Physicochemical characters of oilseeds from selected mustard genotypes

To evaluate the physicochemical characteristics of oilseeds, three varieties of mustard oilseeds were studied their seed weight, moisture, ash, carbohydrate, protein, fat, total energy and minerals composition of oil and cake. Among the varieties the highest thousand grain weight was found in BARI Sarisha-17 (4.24 g) and the lowest thousand grain weight was found in BARI Sarisha-15 (3.17 g). The highest moisture content was observed from BARI Sarisha-15 (4.52%); whereas the lowest moisture content was found in BARI Sarisha-17 (4.10%). The variety BARI Sarisha-17 contained significantly highest amount of oil (41.98%) whereas the variety BARI Sarisha-16 contained lowest amount of oil (40.95%). BARI Sarisha-16 contained highest amount of ash (14.20%). Significantly highest amount of protein was obtained from BARI Sarisha-15 (28.00%) and the lowest amount of protein obtained from BARI Sarisha-17 (22.57%). BARI Sarisha-17 contained highest amount of carbohydrate (18.85%), whereas BARI Sarisha-15 contained the lowest amount of carbohydrate (13.73%). BARI Sarisha-16 contained highest amount of Ca (2.52%) and Zn (66.80%). BARI Sarisha-15 contained highest amounts Cu (13.40%) and Fe (170.30%). Mustard varieties contained erucic acid (51.35-50.67%). Highest amount of total saturated fatty acid (TSFA) contained in BARI Sarisha15 (9.02%) and the lowest amount contained BARI Sarisha-16 (8.28%). Highest amount of total unsaturated fatty acid (TUSFA) contained in BARI Sarisha-17 (90.26%) lowest amount contained in BARI Sarisha-16 (87.04%). The highest amount of gross energy found from BARI Sarisha-15 (543.60 kcal/g; while lowest amount of gross energy recorded from BARI Sarisha-16 (531.20 kcal/g). Highest saponification value found in BARI Sarisha-17 (159.40) and lowest saponification value was found in BARI Sarisha-15 (157.6). The result of this research work confirms the mustard varieties seed to be of good quality and can be used for food preparation. The knowledge of this study could be utilized for various food preparation and selection for breeding purpose.


Introduction
Oilseed crops have occupied an important place in human nutrition as they remain the major sources of calories and proteins for a large proportion of the world population. Oilseeds are those seeds that contain considerably large amounts of oil. The most commonly known oilseeds (conventional oilseeds) are groundnut, soybean, palm kernel, cotton seed, olive, sunflower seed, rapeseed, sesame seed, linseed, safflower seed, etc (Ajala et al., 2014;Aremu et al., 2006). Generally, oils and fats from seeds and nuts constitute an essential parts of man's diet. The chief important of the vegetable oils lies in their food value. Vegetable oils derived from plant seeds have been playing vital roles to provide comfort in human lives in various aspects, they are essential in meeting global nutritional demands are utilized for many food and other industrial purposes (Idouraine et al., 1996). In a balanced diet for human health 20-25% of calories should come from oils and fats. Herbal oils are the main sources of fats and fat-soluble vitamins, which have a substantial role in the human diet (Stuchlik et al., 2002). After cereals, oily seeds are the second food sources throughout the world, whose oil is of rich fatty acid types (Siavash et al., 2005).
Mustard seeds have been highly prized medicinal as well as culinary spice being in use since ancient times. The seeds are obtained from mustard plant (Brassica spp.) which also include cabbage, broccoli, brussels sprouts, etc. Mustard seeds have high energy content, having 28-32% oil with relatively high protein content (28-36%). Well refined mustard oil has a yellow to brown color and its smell and taste are enjoyable. Mustard oil has a special fatty acid composition: 20-28% oleic acid, 10-12% linolic, 9.0-9.5% linolenic acid and 30-40% erucic acid (Abul-Fadl et al., 2011). The protein quality and quantity of B. campestries obtained oil cake is high (Chowdhury et al., 2014). Mustard seeds contain 28-32% protein by weight, although these values can vary slightly between varieties, growing regions and crop years. The amino acid composition of mustard protein is well balanced; it is rich in essential amino acids. Rapeseed and mustard contain relatively lower amount carbohydrate. It consists mainly glucose, sucrose and fructose crude fiber, dry mustard seeds contained 20-23% carbohydrate. Mustards seed are rich source of many health benefiting minerals such as calcium, manganese, copper, iron, selenium, and zinc (Sarwar et al., 2009). Mustard seeds act as a laxative, stimulant to gastric mucosa and increase intestinal secretion (Sarwar et al., 2009;Sarwar et al., 2004). These crops, including rape (B. campestris L., and B. napus L.) and mustard (B. juncea) are the third major source of edible vegetable oils in the world, after soybean and oily palm. The residue left after oil extraction (i.e., oil cake or meal) being rich in protein can be used as livestock feed (Durrani et al., 1990).
Oilseed is critically needed in many developing countries where protein-energy malnutrition remains a major health hazard, especially among children. The protein quantity and quality, caloric value, and overall nutrient content of oilseeds are quite good. Some oil seed cultivars viz. BARI Sarisha-15, BARI Sarisha-16, BARI Sarisha-17, is widely cultivated throughout the country. If the nutritional status of oil seed is known, their consumption and multipurpose uses will be increased. The characteristics of oils from different sources depend mainly on their compositions and no oil from a single source can be suitable for all purposes, thus the study of their constituents is important. Hence the physicochemical properties of these popular selected varieties was undertaken to ensure their nutritional status. The main purpose of our present study is to determine the oil content, protein, carbohydrate and minerals content of different genotypes groundnut seed.

