*2.3. Chemical Analysis*

The analysis of the chemical composition was performed using American Association of Cereal Chemists Approved Methods (AACC) and Association of Official Analytical Chemists International (AOAC) methods [43,44]. The moisture content was determined with the air-oven method (Method AACC 44-15A). The samples (3 g) were placed in a laboratory dryer and dried at 103 ◦C ± 1 ◦ C to constant weight. After cooling in an exsiccator, the samples were weighed and the moisture contents were calculated. The ash content was determined using AACC method 08–01. The samples were measured into ash dishes at 3 g. Then, the samples were placed in a muffle furnace at 550 ◦C. They were incinerated until light gray ash or constant weight was obtained (7 h). After cooling, the samples were weighed and the ash contents were calculated. To determine the total protein content, the Kjeldahl method (Method AACC 46-08) and the Kjeltec 2300 (FOSS, Höganäs, Sweden) apparatus were used. The protein content was calculated from the total nitrogen content using converted factor 5.7. The crude fat content was determined via continuous extraction. The SoxtecTM8000 on application AN 310 (FOSS, Höganäs, Sweden) and hexane as a solvent were used. The total dietary fiber (TDF) content, including insoluble dietary fiber (IDF) and soluble dietary fiber (SDF), according to the enzymatic methods (AACC 32-05, AACC 32-21, AOAC 991.43, and AOAC 985.29) was analyzed. Next, 1 g dried samples were subjected to sequential enzymatic digestion using heat-stable α-amylase, protease, and amyloglucosidase. Megazyme enzymes and analytical procedures were used (Megazyme International Ireland Ltd., Wicklow, Ireland). The digestible carbohydrate content was determined by calculating the difference (weight in grams (protein + fat + TDF + ash) in 100 g of dry matter of pasta or raw material).

#### *2.4. Physical Properties*

The quality parameters of semolina (i.e., wet gluten content, gluten elasticity, and gluten spreadability) were tested in accordance with the Polish Standard (PN-92/A-74021) [45]. The granulometric composition of durum semolina was determined with the sieve method using a laboratory sieve shaker (Sadkiewicz Instruments—Bydgoszcz) and a set of sieves with the following mesh sizes: 400, 315, 250, 160, 125, and 80 µm. Next, 100 g of samples was sieved for 10 min. The percentage share of each fraction in the semolina and the equivalent diameter (weighted average grain diameter in the sample) were calculated. The details of the method were described by Sobota et al. [35]. In the case of fine-grained raw materials (i.e., vital wheat gluten, xanthan gum, and oat β-glucans), the particle size was examined with microscopic image analysis using an optical microscope (×40 magnification) and DLT CAMViewer 3.7.4043 software. Raw material samples were prepared and the diameters of 70 randomly selected raw material particles were measured, and their average diameter was calculated. The water solubility index (WSI) and the water absorption index (WAI) were investigated according to the centrifuge method (AACC 56-20), with slight modification described by Sobota et al. [35]. Further, 2 g of samples were placed in centrifuge tubes and mixed with 30 mL of distilled water. The suspension was left to rest for 5 min then was centrifuged for 15 min at 2200× *g*. Next, 10 mL of supernatant was dried to the solid mass and the WSI was calculated using the Formula (1):

$$\text{WSI} \left( \% \right) = \text{(Weight of dried supernovaant)} \times \frac{30 \text{ mL}}{10 \text{ mL}} / \text{Dry weight of sample} \times 100\%. \tag{1}$$

After 10 mL of the supernatant was downloaded to determine the WSI, the remaining supernatant was carefully decanted. The wet samples were weighed and WAI was calculated using the Formula (2):

WAI (%) = ((Weight of wet sample − Weight of dry sample)/Dry weight of sample) × 100%. (2)

