Temperature Effects on Yields, Fatty Acids and Tocopherols of Prickly Pear (Opuntia Ficus Indica L.) Seed Oil of Oriental Region of Morocco

This study focuses on yields, chemical quality, composition, and the stability of the fatty acids of the oil extracted from Opuntia cus indica seeds, collected from the eastern region of Morocco, regardless of the temperature and the extraction method used. The results of this study reveal that prickly pear is a rich source of oil. The obtained oil yields varied from 12.49%±0.09 for the mechanical extraction, 11.46±0.10 for the chemical extraction, and 10.52%±0.09 for the maceration. The main fatty acids found in O. Ficus indica are linoleic acid 75.80%±0.10 (Chemical), 74.07%±0.14 (Maceration) and 71.59%±0.14 (Mechanical), and palmitic acid 17.32%±0.02 (Chemical) 22.419% ±0.06 (Maceration) and 26.58% ±0.00 (Mechanical). So the oil of prickly pear could be classied as a linoleic. Among the Tocopherols founded, a high value of b-tocopherol has been detected in the mechanical extraction with 502.04±0,76 mg/kg followed by the chemical and the maceration extraction with (430.12±0.61mg/kg, 315.47± 0.96 mg/kg) respectively. The ndings of the present study reveal that the oil of O. cus indica could be used in cosmetics and pharmacological products.

. thanks to its ability to adapt in several environmental conditions, the cactus grows in plains, coastal regions and plateaus (Lahsasni et al. 2004).
Opuntia cus-indica has received greater attention over the past few years and has been extensively investigated for its pharmaceutical properties (Bouaouine et al. 2018). It is a source of food but also the origin of products and by-products for industrial, medical and cosmetic use. .
The species O. cus indica, Opuntia robusta, and Opuntia amyclaea are extensively used in Mexico as a basic element for the production of cosmetics, pharmaceutical and agricultural products, textiles, additives for construction, coagulants (in wastewater treatment), and other applications (Orozco et al. 2018 ).
This cactus has multiple edible uses, juste like the preparation of the juices and salads, also its medicinal bene ts supported antioxidant properties and for treatment of hyperglycemia and hypercholesterolemia.
Fruits and young stems are traditionally utilized to treat hypertension, asthma, edema, diabetes, burns, indigestion, and other health disorders (Galati et Maataoui et al. 2006) and, more generally on the nutritional bene ts of this species (Stintzing, 1999).
Seeds represent approximately 10-15% of the comestible pulp and are usually discarded as waste after the pulp is removed. The oil obtained from the seeds is approximately 7-15% of the total weight (Moukal, 2004).
The main objective of this work is to study the effect of temperature on yields, fatty acids and tocopherols of Opuntia cus indica seed oil from the eastern region of Morocco using different extraction methods and temperatures.
Material And Methods

Collection area
Opuntia cus indica seeds are collected from Guercif (Province of Taourirt). This province belongs to the eastern region of Morocco (Fig. 1).

Methods of extraction
Three types of extractions were used:

Chemical extraction
The seeds of O. cus indica were crushed until the obtaining of a ne powder. 40g of this powder is subjected to an extraction in a Soxhlet by 200ml of n-hexane (99%) at a temperature of 60°C for 7 hours.
The solvent was removed using a steam rotator (HEIDOLPH) at a temperature of 40°C. The obtained oil was conserved in a refrigerator at a temperature of 4°C.

Maceration extraction
This technique consists of letting a solid mass remain in a cold liquid to extract soluble compounds. The extracts are prepared from 400g of the seeds powder using 400ml of n-hexane 99% under stirring at room temperature for 2 hours. The extracts are then ltered by a Büchner type lter and the ltrate is then ltered through a crucible with a porosity equal to 4µm. The solvent was removed from the ltrate by a steam rotator to recover the oils which are then conserved at 4°C until their use.

Mechanical extraction: Hot press (120°C)
The mechanical extraction is a biological method used to extract oil from the seeds. The seeds are placed between permeable barriers by increasing the mechanical pressure thus reducing the volume available for the seeds. In general, regardless of the seeds used, the higher the pressure, the higher the oil extraction e ciency.
3. The physicochemical analysis 3.1 Acid index: Method ISO 660: 1996 (AFNOR 2000) 1 g of fatty substance is stirred in an Erlenmeyer ask, then 5 ml of ethanol with a concentration of 99% and 5 drops of phenolphthalein (PP) are added. Neutralization is done by gradual addition using a burette containing the ethanoic solution of KOH (0.1 mol/l) until a persistent pink colour is obtained.

Saponi cation index: Method ISO 3657: 2002 (AFNOR 2000)
In a 250ml round-bottomed ask, 1g of fatty substance is placed with 3ml of solvent (ethanol-ether (v: v)), and 25ml of alcoholic potassium hydroxide with a concentration of 0.5 mol/l. the ask is then placed in a water bath for 45 to 60 minutes. After cooling the ask to room temperature, 2 to 3 drops of phenolphthalein are added. The excess of potassium hydroxide was determined with hydrochloric acid at a concentration of 0.5 mol/l up to the endpoint (discoloration of the solution). Under the same conditions, a blank test was carried out.