Materials and Measurement
The experiment was carried out at the Biochemistry laboratory of the Department of Biochemistry, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh and Post Harvest Technology Division BARI Joydebpur, Gazipur, Bangladesh. Three varieties of mustard (Brassica spp.) namely BARI Sarisha-15 (grain properties is yellow in color, oval in shape and small in size), BARI Sarisha-16 (grain properties is brownish in color and round in shape and large size), BARI Sarisha-17 (grain properties is brownish in color, round in shape and large size) were collected from the Oilseeds Research Centre of BARI, Gazipur, Bangladesh. Seed were cleaned sun dried and stored into plastic container in a cool place until used for the chemical analysis. The nutritive value of a food-grain is indicated by its composition shown by chemical analysis. The degree to which the chemical analysis indicates the nutritive value is dependent on the constituents determined. All the chemicals used were collected from Merck (Germany), Wako Pure Chemicals Industries Ltd. and JHD (China). These chemicals were used analytical and spectroscopic grade without further purification.

Determination of Physical Properties of Mustard Varieties
Determination of grain weight: Grain weight was determined by randomly selecting thousand seed sample weighted by electric balance (KEY: JY-2003; China).

Determination of moisture:
Moisture content of mustard sample was determined by a thermostat controlled oven. Empty aluminum moisture dish was weighted (W 1 ) and 2.5 g sample was taken in a moisture dish and weighted (W 2 ). The sample was spread evenly and placed without lid in oven and dried samples overnight at 100 °C temperature. The dishes were transferred to desiccators to cool. Aluminum dish was weighed after cooling (W 3 ). The percentage of the moisture was then calculated by the following formula.

Determination of ash:
The sample is ignited at 600 °C by muffle furnace (Nebertherm: Mod-L9/11/c6; Germany) to burn off all organic material. The inorganic material which does not volatilize at that temperature is called ash. The temperature of the muffle furnace was fixed to 600 °C and crucible was heated for 1h and transferred into desiccators; cooled them to room temperature and weighted (W 1 ).
About 2 g sample was put into the crucible weighted (W 2 ). The sample was burned in a muffle furnace at 600 °C for about 2 h. The crucibles were transferred into the desiccators and cooled them to room temperature and weighted (W 3 ). It was done immediately to prevent moisture absorption. The incineration repeated until constant weight was obtained. The percentage of the ash was then calculated by the following formula (Ranganna et al., 1986).

