Evaluation of canola seeds of different cultivars with special emphasis on the quantification of erucic acid and glucosinolates

This study reports the characterization of the seeds and seed oils of five locally grown canola cultivars: Zafar-2002, Bulbul (Frontier), Dunkeld, Oscar and Con-11. The oil contents from canola seeds ranged from 34.3 to 39.3%. The levels of protein, fiber, ash and moisture were found to be 22.1-41.0, 12.0-14.0, 3.0-3.5 and 4.0-7.5%, respectively. The glucosinolate (GSL) contents in the canola seeds examined ranged from 49.7 to 78.1 mmol g -1 . The extracted canola seed oils revealed an iodine value of 118.2-124.6 g of I/100g of oil; refractive index (40 °C), 1.460-1.464; density (24 °C), 0.914-0.919 mg mL–1; saponification value, 187-195; unsaponifiable matter, 0.51-1.10%; acidity (% as oleic acid), 0.40-1.40, and color (1-in. cell), 1.35-1.73 R + 21.0-38.0 Y. Peroxide value (meq/ kg of oil) and specific extinctions at 232 and 270 nm were determined to be 2.00-7.08, 2.17-3.16 and 0.44-0.91, respectively. The seed oils of the five canola cultivars mainly consisted of oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids at levels of 49.16-62.14, 14.61, 23.45 and 6.97-9.10%, respectively. The concentrations of palmitic (C16:0), stearic (C18:0), erucic (C22:1) and gadoleic (C20:1) acids ranged from 3.47 to 6.00, 1.51 to 2.10, traces to 13.03 and 1.30 to 10.63%, respectively. A small amount of arachidic acid (20:0) with a contribution below 1% was also detected. The contents of tocopherols (α, γ, and δ) in the canola oils accounted for 77.1-270.3, 191-500, 3.5-15.6 mg kg -1 , respectively.The presence of rather high levels of erucic acid and GSL in the present analysis of canola emphasized the need to further reduce the contents of these two antinutritional constituents in the investigated cultivars.


INTRODUCTION
Supra optimal levels of erucic acid in seed oil and glucosiniolates in seed meal of regular rapeseed have almost prohibited the utilization of this crop from human and animal consumption.Some earlier studies on animals show that erucic acid (C22:1), when consumed in large quantities may adversely affect heart tissues and thus promotes myocardial lesions (Kramer et al., 1973;Clandinin and Yamashiro 1982).Glucosinolates (GSL) and their decomposition products can cause several health hazards in animals, including enlargement of the thyroid, and negative effects on adrenal glands and kidneys (Zukalova and Vasak 2002;Barillari et al., 2005).
Canola, a "double-low" variety of the rapeseed (Brassica napus or Brassica campestris), contains less than 2% erucic acid (C22:1) in the oil and less than 30 micromoles per gram of glucosinolates in the seed meal.Due to these characteristics, it has gained considerable recognition as a potential oilseed crop worldwide.(Hu et al., 1997;Neff et al., 1997).
Currently, canola oil which is generally available in the market contains only traces of erucic acid, 5 to 8% saturated, 60 to 65% monosaturated, and 30 to 35% polyunsaturated fatty acids.Canola oil is the preferred oil for health-conscious consumers because it is the lowest in saturated fat among the major edible oils (Shahidi and Shukla, 1996).Canola oil contains appreciable amounts of antioxidants such as vitamin E, phenolic acids, flavonoids and insoluble condensed tannins, all of which are beneficial to human health (Zambiazi, 1997).Rapeseed/canola seed meal, constituting 50-58% of the weight of seeds is a potential source of essential amino acids like lysine, methionine, cystine, threonine and tryptophan (Sarwar et al., 1984;Larsen and Sorensen, 1985).
In Pakistan, after cotton, rape seed/mustard and canola are the major oilseed crops grown in an area of about 365,000 ha with an annual production of 350,000 tons contributing about 19% to the overall domestic production of edible oils (PARC, 2007).The oil-seed producers are in continuous efforts to develop true canola cultivars with a reduced content of erucic acid and GSL in order to fulfill international standards.A number of diverse canola cultivars are currently under trials in Pakistan for attaining maximum seed oil under the native environmental conditions, but it is not certain whether the canola cultivars being grown have maintained their desired oil characteristics.In view of this, we sought to characterize such canola cultivars being grown under indigenous environmental regimes.The primary objective of the present study was to conduct a detailed analysis and to investigate the physico-chemical characteristics of canola seed and the seed oils of five commonly grown cultivars with special emphasis on quantifying and comparing the contents of the oil, erucic acid and glucosinolates.

