1. Introduction
Currently, aromatic and medicinal plants have a considerable asset [
1] due to the continuous discovery of useful applications of their essential and vegetable oils in the field of health along with their employment in other fields of economic interest [
2]. Due to their many uses, they are in ever-increasing demand on world markets. At the national level, Morocco has a variety of aromatic and medicinal plants that can be applied in different fields (pharmacy, perfumery, cosmetics, and food processing) for their therapeutic and fragrant properties [
3], and one of the most popular and cultivated plants in Morocco is the walnut (
Juglans regia L.) plant.
The walnut oils studied in this work are those from the seeds of Azilal province, which are labeled as a geographical indication under the name of «AZILAL WALNUT» [
4], and according to the Moroccan Official Bulletin N°6336 offered by the same reference, «AZILAL WALNUT» has a high quality in terms of the physicochemical characteristic of 100 g seeds, with a content of proteins (N × 6.25) ≥ 12 g, lipids ≥ 66 g, carbohydrates ≥ 15 g, and Vitamin B1 (0.15 to 0.18 g) and Vitamin E ≥ 2.5 mg [
5], which is what makes it a solid source of nutritional compounds. They are also regarded as oil crops based on their high oil content, which holds various bioactive and health-promoting components, such as tocopherols, phytosterols, chlorophylls, and carotenoids [
6], including minor components. Furthermore, walnut oil is rich in polyunsaturated fatty acids (PUFAs), especially linolenic acid, which plays an influential role in coronary heart disease prevention, as well as hypertension and blood lipid regulation [
7,
8,
9].
Upon recent reports, walnuts appear to be gaining considerable attention for their beneficial health properties [
10], including antidepressant, anti-inflammatory, as well as antidiabetic activities [
11], primarily due to the availability of bioactive compounds such as phytosterols and plant polyphenols [
12,
13,
14,
15,
16]. In addition, consuming walnuts has been associated with many health benefits, as they are an excellent source of omega-3 fatty acids, with a percentage up to 10% [
17], vitamin E [
18,
19,
20], as well as other antioxidants that are involved in cardiovascular health and reducing cancer cell growth [
21].
There are many different factors involved in determining the quality of vegetable oils, from the time the seeds are harvested to the time the oils are consumed, and one of the most important parameters that directly affect the quality of the oil is the extraction method. A reliable oil extraction method is one that can safeguard the quality and preserve more nutrient components in the oil. Many types of extraction techniques exist, including conventional solvent and mechanical extraction [
22,
23], as well as nonconventional techniques such as supercritical fluid extraction, ultrasonic extraction, microwave extraction, and enzyme-assisted extraction, which have been optimized and applied to improve the oil extraction rates in a shorter time and with minimal deterioration of the oil quality [
24,
25,
26,
27].
In the current study, we tried to provide a comparative study between three different extraction techniques: the first one is extraction by the cold press, which is an environment-friendly method that requires less energy [
28]; the second method is extraction using the soxhlet system, which is the classical method for solid–liquid extraction; the last extraction method used in this study is the ultrasonic-assisted solvent, and this method has allowed us to obtain high-quality edible oils, with a higher yield and a reduced processing time [
29].
The aim of this study was to evaluate the effect of the extraction method (cold press, soxhlet extractor, and ultrasonic extractor), of walnut seeds oil (Juglans regia L.), on the physicochemical properties (acid value, free fatty acid value, peroxide value, iodine value, saponification value, K232, and K270), the content of chlorophyll, carotenoid, sterols, and tocopherols, as well as the fatty acid profile of walnut seed oil.
2. Materials and Methods
2.1. Plant Materials
Harvesting of walnut (Juglans regia L.) seeds was carried out in September 2020, in the regions of Beni Mellal-Khenifra, Morocco, exactly from Agnsou N’Ouargue (31°27′29.5″ N 6°52′58.1″ W). After the harvest of the fruits, they were naturally dried, peeled from the husks, and then shelled manually to obtain the kernel only.
2.2. Oil Extraction
Soxhlet Extractor (SE). The soxhlet extraction was carried out from fifty grams of powdered seeds samples for 8 h using n-hexane at 80 °C, with a liquid-to-solid ratio of 5:1, followed by removal of solvent at 50 °C under reduced pressure in a rotary evaporator (Heidolph Hei-VAP Precision motor, Schwabach, Germany).
Cold Press (CP). The cold pressing of the walnut seeds was carried out from fifty grams of crushed walnut seeds using Komet DD 85 G presses (IBG Monforts Oekotec GmbH, Mönchengladbach, Germany) at a temperature of 80 °C, which is the temperature that refers to a heating element that comes into contact with the extraction tube to apply heat to the sample to facilitate the extraction procedure.
Ultrasonic Extractor (UE). The ultrasound extraction was performed from fifty grams of powdered walnut seeds using the ultrasonic processor UP100H (100 W, 30 kHz) from (Hielscher Ultrasound Technology, Teltow, Germany). However, the solvent used in this experiment was n-hexane at 45 °C, with a liquid-to-solid ratio of 5:1, and the duration of the extraction did not exceed 30 min. After the leaching was completed, the mixture (walnut powdered seeds and n-hexane) was filtered, and the solvent was eliminated at 50 °C under reduced pressure using a rotary evaporator (Heidolph Hei-VAP Precision motor, Germany).
The three extractions were performed in triplicate, and all extracted oils were immediately filled into brown bottles and stored at 3–5 °C until analyzed.
