Bioactive Compounds Assessment in Six Moroccan Rapeseed (Brassica napus L.) Varieties Grown in Two Contrasting Environments

Abstract

Rapeseed (Brassica napus L.) is the world’s second-largest oilseed crop after soybean. It contains functional compounds, including fatty acids and phenols, which are known for their health and nutritional benefits. In Morocco, rapeseed was introduced as a promising oilseed crop that has shown good adaptation and great potential. Six genotypes were registered and released as the most high-performance Moroccan varieties in terms of both seed yield and oil content. Apart from their ‘00′ quality, i.e., oil without erucic acid and meal with very low glucosinolate content, there is no information on other important quality traits. Therefore, this research aimed to characterize the lipochemical and phenolic attributes of those varieties, namely Baraka, Narjisse, Moufida, Lila, Alia, and Adila, grown at two contrasting sites (Allal Tazi and Douyet) so as to assess the environmental impact on oil quality. A set of 14 parameters were analyzed, comprising seed yield, oil content (Oil C), protein content (PC), acidity index (IA), peroxide index (IP), refractive index (IR), iodine value (IO), total phenolic content (TPC), total flavonoids, total carotenoid contents, free-radical scavenging activity (FRSA), half-maximal inhibitory concentration, fatty acid composition, and omega 6/omega 3 ratio. The results displayed significant differences (p < 0.001) between the two sites for all the abovementioned traits, except for IA, with an outperformance of Allal Tazi over Douyet. Additionally, variety and variety by site interaction had a significant effect on all the studied parameters, except for IA, IP, IR, and TPC. Over both environments, the varietal effect generated variations in yield of 10.9 to 17.1 q/ha, Oil C of 36.7 to 39.9%, PC of 21.3 to 25.9%, IO of 94.1 to 100 g I2/100 g, FRSA of 22.7 to 42.9%, and an omega6/omega3 ratio of 1.8 to 2.3%. It is noteworthy that the Moufida and Alia varieties displayed a low-magnitude effect of the environment, as they maintained similar high performance over both sites. They, thus, represent Moroccan genetic material of interest from an agronomic and nutritional perspective. Therefore, they should be promoted and encouraged for cultivation in Morocco, mainly in the Allal Tazi area and similar regions.

1. Introduction

Vegetable oil is an essential component of the human diet and a major source of edible lipids, accounting for more than 75% of the total lipids consumed in the world. It is a natural source of essential fatty acids. As an important basic food item used in cooking, vegetable oil does not only provide energy but also maintains normal body temperature, protects body tissues, carries fat-soluble vitamins, and performs other important functions [1]. Oilseed rape (Brassica napus L., also known as rapeseed or canola) is the second largest oilseed crop in the world after soybean [2]. The global production of rapeseed reached over 72 million tons in 2020 [3].

Due to their food and non-food uses, the demand for oilseeds, especially rapeseed oil, is still increasing [4]. In general, products derived from rapeseed oil are used in healthy diets as they record the highest growth rates in food retail sales [5]. The fatty acid composition along with other bioactive components, contained in rapeseed oil, contribute to its high nutritional value. In fact, rapeseed oil is characterized by low amounts of saturated fatty acids (<7%), known to have negative health effects, and relatively high amounts of polyunsaturated linoleic (ω-6) and α-linolenic (ω-3) fatty acids which are linked to the prevention of cardiovascular diseases [6]. In addition, the high ratio of linoleic to α-linolenic acids in rapeseed oil is nutritionally beneficial because it has proved to be associated with the prevention of atherosclerosis [7]. Although the high content of polyunsaturated fatty acids makes rapeseed oil nutritious, it can make it unstable to oxidation. Nevertheless, the natural presence of bioactive compounds such as tocopherols, phytosterols, and polyphenols plays a key role in preventing the occurrence of oxidation reactions [8]. In particular, rapeseed oil contains a high amount of tocopherols (430–2680 mg/kg), thus being among the main sources of these antioxidant compounds in the human diet [9].

The expression of seed traits, including all the abovementioned, is significantly influenced by genotype (G), environment (E), and genotype–environment (G × E) interaction [2,10]. The seed oil and its fatty acid composition are driven by environmental factors such as water availability, temperature, and duration of the growing period [11,12]. Therefore, many attempts are being undertaken worldwide to understand the effect of genotype and environmental stressors on the oilseeds’ quality. Many researchers have demonstrated the impact of environmental factors on oilseeds’ fatty acid composition [13,14,15]. This raised the importance of including rapeseeds in plant-breeding programs and many research approaches in order to meet the emerging global challenges, such as the satisfaction of the increasing demand for higher quality and nutritious food, the reduction in waste, and the production of predictable yields while considering cultivars’ specific adaptations for targeted environments. Today, the development of oilseed rape cultivars with good quality, i.e., high oil and protein content, zero erucic acid, low glucosinolate content, and high fiber content is a challenging endeavor for many rapeseed-breeding programs.

In Morocco, during the last 20 years, many rapeseed germplasms and inbred lines have been developed, in which only six are registered varieties with good agronomic performance. In addition, their seeds are characterized by high oil content, without erucic acid, and very low glucosinolate concentration in their meal. However, so far, these varieties have not been assessed for their biochemical attributes and the impact of the magnitude of environmental conditions on their expression. Hence, it is important to start evaluating the oil quality of each Moroccan rapeseed variety, including its stability to gain an understanding of the potential of these lines, based on several quality properties that could offer an added value to their agronomic performance. This characterization will be helpful for Moroccan farmers and industries, as consumers begin to show interest in features related to the origin, health, and quality of the foods they buy. Thus, providing products with proven good biochemical traits alongside high antioxidant potency would meet their expectation. Therefore, the current study was designed to identify the major oil-quality parameters of these varieties, as well as to evaluate the effect of the cropping environment (site) on those parameters to assort varieties according to their performance.

