FATTY ACID COMPOSITION OF OIL FROM GRAIN OF SOME TETRAPLOID WHEAT SPECIES

56 The consumption of vegetable oils, including human and pet food production, numerous industrial uses, perfumery/ cosmetic and pharmaceutical industries, fuel manufacturing, etc., has increased dramatically in the past century. Although wheat has never been considered an oil crop (oil from wheat germs makes up around 2.5% by weight of the kernel [1], oil from wheat germs and bran is valuable because it contains important bioactive compounds such as octacosanol [2], tocopherols [3], carotenoids [4] and unsaturated fatty acids [3]. Wheat germ oil is in demand in the cosmetics industry. Wheat bran oil contains carotenoids [5] and tocopherols [6]. Most of studies in this area are conducted on traditional commercial wheat varieties. However, there is an opinion that the value of wheat oil reduced in the course of domestication, in particular, domestication of emmer [7]. At the same time, the interest of breeders, producers and consumers in the UDC 633.11:581.16 https://doi.org/10.15407/biotech13.02.056

Although wheat has never been considered an oil crop, oil from wheat germs and bran is valuable because it contains important bioactive compounds. Most of studies in this area were conducted with traditional commercial wheat varieties. At the same time, the interest of breeders, producers and consumers is going back to ancient and underutilized wheats species. In this respect, we set the purpose to evaluate tetraploid wheat species (Triticum. dicoccoides var. pseudojordanicum, Triticum dicoccum, Triticum timofeevii, Triticum persicum var rubiginosum, Triticum durum var. falcatamelanopus, Triticum polonicum var. pseudocompactum and Triticum aethiopicum var. densimenelikii) for fatty acid composition. Grain was harvested in 2015, 2016, 2017, 2018 and 2019. Fatty acid methyl esters were prepared by the modified Peisker method. Fatty acid composition was analyzed by gas chromatography. Six major fatty acids were found in grain of tetraploid wheat species, with linoleic acid being the most abundant. The ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum was slightly lower than in the domestic emmer varieties. T. timofeevii, emmer varieties Holikovska and Romanivska and radium wheat variety Spadschina had the most beneficial unsaturated/ saturated ratios. As conclusion there was no evidence of deterioration in the grain quality in terms of unsaturated fatty acid levels, and we observed no patterns in variability of fatty acid contents across the species under investigation.
Fatty acid composition was analyzed by gas chromatography. Six major fatty acids were found in grain of tetraploid wheat species, with linoleic acid being the most abundant. The ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum was slightly lower than in the domestic emmer varieties. T. timofeevii, emmer varieties Holikovska and Romanivska and durum wheat variety Spadschina had the most beneficial unsaturated/saturated ratios.
There was no evidence of deterioration in the grain quality in terms of unsaturated fatty acid levels. We observed no patterns in variability of fatty acid contents across the species under investigation. 21 st century is going back to ancient wheats, spelt, emmer, einkorn, as well as to domestic, but underutilized species [8]. They are valuable especially for resistance to fungal diseases, unpretentiousness to cultivation conditions and grain quality. Grain quality parameters are primarily the protein content and composition; contents of antioxidants, vitamins and minerals; and these wheat species often outperform commercial varieties by these parameters. In the literature, there is very little information on the quality of ancient wheat oil. It was found that the lipid content in einkorn grain was 50% higher than that in wheat bread grain (4.2 and 2.8 g/100 g of dry weight, respectively) [9].
It should be noted that this comparison cannot be considered quite correct, since ploidy of these wheat species is different (einkorn is diploid, and bread wheat is hexaploid), yet such a comparison can be valuable for evolutionists and producers. It was demonstrated that grain of Triticum monococcum L. ssp. monococcum was rich in polyunsaturated fatty acids [10]. Linoleic acid (polyunsaturated omega-6 fatty acid) was one of the predominant acids among 14 identified fatty acids in T. monococcum grain [11].
In this respect, it is expedient to evaluate underutilized tetraploid wheat species for oil quality, in particular, to focus on breeding accessions of wild emmer  Fatty acid methyl esters were prepared by the modified Peisker method [12]. Chloroform (Thermo Fisher Scientific Inc., USA) -methanol (Honeywell Research Chemicals, Romania) -96% sulfuric acid (Dneprochem, Ukraine) mix ture in a ratio of 100:100:1 was used for methyla tion. 30-50 μl of lipid extract was placed in a glass ampoule; 2.5 ml of methylation mixture was added, and the ampoule was sealed. Ampou les were incubated in a thermostat at 105 C for 3 hours. After methylation, ampoules were opened, the contents were transferred to test tubes, a pinch of powdered zinc sulfate (ChemElements, Ukraine) was added, and then 2 ml of distilled water and 2 ml of hexane (MOL Group, Hungary) were poured to extract methyl esters. After thoroughly stirring and settling, the hexane extracts were filtered and analyzed by gas chromatography [13].

