Chemical Industry & Chemical Engineering Quarterly www.ache.org.rs/CICEQ
Chem. Ind. Chem. Eng. Q. 21 (1) 71−76 (2015) CI&CEQ
ĐORĐE B. PSODOROV1 MARIJANA M. AČANSKI2 ĐURA N. VUJIĆ1 JOVANA S. BRKLJAČA1
DRAGAN Đ. PSODOROV3
1FINS, University of Novi Sad, Novi Sad, Serbia, 2Faculty of Technology, University of Novi Sad, Novi Sad, Serbia 3College of Management and business communications, Sremski Karlovci, Serbia
SCIENTIFIC PAPER
UDC 582.661.21:66:664:543.544 DOI 10.2298/CICEQ131104007P
HOMOGENITY OF OIL AND SUGAR
COMPONENTS OF FLOUR AMARANTH
INVESTIGATED BY GC-MS
Article Highlights
• GC-MS and correlation analysis have been used
• GC-MS showed that the lipid composition of different varieties of amaranth are very similar
• GC-MS showed that the sugar composition of different varieties of amaranth are less similar
Abstract
Gas chromatography with mass spectrometry (GC-MS) was used for per-forming a qualitative analysis of liposoluble and hydrosoluble flour extracts of three genotypes of Amaranthus sp. All three samples were first defatted with hexane. Hexane extracts were used for the analysis of fatty acids of lipid components. TMSH (trimethylsulfonium hydroxide, 0.2 M in methanol) was used as the transesterification reagent. With transesterification reaction, fatty acids were esterified from acilglycerol to methyl-esters. Defatted flour samples were dried in the air and then extracted with ethanol. Ethanol extracts were used for the analysis of soluble carbohydrates. TMSI (trimethylsilylimidazole) was used as a reagent for the derivatisation of carbohydrates into trimethyl-silylethers. The results show that the dominant methyl-esters of fatty acids are very similar in all the three samples. Such a similarity was not detected in the analysis of soluble sugars. The following test cluster analysis was used for the comparison of liposoluble and hydrosoluble flour extracts of three genotypes of Amaranthus sp.
Keywords: Amaranthus sp., GC-MS, liposoluble and hydrosoluble com-position, correlation.
In ancient times, amaranth was cultivated by the Aztecs and Incas. Today, it is grown in many tropical, subtropical, and temperate countries [1,2].
Amaranthus includes over 75 wild and weedy species native to tropical and temperate regions of the whole world but is most diverse in the Americas [3]. Pre-sently, cultivated grain is grown in many areas of the world, including Central and South America, Africa, India, China, and the United States [4].
Amaranth oil contains about 18.6–23.4% palmitic (C16:0), 22.7–31.5% oleic (C18:1n–9), and 39.4–49.8% linoleic (C18:2n–6) acids [5-7]. The lipid content in
Correspondence: M.M. Ačanski, Faculty of Technology, Univer-sity of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia.
E-mail: macanski@tf.uns.ac.rs Paper received: 4 November, 2013 Paper revised: 25 January, 2014 Paper accepted: 15 November, 2014
amaranth is between two and three times higher than in common cereals such as wheat [8,9]. Amaranth is a robust crop and can withstand a wide range of climatic conditions. Amaranth is rich in proteins, which have a balanced amino acid profile and are rich in lysine [10,11]. Several studies have reported signi-ficant amounts of important unsaponifiable
compo-nents in amaranth oil[3-8] such as tocopherols,
phy-tosterols, and squalene. Tocopherols are well-known for their cardiovascular benefits and antioxidant cap-acity, whereas phytosterols and squalene have the ability to decrease serum total cholesterol [5]. Squa-lene has also been studied for its anticarcinogenic and antioxidant activities [4,10,12].
constitute series of compounds among which sugars, sugar derivatives and sugar polymers are the most important ones [13]. The content of starch in ama-ranth flours is about 61.4% [8]. Considering that the starch was investigated in many studies [14-16], but there are not many studies about soluble carbo-hydrates, it was necessary to determine the content of the samples.
The purpose of this study is to determine lipo-soluble and hydrolipo-soluble components in three
geno-types of Amaranthus sp. and obtain preliminary
infor-mation on their variability, and then to determine the possibility of differentiation of three genotypes of
Amaranthus sp. flour by creating the dendrograms of liposoluble and hydrosoluble extracts. Such studies have been conducted on wheat [17,18], spelt [19,20] and buckwheat [submitted for publication]. It was detected that each plant species has a characteristic composition of liposoluble and hydrosoluble extracts. The final objective of the study is to easily and quickly determine flour authenticity on the basis of liposoluble [submitted for publication] and hydrosoluble compo-sition of obtained chromatograms without an analysis of individual components [21]. Results should also be applicable in the quality control of finished products [submitted for publication].
