FATTY ACID COMPOSITION AND NATURAL ANTIOXIDANT CAPACITY OF TEN SERBIAN LINSEED CULTIVARS

Literature data about nutritional characteristics of linseed cultivars from some specific geographical area or country is scarce. For that very reason, following paper is presenting fatty acid (FA) compositions and antioxidant capacity of lipid-soluble (ACL) components of ten native linseed cultivars from Serbia. These characteristics can be interesting, especially due to the increasing trend of linseed usage in human diet. Presented results show that there were statistically significant (p<0.05) differences between linseed kernels in FA composition. Negative correlation was found between FA C18:0 and αlinolenic acid (ALA). The cultivar with the highest ACL value was No. 10 (342.66 μmol trolox/kg d.m.), meaning that it had very strong protection against oxidation of polyunsaturated FAs. Nevertheless, correlation between ACL and polyunsaturated FA content in cultivars was not statistically significant (p=0.84). ACL of the samples did not depend on FA composition of linseed, but it might depend on characteristics of a specific cultivar. The aforementioned results show potential usage in storage of linseeds or its products, while FA composition of linseed kernels might be one of criteria for authentication of linseed origin, and can be of great help in future selection of the cultivars, depending on purpose of linseed production.


INTRODUCTION
Not so many commercial crops are so intensively exploited during the history such as linseed.In fact, its Latin name (Linum usitatissimum) means "useful."It was primarily grown (about 3000 B.C.) for medicinal purposes and for the fibres of which were made linen.Nowadays, linseed oil is mainly used, whether it comes to food or chemical industry (Bhatty and Cherdkiatgumchai, 1995).This plant grows to a height up to 60 cm, with slim and very fibrous stems, leaves with three veins, and its light blue flowers.The seeds are rich source of both edible and non-edible oils (Rubilar et al., 2010;Matheson, 1976), containing approximately 40% of oil, of which more than 70% are unsaturated fatty acids (FAs).In addition to a large amount of oil, linseed contains approximately 20-30% of crude protein (Ivanov et al., 2012a; Daun et al., 2003;Karlović and Andrić, 1996).Raw linseed oil is dark yellow in colour, with strong specific smell and taste (Dimić, 2005).This oil is well known as one of the most unsaturated vegetable oils with high content of ALA, which amount in total fat is more than 50% (Gunstone, 2001).ALA is a precursor of eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), n-3 polyunsaturated FAs, responsible for the proper brain development in children, as well as for resistance to various allergies, autoimmune diseases, cardiovascular problems, and inflammation (Sierra et al., 2008).These FAs are essential, because mammals, and therefore humans, cannot endogenously synthesize them and must adopt them exogenously from dietary sources (Beare Rogers, 2001).High quality of linseed oil confirms the fact that US National Cancer Institute (NCI) targeted linseed as one of the six plant materials for study as cancer-preventative foods.Linseed oil is a potent inhibitor of pro-inflammatory mediators even when used in domestic food preparation.This is a great advantage of linseed oil, which make it suitable for application in the development of novel anti-inflammatory therapies with or without pharmaceutical products for target populations (Oomah, 2001).
There are a lot of examples of positive linseed effects on animal health and wellbeing.The immunomodulating effects of omega-3 FAs combined with the potential hormonal effects of the phytoestrogens may have a positive effect on sow productivity and the health of the piglets.Due to the positive impact of including omega-3 FAs in human diets, there is significant interest in enriching the omega-3 FA content of meat products produced by beef cattle and swine.Linseed can be included in poultry diets if used in the proper proportions and formulated appropriately.The most popular commercial poultry product is omega-3 eggs (Newkirk, 2008).The aim of this study was to determine FA composition and ACL of ten autochthonous linseed cultivars cultivated in the northern Serbian province Vojvodina, in order to detect some mutual characteristics, and to determine whether any significant variation existed in their compositions.

Material
Examined cultivars of linseeds were developed at the Institute of Field and Vegetable Crops in Novi Sad, Department for Alternative Crops in Bački Petrovac, Serbia.The cultivars were labelled with number from 1 to 10.All of them were cultivated in Vojvodina, the northern Province of Serbia.After harvesting, grains were transported to the laboratory in polypropylene bags and held at room temperature.They were clea-ned on an air screen cleaner to remove all foreign matter and impurities such as dust, dirt and immature and broken kernels.