Materials and Methods
Fatty acid composition was determined using a gas chromatograph Selmikhrom 1 (OAO SELMI, Ukraine) equipped with a flame ionization detector (FID). The stainless steel column, 2.5 m length4 mm i.d., was packed with a stationary phase, Inerton AW-DMCS (0.16-0.20 mm) (Lachema, Czechia) processed with 10% diethylene glycol succinate (BOC Sciences, USA). 2 l of hexane solution of fatty acid methyl esters was injected. Gas chromatography was operated under the following conditions: nitrogen flow 30 ml/min; hydrogen flow 30-35ml/min; air flow 300 ml/min; column temperature 180 C; injector tempe ra ture 230 C and FID temperature 220 C. The fatty acids were identified by comparing the retention times of the peaks with those of reference fatty acid methyl esters (Sigma-Aldrich, USA).
The percentages of fatty acid methyl esters were calculated by internal normalization.
The data were statistically processed in STATGRAPHICS PLUS, using ANOVA method. The results in the Table are presented as mean ± standard deviation (SD) and reported to three significant figures. Graphs were plotted in Statistica 10.
Bottari et al. [15] obtained more than 60 peaks by gas chromatography and mass spectrometry and identified fatty acids with even numbers of carbon atoms from C12 to C30 as well as with odd numbers of carbon atoms C15 and C17. The database of the United States Department of Agriculture (USDA) also reports small levels of C14:0 in durum wheat kernels (0.003 g/100 g fresh matter). There are also publications reporting minor fatty acids (C17, C20, C22 and C24) both in kernels [16] and in germ oil [17,18]. Myristic acid (C14:0) was present in negligibly small amounts and irrelevant for calculation of fatty acid percentages. We detected no other minor fatty acids, as they were below limit of quantification for our method. Only C16:0, C18:0, C18:1, C18:2 and C18:3 accounting for around 90% of the total fatty acid content in durum wheat grain are constantly reported by all researchers and considered as the most important ones in durum wheat, while others amount to approximately 1-2% in total [19].
Wheat and other cereals lack  6 desaturase, the enzyme responsible for catalytic conversion of linoleic acid to -linolenic acid [20] and conversion of -linolenic acid to stearidonic acid (C18:4). Therefore, we expectedly found no stearidonic acid, and all the linolenic acid in our samples should be considered as -linolenic acid.
There is an idea that some parameters of grain quality can deteriorate during domestication. For example, Chatzav et al. reported that domestic emmer was inferior to its wild ancestor in terms of protein, iron and zinc contents [21]. Unsaturated fatty acid levels are obviously not the case, since the ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum is even slightly lower than in the domestic emmer varieties bred at the PPI (Table).
It is noteworthy that there were no significant differences for 4 (palmitic, linoleic, oleic and palmitoleic) of 6 major fatty acids between T. dicoccoides var. pseudojordanicum and T. dicoccum var. serbicum, which is considered to have not been crossed with other tetraploid species and have undergone the least changes in the breeding process. On the other hand, T. dicoccum var. atratum accessions from different locations, which are morphologically very close, in many cases differ one from another in contents of 5 of 6 major fatty acids (except palmitoleic acid).
Increased unsaturated fatty acid contents is known to be associated with cold tolerance and considered as a general biological pattern. However, there are data that increased unsaturated fatty acid contents are due rather to cold hardening than to genetic differences between cold hardy and less hardy varieties, as the fatty acid profiles did not differ between the varieties under investigation [22]. For oil crops, Chernova et al. [23] reported that winter-type rapeseed seeds contained triglycerides with a lower degree of saturation, while in spring-type rapeseed highly saturated lipids were the most abundant. We found that the unsaturated/saturated ratio in grain was not associated with growth habit (winter vs. spring).
