QUALITATIVE ANALYSIS OF HEXANE FLOUR EXTRACT OF SPELT
Đura N. Vujića, Đorđe B. Psodorova
,Marijana M. Ačanskib, Marija I. Bodroža-Solarova and Jovana S. Brkljačaa
a University of Novi Sad, Institute for Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia b
University of Novi Sad, Faculty of Technology Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
Gas chromatography with mass spectrometry (GC-MS) was used for performing a qualitative analysis of the hexane flour extract of three samples of spelt. All the three samples were first treated with hexane and the obtained extracts were used for the analy-sis of the fatty acid lipid components. The transesterification reaction was performed using TMSH (trimethylsulfonium hydroxide, 0.2M in methanol), and the fatty acids were esterified from acylglycerol to methyl-esters. In all analyzed extracts, the predominant component was methyl linoleate, followed by methyl oleate and methyl palmitate. The subsequent tests, performed by cluster analysis, were used to compare the hexane flour extracts of different types of spelt.
KEY WORDS: spelt, GC-MS, hexane extract, correlation
INTRODUCTION
Spelt (Triticum aestivum ssp. spelta) is a breadmaking cereal, alternative to widely grown common soft wheat (Triticum aestivum ssp. Vulgare). Contrary to its close rela-tive, spelt is harvested as a hulled grain and must undergo a costly dehulling procedure before being introduced into the milling process. Authentic spelt flour is therefore much more expensive to produce than wheat flour, and spelt use has long been confined to ani-mal feeding, owing to the large amount of insoluble dietary fiber provided by such a hul-led grain when not dehulhul-led (1). Still, an increased interest for ecoalternative cereals and organic products is at the origin of a new success story for spelt, which is an environmen-tally friendly, low-input crop cultivated in Europe (Belgium, Austria, Germany, Switzer-land, Italy, Spain Czech Republic and Hugary) and North America (United States and Canada) (2, 3). Over the past decade, folk knowledge pointing to nutritional and even therapeutic benefits prompted unconventional medicine to recommend the use of spelt (1) rather than wheat.
The term lipids imply fats and fat-like substances usually contained in flour in con-centration of about 1.5-2.5%. Although they are present in small quantities, lipids play an
important part in creating physical properties of dough and to a large extent have a positi-ve influence on the technological quality of flour (4-7). They affect surface properties of starch grains, improve interaction between starch and gluten, and impact on improve-ments in dough (8). Lipids are also necessary for physiological functions such as fat-solu-ble vitamin absorption and in hormone and eicosanoid synthesis.
Wheat flour lipids contain 30-35% of tryglycerides, 15-20% of mono- and di-glyceri-des, 5-10% of free fatty acids, about 25% of phospholipids and about 15% of glycolipids. In terms of solubility in different solvents, flour contains free and bound lipids. Free li-pids are, due to its polar nature, easily and quickly extracted by non-polar solvents (petro-leum ether, diethyl ether, acetone, hexane, etc.), whereas bound lipids (with proteins and partially with starch) can be extracted fully or partially with polar solvents, i.e. their mix-tures (n-butanol/water, water/ethanol/ether and others) (8-11).
Most studies on free lipids show that spelt is richer in lipids than wheat (1, 12-17). Ruibal-Mendieta et al. (2002) found that total lipid content was also higher in spelt than in wheat. With regard to fatty acids, studies show that the major fatty acids in spelt and wheat wholemeal are linoleic, palmitic, oleic and linolenic acids (1, 14, 18).
The purpose of this study was to determine liposoluble components in three genoty-pes of spelt and obtain preliminary information on their variability, and then to determine the possibility of differentiation of three types of spelt flour by creating the dendrogram of liposoluble extracts.
