ABSTRACT
Estimation of quality assessment of Brassica germplasm provides the basis for rapeseed/mustard genetic improvement. Studies were undertaken to assess the B. napus genotypes for quality characteristics, and to evaluate B. napus germplasm for erucic acid and Glucosinolates using Near Infrared Reflectance Spectroscopy (NIRS). A total of 30 B. napus genotypes of local and exotic origin were characterized using molecular and biochemical approaches. The Biochemical assessment revealed the range of 6.3 to 8.1 for moisture, 21.5 to 30.3 for protein, 41.6 to 50.1 for oil content, 48.3 to 59.1 for oleic acid and 7.3 to 10.2 for linolenic acid, all expressed as percent of fresh seed, with the mean value of 7.1, 26.1, 45.5, 53.7 and 9.1 percent respectively. Almost all the genotypes were rich in oil and protein contents and oleic acid. All the genotypes showed high percentage of erucic acid and glucosinolates. It is recommended that among the thirty B. napus genotypes, genetically distinct lines pointed out in the present study, should be used in future breeding programs for improving yield and quality characteristics of Brassica.
Keywords: Brassica napus, Quality characteristics, fatty acids, Glucosinolates
INTRODUCTION
The quality of oil derived from rapeseed is determined by its biochemical composition. Edible oils are the most concentrated sources of energy, acts as vehicles for important vitamins and provide essential fatty acids. Nutritional benefits associated with the low levels of saturated fatty acids found in Brassica oil have been a significant factor in the success of canola oil in Canada, Australia and United States. The adverse effects of saturated fat on blood cholesterol and its implications for cardiovascular disease, is well documented. There are two different classes of serum cholesterols based on its carrier, the high-density lipoproteins (HDL) and the low-density lipoproteins (LDL). HDL is beneficial as it is associated with the removal of cholesterol from the blood stream, while LDL is undesirable as it is responsible for the movement of cholesterol within the bloodstream. Saturated fatty acid, palmitic acid in particular, has the most significant LDL cholesterol raising effect (Zock et al., 1994).
Brassica oil contains around 35% polyunsaturated fatty acids. It is normally hydrogenated in order to achieve the stability required for food applications requiring prolonged shelf life and for high temperature frying of foods. However, partial hydrogenation may lead to the formation of trans-fatty acids, which can raise LDL in a manner similar to palmitic acid. On the other hand, the monounsaturated oleic acid can lower LDL-cholesterol and at the same time it can confer high stability required by the food industry without the need for hydrogenation. Therefore the ideal healthy cooking oil, especially those used for industrial food applications, should be rich in oleic acid and low in palmitic acid.
Polyunsaturated fatty acids are highly susceptible to autoxidation, which involves the production of free radicals, implicated in a number of diseases, tissue injuries, and in the process of aging (Shahidi, 1996). As a consequence, the reduction of the levels of polyunsaturated fatty acids and their substitution by the monounsaturated oleic acid is an important goal for the development of higher quality oil (Scarth and McVetty, 1999).
Natural rapeseed oil contains erucic acid, a 22-carbon fatty acid which constitutes 40-50% of the fatty acid content in rapeseed, however, canola has been bred to typically contain less than 2% erucic acid. Because of this, canola oil is thought to have a more neutral taste and to be healthier for human consumption.
Glucosinolates, a type of glycoside, are sulfur-containing compounds found in all members of the Brassica family, including canola. The major function of glucosinolates in plants is as feeding deterrents against insects and mammals. When glucosinolates are hydrolyzed, as when plant tissue is crushed, a compound called isothiocyanate is formed. Although the isothiocyanate compound metabolite of cyanide, yet it does not have the same negative effects as cyanide. In terms of health effects, at high levels glucosinolates can adversely affect thyroid function and at low levels may help with cancer prevention. The main goal for rapeseed breeding is to increase the level of oleic acid and decrease the level of linolenic acid in seed oil. The thermostability of oleic makes it a desired fatty acid as it gives oil a superior cooking quality. Conversely, linolenic acid is an undesired fatty acid due to its high sensitivity to oxidative changes, which negatively affect shelf life, cooking quality and sensory aspects. The present study was undertaken with the objective to assess the B. napus germplasm for quality characteristics, in order to provide necessary information to the Brassica breeders for future breeding programs
MATERIALS AND METHODS
Thirty Brassica napus genotypes were assessed for quality parameters, including oil and moisture content by Nuclear Magnetic Resonance (NMR), protein and fatty acids using Near Infrared Reflectance Spectroscopy (NIRS) and glucosinolates by Spectrophotometer.
NMR is a non destructive technique which does not require any sample preparation or chemicals. Oil and moisture contents were determined in whole seed scanned on NMR system (Oxford instrument MQA 7005). First of all a few standards (sample with known oil and moisture content) were run on NMR in order to standardize the NMR apparatus. Afterwards each sample was run accordingly and readings were obtained.
Near infrared reflectance spectroscopy (NIRS) is the measurement of the wavelength and intensity of the absorption of near infrared light by a sample. Near infrared light spans the 800 nm to 2.5 µm (12,500 to 4000 cm-1) range and is energetic enough to excite overtones and combinations of molecular vibrations to higher energy levels. NIR spectroscopy is typically used for quantitative measurement of organic functional groups, especially O-H, N-H and C=O. The components and design on NIR instrumentation are similar to UV-Vis absorption spectrophotometers. The light source is usually a PBS solid state detector. Sample holders can be of glass or quartz and typical solvents are CCl4 and CS2. Protein and Fatty acid (oleic acid, linolenic acid and erucic acid ) of all 20 rapeseed (B.napus and B.campestris ) were determined on a NIR system FOSS (6500) (Daun et al., 1994; Velasco et al., 1997) at Oilseed Laboratory., Nuclear Institute for Food and Agriculture (NIFA) Tarnab, Peshawar.