The oil value is primarily determined by unsaturated fatty acids. In this respect, T. timofeevii seems the most promising species for crossing with other tetraploid species to improve wheat oil quality via breeding. Nevertheless, the emmer varieties bred at the PPI, Holikovska and Romanivska, and durum wheat variety Spadschina, also developed by the PPI, boast rather high unsaturated/saturated ratios (4.5, 4.7, and 5.1, respectively). These values are higher than those registered for durum wheat in USDA and Italian National Institute for Research on Food and Nutrition (IINRAN) databases (3.0 and 3.5, respectively) and also higher than the average ratio obtained by Narducci et al. for Italian durum wheat varieties [14].
Interspecies comparison showed that among emmer species T. timofeevii and emmer varieties Holikovska and Romanivska had the best unsaturated/saturated ratios (see above). This is attributed rather to the sum of unsaturated fatty acids than to an increased content of one component. Linoleic acid content in oil from T. timofeevii grain was significantly higher than in oil from T. dicoccoides var. pseudojordanicum, T. dicoccum var. atratum (Poland), T. dicoccum var. atratum (USA) and T. dicoccum var. serbicum. Linolenic acid content in oil from T. timofeevii grain was significantly lower than in oil from T. dicoccoides var. pseudojordanicum, but higher than in oil from T. dicoccum var. atratum (USA) and T. dicoccum var. atratum (Poland). Oleic acid content in oil from T. timofeevii grain was significantly lower than in oil from T. dicoccoides var. pseudojordanicum, T. dicoccum var. atratum (Poland) and T. dicoccum var. serbicum, but significantly higher than in oil from T. dicoccum var. atratum (Hungary). Palmitoleic acid content in oil from T. timofeevii grain was significantly higher than in oil from T. dicoccoides var. Linolenic acid content in oil from varieties Holikovska and Romanivska was significantly lower than in oil from T. dicoccoides var. pseudojordanicum, but higher than in oil from T. dicoccum var. atratum (Poland) and T. dicoccum var. atratum (USA). Oleic acid content in oil from varieties Holikovska and Romanivska was significantly lower than in oil from T. dicoccoides var. pseudojordanicum, T. dicoccum var. serbicum, T. dicoccum var. atratum (Poland) and T. dicoccum var. atratum (Hungary). Palmitoleic acid content in oil from varieties Holikovska and Romanivska was significantly higher than in oil from T. dicoccoides var. pseudojordanicum, T. dicoccum var. atratum (Poland), T. dicoccum var. atratum (USA), T. dicoccum var. atratum (Hungary) and T. dicoccum var. serbicum. No differences between Holikovska and Romanivska are explained by their close origin in the breeding process.
As to durum wheat and related species, variety Spadschina has the best unsaturated/ saturated ratio. The ratios for T. persicum var. rubiginosum, T. durum var. falcatamelanopus,  densimenelikii oil, respectively). The greatest variability was intrinsic to fatty acids, contents of which were below 1%: the peak variation coefficients amounted to 48.8% for palmitoleic acid in T. dicoccum var. atratum (USA) and 67.7% for eicosenoic acid in T. persicum var. rubiginosum. In Fig. 2, box and whisker plots are presented.
The plots show no patterns in variability of fatty acid contents across the species under investigation. The same species (for example, T. persicum var. rubiginosum with variation coefficients of 15.5% and 4.19% for palmitoleic and stearic acids, respectively) can be characterized by a wide variability in one fatty acid and by a narrow range for another. At the same time, the same fatty acid (for example, palmitic acid) can be very variable within one species (T. dicoccum var. atratum (USA) and variety Holikovska; variation coefficient = 5.45% and 5.49%, respectively) and demonstrate a relatively stable content in another (T. dicoccum var. atratum (Hungary); variation coefficient = 0.75%).
Thus, we can conclude that 1) six major fatty acids were found in tetraploid wheat species, with linoleic acid being the most abundant; 2) there was no evidence of deterioration in the grain quality in terms of unsaturated fatty acid levels, since the ratio of unsaturated acids to saturated ones in grain of wild emmer T. dicoccoides var. pseudojordanicum was even slightly lower than in the domestic emmer varieties; 3) T. timofeevii, emmer varieties Holikovska and Romanivska and durum wheat variety Spadschina had the most beneficial unsaturated/saturated ratios; 4) we observed no patterns in variability of fatty acid contents across the species under investigation, since same species can be characterized by a wide variability in one fatty acid and by a narrow range for another, and, at the same time, the same fatty acid can be very variable within one species.
The work was supported by the National Academy of Agrarian Sciences project 24.01.03.01.Ф. (State Registration Number 0116U001070). The authors declare that they have no conflicts of interest.