EXPERIMENTAL
Preparation of samples for analysis of oil components
About 10 g of the three genotypes of
Amar-anthus sp. were grinded: 2 (A1),16 (A2) and 31 (A3)
[22].
Each sample was homogenized and then treated in the following manner: 0.5 g of flour was poured in 12 mL cuvette for centrifugation with the precision of 0.01 g. The cuvette was additionally filled with 5 mL of n-hexane (Merck) and stirred on Vortex for 2 min, after which the mixture was centrifuged at 2000 rpm for 5 min. After this 3 mL of clear super-natant were poured into a 10 mL beaker and left to steam up on the room temperature. The volume of 10 µL was taken from the oily residue, reconstituted to 400 µL of methanol and additionally 100 µL of trans-esterification reagent-TMSH (Trimethylsulfonium
hyd-roxide, 0.2M in methanol, Macherey-Nagel) were
added, by which derivatisation of fatty acids into methyl esters was performed.
Preparation of samples for analysis of sugar components
Samples of defatted flour were dried in the air. Five mL of 96% ethanol (Merck), were added to the
dried samples and stirred on Vortex for 2 min, after which the mixture was centrifuged at 2000 rpm for 5 min. 2 mL of clear supernatant were separated and dried on a nitrogen flow. The residue was dissolved in 500 µL of pyridine and 50 µL of TMSI
(trimethyl-silylimidazole, Macherey-Nagel) were added, by
which derivatisation of carbohydrates into trimethyl-silyl ethers was performed.
GC–MS Analyses
All the tests were performed on a gas-chroma-tography system.
The GC–MS analyses were performed on an Agilent Technologies 7890 instrument paired with MSD 5975 equipment (Agilent Technologies, Palo Alto, CA, USA) operating in EI mode at 70 eV. A DP-5 MS column (30 m, 0.25 mm, 25 µm) was used. The
temperature programme was: 50–130 °C at 30 °C/min
and 130–300 °C at 10 °C/min. The injector
tempera-ture was 250 °C. 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 μL of the solutions.
WILEY 275 library was used for the mass spec-trum analysis.
PAST programme was used for the statistical data processing [23].
RESULTS AND DISCUSSION
Figure 1 shows the chromatogram of hexane
extracts of all three Amaranthus sp. genotypes, from
10 to 21 min. Chromatograms of all three genotypes are very similar. The peak integration shows a ratio of components areas 1:150.
Table 1 shows the retention time of components from the chromatogram presented in Figure 1.
The profile of fatty acids of analysed samples was the same in all three genotypes.
The following fatty acids have been identified as dominant in the form of methyl esters:palmitic acid, oleic acid and linoleic acid. These results are in accordance with our earlier investigations of lipo-soluble extracts of small grains [18]. The high inten-sity peak in Figure 1 with the retention time 20.40 min is squalene [4].
Figure 2 shows the chromatogram of all three
Amaranthus sp. genotypes, from 10 to 21 min in ethanol extract.
The analysis of amaranth chromatogram in Fig-ure 2 identified trimethylsilyl derivatives of the sugars presented in Table 2. Chromatograms of all three genotypes are also similar, but less than in Figure 1.
Figure 1. Chromatograms of all three genotypes of Amaranthus sp. (А1, А2 аnd А3) in the liposoluble (hexane) extracts.
Table 1. Retention time (Rt) of components of three genotypes Amaranthus sp. in the liposoluble (hexane) extracts
Rt / min Component
10.808 Tetradecanoic acid, methyl ester
11.881 Pentadecanoic acid, methyl ester
12.953 Hexadecenoic acid, methyl ester
13.642 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
15.522 Nonadecanoic acid, methyl ester
16.635 Eicosanoic acid, methyl ester
18.264 Docosanoic acid, methyl ester
19.019 Tricosanoic acid, methyl ester
19.50 Tetracosanoic acid, methyl ester
19.79 Ergost-5-en-3-ol
20.30 Pentacosanoic acid,methyl ester
20.40 Squalene
20.93 Hexacosanoic acid, methyl ester
21.14 γ-Sitosterol
21.32 Ethylcholestanol
revealed the presence of following compounds: from 10 to 12 minutes there is a presence of tetrose TMSI ethers, and from 18 to 21 min there is a presence of pentoso and hexsoso TMSI ethers. GC-MS allows identification of the presence of TMSI esthers of fatty acids, from 12 to 18 min, which remain after incom-plete defattening. However, this is not the focus of the research at the moment.
