All solvents and standards used in the experiments were purchased from Fisher Scientific Company (Ottawa, Ontario, Canada).

The seeds of flaxseed (L. usitatissimum L.) variety, ‘‘I61’’ were supplied by the “Institut National de la Recherche Agronomique de Tunis” (INRAT, Tunisia). Whole flaxseeds (400 g) were germinated on sterile filter paper in Petri dishes. The dishes were incubated for 4 days in the dark at room temperature (20 ± 2°C). The germinated flaxseeds were dried in a dryer at 40 °C and were ground and packed in air tight bags for further analysis.

The dry matter contents of flaxseed were determined by drying in an oven at 105 °C for 24 h to constant weight (AOAC, 1990). The crude protein contents were calculated from the nitrogen contents (N × 6.25) obtained using the Kjeldahl method by AOAC (1990). The crude fat contents were determined by continuous extraction in a Soxhlet apparatus for 5 h using petroleum ether as solvent (AOAC, 1990). The total ash contents were determined by incinerating flaxseed (2 g) in a furnace at 550 °C for 6 h, then weighing the residue after cooling to room temperature in a desiccator (AOAC, 1990). The carbohydrate contents were determined by difference which is by deducing the mean values of other parameters that were determined from 100.

The quantification of fatty acid methyl esters was performed using a gas chromatography-flame ionization detection (GC–FID) apparatus. Fatty acid methyl esters were prepared by simultaneous extraction and methylation following the procedure described by Metcalfe et al. (1966) modified by Lechvallier (1966). Methyl esters were analyzed by GC, using an HP 4890 gas chromatograph equipped with an FID detector on a capillary column coated with Supelcowax^TM 10 (30 m long × 0.25 mm i.d., and 0.2 µm film thickness). Helium was used as the carrier gas at a flow rate of 1mL·min⁻¹. The temperatures of the column, detector, and injector were 200, 250, and 230 °C, respectively. The identification of the peaks was achieved by retention times by means of comparing them with standards analyzed under the same conditions. The area under each peak was measured and the percentage expressed in regards to the total area. To evaluate the efficiency of the desaturation pathway during the maturation process (Mondal et al., 2010) the desaturation ratios from oleic to linoleic (ODR, oleic desaturation ratio) and from linoleic to linolenic acid (LDR, linoleic desaturation ratio) were calculated as follows:

ODR = [% C18: 2 + % C18: 3 / % C18: 1 + % C18: 2 + % C18: 3] × 100

LDR = [% C18: 3 / % C18: 2 + % C18: 3] × 100

The magnitude of desaturation ratios represents the amount of substrate which is successfully desaturated from C18:1 to C18:2 and C18:3, thus providing a proportional measure of the desaturating enzyme activities during seed germination. The Cox value of the oils was calculated based on the percentage of unsaturated C18 fatty acids, applying the formula proposed by Fatemi and Hammond (1980):

Cox value = [1 (18: 1%) + 10.3 (18: 2%) + 21.6 (18: 3%)] / 100

Determinations of Saponification value (SV), Acid value (AV), Free fatty acids (FFA), Iodine value (IV), p-anisidine value (p-AV), Peroxide value (PV), UV absorption characteristics (K₂₃₂ and K₂₇₀), and unsaponifiable matter (UM) of the extracted oil were carried out by standard IUPAC methods for the analysis of fats and oils (Dieffenbacher and Pocklington 1987). Oxidation value (OV) was calculated from Holm's equation, OV = p-AV + 2 (PV), while theoretical flavor scores (F) were obtained from the equation F = 7.7–0.35 (OV) (List et al., 1974). Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland), using an oil sample of 3 g warmed to 100 °C with an air flow of 10 L·h⁻¹.

A 1.5 g sample of germinated flaxseed oil was fully dissolved in 5 mL cyclohexane. Chlorophyll and carotenoid were determined colorimetrically following the method of Minguez-Mosquera et al. (1991). The maximum absorption at 670 nm is related to the chlorophyll fraction and at 470 nm is related to the carotenoid fraction. The values of the coefficients of the specific extinction applied were E₀ = 613 for the pheophytin as a major component in the chlorophyll fraction and E₀ = 2,000 for lutein as a major component in the carotenoid fraction. Thus the pigment contents were calculated as follows:

Chlorophyll (mg·kg⁻¹) = (A₆₇₀ × 10⁶) / (613 × 100 × d)

Carotenoid (mg·kg⁻¹) = (A₄₇₀ × 10⁶) / (2, 000 × 100 × d)

Where A is the absorbance and d is the spectrophotometer cell thickness (1 cm). The data reported is based on oil weight (mg·kg⁻¹ flaxseed oil).