2. Materials and Methods

2.1. Plant Material and Sampling

The plant material used in this study was composed of six Moroccan rapeseed varieties (‘Baraka’, ‘Narjisse’, ‘Moufida’, ‘Lila’, ‘Alia’, and ‘Adila’) developed by the National Institute of Agricultural Research (INRA) of Morocco.

Table 1. Phenotypic description of the six Moroccan rapeseed varieties studied.

Variety	Type/Genetic Structure	Year of Release	Pedigree	Main Phenotypic Characteristics
Narjisse	Pure line	2008	Individual plant selection from an open-pollinated population	High plant with developed leaves, strong lobes, and a blade with a bright-green color. Mid–early flowering and high branching.
Moufida	Pure line	2009	Cross between two introduced varieties: Westar/Optima	Medium plant height. Developed leaves with medium serration, strong lobes, and a medium green blade. Flowers of bright-yellow color. Siliques with long beak.
Adila	Synthetic variety	2015	Intercrossing of four inbred lines: INRA-L615/INRA-L455 + INRA-L315 + INRA-L515	Leaves quite developed with medium serration, strong lobes, and an intermediate green blade. Main stem of medium height. Pale-yellow flowers. Long beaked siliques.
Lila	Synthetic variety	2015	Intercrossing of four inbred lines: INRA-L455/INRA-L615 + INRA-L315 + INRA-L515	Leaves quite developed with strong serration, very strong lobes, and a light-green blade. Main stem of medium height. Pale-yellow flowers. Long beaked siliques.
Alia	Pure line	2017	Cross between two introduced varieties: Fantasio/Kabel	High plant with less developed leaves, strong lobes, and a reduced blade with bright-green color.
Baraka	Pure line	2018	Cross between two inbred lines: INRA-L115/INRA-L455	Very high plant with developed leaves, strong lobes, and a blade of light-green color. Late flowering and high branching.

presents the type, origin/pedigree, and main phenotypic characteristics of these varieties. ‘Adila’ and ‘Lila’ are both synthetic varieties with the same parental lines (four inbred lines) but different intercrossing ways as described by Nabloussi et al. [16]. The varieties ‘Alia’, ‘Baraka’, and ‘Moufida’ are pure lines derived from simple crosses between either introduced varieties or inbred lines selected from our germplasm. The entire cross parents have different origins. Finally, the variety ‘Narjisse’ is a pure line selected from an open-pollinated population derived from numerous introduced varieties under massive bee presence conditions [16]. They were conducted in the cropping year 2019/2020 at two locations. The first one was the INRA-Experimental Station of Douyet (DYT), located 10 km from Fez city (34°04′ N, 5°07′ W) and 416 m above the sea, with an average rainfall of 350 mm. The soil is a vertisol with an average composition of 29% in clay, 58% in silt, and 12% in sand. The climate is of Mediterranean type, with a cold and rainy winter and a hot and dry summer. This experimental station is also characterized by the prevalence of sirocco winds which could be, to some extent, harmful for crop growth. The second one was the INRA-Experimental Station of Sidi Allal Tazi (ATZ), located 30 km from Kenitra City (34°31′ North, 6°19′ West), at an elevation of 10.5 m above the sea, with an average annual rainfall of 475 mm. The soil is little-evolved, with a vertic character and a clay–silty texture (average composition of 39% in clay, 34% in silt, and 25% in sand). The monthly rainfall and mean temperature recorded in both locations during the crop cycle are shown in the

Table A1. The monthly rainfall and mean temperature recorded in Sidi Allal Tazi and Douyet locations during the cropping year 2019/2020.

		Sept.	Oct.	Nov.	Dec.	Jan.	Feb.	Mar.	Apr.	May	June	July
Sidi Allal Tazi (ATZ)	Rainfall (mm)	7.5	12.3	38.6	73.2	201.1	92.2	50.1	0	0	0	0
	Temperature (°C)	25.0	21.1	15.5	13.4	12.4	15.7	14.9	17.9	19.5	20.8	25.7
Douyet (DYT)	Rainfall (mm)	0	58.1	73.4	73.3	32.2	0	37.1	32.8	34.9	7.2	0
	Temperature (°C)	26.8	23.0	11.6	11.9	11.5	14.8	14.0	17.2	18.4	21.7	32.1

.

The field trial was set and conducted, on a crop duration of about six months (from November to May), according to a complete randomized blocks design (CRBD) with three replications. Crop management was carried out according to the conventional cultivation technique adopted at our experimental stations. Before planting, deep fertilization was carried out at a rate of 60 units of nitrogen, 80 units of potassium, and 80 units of phosphorus. After emergence, at the stage of two pairs of leaves, thinning was carried out so as to leave an inter-plant space of 4–5 cm. Weeding was carried out manually at the rosette stage, while insecticide treatment was only performed when necessary. Cover fertilization (60 nitrogen units) was divided between the rosette and flowering stages.

To determine seed yield and perform the different oil analyses for the six varieties, three random samples per variety and per replication (block) were considered (A total of nine replicates).

