Hp 6890 2

A basic storage design was performed to study shelf life [10 (link)] in pasta developed throughout the determination of fatty acid profiles and chemical parameters (TBARS and acidity index). Fatty acid profiles were determined according to Bligh and Dyer, 1959 [29 (link)]. Firstly, each sample was homogenized with an Ultraturrax device (IKA-WERKE, T-25 basic) using different solvents such as chloroform, methanol, potassium chloride, and water. This mixture was centrifuged for 10 min at 4000 rpm, and the fat was extracted from the top, and afterwards, after incorporating BHT, butylated hydroxytoluene, as an antioxidant substance, solvents were evaporated with nitrogen gas. Afterwards, methylation was performed using 0.03 g of this previous fat. This fat was mixed with an intern pattern, C23:0, which does not caused interferences in the matrix. Then, 2 ml of hexane and 1 ml of potassium hydroxide saturated solution in methanol was added. The upper phase was extracted to measure. To analyze the fatty acid profile, a gas chromatograph (HP-6890 II Hewlett-Packard) was employed with a column SP-2380 (100 m × 0.25 mm × 0.20 µm). The temperature program was 140–165 °C at 3 °C/min during 10 min and 165–220 °C at 5 °C/min during 50 min. Fatty acid content was quantified as total area (%) of identified fatty acids.

This determination made it possible to observe probable changes in the fatty acid composition due to the MDDM obtention process [39 (link)]. Firstly, 10 g of raw material or 2 g of MDDM was homogenized with an Ultraturrax device (T-25 basic, IKA-WERKE) using different solvents: chloroform (Carlo Erba), methanol (Carlo Erba), potassium chloride (Panreac), and water. This mixture was centrifuged for 10 min at 4000 rpm (Mod. Universal 320 R, Hettich, Tuttlingen, Germany), and the fat was extracted from the top. After incorporating BHT (Sigma-Aldrich) as antioxidant, solvents were evaporated with nitrogen gas. Afterwards, methylation was performed using 0.03 g of this previous fat. This fat was mixed with an intern pattern, C23:0 (TCI), which does not caused interferences in the matrix. Then, 2 mL of hexane (Carlo Erba) and 1 mL of potassium hydroxide (Panreac) saturated solution in methanol was added. The upper phase was used. To analyze the fatty acid profile, a gas chromatograph (HP-6890 II, Hewlett-Packard, Palo Alto, CA, USA) was employed with a column SP-2380 (100 m × 0.25 mm × 0.20 µm). The temperature program was 140–165 °C at 3 °C/min during 10 min and 165–220 °C at 5 °C/min during 50 min. Fatty acid content was quantified as total area (%) of identified fatty acids.

The determination of fatty acid composition was conducted by the GC method (HP 6890 II with a flame-ionization detector, Hewlett-Packard, USA) according to PN-EN ISO 12966-1:2015-01 [37 ]. For the separation of esters, a high polar capillary BPX 70 column (60 m × 0.25 mm, 25 µm) was used. The injector temperature was 240 °C with a split ratio set to 100:1 and the FID temperature was 250 °C. The oven temperature was ramped from 130 °C (1 min) to 210 °C (7 min) at a rate of 1.5 °C min⁻¹. Helium was used as a carrier gas with a constant pressure of 40 psi at a flow rate of 0.3 mL min⁻¹ and an injection volume of 1 µL. GC-FID was used to determine the relative percent areas of the FAME components present in the analyzed samples as the reference method. The total analysis time was 61 min. The results of the analysis were automatically calculated according to the principle of internal normalization by the ChemStation software version A 03.34^® 1989–1994. Peaks of fatty acid methyl esters resolved by GC were identified by comparison to standard FAME mixtures of known composition.