Collection of canola seeds
The seeds of five, indigenously grown canola cultivars : Zafar-2002, Bulbul (Frontier), Dunkeld, Oscar and Con-II were obtained from the Ayub Agricultural Research Institute (AARI), Jhang Road, Faisalabad, Pakistan.Three seed samples for each cultivar were assayed.

Moisture determination
Moisture content of the seeds was determined at 105°C according to ISO method 666 (ISO 1977) using an electric oven (VOC-300 SD; EYELA, Tokyo, Japan).

Extraction of oil
The seeds of all five canola cultivars were crushed in a domestic electric grinder.The crushed seed material was conditioned (100°C) in an electric oven (VOC-300 SD; EYELA, Tokyo, Japan) for 30 minutes.After conditioning, the ground seed material (~75 g) was fed into a Soxhlet extractor.The extraction was performed with 300 mL of nhexane (B.P. 68°C) on a water bath for 6 h.The oils extracted were stored in a refrigerator at 4 o C until further analysis.

Determination of glucosinolates in canola seeds
The glucosinolates were determined after their hydrolysis with myrosinase by milling the whole canola seeds separately with water and acidified 40% methanol to liberate bound and free glucose.The extracts were filtered through activated charcoal paper to remove any interfering phenolics.The total glucosinolate contents were estimated following a glucose oxidase/peroxidas calorimetric method (Smith and Dacombe 1987).

Analysis of oilseed residues
After oil extraction, the canola oilseed residues were analyzed for protein, fiber, and ash contents.Protein content (N ϫ 6.25) was determined according to the AOAC method 954.01 (AOAC, 1990).Fiber content was estimated by an ISO method 5983 (ISO, 1977).Briefly, a fat-free, finely ground sample (2.5 g) of meal was weighed and boiled with sulfuric acid (0.255 mol L -1 ), followed by separation and washing of the insoluble residue.The residue was further boiled with sodium hydroxide (0.313 mol L -1 ), followed by separation, washing, and drying.The dried residue was weighed and ashed in a muffle furnace (TMF-2100, Eyela, Tokyo, Japan) at 600°C, and the loss of mass was determined.The determination of ash content was made following the ISO method 749 (ISO, 1977).Two grams of meal were carbonized by heating on a gas flame.The carbonized material was then ashed in an electric muffle furnace (TMF-2100, Eyela, Tokyo, Japan) at 550°C, to achieve a constant mass.

Fatty acid composition
Canola oils were transmethylated according to the standard IUPAC method 2.301 (IUPAC, 1987) and analyzed on a SHIMADZU gas chromatograph model 17-A, fitted with an SP-2330 (SUPELCO Inc.Supelco Park Bellefonte, PA 16823-0048, USA) polar capillary column (30 m ϫ 0.32 mm; 0.20 µm), and a flame ionization detector.Oxygen-free nitrogen was used as a carrier gas at a flow rate of 3.0 mL min -1 .The column temperature was programmed from 180 to 220°C at a linear increment of 5°C min -1 .The initial and final retention times were 2 and 10 minutes, respectively.Injector and detector temperatures were maintained at 230 and 250°C, respectively.A sample volume of 1.0 µL was injected using split mode (split ratio, 1:65).Fatty acid methyl esters (FAMEs) were identified by comparing their relative and absolute retention times to those of authentic standards.A datahandling software ((Data APEX Ltd., Prague, The Czech Republic), Chromatography Station for Windows (CSW32) was used for the quantification.

Tocopherols content
Tocopherols (α, γ and δ) in the oil samples were analyzed by high performance liquid chromatography following Lee et al. (2003).An HPLC (Sykam GmbH, Kleinostheim, Germany) equipped with an S-1121 dual piston solvent delivery system and S-3210 UV/Vis diode array detector was used.One gram of oil was made up to a volume of 10 mL with acetonitrile in a volumetric flask, wrapped in aluminum foil to inhibit photoxidation.20-µL of filtered sample were injected into an analytical Hypersil (Thermo Hypersil, GmbH, Germany) ODS reverse phase (C18) column (250 ϫ 4.6 mm; 5µm particle size) fitted with a C18 guard column.The mobile phase comprising a mixture of HPLC grade methanol and acetonitrile (65:35 v/v) was applied at a flow rate of 1.3 mL min -1 at 30°C under isocratic elution.Detection was made at 292 nm.Tocopherols (α-, γ-, and δ) were identified by comparing the retention times and quantified on the basis of the peak area percent of the unknowns compared with those of pure standards (Sigma Chemical Co., St. Louis, MO).The quantification was done on a computer using SRI peak simple chromatography data acquisition and integration software (SRI Instrument, Torrance, California, USA).