The extraction and determination of total phenolic acid and flavonoid contents were carried out according to the method of Gutfinger (1981).

The Higher Heating Value (HHV), known as the gross calorific value or gross energy, represents the heat released by the oxidation of a fuel in air. The HHV is the amount of heat produced by the complete combustion of a unit quantity of fuel. The HHV of germinated flaxseed oil was calculated from the iodine value (IV) and saponification value (SV) derived using the following formula adopted by Demirbas (1998):

HHV = 49.43-(0.041× SV)-(0.015 × IV)

The cetane number of the oil was determined according to Bose (2009):

CN = 46.3 + 5458/SV–0.225 × IV

The oil obtained was subjected to screening for its possible antioxidant activity. The oil was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay. All the data were the averages of triplicate determinations of three tests. The DPPH free radical-scavenging activity of the oil was measured using the method described by Gorinstein et al. (2004). A 0.1 mM solution of DPPH in methanol was prepared. An aliquot of 0.2 mL of sample was added to 2.8 mL of this solution and kept in the dark for 30 min. The absorbance was immediately measured at 517 nm. The ability to scavenge the DPPH radical was calculated with the following equation:

Inhibition percentage = (I%) = [(A₀-A₁) / A₀] × 100

Where A₀ is the absorbance of the control, A₁ is the absorbance in the presence of sample.

The analyses were performed with three replicates. All data are reported as means ± standard deviation of three samples. Differences were tested for significance using the ANOVA procedure, using a significance level of p<0.05.

The moisture content of flaxseed was significantly (p<0.05) affected by germination (Table 1). It ranged from 5.22% to 11.27% during the germination period. During the study period, the oil content of flaxseed progressively decreased, suggesting that oils are the major source of energy during germination and the early periods of seedling growth (Graham, 2008). The protein content increased during germination and showed no significant difference (p<0.05). The increase in protein content may be attributed to the synthesis of cell constituents and enzymes, which lead to the degradation of other constituents (Lee and Karunanithy, 1990). The ash content remained at the same level before and after germination. During germination, the ash content showed no significant difference (p<0.05). The carbohydrate content increased after 4 days of germination, reaching 37 mg per 100 g, which is significantly (p<0.05) higher than that of sesame (30 mg·100 g⁻¹ ) (Hahm et al., 2009). Hahm et al., (2009) reported that the degradation of reserve nutrients (lipids and carbohydrates) during germination is a process whose essential purpose is to provide the energy required for protein synthesis in plant growth. As shown in Table 1, the ratio between unsaturated and saturated fatty acids (USFA/SFA ratio) and the Cox value of flaxseed were statistically (p<0.05) the same during the germination period. Germinated Flaxseed oil was characterized by its higher polyunsaturated fatty acids (PUFA) (linoleic acid, C18:2) and lower SFA percentages (Palmitic acid, C16:0, and stearic acid, C18:0), which make it particularly prone to oxidation. Indeed, the higher value of LDR indicates higher linolenic acid production during the germination period. This suggests that the biosynthetic pathway of fatty acids is efficient in the formation of linolenic acid, justifying therefore the higher amount of the latter fatty acid. The higher ratio of n-3/n-6 (2.63 vs. 2.87) indicated that flaxseed oil had greater nutritional value.