2.2. Seed Yield, Oil Content, and Total Protein Content

After harvest, seed yield (q/ha) was determined for each variety under both environments herein investigated. Oil was extracted using the Soxhlet apparatus, model SER 148/6 (VELP SCIENTIFICA, Usmate Velate, Italy). Hence, 20 g of ground seeds (moisture 6.09 ± 0.27%) was mixed with 150 mL of hexane for 3 h at 130 °C [17]. The solvent was then evaporated at 40 °C using a rotary evaporator, model HAHNVAPOR (HAHNSHIN, Gyeonggi, Korea). Oil content (Oil C) was determined as follows:

Oil content (%) = [(M₁ − M₀)/M₂] × 100

(1)

where M₀ is the weight of the empty flask (g), M₁ is the weight of the flask after evaporation (g), and M₂ is the weight of the ground seeds (g).

The resulting oil was kept away from light and stored at 4 °C until its analysis for various physicochemical and biochemical parameters as described below.

After oil extraction using the Soxhlet apparatus, the remaining meal was analyzed for the total protein content (PC), according to the official AOAC 979.09 Kjeldahl method. In brief, 0.5 g of meal was digested with 6 mL of concentrated sulfuric acid (H₂SO₄) in the presence of a spatula of catalyst Cu-Se (AppliChem, Gatersleben, Germany). The mineralization step was carried out by heating the flasks at 450 °C for 90 min using a Digester, model DK-20 (VELP SCientifica, Usmate Velate, Italy). Then, the distillation step took place in an automatic distillation unit, model UDK-149 (VELP SCientifica, Usmate Velate, Italy) with sodium hydroxide 32% and boric acid prepared in a solution at 3%. The titration was carried out with 0.01 M HCl in an automatic titrator, model TitroLine (VELP SCientifica, Usmate Velate, Italy). Crude protein content (%) was calculated according to the following formulas:

$$\mathrm{Kjeldahl} \mathrm{Nitrogen} \left(\%\right)=\frac{\left(\mathrm{VS} – \mathrm{VB}\right)\times \mathrm{M} \times 14.01}{\mathrm{W}\times 10}$$

(2)

Protein Content (%) = % Kjeldahl Nitrogen × 6.25

(3)

where VS = volume (mL) of standardized acid used to titrate a sample; VB = volume (mL) of standardized acid used to titrate reagent blank; M = molarity of standard HCl; 14.01 = atomic weight of N; W = weight (g) of sample; 10 = factor to convert mg/g to percent; and 6.25 factor to convert N to protein.

2.3. Physicochemical Analysis

2.3.1. Acidity Index (IA)

IA was determined according to Method: NF EN ISO 660—September 2009, by the titration of a solution of oil in ethanol with ethanolic KOH and is expressed as (mg KOH/g oil).

2.3.2. Peroxide Index (IP)

IP was determined according to Method: NF ISO 3960—March 2004 and is expressed in milliequivalents of active oxygen per kilogram of oil (meq O₂/kg).

2.3.3. Refractive Index (IR)

IR was determined according to Method: ISO 6320—2017, by using Abbe refractometer, model BK-R2S (BIOBASE, Jinan, China).

2.3.4. Iodine Value (IO)

IO was expressed as (g of I₂/100 g of oil); it was determined by treatment with Wij’s reagent followed by titration of the iodine excess with Na₂S₂O₃ according to Method: ISO3961—2018.

2.4. Biochemical Analysis

2.4.1. Phenol Extraction

The extracts used to determine the total phenolic content, total flavonoid content, and antioxidant activity were obtained according to the method described by Tsimidou et al. [18]. Two grams of oil sample was dissolved in 10 mL of hexane and then added to 4 mL of methanol 60% (v/v). The mixture was subjected to agitation for 2 h at room temperature in darkness and then the supernatant collected was filtered through Whatman No. 1 filter paper (Whatman International, Brentford, UK). All extractions were performed in triplicate. The three filtrates were combined, washed with 10 mL of hexane, and stored at 4 °C until analysis.

2.4.2. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu colorimetric method described by Singleton et al. [19] with slight modifications. Briefly, 50 μL of the extract was mixed with 3 mL of distilled water, 250 μL of Folin–Ciocalteu reagent, and 750 μL of sodium carbonate (7%). The mixture was stirred for 8 min at room temperature, and then 950 μL of distilled water was added. After 2 h of incubation in darkness, the absorbance was measured at 765 nm using UV-Vis spectrophotometer model V-530 (Jasco, Groß-Umstadt, Germany) against a blank. Gallic acid was used as a standard for the calibration curve. The results are expressed as milligram equivalent of gallic acid (GAE) per 100 g of oil.

2.4.3. Total Flavonoid Content (TFC)

TFC was determined using the method described by Favati et al. [20]. A volume of 0.5 mL of the sample extract was mixed with 0.5 mL of the 2% aluminum chloride MeOH solution. After 15 min at room temperature, the absorption was measured at 430 nm for which MeOH was used as a blank sample.

2.4.4. Total Carotenoid Content (TC)

TC was photometrically analyzed with a UV-Vis spectrophotometer model V-530 (Jasco, Groß-Umstadt, Germany) according to the BSI method reported by Guizhen et al. [21]. Hence, 2 g of the extracted oils was diluted in 2 mL of cyclohexane and the mixture was measured at 445 nm. The absorption of cyclohexane served as a blank of which the optical density was deducted from the samples’ absorption. Carotenoid content (mg/kg) was calculated according to the formula:

Carotenoid Content = 383 × A445/L × C

(4)

where A is the absorbance at the indicated wavelength (445 nm), L represents the length of the cuvette in cm (1 cm), and C is the oil concentration in cyclohexane (g/100 mL).