Peroxide index: Method ISO 3960: 2001 (AFNOR 2000)
In a 250 ml Erlenmeyer ask, 1 g of fatty substance is stirred with 25 ml of the solution (chloroform / acetic acid (2: 3 (v: v))) then 1 ml of the potassium iodide solution is added (KI) of concentration (0.01N). The mixture is placed in the dark for 5 min at a temperature between 15 and 25°C. Afterwards, 75 ml of distilled water is added and the mixture is stirred. The iodine released by sodium thiosulphate is titrated in the presence of starch as an indicator.

Relative density: IUPAC Method 2.101 (AFNOR 2000)
It is the ratio of the mass of one volume of oil to the mass of the same volume of distilled water at a temperature of 20°C.

Chromatographic analysis CPG-MS
The chromatographic analyses were carried out after transesteri cation by gas chromatography coupled with a mass spectrometer (SHIMADZU series GCMS-QP2010), equipped with a split/splitless injector and a column (LxDI :30m x 0.25 mm) apolar (Stationary phase : 95% dimethylpolysiloxane : 5% phenyl; Thickness ; 0.25µm). The carrier gas used was the Helium.

Tocopherols analysis (HPLC)
The tocopherol analyses were performed by HPLC-FLD, (Agilent Technologies 1200 series system, Agilent Technologies) using the o cial method, AOCS 8-89 (AOCS 1989). It was equipped with an automatic injector, on an Uptisphere 120A° NH2 column (150 mm * 3 mm, 3 µm) Interchim (Montluçon, France). The temperature was maintained at 30°C. The mobile phase was hexane/2-propanol (99:1, v/v) with a ow rate of 1 ml min-1. The different isoforms of tocopherol (α-, β-, γ-and δ-tocopherols) were estimated using an oil/hexane solution of the oils of the three extraction types of Opuntia cus indica. Tocopherols obtained from Sigma-Aldrich (Steinheim, Germany) were used as external standardization for the identi cation of tocopherols at 292 nm. The homologues of the tocopherols were quanti ed by comparing the peak response of each sample with that of the corresponding standard.

Statistical analysis
Data were statistically tested by a unidirectional analysis of variance (ANOVA) followed by a Tukey test to compare means that showed a signi cant variation (P ≤ 0.05) using SPSS 20 software.
Results And Discussion

Yields
The oil yields obtained from Opuntia cus indica seeds using the tree extraction methods are presented in (Fig. 2). The results varied from 12.46% and 11.49%. The oil yields present a highly signi cant difference (P < 0.05). The mechanical extraction shows a high oil content with 12.49 ± 0.09 followed by the chemical and the maceration extractions (11.46%±0.10, 10.52 ± 0.09) respectively. Gharby et al (2015) reveal that the oil yields of cold press extraction are ranged from 6 to 7%. However, the ndings of Bertrand Matthäus et al (2015) showed that the oil contents of the seeds of Opuntia cus indica in Turkey varied among the different localities ranging from 5.0% (Ortaören) to 14.4% (Eskioba) wish is in according with our results, therfore the result of Sawaya and Kahn (1982), which found that the seeds of Opuntia cus indica represented only about 12-15% of the whole fruit and that the oil yield of the seeds was about 13.6% whereas El Hachimi et al (2015) shows that prickly pear oil is a uid oil with a relatively low extraction yield of (7.81 ± 0.78% to 10.45 ± 1.34%).

Prickly pear oil
Opuntia cus indica oil obtained by mechanical extraction has a lower acid index (4.37 ± 0.10 mg KOH/g oil) compared to the chemical extraction (5.85 ± 0.03 mg KOH/g oil) and the extraction by maceration (5.66 ± 0.07 mg KOH/g oil). This signi cant difference (P < 0.05) is probably due to the solubility of the acids in the solvents (Table 1). This high acidity value could indicate a strong enzymatic hydrolysis of the seeds during harvesting, handling or processing of the oil or could also be due to the wrong storage. Compared to our results, the study carried by Kandji (2001) and R'bia et al (2017), showed a low acidity index of OFI oil produced in Tunisia, which was 1.28 ± 0.007%.
The results of saponi cation index obtained from Opuntia cus indica show a signi cant difference (P < 0.05) between the three types of extractions (mechanical, chemical and maceration) which varies from (181.12 ± 0.18, 183.77 ± 1.23 and 179.08 ± 3.45) respectively (Table 1). This index was in accordance with the CODEX STAN 210-1999 standard and these results are comparable to that of the castor oil The peroxide value (PV) of Opuntia cus indica oil showed a signi cant difference (P < 0.05) for the three extraction types (mechanical, chemical and maceration) with 5.75 ± 0.08, 6 ± 0.06 and 5.97 ± 0.04 meq/kg respectively. Compared to our ndings, the research performed by Brahmi et al (2020) shown a higher PV value (12.0 ± 0.4 meq O2/Kg, P ≤ 0.05) of OFI samples.
This difference may be due to the extraction time and the solvent used ( Table 1). The PV may also be affected by the oxidation of the oil studied under the extraction and conservation conditions. This can lead to the oxidation of unsaturated fatty acids, and thus their reduction.
The density of Opuntia cus indica oil for the three types of extraction (Mechanical, Chemical and Maceration) are (0.926 ± 0.003, 0.925 ± 0.001, 0.919 ± 0.005) respectively, which is in accordance with CODEX STAN 210-1999. Therefore, the density of this oil is comparable to both olive oil (0.910) and The refractive index shows that there is a signi cant difference (P < 0.05) between the values of the three types of extraction (mechanical, chemical, maceration) with (1.475 ± 0.001, 1.476 ± 0.003 and 1.476 ± 0.001) respectively, which is in accordance with CODEX STAN 210-1999.