The carbohydrate content was much lower than the unbound triglyceride content [17], and therefore
the remains of triglycerides are higher than the con-tent of carbohydrates.
The purpose of the study was to identify lipo-soluble and hydrolipo-soluble components and to com-pare presence of components in samples of hexane and ethanol extracts from amaranth flour. Cluster analysis was used for comparing the samples. A single linkage algorithm and similarity measure type of correlation has been used [22]. Figures 3 and 4
show dendrograms of Pearson’s r correlation of
Figure 2. Chromatograms of three genotypes of Amaranthus sp. (А1, А2 аnd А3) in the hydrosoluble (ethanol) extract.
Table 2. Retention time (Rt) of components of three genotypes Amaranthus sp. in the hydrosoluble (ethanol) extract
Rt / min Compound
12.401 1,2,3,5-Tetrakis-O-(trimethylsilyl)-tetrose isomer
12.63 1,2,3,5-Tetrakis-O-(trimethylsilyl)-tetrose isomer
18.01 1,2,3,5-Tetrakis-O-(trimethylsilyl)-pentose isomer
18.514 D-Ribofuranose, 1,2,3,5-tetrakis-O-(trimethylsilyl)- (CAS)
18.98 Gluconic acid, 2,3,5,6-tetrakis-O-(trimethylsilyl)-, lactone
19.43 Glucofuranoside, methyl-tetrakis-o-(trimethylsilyl)-
19.547 2,3,5-tris-O-(trimethylsilyl)-hexose isomer
19.70 2,3,5-tris-O-(trimethylsilyl)-hexose isomer
20.046 PER-TMS D-Hexose isomer
Figure 3. Dendrogram of component correlations from Table 1 of three genotypes of Amaranthus sp. (А1, А2 аnd А3).
A correlation coefficient is shown on the ordinate (Y-axis). X-axis is labelled distance and refers to a distance measure between clusters.
The dendrograms of Pearson’s r correlation
show that the similarity in the composition of lipophilic substances of amaranth (components listed in Table 1) is significant (r> 0.9942, Figure 3).
Similarity in the composition of carbohydrates of three types of amaranth components listed in Table 2 is much smaller (r> 0.64, Figure 4).
CONCLUSION
Gas chromatography-mass spectrometry was used to show that the lipid compositions of all three
genotypes of Amaranthus sp. are, from the nutritional
aspect, very similar.
Gas chromatography-mass spectrometry also showed that the sugar compositions of all three
genotypes of Amaranthus sp. are, from the nutritional
aspect, less similar.
Three genotypes of Amaranthus sp. have a
characteristic composition of liposoluble and hydro-soluble extracts. This was also detected in other plant species (wheat, spelt and buckwheat).
Therefore, the final objective of this study is to determine the authenticity of the flour and finished products without an analysis of individual components of the extracts in a relatively simple and quick manner.
Acknowledgements
The authors gratefully acknowledge the financial support from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Projects TR 31066 and III 46005) and Provincial Secretariat for Science and Technological Develop-ment of Vojvodina (Project no. 114-451-2373/2011).
REFERENCES
[1] W. Breene, Cereal Foods World 36 (1991) 426-429
[2] National Research Council (US), Amaranth: modern prospects for an ancient crop, National Academy Press, Washington DC, 1984, p. 1
[3] J.D. Sauer, Amaranthaceaes - Amaranth family. In Histo-rical Geography of Crop Plants: a Select Roster; CRC Press, Boca Raton, FL, 1993, p. 9
[4] H.P. He, Y. Cai, M. Sun, H. Corke, J. Agric. Food Chem. 50 (2002) 368-372
