2.2. Analyses and tests

Base flours (wheat, brown rice, polished rice and bean), as well as the crumbs obtained from crushing baked biscuits of each formulation, were tested for their physical and chemical parameters, with regard to contents of moisture, according to the gravimetric method (AOAC, 2000); ash, according to method 08–12.01 (AACC, 2010); protein, through method 46–13.01 (AACC, 2010) with a conversion factor of 5.7 for whole wheat products, 6.25 for white bean flour, and of 5.95 for any rice products; total fat, through the Bligh and Dyer (1959) method; dietary fiber, according to method 991.43 (AOAC, 2000); and carbohydrates by difference. All those experiments were performed in triplicates for each food matrix and formulation.

The color of each flour and biscuits (crushed to flour) was assessed using the Hunterlab ColorQuest II colorimeter (Reston, Virginia, USA). The device was set at a standard Illuminant D65 (representing the typical spectral power distribution of the midday light) at 10° (field of vision) standard observer. Samples were placed in a Petri dish to measure the color in the quadruplicate surface. CIEL∗a∗b∗ scales were used and the measured color parameters were: L∗ = Lightness (0 = black, 100 = white), +a∗ = red, and -a∗ = green; +b ∗ = yellow, and -b∗ = blue (Hunterlab, 1996). This analysis was performed in triplicates for each food matrix. Calculations for Δ used subtraction between results of color results of every sample and any other sample, in all the possible paired combinations, and ΔE used the formula: ΔE∗=((ΔL∗)²+(Δa∗)²+(Δb∗)²)1/2 as described by Moritz (2011).

The viscoamilographic behavior of flours was assessed using ICC method 162 (1996), in RVA (Rapid Visco Analyzer) viscometer, model RVA 4500 (Warriewood, Australia) and the curves were analyzed by the software TCW 3.15.1.255. The crackers samples were previously crushed and homogenized to flour in the blender, model OBL 10/2 (OXY, Santana de Parnaíba, BRA), to 25000 rpm by 1 min. In the sequence, the flours were passed through sieves of 60 (250 μm) and 80 (177 μm) mesh to obtain a more refined flour. Four replicates for each sample was analysed, having parameters results of pasting properties including peak viscosity, trough, breakdown viscosity, final viscosity and setback, pasting temperature and peak time, registered in RVU (Rapid Viscosity Units).

Weights of biscuits were measured with an analytical scale right before and after the baking process. The area was calculated multiplying the diagonal length by diagonal width of each biscuit sample and dividing by two. Thickness, the biscuit height, was ascertained with a caliper. All those measurements were done in triplicate.

Using a texture analyzer (TA-XT2i, Stable Micro Systems, Surrey, UK) with the option of measuring hardness, a force in compression registering a maximum peak, and distance, the linear distance was taken as an indication of fracturability, were measured with 12 replicates for each formulation. The test conditions were: pre-test speed at 1 mm/s, test speed at 1 mm/s and post-test speed at 10 mm/s, and the penetration distance was 3mm.

For shelf-life analysis purposes, the biscuits were stored in closed bags inside hermetically sealed polypropylene jars for the same instrumental texture measurements after about 3 h, 7, 14, 30 and 60 days, at room temperature (25 °C).

This experimental study was approved by the Research Ethics Committee of the State University of Campinas, under CAAE No.: (65581517.8.0000.5404), and was designed in a way that 120 participants could try the acceptance of six biscuit samples (five made of rice and beans and one made of wheat).

The 120 subjects [51 men, 69 women, average age (M) = 25.08 years, standard deviation (DP) = 9.43] filed a sensory evaluation sheet, while they tried and judged each corresponding sample, using a 9-point hedonic scale (from 1-dislike extremely to 9-like extremely) to evaluate attributes such as appearance, aroma, taste, texture and overall impression (Stone and Sidel, 1993). In the same sheet, a field would question about the consumer's purchasing behavior, using a five-point purchase intent scale (Meilgaard et al., 1999) for each sample.

The test was applied in a laboratory room (see Figure 2) with 15 cabins where participants could find: 6 biscuit samples displayed in complete balanced blocks (MacFie, 1989), to be managed by the participants themselves, served at room temperature over a plate lined with white paper napkin, a glass of filtered water (200 mL), a sensory evaluation sheet, and a pen. Participants were requested to drink water between each sample to minimize the first-order carry-over effect when a sample assessment influences the evaluation of the next sample (Walkeling and MacFie, 1995). Participants took approximately 10–20 min to conclude the test, with their notes.

Graphic representation of the RVA analysis results for the biscuits' formulations. (cP - centipoise unit, a dynamic viscosity measure). B1: WWF (Whole wheat flour as primary ingredient). B2: WBF + BRF (White beans flour + Brown rice flour as primary ingredients). B3: CB + PRF (Cooked beans + Polished rice flour as primary ingredients). B4: CB + BRF + PRF (Cooked beans + Brown rice flour + Polished rice flour, as primary ingredients). B5: WBF + BRF + CPR (White beans flour + Brown rice flour + Cooked polished rice, as primary ingredients). B6: CB + BRF + CPR (Cooked beans + Brown rice flour + Cooked polished rice, as primary ingredients).

The statistical analysis was carried out using the SAS software system version 9.4 (SAS Institute Inc., Cary, NC, USA). Physicochemical and sensory results were evaluated using a univariate statistical analysis – one-way variance analysis (ANOVA), to ascertain the effects of the sample and the taster; as well as Tukey's test to run multiple comparisons of the averages of the participants by attribute in each sample, in order to ascertain which samples would diverge from each other at a 5% significance level.