Determination of Chemical Constant of Mustard Varieties
Estimation of fatty acid composition: Fatty acid composition was determined by gas-liquid chromatographic method (Uppstrom et al., 1978). About 12 mg of oil or equivalent amount of oil seeds was taken (seed was crushed in an oil paper and then transferred into a test tube). The sample was extracted and transesterified at the same time with 5 ml ethylated reagent (petroleum ether/0.02M sodium hydroxide in ethanol (2/3) and shake. The samples were kept for overnight at room temperature. 10 ml salt solution (80 g NaCl and 3 g sodium hydrogen sulphate in 1 liter water) was added and shake. As soon as the two layers were separated, the benzene phase was transferred to small test tubes. A Philips PU 4500 chromatograph instrument was used with flame ionization detector (FID). A glass column (1.5m x 4mm) was packed with BDS. With this column the injection post, column and detector temperature was set at 220 °C, 185 o C and 240 °C, respectively. Nitrogen flow (used as career gas) rate was 22 ml/min, the injection volume was 2 µl. Peak areas were measured with an electronic digital integrator (Shinadzu C-R6A chromatopac).
Saponification value: At first, 2 g fat was taken in 250 ml round bottom flask and 25 ml, 0.5 N alcoholic potassium hydroxide solution added in same flask. The flask was fitted with a long air condenser and heated solution at reflux temperature about 30 minutes. Finally, the flask was cooled and added 1 ml of 1% phenolphthalein solution and titrate the excess of the alkali against standard 0.5 N HCl acid. At the same time and under similar conditions carry out a blank titration without fat (25 ml, 0.5 N same alcoholic KOH solution was taken in another round bottom flask and heated in a similar way and titrated, against 0.5 N acid). 1 ml of 0.5 N HCl acid was equivalent to 0.02805 g of KOH.

Chemical analyses
Estimation of oils/fats: The oil content was determined by the ether soluble extract method. Dried sample was weighed out into an extraction thimble. Weight of thimble and sample were recorded in laboratory book. The thimble was placed into the soxhlet. 100 ml ethyl ether was added to the soxhlet flask then it was connected to holder and condenser. Soxhlet flask was placed on hot plate and distilled at low temperature for 20 hours. After extraction it was turned off and allowed to cool. When distillation was ceased, the extraction thimble was removed and allowed to air dry for 40 minutes the thimble was weighed out. The fat determined by the above procedure contains usual lipids including waxes pigments, certain gums and resins (Thiex et al., 2003). A better name for these constituents would be "ether soluble extract." 100 extraction before sample of Weitht

Estimation of total protein content by microkjeldhal method:
The protein content of food stuff is obtained by estimating the nitrogen content of the material and multiplying the nitrogen value by 5.5 (according to the fact that nitrogen constitutes on average 18% of a protein molecule). The estimation of nitrogen is done by Kjeldhal method (AOAC, 2010) which depends upon the fact that organic nitrogen when digested at 380 °C temperature with sulphuric acid in the presence of catalyst selenium oxide, mercury or copper sulfate is converted into ammonium sulphate (AOAC, 2010). Ammonia liberated by making the solution alkaline is distilled into a known volume of a standard acid which is then back titrated. A known quantity of the finely ground sample (100 mg) weighted out in an Mkj digestion flask. About 2 g digestion mixture (100 g of K 2 SO 4 was thoroughly mixed with 20 g of CuSO 4 .5H 2 O and 2.5 g selenium dioxide was added with it) was added with it 2 ml of concentrated sulphuric acid was dispensed into the flask. Then it was digested for about 2 hrs at 380 °C temperature in Mkj digestion set and was cooled the clear digest. The digest was dissolved in minimum amount of distilled water and carefully transferred to an Mkj distillation set 60%, 10 ml of NaOH solution was added and distilled it. The distillate was collected for 5 min into 4%, 5 ml boric acid containing 2 drops of mixed indicator (methyl red and bromocresol green) in a 50 ml conical flask, till the color of solution was changed. The distillate was titrated against a standard 0.02 N HCl acid and noted the titer value (TV).
(mg) sample of Weight volume) (Titer 0.02) Acid, the of (Normality (14.007) % N Where, 14.007 is the equivalent weight of nitrogen Nitrogen % is converted into protein by multiplying with a factor 5.5 for cereals and pulses.

Estimation of minerals
Mineral content was determined using absorption spectrophotometer.
Reagent for Ca and Mg determination: 1% lanthanum solution: 59 g of lanthanum oxide (La 2 O 3 ) were added with about 50 ml of water. Slowly and cautiously, 250 ml concentrated HCl was added to dissolve the La 2 O 3 . It was made to 5 liters with water.

