2. Materials and Methods

Dried apricot kernels were obtained from local farmers in Batken, Kyrgyz Republic, in March 2024. The apricot cultivars used in this study and their corresponding abbreviations are presented in Table 1. Kernel samples from both sulphured and unsulphured fruits of the Kurmayi cultivar were additionally analysed as a case study to evaluate the effects of SO2 treatment on the nutritional value of apricot kernels. For each cultivar, 250 g of fruit stones was manually cracked, and the edible kernels were separated from the shells, weighed, ground into a fine powder and thoroughly homogenised. The resulting kernel powder was used for all subsequent analyses. Before analysis, the samples were stored at −20°C.

Cultivar and sample name | Treatment and drying technology | Country of origin
Suhany | Sun‐dried kernel, unsulphured | Batken, Kyrgyzstan
Suhany, oil | Pressed oil from sun‐dried, unsulphured kernels | Batken, Kyrgyzstan
Kandek | Sun‐dried kernel, unsulphured | Batken, Kyrgyzstan
Kurmaiy | Sun‐dried kernel, unsulphured | Batken, Kyrgyzstan
Kurmaiy, sulphured | Sun‐dried kernel, sulphured | Batken, Kyrgyzstan

2.1. Analyses of Macronutrients

Moisture content was determined by heating 5.0 ± 0.5 g of ground, air‐dried apricot kernel samples in a drying oven at 105°C until constant weight was achieved. Nitrogen content was determined using the improved Kjeldahl method (AOAC 950.48) with a Büchi Labortechnik AG analytical system (Flawil, Switzerland) and converted to crude protein using a nitrogen‐to‐protein conversion factor of 5.30 [15].

Crude fat content was determined using the Soxhlet extraction method (AOAC 2003.05), with petroleum ether as the extraction solvent [15].

Crude fibre content was determined by first defatting 2 g of the ground sample with petroleum ether, followed by digestion with 1.25% (v/v) H2SO4 and 1.25% (v/v) NaOH for 30 min. The digested material was filtered through a cotton filter, and the residue was ashed at 550°C for 4–6 h, cooled in a desiccator and weighed (AOAC 962.09) [15].

Ash content was determined according to the AOAC method 942.05 [15]. All analyses were performed in triplicate.

2.2. Determination of Glucose, Fructose and Sucrose Contents

Glucose, fructose, sucrose and sorbitol contents were determined by extracting approximately 10–13 mg of each ground sample with 10 mL of ultrapure water in an ultrasonic bath for 15 min. The extracts were filtered through Whatman No. 4 filter paper and subsequently diluted 20‐fold before analysis. Carbohydrates were quantified using high‐performance anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) on a Dionex AS‐AP system (Thermo Scientific, Waltham, MA, United States), following the method described by Barber et al. [16]. Separation was performed on a Dionex CarboPac PA1 column (2 × 250 mm). The injection volume was 10 _μ_L. Isocratic elution was carried out using 200 mM NaOH as the mobile phase at a flow rate of 0.25 mL/min. Calibration curves were prepared using external standards of glucose, fructose and sucrose at concentrations ranging from 1 to 200 _μ_mol/L. Data acquisition and chromatographic analysis were performed using Chromeleon 7 software.

2.3. Determination of Ascorbic Acid Content

Ascorbic acid content was determined by reversed‐phase HPLC with UV detection, according to the method described by [17]. Ground sample (1.250 g) was mixed with 15 mL of 1.5% metaphosphoric acid, shaken for 15 min and sonicated for 3 min. The extract was then diluted to a final volume of 25 mL with the same solution (1.5% metaphosphoric acid). The resulting solution was filtered through Whatman No. 4 filter paper, then through a 0.45 _μ_m membrane filter (RC), before chromatographic analysis.

Analysis was performed using an Agilent 1100 HPLC system (Agilent Technologies, United States). A stock solution of vitamin C (1 g/L) was prepared using 1.5% metaphosphoric acid, and calibration standards were prepared in the concentration range of 50–1000 μ_L/mL. The calibration equation was y = 16.183_x − 20.348, with a coefficient of determination (R 2) of 0.9974.

The mobile phase was prepared by dissolving 1.361 g of KH2PO4 in 600 mL of bidistilled water, followed by the addition of 200 mg of tetrabutylammonium hydrogen phosphate. The pH was adjusted to 2.5 using dilute H3PO4 solution, and the final volume was adjusted to 1000 mL with distilled water. The mobile phase was filtered through a 0.45 _μ_m membrane filter (RC 55) and degassed in an ultrasonic bath for 5 min. Chromatographic separation was performed on a ProntoSIL 60‐5 Phenyl column (250 × 4.6 mm; Knauer, Wissenschaftliche Geräte). The flow rate was set at 0.8 mL/min, and detection was carried out using a diode‐array detector at 243 nm, with the column temperature maintained at 40°C. Each sample was analysed in duplicate.

2.4. Determination of Antioxidant Activity

Antioxidant activity was evaluated using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical‐scavenging assay according to the method described by Hangun‐Balkir and McKenney [18]. A 0.01% (w/w) DPPH solution in 80% ethanol was prepared as the free‐radical reagent. For extraction, 1 g of ground apricot kernel was mixed with 20 mL of 80% ethanol and stirred for 20 min. The mixture was then centrifuged at 5000 rpm for 3 min. The supernatant was collected and diluted to obtain five concentrations (1, 2.5, 5, 7.5, 10, 15 and 20 mg/mL) using 80% ethanol.