2.4.5. Free-Radical Scavenging Activity (FRSA)

FRSA was determined following the DPPH (2,2-diphenyl 1-picrylhydrazyl) method originally described by Brand-Williams et al. [22] with slight modifications. An amount of 50 μL of oil extract was mixed with 950 μL of DPPH methanolic solution (0.030 mg mL⁻¹). After 60 min of incubation in darkness, the mixture absorbance was measured at 515 nm against a reagent blank (ultrapure water instead of the sample). The formula [(Ac−Ae)/Ac] × 100 was used to calculate the percentage of the free-radical inhibition, where Ac is the absorbance of the control and Ae is the absorbance of the sample.

2.4.6. The Half-Maximal Inhibitory Concentration (IC50)

IC50 was determined as the minimum concentration of sample extracts necessary for 50% of the DPPH radical scavenging potency, and is expressed as a percentage. Thus, a lower IC50 value indicates higher DPPH radical scavenging activity. All measurements were performed in triplicate.

2.4.7. Fatty Acid Composition (FA)

FA was determined using capillary gas chromatography (CGC) following the ISO standard (ISO 12966-2, 2017) gas–liquid chromatography analyses. Fatty acids were first converted into fatty acid methyl esters (FAMEs) by mixing 60 mg of oil and 3 mL of hexane with 0.3 mL of 2 N methanolic potassium hydroxide, and all were shaken continuously for 25 min. FAs were carried out using a Varian CP 3380 Chromatograph (VARIAN, Palo Alto, California, United States), with a capillary column (CP-Wax 52 CB: L = 25 m; Φ = 0.25 mm; Ft = 0.20 μm), using a split–splitless injector equipped with a CP-8400 autosampler and an FID detector. The chromatographic parameters were as follows: the temperature of the injector was 220 °C, the detector temperature was 230 °C, and the oven was held at 190 °C. The carrier gas was nitrogen. The injected volume was 1 μL with a split ratio of 1:50. Each sample was analyzed in triplicate and the results are expressed as the relative percentage of each fatty acid, calculated by the internal normalization of the chromatographic peak area. The calibration and identification of the FAME were carried out using the FAME reference standard mixture (C4–C24, FAME Mix 37) based on their retention times.

2.5. Statistical Analysis

For each experimental site, three random samples per replication (block) and per variety were considered, which corresponded to a total of nine replicates per variety. Statistical analyses were performed using the software IBM SPSS Statistics V21. All analyses were conducted on multiple-test ANOVA followed by Duncan’s multiple range test (DMRT) to examine significant differences among the studied varieties and between the two environments (sites). Mean differences were considered significant at the p < 0.05 level.

Principal components analysis (PCA) was also used to determine the most discriminating variables among those herein studied and then to classify the investigated varieties based on their similarities in response to the environmental factor.

3. Results and Discussion

3.1. Seed Yield, Oil Content, and Total Protein Content

The seed yield (q/ha) of the six Moroccan rapeseed varieties is shown in

Table 2. Variation in seed yield, oil content, and other physicochemical parameters in six Moroccan rapeseed varieties evaluated in two contrasting environments.

		Parameters
		Seed Yield (q/ha)	Oil Content (%)	Protein Content (%)	Acidity Index (mg KOH/g)	Peroxide Index (meq O2/kg)	Refractive Index	Iodine Value (g I2/100 g)
Rapeseed Varieties	Adila	11.4 ± 3.0 b	38.9 ± 1.4 a	25.75 ± 1.42 a	2.1 ± 0.14 a	2.8 ± 0.6 a	1.469 ± 0.006 a	100.01 ±5.8 c
	Alia	16.2 ± 6.3 a	37.9 ± 2.1 a	24.17 ± 1.01 a,b	2.1 ± 0.11 a	2.3 ± 0.6 a	1.467 ± 0.013 a	97.37 ± 4.0 b
	Baraka	10.9 ± 4.5 b	37.5 ± 2.2 b	25.18 ± 1.46 a	2.1 ± 0.14 a	2.6 ± 0.7 a	1.466 ± 0.010 a	99.48 ± 3.1 c
	Lila	12.7 ± 3.4 b	37.9 ± 1.8 a	25.29 ± 1.31 a	2.2 ± 0.36 a	2.5 ± 0.5 a	1.467 ± 0.010 a	98.43 ± 4.5 b,c
	Moufida	17.1 ± 6.9 a	39.9 ± 2.4 a	25.93 ± 1.95 a	2.0 ± 0.14 a	2.3 ± 0.3 a	1.468 ± 0.007 a	94.13 ± 6.3 a
	Narjisse	15.1 ± 4.0 a	36.7 ± 2.6 b	21.26 ± 1.04 b	2.5 ± 0.14 a	3.2 ± 0.7 a	1.465 ± 0.009 a	94.19 ± 4.6 a
Codex Limits CXS 210-1999 (Amended in 2019)		-	-	-	Virgin oils: 4.0 mg KOH/g Oil	Virgin oils: up to 15 milliequivalents of active oxygen/kg oil	Refractive index (ND 40 °C) of Rapeseed oil: 1.465–1.469	Iodine value of Rapeseed oil: 94–120
Environment (E) effect p-value		<0.001	<0.001	<0.001	0.225	<0.001	<0.001	<0.001
Variety (V) effect p-value		<0.001	<0.001	<0.001	0.059	0.313	0.733	<0.001
E × V interaction effect p-value		<0.001	<0.001	<0.001	0.069	0.227	0.199	<0.001

Values are given as means of eighteen replicates of the two environments ± standard deviation. Mean values followed by the same small letters in a column are not significantly different at p < 0.05 according to Duncan’s multiple range test.