Apparent viscosity was tested in accordance with the method developed by Zarzycki and Sobota [46]. A rotary rheometer RM 180 (Mettler-Toledo AG, Switzerland, software RSI Orchestrator, ver. V6.5.8.) with coaxial cylinders without the bottom cylinder guard (shear rate of 1000 s−<sup>1</sup> ) was used. A 5% suspension in distilled water at 30 ◦C was prepared from ground pasta samples. The suspensions were held for 30 min at 30 ◦C with constant stirring. The suspensions (300 mL) were heated in a laboratory shaker (Elpin type 357, Elpin Plus s.c., Lubawa, Poland). The viscosity was measured at a temperature ranging from 65 to 95 ◦C; next, they were kept at 95 ◦C for 20 min, cooled to 50 ◦C, and kept for 30 min at this temperature. A constant temperature gradient of 1 ◦C min−<sup>1</sup> was maintained during the heating and cooling processes. When the viscosity measuring agitation was stopped, the cylinders were immersed in the suspension, and five consecutive readings were taken every 10 s. The measurements were made in 3 replications for each sample.

#### *2.5. Cooking Quality of Pasta Samples*

The optimal cooking time (OCT, min) was measured according to Method AACC 16–50 [43]. Next, 50 g of pasta was boiled in 500 mL of distilled water. Every 30 s, the pasta was removed and squeezed between two glass plates until the mealy core disappeared. The time needed for this process was assumed as the optimal cooking time (OCT). The weight increase index (WI) was calculated by dividing the weight of the pasta sample after cooking by the weight of the uncooked pasta sample (50 g) [47]. In order to determine the volume increase index (VII), the volume of the pasta was tested by dipping a 50 g sample of an uncooked product in a measuring cylinder filled with 400 mL of vegetable oil. The volume increase was equal to the volume of the tested pasta sample. A sample of pasta (50 g) was then cooked and the volume of the cooked product was determined in an analogous manner. VII was calculated by dividing the volume of cooked pasta by the volume of an uncooked product. Cooking loss (CL, g/100 g d.m.) was determined by testing the dry matter content in water after cooking a 50 g pasta sample. The dry matter content in water was determined according to the AACC 44-15A method [43].

#### *2.6. Color of Pasta*

The color of the cooked and uncooked pasta samples was measured using a colorimeter (X-Rite 8200, Inc., Grand Rapids, MI, USA) with a standard light source (D65), a standard colorimetric observer (10◦ ), and a 12.3 mm diameter hole. White and black calibration references were applied to standardize the instrument before analysis. The following CIE parameters were recorded: L\* (lightness, indicates the level of light 100 or dark 0), a\* (−a\* = indicates greenness, +a\* = indicates redness), and b\* values (−b\* = blue, +b\* = yellow). The measurements were performed repeatedly 10 times per each sample.

#### *2.7. Sensory Analysis*

The sensory analysis was carried out in accordance with the method described by Sozer et al. [48]. The analysis involved 12 people (8-females and 4-males, 23–48 years old), who had adequate taste sensitivity. The panelists had been previously trained how to evaluate the sensory parameters of pasta: appearance (regularity of shape, lack of deformation, cracks and scratches), color (should be regular and light-yellow), odor and taste (should be characteristic and similar to that of durum semolina pasta), hardness (evaluated as a resistance of cooked pasta to compression by the teeth), adhesiveness (evaluated by placing in the mouth, pressing it against the palate, and determining the force required to remove it with the tongue), springiness (was measured as the degree to which the product returns to its original shape after partial compression). Properly coded samples were cooked for OCT in random order and evaluated within a time no longer than 5 min after cooking.

A 5-point rating scale was used, in which 5 was the maximum score. Assuming that all evaluated parameters had equal weight, the average sensory rating was calculated for each pasta sample.

#### *2.8. Statistical Analysis*

The obtained results were subjected to statistical analysis using the statistical program STATISTICA 13.1 (StatSoft ©, Inc. Tulsa, USA). All experimental results were means (± S.D) from at least three

assays. One-way analysis of variance (ANOVA) and Tukey's post-hoc test were used to compare the groups. The results were statistically different for *p*-values < 0.05.