Fatty acid analysis
Supercritical fluid extraction (SFE) with CO 2 was used for extraction of lipids from the samples, since it showed good results as a preparative technique for FA analysis.Extraction was performed on LECO TFE-2000 fat analyzer and extraction conditions were adjusted as explained in the paper of Ivanov et al. (2012b).FA methyl esters were prepared from the extracted lipids by transmetilation method that use 14% wt.boron trifluoride/methanol solution, as recommended method for this type of substrates (Ivanov et al., 2012b;Karlović and Andrić, 1996).Nitrogen gas (99.99%) was used for removing boron trifluoride/methanol solution and n-heptane (99.99%) from FA methyl esters.Obtained samples were analyzed by a gas chromatograph Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA) with flame ionization detector (GC-FID), auto-injection module for liquid, equipped with fused silica capillary column (DB-WAX 30 m, 0.25 mm, 0.50 um).Carrier gas was helium (purity>99.9997vol %, flow rate=1.26ml/min).The FAs peaks were identified by comparison of retention times with retention times of standards from Supelco 37 component FA methyl ester mix and with data from internal data library, based on previous experiments and FA methyl ester determination on GC-MS.Results were expressed as mass of single FA or FA group (g) in 100 g of total FAs (relative content).

Antioxidant capacity of lipid-soluble substances analysis
Estimation of the antioxidant capacity in lipid-soluble substances (ACL) was done by photochemiluminescence method using an antioxidant analyser Photochem instrument (Jena Analitik, Germany).In the ACL assay, the photochemical generation of free radicals was measured with a sensitive detector by using chemiluminescence.Free radicals were produced from the luminol, which worked partly as a photosensitizer and partly as an oxygen radical detection reagent.The lipid-soluble antioxidants were measured with the ACL kit, according to manufacturer's protocol (Jena Analytik, 2004).The working solution consisted of the following reagents: methanol (Reagent 1) -2.3 ml, buffer solution (Reagent 2) -200 μl, photosensitizer (Reagent 3) -25 μl.
Antioxidant capacities of the samples were determined from calibration curve by extrapolation method.Trolox standard with the concentration of 100 μmol/dm 3 was used for the preparation of calibration curve.All measurements were done in duplicates.

Statistical analysis
Statistical analysis of experimental data was performed using STATISTICA software version 10 (Statsoft, Tulsa, OK, USA).One way analysis of variance -ANOVA and Tukey's HSD comparison of means of samples were used for analysing variations.Differences among means with probability p≤0.05 were accepted as representing statistically significant differences.Correlations between FAs and ACL pairs were evaluated at p≤0.05.
Another interesting fact is that C18:0 FA had significant (p≤0.05)negative effect (r=-0.86) on the content of essential ALA (see Table 2).It means that with the increase of C18:0 FA content, content of ALA generally decrea-sed, which was especially obvious in cultivar 1, as already mentioned.N-6/n-3 ratio in the linseed grains ranged from 0.22 (cultivar 5) to 0.35 (cultivar 1), as presented in Figure 1.Western diets are deficient in n-3 FAs, while having extortionate amounts of n-6 FAs in comparison with the diet on which humans evolved.Recommended values of n-6/n-3 ratio should be at least less than 4/1 in the secondary prevention of cardiovascular disease, and a ratio of 2-3/1 suppressed inflammation in patients with rheumatoid arthritis (Simopoulos, 2004; Scollan et al., 2006).FA composition of all investigated cultivars can be considered as nutritionally desirable.Although there were significant differences in n-6/n-3 ratio between the cultivars, all values were far below 2-3/1.
The significance of lipid soluble antioxidants is crucial in protection of lipid-rich food from fast oxidative deterioration.Since linseed is an oilseed, ACL was chosen as an appropriate criterion for determination of antioxidant capacity in linseed kernels, and the results are presented in Table 3.

Table 1 .
Fatty acid composition of extracted linseed oils

Linseed cultivar Fatty acid content (% of total fatty acids)
a-c Different superscripts within the same column indicate significant differences (p≤0.05)

Table 2 .
Correlation coefficients between fatty acids of various linseed cultivars

Table 3 .
Antioxidant capacity of lipid-soluble substances in linseed kernels Linseed