. Statistical analysis revealed a highly significant difference (p < 0.001) among the varieties and the overall mean values. In fact, seed yield ranged from 10 to 17 q/ha. The highest seed yield (17 q/ha) was recorded in ‘Moufida’, while the lowest (10 q/ha) was observed in ‘Baraka’. Under the favorable Sidi Allal Tazi conditions, the variety ‘Moufida’ produced 23.5 q/ha (Figure 1A). According to Nabloussi et al. [15], this variety has a yield potential that exceeds 35 q/h. In Canada, the seed yield of oilseed rape varies between 27 and 35 q/h [23], while in China, the top national average yield is 27.3 q/ha [24]. This indicates that Moroccan varieties display a comparable yield to those of other countries.

Seed yield per plant is one of the main yield components that are greatly influenced by genotype, environment, and their interaction [13,25,26]. In this study, the growth environment markedly influenced the seed yield. Overall, the studied varieties exhibited higher performance under Allal Tazi conditions than Douyet (17 q/ha vs. 10 q/ha) (Figure 1D). For the variety ‘Moufida’, the propitious conditions in Sidi Allal Tazi (ATZ) relative to Douyet (DYT) generated a 12.5% increase in seed yield (Figure 1A).

For the seed oil content (%), the mean values registered in the six Moroccan rapeseed varieties are presented in Table 2. There were significant differences among these varieties, with a variation between 36.7% and 39.9%, which is in agreement with findings of previous studies [17,27,28,29]. By changing their growth environment, the investigated varieties exhibited higher oil content under ATZ conditions than DYT (39.6 vs. 37.3% on average) (Figure 1D). Under ATZ conditions, the variety ‘Moufida’ recorded the highest seed oil content (40.7%), followed by ‘Alia’ with 39.7%, while the lowest value was found in the variety ‘Narjisse’ (35.8%) (Figure 1B).

Regarding seed yield and seed oil content, our findings are in agreement with the available literature [29,30], demonstrating that both traits are quantitative with low heritability, which make them highly influenced by environmental conditions. In a more recent study, the environmental main effect represented 56.41% of the total variation in oil content, while the effect of genotype explained only 15.50% of this variation [2]. Moreover, the quality of fats and oils is dictated by several physical and chemical parameters that are closely linked to the source of oil, namely geographic, climatic, and crop management variables, as well as processing and storage conditions [31]. The results, as shown in Figure 1A,B, indicate that the influence of the environmental conditions was not similar among the varieties. As mentioned above, ATZ’s environment was more favorable than the DYT one in which a decline in seed yield, from 2.1% in the variety ‘Baraka’ to 12.5% in ‘Moufida’, was recorded (Figure 1A). However, the variety ‘Moufida’ maintained the greatest yield, regardless of the growing location, indicating its higher performance compared to the rest of varieties. Even though ‘Baraka’ was the most stable, with the smallest percentage decrease, it exhibited the lowest average seed yield across both environments. For seed oil content, the observed decline varied from 1.6% in ‘Moufida’ to 2.7% in ‘Adila’ (Figure 1B). Besides being the most stable, ‘Moufida’ was the richest in terms of this important trait over both environments.

Similarly to seed yield and oil content, protein content was significantly influenced by the genotype and the growing environment (Table 2). The highest protein content (25.93%) was recorded in ‘Moufida’, while the lowest one (21.26%) was observed in ‘Narjisse’. According to several previous studies [2,32,33,34,35], protein percentage in rapeseed ranges from 16 to 27%, which indicates that Moroccan varieties are very interesting for this trait and, hence, may be a beneficial and valuable source for the animal feed industry. Nevertheless, the total protein content in these varieties was found to be markedly affected by the environment. In fact, the overall value registered in Sidi Allal Tazi was 28%, which was significantly higher than the 22% recorded in Douyet (Figure 1D). The decline observed in the different varieties varied from 3.9% in ‘Alia’ to 9.2% in ‘Moufida’ (Figure 1C). Protein content in rapeseed could be influenced by several factors including the method of analysis, nitrogen fertilization, harvest management, and environmental conditions during the seed development and filling stage [2,36,37,38]. In particular, moderately high temperature during this stage causes an increase in protein content [35,39,40]. By comparing Sidi Allal Tazi and Douyet for the average temperatures recorded in the April–May period, which corresponds to seed development and filling, one could notice moderately higher temperatures in Sidi Allal Tazi than Douyet (Table A1), which might explain the variation in protein content between both the environments.

3.2. Physicochemical Analysis

Table 2 presents the physicochemical traits investigated on the six varieties studied herein. According to the CODEX ALIMENTARIUS (STANDARD FOR NAMED VEGETABLE OILS-CXS 210-1999—Revised and amended in 2019) normalizing quality characteristics of vegetable oils, our results showed that all studied varieties comply with all rapeseed oil limits.

Moreover, the statistical results showed that all varieties were comparable for acidity index, peroxide index, and refractive index, while they displayed significant differences regarding iodine value (Table 2). Once again, the variety ‘Moufida’ was most interesting, showing the lowest iodine value. According to Knothe et al. [41], the iodine value has been used beyond analytical purposes, as it was related to various physical and chemical properties. In addition, it served as a quality-control method in hydrogenations and is occasionally used in assessing the oxidative stability of oils and fats and their derivatives. Since we have found a significant variation among varieties for this parameter, this means that the variety ‘Moufida’ could reveal the best oxidative stability.

No significant effect of the interaction variety x environment was observed for the acidity index, peroxide index, and refractive index (Table 2). This could indicate that these three parameters are oil-quality characteristics that are mainly related to extraction conditions and not to the genotype or its interaction with the environmental conditions.

3.3. Biochemical Analysis

Table 3. Variation in the biochemical oil parameters of six Moroccan rapeseed varieties evaluated in two contrasting environments.

