Total dietary fiber assay kit

Dietary fiber was determined through a combination of enzymatic and gravimetric methods. Dietary fiber content was determined using the total dietary fiber assay kit (Sigma‐Aldrich Co., USA) based on the AOAC 985.26 method. Defatted samples were weighed into beakers (0.5 g) and gelatinized using α‐amylase. Then, they were enzymatically digested with 5 mg of protease and 0.1 mL of amyloglucosidase to remove the protein and starch present in the sample. Ethanol (200 mL) was then added to precipitate the soluble dietary fiber. The residue was then filtered and washed with ethanol and acetone. After drying in a vacuum oven at 70°C, the residue was weighed. Half of the samples were analyzed for protein and the others were ashed. Total dietary fiber is the weight of the residue minus the weight of the protein and ash.

Total nitrogen concentrations were measured in a 500 mg sample of cooked beans by the Dumas combustion method at A&L Great Lakes Laboratories (Fort Wayne, IN, USA) in accordance with AOAC method 968.06 [34 ]. The percentage of crude protein was estimated by multiplying the dry weight total nitrogen concentration by a factor of 6.25 [35 ]. Insoluble, soluble and total fiber concentrations were determined by the enzymatic-gravimetric AOAC method 985.29 [36 ], using enzymatic hydrolysis with heat-resistant amylase, protease, and amyloglucosidase (Total Dietary Fiber Assay Kit, Sigma Aldrich Co., St. Louis, MO, USA).

The samples of jussara before and after the fermentation process were evaluated in triplicate according to the Association of Official Agricultural Chemists methodology (AOAC, 2005 ). The fat contents were determined by the Rose–Gottlieb method. Total solids contents were determined by drying the sample in a vacuum oven at 70°C for 24 h. Protein analyzes were performed based on the determination of nitrogen by the micro-Kjeldahl method. The protein content was calculated by multiplying the nitrogen value by 6.25. The incineration of the sample determined the ashes in muffle at 550°C. The carbohydrate content was calculated by the difference between the total solids and the sum of the fat, protein, moisture, and ash contents. The enzymatic–gravimetric method 991.43 (AOAC, 2005 ) was used for fiber determination, which was performed in quadruplicate and using Sigma-Aldrich reagents and Total Dietary Fiber Assay Kit (Megazyme brand). The pH values were measured using a digital potentiometer.

Kjeldahl tablets (without addition of Se and Hg) were obtained from Merck (Darmstadt, Germany). The standards of chlorogenic acids (5-, 4-, and 3-caffeoylquinic acids), caffeine, 5-hydroxymethylfurfural, Folin–Ciocalteu reagent, sodium nitrite, epicatechin, ferrous sulfate heptahydrate, and trolox, as well as 2,2 diphenyl-1-picrylhydrazyl radical (DPPH^•), ferric chloride, aluminum chloride, 2,4,6 tripyridyl-S-triazine (TPTZ), sodium acetate, and total dietary fiber assay kit were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Absolute ethanol (≥99.8%) and HPLC-grade methanol were obtained from Honeywell Riedel-de Haën (Seelze, Germany). Glacial acetic acid was from VWR Chemicals (Fontenay-sou-Bois, France). A Seralpur PRO 60 CN and Seradest LFM 20 water purification system was used to obtain ultrapure water. All other chemicals were of analytical grade from several suppliers: Panreac (Barcelona, Spain), Merck (Darmstadt, Germany), and Carlo Erba (Emmendingen, Germany).

Moisture content of CBS was determined using a variation of AOAC method 927.05 by drying for 18 h at 70 °C in vacuum oven. Total fat content of blended CBS was determined using a modified AOAC method 945.16. In this last method, Soxhlet extraction of CBS (1 g with 150 mL) was carried out using petroleum ether as solvent for 6 h. Ash content was measured by AOAC method 942.05, charring 2 g of blended CBS for 30 min, followed by incineration in a muffle furnace (5 h at 525 °C). Crude protein content was determined on 0.1 g of blended CBS by Dumas method [24 (link)] with a FP 628 analyser (LECO Corporation, St Joseph, MI, USA). Carbohydrate content (% dry weight) of blended CBS will be determined as: $$\mathbf{100}-\left(\mathit{crude} \mathit{fat}+\mathit{ash}+\mathit{crude} \mathit{protein}\right)$$
Dietary fiber content was determined on 1 g of blended CBS using the total dietary fiber assay kit (Sigma-Aldrich Co., St. Louis, MI, USA) and a modified AOAC method 985.29 [25 ], in which α-amylase, protease and amyloglucosidase activities were used to digest the starting material. The remaining fiber was precipitated with ethanol, washed with ethanol and acetone, filtered, and, once dry, its protein and ash content were determined and subtracted from the dry weight. All analyses were performed in triplicate.

Proteins and soluble and insoluble fibers were determined by the gravimetric-enzymatic method, AOAC International, using a total dietary fiber assay kit, Sigma^®, San Luis, MO, USA [55 ]. More specifically, the analysis of protein content was performed in three repetitions and determined by the micro-Kjeldhal method, while total dietary fiber was performed in two repetitions.