Gas chromatography analysis
The fatty acid composition of vegetable oils depends on several factors: plant origin, genetic factors, fruit ripening and speci c climatic conditions (Tlili et al. 2011).
The chromatographic analysis of prickly pear oil for the three types of extraction clearly shows that the fatty acids are stable whatever the extraction and the temperature used. This stability is remarked for the three major fatty acids. The proportion of linoleic acid always remains around (70%) despite the different temperature extraction (maximum 120°C), it is also the case of the saturated fatty acids where we observe no degradation of those acids (palmitic, stearic and arachidic).
The SFA content varies slightly between 24.19% (chemical extraction), 25.92% (extraction maceration) and 28.41% (mechanical extraction), while there is a clear predominance of UFA with slightly variable proportions and the dominance of linoleic acid (Table 2) Moreover, according to Ennouri et al (2005;, the rate of linoleic acid overtakes 70% and oleic acid 12% in OFI seed oil of Sfax (Tunisia).
Other parameters related to nutritional aspects are the unsaturated/saturated ratio, in fact, OFI oil has an average value of 3.13 due to its high linoleic acid content. In addition, linoleic acids (omega-6) can be transformed by organisms into a series of long-chain fatty acids (Letawe et al. 1998), a precursor of the biosynthesis of arachidonic acid, a substrate for the synthesis of eicosanoids (Ghazi et al. 2013) which play an important role in vascular level and blood coagulation. Also, this oil has useful properties for the skin that's why it is used in the cosmetics industry (Ramadan and Mörsel 2003).
However, the work of Chaaben et al. 2015 shows that the average content of SFA in prickly pear oil is only 16.64%. This difference is probably due to the genetic, climatic and soil type of the cultivated plant which is in agreement with the work of Ramadan and Mörsel (2003), which shows that the fatty acid composition of the oil of Opuntia cus indica, is very much in uenced by climatic factors, soil type and genetic factors in which they are cultivated.

Tocopherols
The major tocopherol founded in Opuntia cus indica oil is β-tocopherol, it represents 502,04 ± 2,7mg/kg for the mechanical extraction followed by the chemical and the maceration extractions 499,6 ± 1,56 mg/kg and 315,47 ± 1,11mg/kg respectively (Fig. 3). Our results also shown that γ-tocopherol was detected in the chemical and the maceration extractions (257,86 ± 2,01 mg/kg and 260,61 ± 2,9 mg/kg) respectively, but missing in the mechanical extraction. The extraction by maceration presents a high value of α-Tocopherols with 237,12 ± 0,33, followed by the chemical and the mechanical extractions (98,71 ± 1,06 and 93,49 ± 0,89) respectively. The γ-tocopherol was missing in mechanical extraction and that may be due to temperature of extraction or the absence of the solvent.
Our results are in accordance with those of Matthäus and Özcan (2011) who nd that β-tocopherol contents of Opuntia seed oil varied between 3.9% (Eskioba) and 50.0% (Adana). But less than  shown that prime level of tocopherols in cactus seed cold press oil 946 mg/kg decided, it's much above that of the Tunisian (447 mg/kg ) and therefore the Germany cactus seed oil (403 mg/kg). Total tocopherols content of cactus oil is almost that of sun ower-seed oil (490 mg/kg), however, much above that of vegetable oil (220 mg/kg), and less than that of the soya bean (650 mg/kg) This study demonstrates the stability and richness of Opuntia cus indica oil in tocopherol, satured and unsaturated fatty acids with the dominance of linoleic acid. This richness may interest the production units of food, cosmetics and pharmaceutical products and allow all units exploiting this oil in their artisanal or industrial preparations, and their commercialization on the national and international market, either in full or by the manufacture of new products based on these Moroccan oils.

Declarations
Ethics approval Not applicable.

Consent to participate
All authors were participated in this work.

Consent for publication
All authors agree to publish.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Oil yields extracted from Opuntia cus indica seeds using different extraction methods.

Figure 3
Tocopherols extracted from Opuntia cus indica oil using different methods of extraction.