Statistical Analysis
Three canola oilseed samples were assayed for each cultivar and analyzed individually in triplicate.Data is reported as mean (n = 1 ϫ 3 ϫ 3) ± SD (n = 1 ϫ 3 ϫ 3).One way ANOVA was used to determine significant differences among the cultivars considering a level of significance less than 5% (p < 0.05), using the statistical software STATISTICA (StatSoft, OK, USA).

RESULTS AND DISCUSSION
The data for the analysis of seeds and seed oils of five indigenously grown canola cultivars: Zafar-2002, Bulbul (Frontier), Dunkeld, Con-II and Oscar are summarized in Tables 1-5.The hexane-extracted oil content of canola seeds from the five investigated cultivars ranged from 34.3% (Con-II) to 39.3% (Dunkeld) (Table 1).The variation in the oil content within different canola oilseed cultivars may be attributed to their varied genetic make-up, and to different growing and agro-ecological conditions.The range of oil content (34.3-39.3%) in the present analysis of canola oilseeds was comparable to that reported in the literature (Pritchard, 1991).Our findings showed the highest concentration of oil in Dunkeld and the lowest in Con-11, however, Hassan et al., (2005) reported somewhat higher oil contents in Bulbul (Frontier) and lower in Zafar-2002.The variations in the oil contents of different canola cultivars in the present study could be supported from the investigations of Pritchard et al. (2000).
The glucosinolate (GSL) contents of the canola seeds of the different cultivars ranged from 49.7 to 78.1 mmoles g -1 of dry seed weight (Table 1).The lowest content of GSL (49.7) was detected in cultivar Zafar-2002, whereas, that of Con-II offered the highest level (78.1).The present levels of GSL for Oscar, Dunkeld, and Con-II were in agreement with the findings of Hassan et al., (2005), while for Zafar-2002 and Bulbul (Frontier) the present values were quite different.True canola cultivars should have GSL contents less than 30 mmoles/g of airdried meal (Pritchard, 1991).However, the present analysis showed that none of the canola cultivars examined here were a true canola because of the presence of quite high contents of GSL.The significant differences (p < 0.05) of GSL contents among the cultivars examined could be due to the diverse genetic makeup of the cultivars, and to specific agroecological and environmental conditions of the regions.Glucosinolates are antinutritional components present in high amounts in rapeseed/canola meal, but not in considerable amounts in canola oil.Glucosinolate accumulation is known to be affected by water/moisture availability, particularly at the seed development and maturation stages.The glucosinolate levels in seeds can be controlled during growth stages through applications of jasmonic acid (Pritchard et al., 2000;Harbans and Hamama 2003).
The color values of the canola seed oils were found to be 1.35-1.71red units and 21.0-38.0yellow units.Color growth in vegetable oils is mainly due to the occurrence of a variety of pigments such as chlorophyll and carotenoids.Such pigments are efficiently removed using different treatments like refining and bleaching of oils.From the viewpoint of commercial applications, the oils with the least coloration are more appealing.
The peroxide value, which is generally used to express the magnitude of primary oxidation products in oils (McGinely, 1991), ranged from 2.00 to 7.08 meq kg -1 in the canola seed oils investigated (Table 3).The specific extinctions at 232 and 272 nm, which are important indicators of oxidative deterioration of oils (McGinely, 1991; Manzoor et  al., 2007), ranged from 2.17-3.16and 0.44-0.91,respectively (Table 3).There are no previously reported data on these canola cultivars to compare the results of peroxide value and conjugated diene and triene contents with our findings reported here.
The levels of C16:0 and C18:0 in the present analysis of canola seed oils was quite comparable with those for canola oil reported in the literature (Rossell, 1991).The contents of oleic acid (C18:1 n-9), ranging from 49.16-62.14%,varied significantly (p < 0.05%) among the canola cultivars analyzed.The highest amount of oleic acid was detected in Con-11 (62.14%), whereas, the lowest in Bulbul (Frontier) (49.16%).The content of oleic acid in our present analysis was slightly different from that reported by Hassan et al., (2005).However, the present range of oleic acid was in agreement with those reported in the literature (Rossell, 1991;Pritchard et al., 2000).Oleic acid is the most abundant fatty acid in nature and is present in almost all vegetable oils and fats.Oleic acid is gaining importance in human nutrition owing to its potential health benefits against cardiovascular heart diseases and its cholesterol lowering effects (Kris-Etherton 1999;Lovejoy 2002).