EXPERIMENTAL
About 10 g of the following grains was ground: Austrija (S1), Eko-10 (S2) and Nir-vana (S3). 70% of particle size of ground material were below 200 µm and 30% were above 200 µm. Each sample was homogenised and further treated in the following man-ner. A 12-mL cuvette for centrifugation was used for pouring 0.5 g of flour with the pre-cision of 0.01 g. The cuvette was additionally filled with 5 mL of n-hexane and stirred on Vortex for 2 minutes, after which the mixture was centrifugated at 2000 rpm for five mi-nutes. After this, 3 mL of clear supernatant was poured into a 10 mL glass and left to steam up at the ambient temperature. From the oily residue an amount of 10 µL was ta-ken, reconstituted to 400 µL of methanol and additionally added 100 µL of transesteri-fication reagent: TMSH (trimethylsulfonium hydroxide, 0.2M in methanol, Macherey-Nagel). By such transesterification reaction, fatty acids from acylglycerol esterified to their methyl esters.
All the testings were conducted on a gas-chromatography system.
The GC–MS analyses were performed on an Agilent Technologies 7890 instrument coupled with MSD 5975 equipment (Agilent Technologies, Palo Alto, CA, USA) opera-ting in EI mode at 70 eV. A DP-5 MS column (30 m 0.25 mm, 25 µm) was used. The temperature programme was: 50-130oC at 30oC/min and 130–300oC at 10oC/min. Injector temperature was 250oC. The flow rate of the carrier gas (helium) was 0.8 mL/min. A split ratio of 1:50 was used for the injection of 1 l of the solutions.
RESULTS AND DISCUSSION
Figure 1 shows the chromatogram of the hexane extract of spelt samples from 10.00 to 21.00 minutes. As is evident, the chromatograms of all three spelt samples are very si-milar. The peak integration shows that the ratio of the areas of the components is 1:150.
Figure 1. Chromatograms of all spelt samples
Table 1 shows the retention times of the components from the chromatogram presen-ted in Figure 1.
Table 1. Retention times (Rt) of spelt components
Rt/min Components
10.808 Tetradecanoic acid, methyl ester 12.953 Hexadecanoic acid, methyl ester 13.907 Heptadecanoic acid, methyl ester
14.569 9,12-Octadecadienoic acid (Z,Z)-, methyl ester, Methyl linoleate 14.675 9-Octadecenoic acid, methyl ester
14.887 Octadecanoic acid, methyl ester 16.516 3-Heptadecenal
18.025 Heneicosane
18.264 Docosanoic acid, methyl ester 19.50 Tetracosanoic acid, methyl ester 20.40 Squalene
20.93 Hexacosanoic acid, methyl ester
acid, linoleic acid and oleic acid. The retention time of the three methyl esters is in the range from 12 to 15 min, Figure 1. These fatty acids and their methyl esters comprise about 90% of the integrated area of the chromatogram.
The presence of squalene was registered in all three samples .
Generally, the similarity and specificity in the composition of lipophilic components was detected in all three types of spelt, which is consistent with previous studies with small grains (20).
The purpose of the study was also, to compare the presence of the components in the samples of hexane extracts from spelt flour. A cluster analysis was used for comparing the samples. A single linkage algorithm and similarity measure type of correlation were used (19). Figure 2 shows the dendrogram of Pearson’s r correlation of three liposoluble samples. The correlation coefficient is shown on the Y-axis, while the X-axis gives the measure for the distance between the clusters.
Figure 2. Dendrogram of the correlation of the components from Table 1 for different types of spelt
CONCLUSION
The analysis carried out by gas chromatography-mass spectrometry showed that the lipid composition of different varieties of spelt is very similar.
This paper suggests that it is possible to compare types of plants through their content of lipids using the GC-MS chromatography and correlation analysis.
Acknowledgɟments
The authors gratefully acknowledge the financial support from the Ministry of Edu-cation, Science and Technological Development of the Republic of Serbia (Project III 46005 and Project TR 31066 and Provincial Secretariat for Science and Technological Development of Vojvodina (Project no. 114-451-2373/2011).
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