		TPC * (mg GAE/100 g)	TFC * (mg/100 g)	TC * (mg/kg)	FRSA * (%)	IC50 * (%)
Rapeseed Varieties	Adila	4.07 ± 0.3 a	17 ± 0.16 b	18.78 ± 4.2 a	24.51 ± 7.7 d	31.38 ± 5.1 b
	Alia	4.09 ± 0.3 a	29 ± 0.12 a	15.29 ± 1.9 b	35.72 ± 6.8 b	34.48 ± 2.3 a
	Baraka	4.14 ± 0.4 a	16 ± 0.17 b	21.45 ± 5.3 a	30.94 ± 6.3 c	31.62 ± 1.3 b
	Lila	4.00 ± 0.2 a	20 ± 0.19 b	16.54 ± 6.2 a,b	22.74 ± 8.4 d	28.84 ± 6.5 d
	Moufida	4.16 ± 0.4 a	33 ± 0.30 a	11.35 ± 6.0 c	42.85 ± 6.6 a	32.89 ± 2.3 a
	Narjisse	4.06 ± 0.3 a	29 ± 0.14 a	21.54 ± 5.5 a	29.59 ± 7.1 c	30.25 ± 6.2 c
Environment (E) effect p-value		<0.001	<0.001	<0.001	<0.001	<0.001
Variety (V) effect p-value		0.890	<0.001	<0.001	<0.001	<0.001
E × V interaction effect p-value		0.225	<0.001	<0.001	<0.001	<0.001

Values are given as means of the two environments ± standard deviation. Mean values followed by the same small letters in a column are not significantly different at p < 0.05 according to Duncan’s multiple range test. * Total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TC), free-radical scavenging activity (FRSA), half-maximal inhibitory concentration (IC₅₀).

presents average values of the biochemical attributes for the six varieties over the both environments. The results showed significant differences among varieties for total flavonoid content, total carotenoid content, free-radical scavenging activity, and half-maximal inhibitory concentration. However, the investigated varieties were comparable for total phenolic content (TPC).

The TPC found in the studied varieties varied between 4 and 4.16 mg GAE/100 g. It differs according to the oil and its value is usually between 1 and 3 mg/100 g [1]. For the rapeseed oil, it was reported that TPC varied from 1.35 mg GAE/100 g to 4.35 mg GAE/100 g [17,42,43]. Our results demonstrate that the Moroccan varieties show a higher phenolic content that can contribute to the antioxidant effect and appear to be beneficial for shelf life and oil stability [44]. In another work, it was found that the TPC in sesame oil was about 0.26 mg GAE/100 g, indicating that rapeseed holds a much higher level of total phenols compared to sesame [45]. Moreover, it was also indicated that the TPC in rapeseed flour is much higher than other oleaginous seeds, exceeding about 28 times the level of phenolics in soybean flour [46].

The statistical analyses indicate that the studied varieties were comparable for TPC, while the environment’s main effect on this parameter was highly significant (Table 3). Other previous studies have also shown that the environmental effect had a significant impact compared to that of the genotype on some oil quality in canola seeds including the TPC [14,30,39]. It was reported also that only a small variation attributed to the effect of the genotype [17,43].

Typically, when discussing phenolics in plant foods, flavonoids are the predominant class described because they account for approximately two-thirds of the dietary phenols [46]. Therefore, in our study, we intended to study the total flavonoid content (TFC) also. The results showed significant differences among varieties. The TFC average varied from 16 mg/100 g in ‘Baraka’ to 33 mg/100 g in ‘Moufida’. The TFC in some cold-pressed seed oils (soybean, sunflower, rapeseed, corn, flax, and rice bran) exhibited a variation that ranged from 12.7 to 25.6 mg/100 g, which confirms the relevance and the good performance of the Moroccan varieties studied herein, in general, and ‘Moufida’ in particular. The environment main effect on this parameter was highly significant and, according to Figure 2, TFC increased from 21 to 27 mg/100 g.

Regarding the total carotenoid content (TC), the results show a variation from 11.35 mg/kg in ‘Moufida’ to 21.54 mg/kg in ‘Narjisse’, which is much higher than the one from 0.5 to 15.2 mg/Kg detected in two cold-pressed rapeseed oils [47]. Moreover, the color changes observed in the oil models (butter, rapeseed oil, and their blends) are closely related to changes in carotenoid levels [48]. Therefore, TC may present an important parameter that could help consumers with their oil selection as it affects nutritional and sensorial-quality parameters.

The studied varieties showed an FRSA variation ranging from 22.7% in ‘Lila’ to 42.9% in ‘Moufida’. However, in another Moroccan work, designed to develop rapeseed mutants with improved fatty acid composition and increased levels of polyphenols and antioxidant capacity, the FRSA varied from 40.5 to 59.28% [17]. It was confirmed that the antioxidant activity of rapeseed oil extracts scavenged 51.2% of DPPH radicals [43]. In the present study, we found that both the variety and environment significantly affected FRSA, which was in accordance with Kraljić et al. who demonstrated that both rapeseed cultivars and storage conditions significantly affected the reduction in the amount of DPPH radicals [6]. Therefore, the small differences between our results and those mentioned above may be owed to the studied genotypes and environmental conditions (geographic, climate, storage, and analysis conditions).

The relationship between total phenolic content, total flavonoid content, total carotenoid content, and DPPH scavenging activity was performed using the Pearson correlation coefficient. Pearson’s correlation coefficients among the evaluated parameters are shown in

Table 4. Correlation coefficients among characters of biochemical properties of six Moroccan rapeseed varieties.