The contents of C18:2 n-6 in the tested canola seed oils (14.61-23.45%)Hassan et al., (2005).Both, C18:2 n-6 and C18:3 n-3 are the essential fatty acids that the human body is unable to synthesize.The potential health benefits of essential fatty acids are well documented elsewhere (Markwell et al., 1998;Schaefer 2002).Erucic acid (C22:1) was detected in the range of traces to13.03% with the highest level (13.03%) in Bulbul (Frontier), with the lowest (traces) in Oscar and Con-11.True canola contains less than 2% erucic acid (C22:1).It was observed from the present fatty acid data that only two of the five tested canola cultivars exhibited erucic acid levels below 2% and could therefore be defined as true canola.
The data also revealed significant (p < 0.05) differences among cultivar means for gadoleic acid (C20:1), ranging from 1.30% in Con-11 to 10.63% in Bulbul (Frontier).The exceptionally high amount of gadoleic acid (10.63%) determined in the present analysis of Bulbul (Frontier) as compared to the other canola cultivars tested might be attributed to the presence of higher levels of erucic acid.This fact is supported by the findings of Rossell (1991), who reported higher levels of gadoleic acid (2.6-9.4%) in those canola cultivars, which possess elevated amounts of erucic acid (6.5-51.6%)as compared to those containing low erucic acid (tr-4.7%).
Table 5 presents the levels of tocopherols in the seed oils of different canola cultivars.The contents of α tocopherol, γ-tocopherol, and δ-tocopherol ranged from 77. 1-270.3, 190.6-500.0, 3.5-15.6mg kg -1 of oil, respectively.The concentration of α-tocopherol, which has greater vitamin E effectiveness (Rossell, 1991) was found to be the lowest in Zafar-2000, and the highest in Dunkeld.The amount of γ-tocopherol was noted to be higher in Oscar (500.0 mg kg -1 ) and Con-11 (420.0 mg kg -1 ), whereas Dunkeld offered the lowest level of this isomer of tocopherol (190.0 mg kg -1 ).The concentration of δ-tocopherol having superior antioxidant activity over γor α-tocopherol (Anwar et al., 2006), was quite low (3.5-15.6 mg kg -1 ).No previously reported data on the tocopherol contents of these cultivars of canola seed oils with which to compare the values of the present analysis have been reported.Overall, the tocopherol profiles of Oscar and Con-11 seed oils were quite comparable to those reported for canola oils in the literature (Rossell, 1991).
From the values of erucic acid and GSL contents of the investigated canola cultivars, grown in Pakistan, it is possible to declare that none of the tested cultivars is true canola because the contents of both of these antinutritional components are above the permissible limits as established by current specifications of the Canola Council of Canada.Of course these cultivars are in the developing stage in Pakistan and such variation in the contents of components may be attributed to the varied geographical and cultivation regimes.This necessitates the need to further reduce and control the contents of GSL and erucic acid in these locally grown canola cultivars, where applicable.The present information might be useful in controlling and maintaining the required attributes of growing canola cultivars under indigenous conditions and environment for the on-going breeding programs and for researchers.
± SD) are from three canola seed samples of each cultivar, analyzed individually in triplicate.Mean values followed by different superscript letters in the same row indicate significant difference (p < 0.05) among cultivars ± SD) are of three canola oil samples from each cultivar, analyzed individually in triplicate.Mean values followed by different superscript letters in the same row indicate significant difference (p < 0.05) among cultivars.

Table 3 Oxidation state of canola seed oils from different cultivars
Values (mean ± SD) are of three canola oil samples from each cultivar, analyzed individually in triplicate.Mean values followed by different superscript letters in the same row indicate significant difference (p < 0.05) among cultivars.

Table 4 Fatty acid (FA) composition (g/100g of FA) of canola seed oils from different cultivars
Values (mean ± SD) are of three canola oil samples of each cultivar, analyzed individually in triplicate, (tr traces)?Mean values followed by different superscript letters in the same row indicate significant difference (p < 0.05) among cultivars.