arabinan, and lichenan were purchased from Megazyme (Bray, Ireland). Microcrystalline cellulose was purchased from ACROS Organics. Formic Acid (FA) was purchased from Fisher Scientific (Belgium, UK). Acetonitrile (HPLC grade) was purchased from Honeywell (Muskegon, MI). Nano-pure water was used for all experiments.

Cellulose (Avicel PH101), xylan (beechwood) and endo-(1,4)-β-xylanase derived from Trichoderma longibrachiatum are purchased from Sigma-Aldrich, USA. Arabinoxylan (wheat) is purchased from Megazyme, and lignocellulose (Bambusa bambos) is collected from local area. Sodium acetate anhydrous, glacial acetic acid, 3,5-dinitrosalysilic acid, phenol, potassium sodium tartrate tetrahydrate, and sodium sulfite are purchased from Merck. D-xylose, sodium hydroxide (pellets), benzoic acid, thiourea, potassium bromide, and borax are purchased from SRL, Mumbai, India, and ortho-toluidine is purchased from Loba Chemie, Mumbai, India. All the chemicals used in this study are of analytical grade.

Cellulosic composites were produced following the method described by Chanliaud et al. [23 (link)] and Mikkelsen et al. [24 ]. Briefly the Gluconacetobacter xylinus frozen strain ATCC 53524 (Manassas, VA, USA) was cultivated in Hestrin and Schramm medium at pH 5. To prepare 1% (w/v) xyloglucan solution, tamarind xyloglucan (Lot 100402, Megazyme International Ireland Ltd., County Wicklow, Ireland) was dissolved in deionised water at room temperature under sterilised conditions. To produce cellulose/xyloglucan composites, a 1% xyloglucan solution was mixed with double concentrated Hestrin and Schramm medium (1:1) before inoculation, leading to a final xyloglucan concentration of 0.5% w/v. Similar preparation method and concentrations were used for the cellulose/arabinoxylan composites (medium viscosity wheat arabinoxylan, Lot 40302b, Megazyme International Ireland Ltd., County Wicklow, Ireland). Composites were cultivated statically at 30°C for 72 hours in 40 mm diameter containers. After cultivation they were harvested and washed 6 times with ice-cold water under agitation at 100 rpm. Samples were stored in 0.02% NaN₃ solution and kept at 4°C until further analysis.

Myceliophthora thermophila M77 was grown in Erlenmeyer flasks on Mandels & Sternberg salts, 0.1% peptone containing SCBIN (natural sugar cane bagasse, milled at 200 μm particle size) as well as modified sugar cane bagasse versions such as SCBDL (delignified with sodium hydroxide), SCBSE (steam exploded), purified celluloses containing 0.5% of avicel and 0.5% carboxymethylcellulose (Sigma Aldrich, St Louis MO), purified hemicelluloses containing 0.2% of each; birchwood-, beechwood-, oat spelt-xylan, arabinan and arabinoxylan (Megazyme International, Wicklow, Ireland) and glucose (control).
Secreted proteins were collected after a 36 h cultivation period at 45 °C, 200 rpm supernatants cleared by centrifugation (5000×g), concentrated by ultra-filtration (10,000 MWCO, PES membrane, Vivaspin, Littleton USA), rinsed twice with 5 mL of sodium acetate buffer 50 mM pH 5 and the proteins were separated by SDS-PAGE (Weber and Osborn 1969 (link)).

Commercially available soluble apple pectin (classic CU-L 051/13, lot 01307706, Herbstreith & Fox KG, Pektin-Fabrik Neuenbürg, Germany), β-glucan (medium viscosity barley, lot 90802, Megazyme, Bray, Ireland) and arabinoxylan (medium viscosity wheat, lot 40302a, Megazyme, Bray, Ireland) have been previously analyzed within our research group and have been shown to function as a mimic for creating plant cell wall models^{45 (link)–47} . Soluble polysaccharides were fluorescently labelled with 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF) reactive dye. Briefly, 0.2 mM 5-DTAF was added to polysaccharide solutions in deionized water (10 mg/mL) and allowed to dissolve at room temperature before 10 mM Na₂SO₄ was added slowly over 2 minutes. To initiate the reaction, pH was raised to 10 using 10% (w/v) NaOH and monitored at room temperature. After two hours, the reaction was quenched by the addition of two volumes of ethanol/sodium acetate buffer mixture (1:2 ratio of 0.055 M sodium acetate (pH 5.4): ethanol). Unbound 5-DTAF was washed with sodium acetate: ethanol solution using a Buchner funnel with a 0.45 μM PTFE membrane under vacuum until the filtrate appeared clear. Fluorescently-labelled polysaccharides were then progressively dehydrated with increasing concentrations of ethanol before a final wash with acetone and dried under vacuum overnight in the dark.

All foods and food products were purchased from local markets (Davis and Sacramento, CA, USA) including Safeway, Trader Joe’s, Davis Food Co-op, Whole Foods, Nugget Markets, Target, and online (Amazon). Trifluoroacetic acid (TFA, HPLC grade), 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP), chloroform (HPLC grade), ammonium hydroxide solution (NH₄OH) (28–30%), ammonium acetate, sodium acetate, glacial acetic acid, methanol (HPLC grade), D-fructose, D-mannose, D-allose, D-glucose, D-galactose, L-rhamnose, L-fucose, D-ribose, D-xylose, L-arabinose, N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), D-glucuronic acid (GlcA), and D-galacturonic acid (GalA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arabinoxylan and polygalacturonic acid were purchased from Megazyme (Bray, Ireland). 96-well Nunc plates and lids were purchased from Thermo Scientific. Viscozyme was provided by Novozyme (Davis, CA, USA). Acetonitrile (ACN) (HPLC grade) was purchased from Honeywell (Muskegon, MI, USA). Nanopure water was used for all experiments.

The fruiting bodies of D. indusiata were purchased from a local market in Ya’an, China. The sample was dried at 50 °C for 2 days and then milled into flour. Subsequently, the flour was passed through a 60-mesh screen and stored at −20 °C for further analysis.
Aniline blue, pectin (galacturonic acid ≥74.0%), chitosan, glucose, soluble starch, dextran (molecular weight, 64,000–76,000 Da), fucoidan, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carboxymethyl cellulose (CM-cellulose), β-glucan from barley (purity, ≥95%), arabinoxylan (purity, ~90%), inulin (purity, ≥95%), glucomannan (purity, ≥98%), pectic galactan, and curdlan (1,3-β-glucan; purity, >95%) were obtained from Megazyme (Wicklow, Ireland). All other reagents and chemicals used were of analytical grade.