	TPC (mg GAE/100 g)	TFC (mg/100 g)	TC (mg/kg)	FRSA (%)	IC50 (%)
TPC (mg GAE/100 g)	1	0.251 **	0.324 **	0.342 **	−0.337 **
TFC (mg/100 g)			0.192 *	0.470 **	−0.532 **
TC (mg/kg)				0.294 **	−0.265 **
FRSA (%)					−0.763 **
IC50 (%)					1

* Correlation is significant at the level p < 0.05. ** Correlation is significant at the level p < 0.01. Total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TC), free-radical scavenging activity (FRSA), half-maximal inhibitory concentration (IC₅₀).

. As expected, TPC, TFC, and TC were positively and significantly (p < 0.01) correlated with the antioxidant capacity (FRSA). Phenolic acids naturally present in plants play an important role in food preservation and supplementation due to their antioxidant potential [49]. They have been reported to exhibit a potent antioxidant effect and to scavenge free radicals through hydrogen or electron donation [17]. In the case of rapeseed, high phenolic content is an important factor in determining the antioxidant capacity. Concordantly, a significant correlation between antioxidant capacity using different assays (DPPH, FRAP, and ORAC) and TPC in seven winter rapeseed varieties has been previously documented by Szydłowska-Czerniak et al. [50]. Another study, conducted on Brassica napus L. Var. Oleifera oil, revealed a positive correlation between the total phenolic and flavonoid content under different conditions [51], which is corroborated by our findings displayed in Table 4. Moreover, a negative and highly significant correlation was observed between the IC₅₀ values of DPPH, TPC, TFC, and TC (Table 4). This suggests the intervention of flavonoids and phenolic acid in the radical scavenging activity of the examined extracts. Besides flavonoid and phenolic compounds, the most likely suggestion of the shown correlation may be related to the presence of other antioxidant compounds that could be involved in the observed free-radical-scavenging effect such as terpenoids, ascorbic acid, and other micro-/macro-antioxidant nutrients.

The edible and processing quality of rapeseed oil is fundamentally related to the fatty acid composition of triacylglycerol lipids in the seeds [52].

Table 5. Fatty acid composition (% of total fatty acids) in seed oil of six Moroccan rapeseed varieties evaluated in two contrasting environments.

Variety	C16:0	C16:1	C18:0	C18:1	C18:2	C18:3	C20:0	C20:1	Ratio ꞷ-6/ꞷ-3
Adila	4.08 ± 0.2 a,b	0.19 ± 0.04 b	1.60 ± 0.2 c	62.76 ± 1.1 b	20.20 ± 1.1 b	9.61 ± 0.6 b	0.46 ± 0.21 a,b	1.11 ± 0.3 b,c	2.11 ± 0.2 b
Alia	3.87 ± 0.1 c	0.22 ± 0.04 b	1.79 ± 0.1 a,b	62.79 ± 1.9 b	19.20 ± 0.9 c	10.78 ± 1.3 a	0.42 ± 0.15 b	0.93 ± 0.2 d	2.26 ± 0.2 a
Baraka	3.95 ± 0.3 b,c	0.22 ± 0.02 b	1.55 ± 0.3 c	61.77 ± 1.7 b	21.59 ± 1.6 a	9.47 ± 0.6 b,c	0.41 ± 0.20 b	1.05 ± 0.1 c,d	2.15 ± 0.2 b
Lila	4.15 ± 0.2 a	0.20 ± 0.03 b	1.66 ± 0.2 b,c	62.38 ± 1.0 b	20.98 ± 1.0 b	9.28 ± 0.3 b,c	0.43 ± 0.15 b	1.02 ± 0.3 c,d	2.09 ± 0.2 b
Moufida	3.90 ± 0.2 c	0.25 ± 0.04 a	1.81 ± 0.2 a	64.67 ± 2.1 a	18.66 ± 1.0 c	9.01 ± 1.1 c	0.45 ± 0.16 a,b	1.25 ± 0.2 a,b	2.29 ± 0.1 a
Narjisse	4.17 ± 0.3 a	0.21 ± 0.06 b	1.74 ± 0.2 a,b	61.87 ± 1.9 b	20.56 ± 1.4 b	9.56 ± 0.4 b,c	0.56 ± 0.24 a	1.34 ± 0.3 a	1.80 ± 0.1 c

Values are given as means of the two environments ± standard deviation. Mean values followed by different small letters in a column are significantly different at p < 0.05 according to Duncan’s multiple range test.

presents the fatty acid (FA) composition of the studied rapeseed varieties. The results show that the erucic acid (C22:1) was not detected in these varieties, which confirms they are all canola. There were significant differences among the varieties for all fatty acid content and the ratio of omega6/omega3 (ω-6/ω-3). Oleic acid (C18:1) was the predominant FA among all of them and ranged from 60.98% in ‘Baraka’ to 65.37% in ‘Moufida’. One of the main traits that enhances oil quality is low levels of polyunsaturated fatty acids with their substitution by monounsaturated. In fact, high C18:1 content in rapeseed oil is desirable for long-shelf life and stability under high temperature during deep frying [53]. On the other hand, linolenic acid (C18:2) is a highly unsaturated fatty acid and its presence in high levels makes the oil sensitive to oxidation in air and unstable during frying. Therefore, a reduced linolenic acid level in rapeseed oil, associated with an elevated oleic acid level, is beneficial for storage and the extension of the shelf life of this oil [1,52]. Moreover, FAs are widely reported for their natural preventive function in cardiovascular diseases and effective plasma-cholesterol-lowering activity, and are well-known for their ability to reduce total and LDL cholesterol, with a minor reduction in HDL cholesterol [6,8,17]. Being superior in C18:1 and inferior in C18:2, the variety ‘Moufida’ may present an interesting source of edible oil with a possibly high-standard rapeseed oil quality, which may contribute to a health-promoting human diet.

