SUPPLEMENTARY MATERIAL
Composition of fatty acids, triacylglycerols and polar compounds of
different walnut varieties (Juglans regia L.) from Tunisia
I. Bouabdallaha*, I. Boualia, E. Martinez-Forceb, A. Albouchic, M. C. Perez Caminob and S.
Boukhchinaa
a
Facullté des Sciences de Tunis, Unité de Biochimie des Lipides, département de Biologie, Université
de Tunis El Manar, 2092 Tunis, Tunisia.
b
c
Instituto de la Grasa, Consejo Superior de Investigaciónes Científicas (CSIC), 41012 Sevilla, Spain.
Laboratoire d’Ecologie Forestière, Institut Nationale des recherches en Génie Rural, Eaux et
Forêts, 2080 Ariana, Tunis, Tunisia.
Abstract
The chemical composition (Total oil content, fatty acids, triacylglycerols (TAG) and polar
compounds) of six walnuts (Juglans regia L.) cultivars (Lauzeronne, Franquette, Hartley, Local pt,
Local gd and Parisienne) collected from Mateur (North of Tunisia) were evaluated. The major fatty
acids found in the walnut oils are linoleic acid (60.42–65.77%), oleic acid (13.21– 19.94%) and
linolenic acid (7.61–13%). The triacylglycerol species were mainly composed by trilinolein (LLL),
dilinoleoyl-linolenoyl-glycerol (LLLn), dilinoleoyl-oleoyl-glycerol (OLL + VLL) and palmitoyldilinoleoyl-glycerol (PLL) classes. The results revealed that Local pt variety has the highest level of
oil (62.56 %), linoleic acid (65.77%) and trilinolein (LLL: 33.48%). In this study significant
differences among oil samples were observed, conducting therefore to a great variability in the oil
composition among cultivars.
Keywords: Juglans regia L.; Fatty acids; Triacylglycerols; Polar compounds; varietal effect
* Corresponding author: email: ikrambouabdallah@yahoo.fr
Telephone: +216225514 20
Experimental
Reagents and standard
Petroleum ether and chloroform were acquired from Fisher Scientific SA (Loughborough.,
Spain). Methanol, n-hexane (95%) solvents of HPLC grade, Isopropanol (2-propanol) and
toluene were purchased from Panreac Quimica SA (Barcelona., Spain). Fatty acid methyl
ester (FAME) standards were purchased from Nu-Chek-Prep (Elysian, MN., USA).
Plant material
The walnut (Juglans regia L.) samples used in this study consists of four introduced
(Franquette, Parisienne, Harteley and Lauzeronne) and two landrace (Local pt and Local gd)
varieties. Walnut fruits were obtained from National Institute of Research Rural Engineering
Water and Forest (INRGREF) of Tunisia. Fruits were harvested from trees planted in an
experimental farm of the INRGREF, in the North of Tunisia Sidi Mbrarek (Mateur, Bizerte).
The planting density was 5m x 5m (400 plants/ha). The soil was sandy clay loam type,
characterized by a pH of 7.4 and a salinity of 0.64 g/l. The harvested plants were identified by
Dr A. Albouchi (INRGREF) Tunisia. The identification numbers of Parisienne, Lauzeronne,
Local gd, Hartely, Local pt, Franquette were WP09, WL09, WLG09, WH09, WLP09 and
WH09, respectively. These voucher specimens were deposited at the Herbarium of
(INRGREF). After that, the kernels were analyzed to determine moisture (by drying the
walnuts at 60 °C to a constant weight). All data presented are the averages of three
measurements, with the standard deviation being less than 10% of the mean value.
Total lipid extraction
The walnut kernels (about 30 g) from each variety were ground into a fine powder with a
hand mortar. Then the oil was extracted using a soxhlet extractor by petroleum ether as a
solvent. After 6 h of extraction, samples were evaporated under vacuum, weighted and their
oil yield was determined.
Fatty acid analysis
Fatty acid methyl esters were obtained from isolated lipids by heating the samples at 80°C for
1 h in 3 ml of methanol/toluene/H2SO4 (88:10:2 by volume) following the method described
by Gunstone et al. (1994). Fatty acid methyl esters were dissolved in 1 mL of heptane and
then analyzed by gas chromatography (GC) using a
Hewlett–Packard 5890A gas
chromatograph (Palo Alto, CA, USA) with a Supelco SP-2380 fused silica capillary column
(30 m length; 0.25 mm i.d.; 0.20 µm film thickness: Bellefonte, PA, USA). Hydrogen was
used as the carrier gas. Detector and injector temperature was 200 ºC, whereas oven
temperature was kept at 170 ºC, and the split ratio was 1:50. The different fatty acids were
identified by comparison their retention times with those of commercial and synthetic
standards (Sigma, St. Louis, MO, USA).
Triacylglycerol analysis by GC
TAGs were separated and quantified by GC with an Agilent 6890 gas chromatograph (Palo
Alto, CA, USA), and hydrogen was used as the carrier gas. The injector and detector
temperatures both were 380°C, the oven temperature was 345°C, and a head pressure gradient
from 70 to 120 kPa was applied, changing this last parameter depending on the column. The
gas chromatography capillary column was a J & W Scientific DB-17HT (15 m length, 0.25
mm i.d., 0.15 µm film thickness) (Folsom, CA, USA), with the linear gas rate being 50 cm/s,
the split ratio being 1:80, and the detector being a flame ionization detector (FID). The
different TAG molecules were identified with respect to known samples.
Analysis of the molecular species of polar compound by HPLC
Non-polar and polar fractions were separated from oil sample by silica column
chromatography as described in the IUPAC Method 2507 (IUPAC 1992), oil samples were
purified by passing the oil through silica SPE (solid-phase extraction). Silica SPE column
(VARIAN, Bond Elut SI) was placed in a vacuum elution apparatus and washed under
vacuum with 6 ml of hexane and then a solution of the oil (100 mg) in 0.5 ml of hexane was
charged into the column. The nonpolar fraction solution eluted with 10 ml of hexanediethylether (87:13, v/v), the second fraction containing the polar compounds was eluted with
10 mL of diethyl ether. The latter fraction was evaporated until dryness in a rotary evaporator
under reduced pressure at room temperature and the residue redissolved in acetone. An aliquot
of 10 µl of the purified oil solution in acetone (5%) was injected onto the HPLC system using
an autosampler Beckman Gold 508 (Beckman-Coulter, Fullerton, CA, U.S.A.). analyses were
done on a Lichrosphere 100 RP-18 (4µm) column (25 cm x 4 mm I.D.) using a Beckman
Gold 126 pumping unit (Beckman-Coulter, Fullerton, CA, U.S.A.), refractive index detector
Perkin Elmer 200 (Perkin Elmer, Norwalk, CT, USA). The mobile phase was propionitrile at
a flow rate of 0.6 mL/min. The column was maintained at 20°C using a Peltier oven.
Statistical analysis
All extractions and determinations were conducted in triplicate. The data were analyzed using
the analysis of variance (Anova). Comparisons of means were achieved using the Statistical
Analysis System XLSTAT (version 2013). Differences between varieties were assessed using
Duncan test. Differences at p < 0.01 were considered to be significant.
References
Gunstone FD, Harwood JL, Padley FB. 1994. The Lipid Handbook, Chapman Hall. London.
IUPAC (International Union of Pure and Applied Chemistry). 1992. Standard Methods for the
Analysis of Oils, Fats and Derivatives.1st Suppl. to 7th Edn., Enlarged Edn. Blackwell
Scientific Publications. Oxford, (UK).