Material and Methods

Material

In 2025, chia seeds were bought from a seed market in Konya district, Turkey. Afterward, they were stored under cold conditions. Dried seeds were then cleaned using an air screen cleaner to remove dust, immature seeds, and broken seeds. Additionally, any remaining broken kernels were discarded.

Methods

Roasting Process

Chia seeds were evenly distributed in drying trays to a layer about 2 mm thick. They were then heat‐treated in an oven at 175°C for 30 min (Ahmed et al. 2024). Prior to analysis, the samples were ground using a grinder (ARZUM AR1034 Clipper, stainless steel, 150 W motor power, 50 g capacity, Türkiye).

Moisture Content

The KERN & SOHN GmbH infrared moisture analyzer was used for analysis of the moisture contents of chia seed samples. The moisture of raw and roasted chia seeds was 3.67% ± 0.43% and 2.44% ± 0.61%, respectively.

Extraction Procedure

Chia seed samples were extracted according to the method described by Zalewski et al. (2020), with slight modifications. Briefly, 3 g of ground chia seed sample was mixed with 10 mL of a methanol–water solution (70:30, v/v). The mixtures were subjected to ultrasonic‐assisted extraction for 0 min (control), 20 min, or 40 min at either 25°C or 40°C, followed by centrifugation at 6000 rpm for 10 min. The resulting supernatants were evaporated at 40°C, and the dried extracts were reconstituted in 10 mL of methanol. For conventional extraction, the same procedure was followed, except that the samples were extracted using a water bath instead of an ultrasonic bath (Bandelin Sonorex, Germany).

Total Phenolic Amount

The total phenolic content of the extracts was determined using the Folin–Ciocalteu (FC) method as described by Yoo et al. (2004), with slight modifications. Briefly, 1 mL of FC reagent and 10 mL of Na2CO3 solution were added to the extract, and the mixture was homogenized using a vortex mixer. Deionized water was then added to adjust the final volume to 25 mL. The samples were incubated in the dark for 1 h to allow color development. Subsequently, the absorbance was measured at 750 nm using a spectrophotometer (Shimadzu UVmini 1240, Japan). A calibration curve was established using gallic acid as the standard reference at concentrations ranging from 0 to 200 mg/mL. The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample.

Total Flavonoid Amount

The extract (1 mL) was sequentially mixed with 0.3 mL of NaNO2, 0.3 mL of AlCl3, and 2 mL of NaOH. The resulting mixture was incubated in the dark for 15 min, after which its absorbance was measured at 510 nm using a spectrophotometer (Shimadzu UVmini 1240, Japan). The results were expressed as mg quercetin equivalents (QE) per 100 g of sample, according to the method described by Hogan et al. (2009).

DPPH Free Radical Scavenging Activity

The free radical scavenging activity of the extracts was evaluated using the DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) assay according to the method described by Lee et al. (1998). Briefly, the extract samples were mixed with 2 mL of a methanolic DPPH solution. Following vigorous shaking, the mixtures were incubated at room temperature for 30 min. The absorbance was then measured at 517 nm using a spectrophotometer (Shimadzu UVmini 1240, Japan). The results were expressed as mmol Trolox equivalents (TE) per kg of sample.

Determination of Phenolic Compounds

The chromatographic separation of phenolic compounds was carried out using a high‐performance liquid chromatography (HPLC) system (Shimadzu, SCL‐10A VP‐Shimadzu, Japan) equipped with a photodiode array (PDA) detector and an Inertsil ODS‐3 column (5 μm particle size; 4.6 × 250 mm). The mobile phase consisted of 0.05% acetic acid in water (solvent A) and acetonitrile (solvent B), which were delivered at a flow rate of 1 mL/min under isocratic/gradient conditions at 30°C. A sample injection volume of 20 μL was used for each analysis. The detection of phenolic compounds was performed at 280 nm using the PDA detector. The gradient elution program was applied as follows: 8% B from 0 to 0.10 min, 10% B from 0.10 to 2 min, 30% B from 2 to 27 min, 56% B from 27 to 37 min, followed by a decrease to 8% B from 37 to 37.10 min and maintained at 8% B from 37.10 to 45 min. The total analysis time for each sample was 60 min.

Oil Extraction

Ground samples (5 g) were placed on filter paper and mixed with 150 mL of petroleum ether in individual flasks. The flasks were then treated in an ultrasonic bath for 20 and 40 min at temperatures of 25°C and 40°C. For conventional extraction, the same procedure was performed using a water bath. After pre‐sonication, the oils were extracted using a Soxhlet apparatus. The solvent was subsequently removed using a rotary vacuum evaporator (Heidolph Laborota 4001, Germany) at 50°C.

Fatty Acid Composition

The oil from chia seeds was esterified according to the method of Multari et al. (2019), and the resulting fatty acid methyl esters were analyzed by gas chromatography (Shimadzu GC‐2010) using a flame‐ionization detector and a capillary column, with both the injector and detector maintained at 260°C.

Calculation

Calculated Oxidizability Value (Cox)

The oxidizability ratio values of the chia seed oils depending on lye concentrations were determined according to the method recommended by Fatemi and Hammond (1980).

Oleic Desaturation and Linoleic Desaturation Ratio Values

The Oleic Desaturation Ratio (ODR) and Linoleic Desaturation Ratio (LDR) values were determined following the methodology proposed by Pleines and Friedt (1988).

Statistical Analyses

Analysis of variance (ANOVA) was conducted using JMP version 9.0. Data are presented as mean ± standard deviation, calculated with MSTAT C for the independent roasting and sonication conditions.