Preparation of standards solution:
For convenience the Cu, Fe and Zn were prepared together in water. The high concentration for these elements was as follows: 2 µg Cu/ml, 10 µg Fe/ml, 4 µg and 2 µg Zn/ml. Ca and Mg were prepared in the same solution with high concentrations as follows; 100 µg Ca/ml, 40 µg Mg/ml.

Digestion of oilseed cake for determination of Ca, Mg, Fe and Zn
Digestion procedure: Weighted 500 g dry seed sample and put into a 50 ml boiling flask. 5 ml of nitricperchloric solution was allowed on cool hot plate and turned temperature to 375°C. It was allowed to digest for 1 hour and 30 minutes. The flask was removed from digestion chamber and was cooled and 15 ml water was added. The flask was agitated and heated to dissolve the ash and filter.
Analytical procedure: By using a combination diluter-dispenser, 1 ml aliquot was taken from filtrate and 19 ml water (dilution 1) was added. The other dilutions were made in the following order. For P, K and Na determination, 1 ml aliquot from dilution 1, 9 ml of water and 10 ml of color reagent were mixed together. It was allowed to stand about 20 minutes and reading was taken of spectrophotometer at 680 nm. For Ca and Mg determination, 1 ml aliquot from dilution 1, 9 ml of water and 10 ml of 1% lanthanum solution were mixed together. It was analyzed by absorption spectrophotometer. For Fe, Mn and Zn determination, the original filtrate was used to analyze these elements by absorption spectrophotometer.

Estimation of energy
The chemical energy content of food ingredients is usually expressed in terms of heat units (since all forms of energy are convertible into heat energy). The gross food energy was estimated by multiplying the crude protein, crude fat and total carbohydrate by at water factors 4, 9 and 4 respectively (Onimawo et al., 1998;Osborne et al., 1998).

Statistical analysis
The recorded data for each character from the experiments was analyzed statistically find out the variation resulting from experimental treatments using MSTAT package program. The mean for all the treatments were calculated and analysis of variance of characters under the study was performed by F variance test. The mean differences were evaluated by least significance difference test.

Results and Discussion
Three varieties of mustard seed were collected for the determination of physical and chemical characteristics.

Physical characteristics of mustard varieties
Grain weight: Seed weights were determined at 13% moisture level. Seed weight varied with their size and shape (Table 1). BARI Sarisha-17 seed size is higher than the other two varieties mustard. Among different mustard varieties highest thousand grain weight was found in BARI Sarisha-17 (4.24 g), followed by BARI Sarisha-16 (3.620 g) and BARI Sarisha-15 (3.17 g). Our studies mustard seed grain weight are consistent with the results reported by Chowdhury et al., (2014) Banga et al., (2013), Siddiqui et al., (2004), Andarhennadi et al., (1991), Biswas (1989, Chowdhury et al., (1987) and Kaul et al., (1986). Banga et al., (2013) and Siddiqui et al., (2004) found that the highest amount of 1000 seed weight were 5.15 g and 3.95 g, respectively. Our studies mustard seed grain weight values are higher than the reported value of Mandal et al., (2002); who found that thousand seed weight varies from 2.50-2.65 g.

Analysis of oil cake
After extraction of oil, the seeds gave the defatted cakes of varying amounts. The results of cake analysis were presented in Table 5.
Protein: Protein content of different varieties of mustard has been presented in Table 5. Significantly highest amount of protein was obtained from BARI Sarisha-15 (28.00%), the lowest amount of protein content found in BARI Sarisha-17 (22.57%). The present studies values are more or less similar with the reported values of Sarker et al., (2015), Chowdhury et al., (2010), Sosulki et al., (1991), and Mirza et al., (1998), Chowdhury et al., (2015. These results are lower than those reported by many other authors. Prapakornwiriya and Diosady (2004) determined the protein 45.0% and 34.0% respectively. Sengupta et al. (2003) revealed that protein content of rapeseed were ranges from 44.2-44.7%. However our studies result is higher than the reported value of many other authors of Asibuo et al., (2008), Ingale et al., (2011), Ayoola et al., (2012. This might be due to the nitrogen fertilizer application, ecology and agronomics practices. Carbohydrate: Carbohydrate content of different released varieties of oil seed were determined moisture free basis. The data have been presented in Table 5. The amount of carbohydrate contained found in BARI Sarisha-17, BARI Sarisha-16, BARI Sarisha-15 were 18.85%, 14.05%, 13.75% respectively. Agronomics practices, environmental factors as well as variation among the species and varieties might be influenced the carbohydrate content. Our studies carbohydrate content are slightly lower than the reported values of Ayoola et al., (2012), Bachheti et al., (2012) and Gopalan et al., (1981). Bachheti et al., (2012) found that mustard seeds contain 23.8% carbohydrate and Gopalan et al., (1981) stated that dry mustard seeds contained 20-23% carbohydrate.