2.3. Physicochemical Quality Parameters
The acid value (AV) and the free fatty acids value (FFA) were determined according to the ISO 660 norm [
30]; the peroxide value was calculated according to the ISO 3960 [
31]; the saponification, the iodine, and the specific extinction coefficients values (K
232 and K
270) were determined according to the American Oil Chemists’ Society (AOCS) recommended practice, respectively, Cd 3a-94, Cd 1c-85, and Ch 5-91 [
32]. The FFA content reflects the amount of free oleic fatty acid; it is expressed in mg KOH/g oil of free oleic acid in relation to the total amount of the oil; the specific extinction coefficients (K
232 and K
270) were expressed as the specific extinction of a 1% (
w/
v) solution of the oil in cyclohexane in a 1 cm cell way length, utilizing an (LLG-uniSPEC 2) UV spectrometer. Saponification and iodine values were expressed in (mg KOH/g oil) and (mg of I
2/100g oil), respectively.
2.4. Chlorophylls and Carotenoids Content
The determinations of chlorophylls and carotenoids were carried out by the preparation of a 1% solution of walnut seeds oil in cyclohexane followed by measuring the absorbance at 670 nm and 470 nm, respectively [
33]. Contents were calculated using the following formulas, Equations (1) and (2).
where
A is the absorbance and
d is the thickness of the spectrophotometer cell (1 cm). The chlorophyll and carotenoid content were expressed in ppm, (mg predominantly pheophytin a by kilogram of oil) and (mg Lutein by kilogram of oil), respectively.
2.5. Fatty Acids’ Composition
Saturated (SFAs), monounsaturated (MUFAs), and polyunsaturated fatty acids (PUFAs) were determined following the recommended practices required by the American Oil Chemists’ Society (AOCS), Ce 1i-07 [
32]. The fatty acids (FAs) underwent a transesterification reaction with methanol to obtain the fatty acid methyl esters (FAMEs). The latter were analyzed on a gas chromatography with capillary column (Varian CP-3800, Varian Inc. Middelburg, The Netherlands) that has a polar stationary phase and split inlet system. The separation was according to the fatty acids chain length (CL), degree of unsaturation, and position of double bonds (which means the position isomers). C17:0 was used as an internal standard for quantification. A fused silica column was used with the following dimensions (l = 30 m; Ø = 0.32 mm). The carrier gas used was helium and the initial and final column temperatures were 170 °C and 230 °C, respectively, with a rising rate of 3 °C/min. The injected volume was 1 μL and the detection was with a flame-ionization detector (FID) at 230 °C. Results are presented as relative percentages of individual fatty acids within the sample.
2.6. Phytosterols Composition
The quantification of sterols composition was carried out according to the AOCS Official Method Ch 6-91 [
32], which necessitates the saponification of each oil sample, followed by the extraction of the unsaponifiable fraction, then separating sterols in this fraction by gas chromatography with a capillary column (Varian CP-3800, Varian Inc. Middelburg, The Netherlands). 5α-cholestanol was used as an internal standard for quantification. The column dimensions were (l = 30 m, Ø = 0.32 mm), helium was used as a carrier gas, and the injection volume was 1 μL for each analysis. The results of this method can be used to verify the purity of the fat or to check compliance with a commercial denomination or an advertised composition.
2.7. Tocopherols Composition
The amounts of tocopherol were quantified following the ISO 9936 standard method using an HPLC equipped with a fluorometric detector on a silica column (25 cm × 4 mm). An isooctane: isopropanol (99%: 1%) mixture was used as an eluent with a rate of 1.2 mL/min for 20 min. Furthermore, the quantification was carried out with the help of external standard curves of α-, β-, γ-, and δ-tocopherols and a quantitative and qualitative daily reference of tocopherol standards [
34].
2.8. Statistical Analysis
IBM SPSS Statistics 26 software was used to affect the ANOVA One-Way Tukey HSD test for the verification of the statistical significance at a confidence level of 95.0%, and the results were expressed as means ± standard error of the mean (Mean ± SE). Correlation analysis was carried out by Pearson’s test using XLSTAT 2021, and the data obtained were analyzed using principal component analysis (PCA).
4. Conclusions
The current study provides an evaluation of the effect of extraction method (cold press, soxhlet, and ultrasonic extraction) of Moroccan walnut oil on the fatty acid profile, physicochemical properties, chlorophyll, carotenoid, sterol content, as well as tocopherol compositions of walnut oil. The results showed that Juglans regia L. oil is a rich source of unsaturated fatty acids, sterol, and tocopherol (Vitamin E), and that linoleic acid (C18:2) is the main fatty acid and that β-sitosterol is the predominant sterol, while γ-tocopherol is the primary isomer of tocopherol. Ultrasonic extraction may be a time- and solvent-saving technique, but it provides oil with a higher PV content compared to what cold pressing produces. Similarly, the extraction by soxhlet gives the highest yield of oil but also with the highest values of AV and extinction coefficients, in addition to traces of hexane that can be harmful to the health of the consumer. We can conclude that the extraction technique has a direct impact on the content of the composition in phytosterols, as well as tocopherols, and that the cold pressing technique was the most effective method to protect the studied compositions from degradation during the extraction process. Overall, this study presents essential information for producers of nutritional oils and, in particular, walnut (Juglans regia L.) oil; this information helps to produce a safe walnut oil with high nutritional value using an eco-friendly technique. It also helps to form a scientific basis for further research on this plant in order to provide a vision for the possibility of exploiting these oils in the pharmaceutical, cosmetic, and food fields.