The physicochemical characteristics of flaxseed oils and their values in the literature are shown in Tables 2A and 2B, respectively. The saponification value is an index of average molecular weight (or chain length) of all the fatty acids present. The observed saponification value increased with germination (182–192 mg KOH·g^–1 oil) and there were statistically significant differences among them (p<0.05). These values indicate the absence of lauric acid in the investigated flaxseed oils, and this range is indicative of oils characterized by medium chain-length FAs. Acid value varied significantly (p<0.05) during the germination period. The lower acidity value of germinated flaxseed oil indicates that the oil has a better quality and longer shelf life. The acid values of all the extracted samples in this study were not different from those reported by Teh and Birch (2013) and Choo et al. (2007), and higher than those determined for hemp oil and canola oil. Peroxide value was found between 1.4 and 2.6 (mequiv O₂·kg⁻¹ oil) and showed significant differences (p<0.05). These results are in accordance with those reported by Teh and Birch (2013) and Choo et al. (2007) and were not different from the results reported for hemp oil and canola oil. According to the Codex Alimentarius Commission (2006) standard for virgin oils and cold pressed fats and oils, a good quality oil should have a peroxide value of less than 10 mequiv O₂·kg⁻¹ oil. Lower acid and peroxide values have indicated that germinated flaxseed oil was more suitable as an edible oil. The iodine value, which indicates the degree of unsaturation of an oil, decreased during the germination period. The IV (172–160) obtained in this study indicates that the oils contain appreciable level of unsaturated FAs, which is confirmed by the FA profile of the germinated flaxseed investigated. The p-anisidine values of germinated flaxseed oils showed slight increases. These may be attributed to the light increase in carbonyl compounds. A good quality oil should have a p-anisidine value of less than two (Subramanian et al., 2000). All the oil samples from the literature had low p-anisidine values (Table 2B), reflecting the p-anisidine value of a good quality oil. The oxidation value of flaxseed oils ranged from 4.1 to 6.4% and showed no significant difference among all the samples (p>0.05). These relatively higher oxidation values prompted a separate study for the lipoxygenase activity of chickpea oil. These values were similar to those reported in the literature (Table 2B). The values of K₂₃₂ and K₂₇₀ extinction coefficient showed a slight increase during germination. Oil stability decreased during flaxseed germination. This decrease (1.4 h-1.0 h) in stability is explained by the loss of natural antioxidants. The relative decrease in the unsaponifiable content observed during the germination period may possibly be due to the initial loss lipids and other major reserves of the seed. A parallel decrease in the content of triglycerides was also observed. The ascorbic acid of flaxseed increased in the early stages of germination and then remained constant until the fourth day of germination. A significant decrease in total phenolic acid content was observed during germination (108.58-73.11 mg ·g⁻¹). The total phenolic acid content showed no significant difference (p<0.05). The decrease in TPC has also been attributed to enzymatic activity during germination (Gujral et al., 2011). Randhir et al. reported that germination causes a decrease in the total phenolic content in Green mung. All the respective values were similar to those reported from the literature (Table 2B). In addition, these results are lower than those reported for hemp oil and higher than those reported for canola oil. The Total flavanoid content increased to 14.10 mg·g⁻¹ on the second day then dropped down at the end of day 3. The highest level of chlorophyll and carotenoids was detected by the end of the germination period (6.27-7.37 mg·kg⁻¹ oil, respectively). The fuel properties shown in Fig. 1 indicate that there was no significant difference for HHV and the cetane number during germination. The HHV, which is one of the most important properties of a fuel, is the amount of heat released during the combustion of 1 g of fuel to produce CO₂ and H₂O at its initial temperature and pressure. The highest heating value (39.39 MJ·kg⁻¹) was obtained from ungerminated flaxseed oil. The heating values are the same for most of the oils (39–40 MJ·kg⁻¹) except for castor oil (37.3 MJ·kg⁻¹). The cetane number (CN) is one of the most commonly cited indicators of diesel fuel quality, especially the ignition quality (Bamgboye and Hansen, 2008). The highest CN (37.87) was obtained for germinated flaxseed oil (1 day of germination). The values of CN of soybean oil-derived biodiesel ranged from 45 to 60, whereas those of rapeseed oil-derived biofuel ranged from 48 to 61.2 (Bamgboye and Hansen, 2008). Higher CNs are associated with greater combustibility, good ignition, and assist in easy engine starting, low temperature starting, low ignition pressures, and smooth operation with lower knocking characteristics (Aminul Islam et al., 2012). Germinated Flaxseed oils with good physicochemical properties will have potential to be biodiesel feedstocks.

Antioxidant activity is expressed as percent DPPH radical scavenging activity with higher values indicating greater antioxidant activity. The antioxidant activity ranged from 40.14 to 52.48% (Fig. 2) with the highest activity exhibited by ungerminated flaxseed and the lowest exhibited by germinated flaxseed oil at the end of day 3. During germination, the antioxidant activity significantly decreased (p<0.05) up to 3 days of germination and then further increased upon 4 days of germination. The decreasing trend of antioxidant activity during germination is similar to the trend observed in lentils (Lens culinaris) while the increasing trend of antioxidant activity is similar to beans (Phaseolus vulgaris) and peas (Pisum sativum) (López-Amorós et al., 2006). Wong et al. (2006) stated that one of the possible reasons for the decreased value obtained from the DPPH assay for plant samples could be due to the presence of compounds which are not reactive towards DPPH free radicals. Polyphenols may be more efficient as reducing agents in reducing ferric iron but some may not scavenge DPPH free radicals as efficiently due to stearic hindrance. All the samples showed significant difference (p<0.05). The scavenging action of plant constituents has been found to relate to polyphenolic compounds (Siger et al., 2008). Although the constituents of germinated flaxseed oil, which show free radical scavenging action are still unclear, it is possible that the antioxidative activity of germinated flaxseed oil is caused, at least in part, by the presence of polyphenols (Azhari et al.,2014; Anwar et al., 2013; Gujral et al., 2011) and other yet to be discovered antioxidant compounds.