NIR also is a non destructive technique which does not require any sample preparation or chemicals. Protein and Fatty acids (C18:1, C18:2, C18:3) were determined in whole seed scanned on NIR system (FOSS 6500 equipped with ISI version 1.02a software program of infra Soft International). The samples of all 30 rapeseed lines were scanned three times to minimize the sampling error. The observations were recorded. The reference calibration for fatty acids (C18:1, C18:2, C18:3) on NIR System was developed by the estimation of fatty acid through Gas Chromatography (UNICAM 610 capillary column).
Spectrophotometer (Shimadzu UV 1700) was used for assessment of glucosinolates in Brassica germplasm. About 1g seed was taken in a 5 ml of plastic scintillation vial containing the small size steel rods and added 3 ml of myrosinase activation buffer (1mM Sodium ascorbate, 0.02 M imidazole, pH5.5) to each vial. The vial containing mixture was vigorously shaken for 10 minutes and an equal volume of 1ml charcoal, was also added to each vial after shaking. Each mixture was then transferred into 2 Eppendorf and aqueous phase was recovered by centrifugation at 10000 rpm for 5 minutes. The supernatant was transferred to a fresh tube containing 2 ml of colour reagent. Also eight glucose standard solutions of known concentration were prepared by serial dilution of the 8 mM glucose solution. The samples and standards were then kept in water bath at 370C for 15 to 20 minutes. The 8 mM glucose solution is equivalent to 160 µ mole glucosinolates per gm seed at absorbance of 546 nm on a spectrophotometer.
A calibration curve was constructed from the standards and the unknown concentration of glucosinolates content was estimated by comparing its intensity with that of the standards.
RESULTS AND DISCUSSION
The protein content of the B. napus genotypes included in the study, ranged from 21.5 to 30.3 % of fresh seed with a mean value of 26.1 percent. These results are in agreement with the findings of Velasco et al. 1999 and Mohammad et al. 1991 who reported the protein value ranging from 13.4 to 28.3 % and 23.8 to 25.5 % respectively in fresh seed of Brassica genotypes.
The oil content among the 30 genotype ranged from 41.6 to 50.1 %, with a mean value of 45.5 %. Similar mean value of oil content (44.3%) was found by Velasco et al., 1999 using NIRS for screening of quality traits in rapeseed. However, comparatively lower levels of oil content ranging from 38 to 44 %, 36 to 46 % and 37 to 41 % were reported by Novoseloy (2002) and Si et al. (1997) respectively. These differences may be due to variation in genotype of germplasms and/or environmental influences.
The oleic acid content in our study ranged from 48.3 to 59.5 % of fresh seed. The level of oleic acid found was lower than that of pallot et al., 1999 who reported a range of 56 to 74 % of oleic acid in Brassica using NIRS. However, Agnihothri and Kaushik (2001) reported comparatively lower values of oleic acid content ranging from 40 to 50 % in fresh seed of B. napus. The monounsaturated oleic acid is important from nutritional standpoint because it lowers the undesirable LDL cholesterol level and also confers high stability required for healthy cooking. The polyunsaturated linolenic acid ranged from 7.3 to 10.2 % of fresh seed in our study. Similar findings were reported by Ishida et al., 1995 who observed 3.3 to 13.1 % linolenic acid in B. napus cultivars, Several studies on recently developed low linolenic acid lines of canola have demonstrated that this type of canola oil has improved storage and frying stability.
Kaushik (1998) reported that erucic acid and glucosinolates are the two toxic substances found in rapeseed mustard seeds. In the present study erucic acid content among the genotypes ranged from 32.3 to 49.8 % of fresh seed. Luhs et al. (1999) reported that a series of alleles have been identified in B.napus and B. rapa which make it possible to breed strains containing almost any level of erucic acid from less then 1 % to about 60 % of the total fatty acids. Bhardwaj and Hamama (2000) reported significant variation among 455 accessions of B. napus for erucic acid content and the mean value observed was 26.1 %.
The glucosinolates content of the 30 B. napus genotypes ranged from 45.8 to 88.5 µmol g-1 of fresh seed with the mean value of 64.8 µmol g-1. Velasco et al. 1999 reported a lower mean glucosinolate value of 51.2 µmol g-1 of fresh rapeseed using NIRS. Bhardwaj and Hamama (2000) reported higher glucosinolates content in B. napus than B. rapa meal, 49.2 verses 43.8 µmol g-1.
A relationship between protein and oil content was also studied using regression analysis. A strong negative correlation was observed between protein and oil contents among all the tested B.napus (Fig. 1) genotypes studied. A linear decrease in oil content was shown with the increase in protein content and vice versa. Charron et al. (2005) and Singh et al. (1996) reported negative correlation betweem oil and protein content in rapeseed and soybean.
CONCLUSION:
The present quality assessment studies in B. napus lines indicated the existence of a wide variation with respect to various parameters among the genotypes, which can serve a prominent role in designing future breeding programs by the Brassica breeders.
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