For the antioxidant assay, 2 mL of each extract solution was mixed with 2 mL of a 0.01% DPPH solution prepared in 80% ethanol. The mixtures were thoroughly shaken and incubated at room temperature for 30 min. The control solution consisted of 2 mL of 80% ethanol and 2 mL of DPPH solution. The absorbance of both the control and sample solutions was measured at 517 nm using a UV‐Vis spectrophotometer (Tecan Infinite 200Pro, Germany). The antioxidant activity was calculated according to Equation (1): where _%_AA represents the antioxidant activity (per cent inhibition), Abs sample is the absorbance of the sample and Abs control is the absorbance of the control. The results were expressed as IC50 values, defined as the concentration of extract required to inhibit 50% of DPPH free radicals. IC50 values were determined from inhibition curves obtained by plotting percentage inhibition against extract concentration. All measurements were performed in triplicate.

(1) %AA=Abscontrol−AbssampleAbscontrol·100

2.5. Determination of Total Phenolic Content (TPC)

The TPC was determined using the Folin–Ciocalteu method according to the procedure described by [19]. A 10% methanolic extract of the kernels was prepared, homogenised and filtered through Whatman No. 2 filter paper. A 20‐_μ_L aliquot of the extract was mixed with 100 _μ_L of Folin–Ciocalteu reagent (Merck, Darmstadt, Germany) and allowed to react for 5 min. Subsequently, 300 _μ_L of sodium carbonate solution (Na2CO3, 20 g/L, Merck KGaA, Darmstadt, Germany) was added, followed by 1580 _μ_L of distilled water to obtain a final volume of 2 mL. The reaction mixture was incubated in the dark at room temperature for 30 min. The absorbance was then measured at 765 nm against a reagent blank using a UV‐Vis spectrophotometer (Tecan Infinite 200Pro, Germany).

A calibration curve was prepared using gallic acid standard solutions in the concentration range of 0.5–5 mg/mL. The calibration equation was y = 0.496_x_ with a coefficient of determination (R 2) of 0.9899. TPC was expressed as milligrammes of gallic acid equivalents per 100 g sample. All analyses were performed in triplicate.

2.6. Determination of Fatty Acid Composition of Kernels

Fatty acid composition was determined by converting triglycerides into fatty acid methyl esters (FAMEs) through alkaline transesterification using potassium methanolate (5% KOH in methanol). The resulting FAMEs were extracted with cyclohexane and dilute sulphuric acid and subsequently analysed by gas chromatography coupled with a flame ionisation detector (GC‐FID; Agilent Technologies 7820A GC System, United States). Separation was performed using a ZB‐FFAP capillary column (30 m × 0.25 mm i.d., 0.25‐_μ_m film thickness) according to the method described in Ulambayar et al. [20].

Identification of individual fatty acids was carried out by comparing retention times with those of a certified FAME standard mixture (Supelco 37 Component FAME Mix, Sigma‐Aldrich, United States). In addition to apricot kernel samples, cold‐pressed apricot kernel oil obtained from the Suhany cultivar was analysed to assess the effect of the oil extraction (pressing) process on fatty acid composition. All analyses were performed in triplicate.

2.7. Determination of the Amino Acid Composition of the Kernel

The amino acid composition of apricot kernels was determined by ion‐exchange chromatography with postcolumn ninhydrin derivatisation using a Sykam S 433 Amino Acid Analyser (Chromatographie Vertriebs GmbH, Fürstenfeldbruck, Germany). Briefly, 0.2 g of the sample was hydrolysed with 10 mL of 6 M HCl in a sealed long‐neck ampoule and incubated at 110°C for 23 h. Following hydrolysis, a 1.5 mL aliquot of the hydrolysate was evaporated to dryness. The residue was reconstituted in 2 mL of lithium citrate buffer (0.12 M, pH 2.2) and filtered through a regenerated cellulose membrane filter (0.45‐_μ_m pore size).

Chromatographic separation was performed on a cation‐exchange column (LCA K07/Li, 7 _μ_m, 4.6 × 150 mm) maintained at 42°C. The flow rates were set at 16 mL/h for the buffer solutions and 10 mL/h for the ninhydrin solution. Four buffer systems were used for amino acid separation: A‐1 (0.12 M lithium citrate, pH 2.9), B‐1 (0.3 M lithium citrate, pH 4.2), C‐4 (0.3 M lithium citrate, pH 8.0) and a regeneration buffer (0.5 M lithium hydroxide).

Identification and quantification of amino acids were carried out using a standard amino acid mixture obtained from Serva Electrophoresis GmbH (Heidelberg, Germany). In this method, glutamic acid was determined as the sum of glutamic acid and glutamine, whereas aspartic acid was determined as the sum of aspartic acid and asparagine. Tryptophan could not be quantified because it is degraded during acid hydrolysis. Likewise, cystine and methionine were not determined due to oxidative losses occurring during sample preparation. Detection was performed at 440 nm for proline and at 570 nm for all other amino acids [21]. Each sample was analysed in duplicate.

2.8. Statistical Analysis

All results were expressed as mean ± standard deviation (SD) based on three independent replicates (n = 3) reported per 100 g of sample. Statistical differences among the three apricot kernel varieties were evaluated using one‐way analysis of variance (ANOVA) followed by Duncan′s multiple range test as a post hoc comparison. Statistical analyses were performed using IBM SPSS Statistics Version 20 (IBM Corp., Armonk, NY, United States). Differences were considered statistically significant at p < 0.05.