Ultraflo xl

Gallic acid (GA), Folin–Ciocalteu (FC) reagent, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), fluorescein, 2,20-diazobis-(2-aminodinopropane)-dihydrochloride (AAPH), 2,20-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS^•+), 2,2-diphenyl-1-picrylhydrazyl (DPPH), iron(III) chloride hexahydrate (FeCl₃∙6H₂O), 2,4,6-tripyridyl-triazine (TPTZ), iron(II) sulfate heptahydrate (FeSO₄∙7H₂O), Gallic acid, apigenin, ferulic acid, avenanthramide C, p-coumaric acid and sinapic acid were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). Amyloglucosidase (EC 3.2.1.3) and glucose oxidase-peroxidase (GOPOD) were provided by Megazyme (Wicklow, Ireland). Glacial acetic acid, sodium acetate and chlorhydric acid were obtained from PanReac AppliChem (ITW Reagents, Darmstadt, Germany). The solvents were HPLC-grade (Sigma Aldrich Co., Madrid, Spain, and Merck KGaA, Darmstadt, Germany). Food-grade enzymes UltraFlo XL and Viscoferm were kindly provided by Novozymes (Bagsværd, Copenhagen, Denmark).

Enzymatic extractions were performed using seven commercial glucanases: Ultraflo XL, Ultraflo Max, Ultimase BWL 40, Viscozyme L, Celluclast 1.5L, Pectinex Ultra Tropical and Shearzyme Plus 2X (Novozymes, Bagsvaerd, Denmark). Enzymatic treatments (100 mL) were performed in water at a solid-to-solvent ratio of 1:20 (w:v) in accordance with previous studies [11 (link)]. Reaction mixtures consisted of a 1% enzyme-to-lentil hull ratio (w:w) according to the manufacturer’s recommendations and were processed at 40 °C for 3 h in a Thermomixer C shaker (Eppendorf Ibérica, Madrid, Spain) at 2000 rpm. The reaction with Pectinex^® Ultra Tropical was monitored at selected times (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h) by taking 2 mL aliquots. Enzymes were deactivated at 95 °C in a water bath for 5 min.

WB was kindly supplied by Emilio Esteban, S.A. (EMESA, Valladolid, Spain) and stored in plastic bags under vacuum until use. WB showed the following composition: 53.9 ± 3.3% of dietary fiber, 4.0 ± 0.5% of fat, 12.6 ± 0.2% of moisture, 13.1 ± 0.2% of protein, and 7.2 ± 0.0% of ash. Total dietary fiber was determined using the rapid integrated total dietary fiber assay procedure (Megazyme, Wicklow, Ireland). Moisture content was analyzed gravimetrically using drying treatment of the samples at 100 °C for 24 h. Fat content was measured using petroleum ether 40–60 °C for over 4 h of extraction and was gravimetrically determined. Nitrogen content was analyzed by the Dumas method, using a nitrogen analyzer (LECO Corp., St. Joseph, MI, USA). Ash content was determined by gravimetry following the Association of Official Agricultural Chemists (AOAC) method 923.03. The commercial food grade enzymes Depol 333MDP, Depol 40L, Depol 667P, Depol 670L, Depol 686L, Depol 740L, Depol 761P, Depol 793L, and Pectinase 62L were obtained from Biocatalyst Ltd. (Cardiff, Wales, United Kingodm). Shearzyme, Ultraflo XL, Viscoferm, and Viscozyme were acquired from Novozymes (Bagsværd, Copenhagen, Denmark). All chemicals of analytical grade were purchased from Sigma-Aldrich (Madrid, Spain), unless otherwise stated.

Hulls from an industrial de-hulling process that produces red football and split lentils were kindly provided by Prairie Pulse Inc. (Vanscoy, Saskatchewan, SK, Canada) in May 2019. Hulls were ground into a fine powder with a mixer mill (MM 400, Retsch, Haan, Germany) for approximately 3 min at maximum speed and stored in sealed plastic bags at 4 °C. Ultraflo XL, Ultraflo Max, Ultimase BWL 40, Viscozyme L, Celluclast 1.5 L, Pectinex^® Ultra Tropical and Shearzyme Plus 2X were obtained from Novozymes (Bagsvaerd, Denmark). Fast Blue BB (FBBB) [4-benzoylamino-2,5-dimethoxybenzenediazonium] chloride hemi-(zinc chloride), 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-diazobis-(2-aminodinopropane)-di-hydrochloride (AAPH) and fluorescein were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Standards such as (+)-catechin, trans-p-coumaric acid, quercetin 3-O-rutinoside, quercetin 3-O-glucoside and kaempferol 3-O-rutinoside were provided by Extrasynthese (Lyon, Genay Cedex, France). Standards of 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), D-glucose, glycerol and gallic acid were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA).

Fluorescein, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,20-diazobis-(2-aminodinopropane)-dihydrochloride (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS^•+), Folin–Ciocalteu (FC) reagent, gallic acid (GA), iron (III) chloride hexahydrate (FeCl₃∙6H₂O), iron (II) sulfate heptahydrate (FeSO₄∙7H₂O), 2,4,6-tripyridyl-triazine (TPTZ), ferulic acid, hidroxybenzoic acid and p-coumaric acid were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). Glucose oxidase-peroxidase (GOPOD) was supplied by Megazyme (Wicklow, Ireland). Sodium acetate, hydrochloric acid, glacial acetic acid and formic acid were purchased from PanReac AppliChem (ITW Reagents, Darmstadt, Germany). Different solvents were HPLC grade (Merck KGaA, Darmstadt, Germany, and Sigma Aldrich Co., Madrid, Spain). Viscoferm and UltraFlo XL food-grade enzymes were kindly supplied by Novozymes (Bagsværd, Copenhagen, Denmark).

Enzymatic hydrolysis was performed following the method of Martín-Diana et al. [30 (link)], with some modifications. OH was resuspended in water (1:20 w/v). The solution was submitted at high hydrostatic pressure (HHP, 6 × 108 Pa, 5 min) using an HHP unit (Wave 6000/135, NC Hyperbaric, Burgos, Spain) with a vessel of 135 L and 200 mm diameter. After the batch was treated using a hydrothermal machine performed at 121 °C, 1.2 × 10⁵ Pa for 15 min using Ilpra Plus 100 autoclave equipment (Ilpra Systems, Barcelona, Spain). Subsequently, 1.5 M malic acid was used to adjust the pH to 5 prior to enzyme incorporation. Immediately thereafter, one part of batch was incubated with Novozymes food grade UltraFlo XL and in the other batch with Viscoferm, both at 1% (enzyme to OH dry weight ratio, w:w), and enzymatic hydrolysis was performed at 47 °C for 20 h using a temperature-controlled water bath with magnetic stirring at 1000 rpm (Unitronic Vaivén C, Selecta S. A., Spain), resulting in enzymatic hydrolysates EH1-OH and EH2-OH, with UltraFlo XL and Viscoferm, respectively. At the end of the incubation period, enzymes were inactivated in a water bath at 95 °C for 10 min. Insoluble residues were removed by filtration using a nylon filter (200 μm-mesh). Finally, OH soluble fractions were filtrated and stored at 4 °C and immediately analyzed.