[5] A.M. Pina-Rodriguez, C.C. Akoh, J. Agric. Food Chem. 57 (2009) 6748-6756
[6] F. Jahaniaval, Y. Kakuda, M.F. Marcone, J. Am. Oil Chem. Soc. 77 (2000) 847-852
[7] T.H. Gamel, A.S. Mesallam, A.A. Damir, L.A. Shekib, J.P. Linssen,. J. Food Lip. 14 (2007) 323-334
[8] L. Alvarez-Jubete, E.K. Arendt, E. Gallagher, Tr. Food Sci. Technol. 21 (2010) 106-113
[9] L. Alvarez-Jubete, E.K. Arendt, E. Gallagher, Int. J. Food Sci. Nut. 60 (2009) 240-257
[10] R.V. Tikekar, R.D. Ludescher. M.V. Karwe, J. Agric. Food Chem. 56 (2008) 10675-10678
[11] S. Gorinstein, E. Pawelzik, E.D. Licon, R. Haruenkit, M. Weisz, S. Trakhtenberg, , J. Sci. Food Agric. 88 (2002) 886-889
[12] H.P. He, H. Corke, J. Agric. Food Chem. 51 (2003) 7913- –7920
[13] Lj. Đaković, Wheat Flour, Institute for Technology of Grain and Flour, Faculty of Technology, Novi Sad, 1997, p. 28
[14] H. Choi, W. Kim, M. Shi, Starch/Stärke 56 (2004) 469- -477
[15] X. Konga, E. Bertoft, J. Baoc, H. Corkea, Int. J. Bio. Mac. 43 (2008) 377-382
[16] J. Pal, R.S. Singhal, P.R. Kulkarni, Carb. Polym. 48 (2002) 49-53
[17] Đ.N. Vujić, M.M. Ačanski, M.I. Bodroža-Solarov, N.S. Hristov, M.N. Krunić, Chem. Ind. Chem. Eng. Q. 18(4) (2012) 555-561
[18] M.M. Ačanski, Đ.N. Vujić, N.S. HristovM.I. Bodroža-So-larov,PTEP 16(3) (2012) 113-115
[19] Đ.N. Vujić, M.M. Ačanski, M.I. Bodroža-Solarov, Đ.B. Psodorov, J.S. Brkljača, in Proceedings of 3nd Internat-ional conference Sustainable postharvest and food tech-nology INOPTEP 2013 and XXV National Conference processing and energy in agriculture PTEP 2013, 2013, Vrnjačka Banja, Serbia, 2013, pp. 241-244
[20] Đ.N. Vujić, Đ.B. Psodorov, M.M. Ačanski, M.I. Bodroža-Solarov J.S. Brkljača, APTEFF 35 (2013) 165-177
[21] M.M. Ačanski, Đ.N. Vujić, Food Chem. 145 (2014) 743- -748
[22] M. Bodroža-Solarov, J. Lević, S. Biliznikas, B. Kokić, N. Spasevski, Š. Kormanjoš, in Proceedings of 2nd Work-shop XIV International Feed Technology Symposium, 2010, Novi Sad, Proceedings, 2010, pp. 209-214
ĐORĐE B. PSODOROV1 MARIJANA M. AČANSKI2 ĐURA N. VUJIĆ1 JOVANA S. BRKLJAČA1 DRAGAN Đ. PSODOROV3 1FINS-Institut zа prеhrаmbеnе
tеhnоlоgiје, Bulеvаr Cаrа Lаzаrа 1, 21000 Novi Sad, Srbiја 2Те
hnоlоški fаkultеt, Univеrzitеt u Nоvоm Sаdu, Bulеvаr Cаrа Lаzаrа1, 21000 Nоvi Sаd, Srbija 3Visoka škola strukovnih studija za
menadžment i poslovne komunikacije, Mitropolita Stratimirovića 110, 21205 Sremski Karlovci, Srbija
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Primеnоm gаsnе hrоmаtоgrаfiје sа mаsеnоm spеktrоmеtriјоm urаđеnа је kvаlitаtivnа аnаlizа lipоsоlubilnih i hidrоsоlubilnih еkstrаkatа 3 genotipa amarantusa (Amaranthus sp.). Svа tri uzоrkа su prvо оbеzmаšćеnа hеksаnоm. Hеksаnski еkstrаkti su kоrišćеni zа аnа-lizu mаsnih kisеlinа lipidnih kоmpоnеnаtа. Rеаgеns zа trаnsеstеrifikаciјu је biо ТМSH (tri-mеtilsulfоniјum-hidrоksid, 0,2 M u mеtаnоlu). Rеаkciјоm trаnsеstеrifikаciје mаsnе kisеlinе su iz аcilglicеrоlа еstеrifikоvаnе u mеtil еstrе. Оbеzmаšćеni uzоrci su zаtim sušеni nа vаz-duhu i zаtim еkstrаhоvаni еtаnоlоm. Еkstrаkti еtаnоlа su kоrišćеni zа аnаlizu uglјеnih hidrаtа. Rеаgеns zа dеrivаtizаciјu biо је ТМSI (trimеtilsililimidаzоl), čimе је izvršеnа dеri-vаtizаciја šеćеrа u trimеtilsililеtrе. Rеzultаti pоkаzuјu dа su dоminаntni mеtil еstri mаsnih kisеlinа svа tri uzоrkа vеоmа slični. Таkvа sličnоst niје uоčеnа pri аnаlizi rаstvоrlјivih šеćеrа.