The omega6/omega3 ratio (ω-6/ω-3) is considered an important index in the nutritional lipid quality and it is usually recommended to approach 4:1 [13]. In this study, the studied varieties exhibited a significant variation in this ratio, ranging from 1.8% found in ‘Baraka’ to 2.29% observed in ‘Moufida’ (Table 5). It was reported that the ω-6/ω-3 ratio in rapeseed-mustard oil is 1.14 which is the best one in comparison to some other major oilseeds (64 for groundnut and 132 for sunflower) [54]. In our case, ‘Moufida’ and ‘Alia’ were found to meet the recommended specification more compared to the other varieties. Generally, most of the vegetable seed oils presently in use fall short of these recommendations; they are usually blended to obtain a healthy oil mixture [55].

With the increasing effects of climate change that alter crop-growing conditions, we have to assess and monitor such effects not only on yield but also on quality. This is particularly true for oilseed crops. According to this study (Figure 2), we have demonstrated that all oil-quality parameters have been increased in the best environment (Sidi Allal Tazi). In fact, by changing their growth environment, the planted varieties exhibited higher performance in terms of TPC (4.2 vs. 3.9 mg GAE/100 g), TFC (27 vs. 21 mg/100 g), TC (20 vs. 15 mg/kg), FRSA (40.3 vs. 21.8%), and ω-6/ω-3 ratio (2.13 vs. 2.09). Many authors have demonstrated that environmental conditions and their interaction with plant genotypes play an important role in the final expression of quality attributes. The most influencing environmental factors are temperature and water availability, in addition to other ones, such as soil type and pH value [2,13,14,29,30,49].

3.4. Principal Component Analysis

Table 6. Eigenvalues and results of the principal component analysis (for the first three PC) of the quality traits in Moroccan rapeseed varieties.

	Components
Variables	PC1	PC2	PC3
Seed yield	0.354	0.028	0.697
Oil C *	0.420	0.144	0.596
IA *	0.666	0.271	−0.499
IR *	0.657	0.276	−0.511
IP *	0.591	0.051	−0.446
IO *	−0.553	0.023	−0.086
TPC *	0.242	−0.115	0.794
TFC *	−0.418	0.596	0.041
TC *	−0.320	0.597	−0.317
FRSA *	−0.124	0.845	−0.257
IC50 *	0.148	0.608	−0.338
Ratio omega 6/omega 3	−0.266	0.603	0.267
Variance (%)	26.19	23.70	15.88
Cumulative variance (%)	26.19	49.89	65.77

Eigenvectors greater than |0.5| are marked in bold. * Oil content (Oil C), acidity index (IA), refractive index (IR), peroxide index (IP), iodine value (IO), total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TC), free-radical scavenging activity (FRSA), half-maximal inhibitory concentration (IC₅₀).

presents the first three principal components that accounted for 65.77% of the total variance (PC1 = 26.19%, PC2 = 23.70% and PC3 = 15.88%, respectively).

PC1 was defined by IA (0.666), IR (0.657), IP (0.591), and IO (−0.553) which means that the first component round lines are based on the parameters linked to the extraction conditions (Table 6). Then, PC2 was correlated to TFC (0.596), TC (0.597), FRSA (0.845), IC₅₀ (0.608), and ratio ω6/ω3 (0.603) which indicates that the second component separates lines for their biochemical parameters first, and then for the FA composition. Finally, the PC3 was highly contributed by seed yield (0.697), oilseed content (0.596), and total phenolic content (0.794).

The 3D plot was made based on PC1, PC2, and PC3 to assess the Moroccan rapeseed variety distribution (Figure 3). First, PCA was performed to determine the variables capturing higher score in plant material discrimination. Second, the 3D plot displays the classification of surveyed varieties based on their similarity or their performances in lipochemical and phenolic traits and then the influence of the environment on the revealed patterns. Checking the 3D plot, over both growing environments, the varieties ‘Moufida’ and ‘Alia’ were distinguished from the others, based on their high levels of biochemical parameters TPC, TFC, FRSA, IC₅₀, and ratio of ω6/ω3, in addition to seed yield and oil content. In the third position, we found the varieties ‘Adila’ and ‘Lila’, while ‘Narjisse’ and ‘Baraka’ were the last ranked. The synthetic varieties, ‘Adila’ and ‘Lila’, found in the same class/group were very close to each other in both environments, particularly in ATZ. This can be explained by their similar pedigree as mentioned above. For the rest of the groups, no relatedness or kinship among the investigated varieties was observed.

4. Conclusions

Significant differences were found among varieties and between their growth environments for most of the quality attributes studied. The environmental conditions and their interaction with genotype determine the final expression of these attributes. The investigated varieties exhibited higher performance under Allal Tazi conditions than those of Douyet in terms of seed yield, oil content, protein content, IP, IR, IO, TPC, TFC, TC, FRSA, and ω-6/ω-3 ratio. By comparing the seed yield, oil content, protein content, and all the physicochemical and biochemical parameters, we can conclude that the varieties ‘Moufida’ and ‘Alia’ are the most interesting, having exhibited the best performances. In particular, ‘Moufida’ was able to maintain its top performance through both experiment environments. Therefore, it can be proposed as a promising variety suited to the Allal Tazi area or other similar regions to contribute to the promotion and development of rapeseed crop. This will actually represent an asset for the development of the Moroccan oilseed sector.