Proximate analysis of minerals content of the different oilseed varieties
Different major and minor minerals were analyzed this work. The amounts of major minerals content of rapeseed have been illustrated in Table 6. It is well known that rape seed contained small amount of minerals.
Magnesium is the major minerals for human nutrition. Magnesium content of different released varieties of oil seed have been presented in Table 6. There is no significant variation in magnesium content among the varieties. Magnesium content of different varieties was ranged from 0.64% to 0.740%. The present investigations were supported by reported value of Sarker et al., (2015), Sengupta et al., (2003), Chowdhury et al.,(2015), Bachheti et al., (2012), Josefson (1988), and Pathak et al., (1973).
Percentage of nitrogen is influenced by nitrogen fertilizer application, ecology and agronomies practices. Significantly highest amount of nitrogen content was obtained from BARI Sarisha-15 (4.48%), the lowest amount of nitrogen content found in BARI Sarisha-17 (3.67%) ( Table-6). The present studies values are more or less similar with the reported values of Sarker et al., (2015), Sosulki et al., (1991), Chowdhury et al., (2010), and Mirza et al. (1998).
Iron contained of different varieties of mustard was ranged from 75.70 ppm to 170.30 ppm (Table 6).
Significantly highest amount of Fe contained was observed in BARI Sarisha-15 (170.30 ppm) which was followed by BARI Sarisha-17 (100.70 ppm). However these results were supported by the reported values of Chowdhury et al., (2015). These might be influenced the different levels of Fe in soil, fertilizer and variation among the varieties. Our studies Fe values were higher than the reported values of Bachheti et al., (2012) and Josefson (1988); who found that Fe content of mustard seed oil were 8.11 g/100 g and 18 ppm respectively.
The zinc content of different varieties of mustard was ranged from 51.45 to 66.80 ppm in (Table 6).

Gross energy
Significantly the highest amount of energy from carbohydrate found in BARI Sarisha-17 (75.41 kcal/g), followed by BARI Sarisha-16 (56.20 kcal/g), BARI Sarisha-15 (54.92) ( Table 7). The highest energy from protein observed from BARI Sarisha-15 (112.00 kca/g); whereas the lowest amount of energy from protein observed from BARI Sarisha-16 (90.27 kcal/g). The highest amount of energy from fat was observed in BARI Sarisha-17 (377.80 kcal/g), significantly same amount of energy obtained from BARI Sarisha-15 (376.60 kcal/g). The study found that Gross energy of different varieties of mustard ranged from 531.20 kcal/g to 543.6 kcal/g. The highest amount of gross energy found from BARI Sarisha-15 (543.60 kcal/g); while lowest amount of gross energy recorded from BARI Sarisha-16 (531.20 kcal/g). Our studies reports were supported by the reports of Chowdhury et al., (2015), Hossain et al., (2015). Gross Energy (kcal/g)

Variety Name
Energy from Carbohydrate (kcal/g) Energy from Protein (kcal/g) Energy from Oil (kcal/g)

Conclusion
The physicochemical characteristics of three varieties of mustard oilseed were analyzed. Among those mustard varieties BARI Sarisha-17 contained highest amount of oil, saponification value, iodine value, carbohydrate contain and highest amount of total unsaturated fatty acid (TUSFA). It was observed that highest amount of gross energy found from BARI Sarisha-15 and it was also found that highest amounts protein, Mg, N, Cu, Fe and highest amount of total saturated fatty acid (TSFA). All mustard varieties contained good amount of erucic acid. BARI Sarisha-16 contained highest amount of Ca and Zn. It indicates that the BARI Sarisha-15 is a good candidate of most of the nutrient containing oil seed.
Farmers are cultivating different varieties of mustard in their field for the consumption oil and oilcake as food, feed and fertilizer. BARI Sarisha-15, BARI Sarisha-16, and BARI Sarisha-17 are grown in large scale as they contained the fair amount of nutrient contents.