ARTICLE
Auteur(s) : Georges
Piombo1, Nathalie Barouh1, Bruno
Barea1, Renaud Boulanger2, Pierre
Brat3, Michel Pina1, Pierre
Villeneuve1
1UMR IATE, Laboratoire de lipotechnie, CIRAD, TA
40/16, 73 rue Jean François Breton, 34398 Montpellier cedex 5
France
2UR IMPACT, CIRAD Montpellier TA 40/16 UPR- IMPACT, 73
rue Jean François Breton, 34398 Montpellier Cedex 5, France
3UR TROPIQUAL, CIRAD Montpellier TA 40/16, 73, rue Jean
François Breton, 34 398 Montpellier Cedex 5, France
Article reçu le 24 Mai 2006, accepté le 26 Juin 2006
Introduction
All fruits are generally regarded as having nutritional value due
to their vitamins and carbohydrates content of the pulp, however
very little is known regarding the lipid quantity and quality found
in the respective seeds. Increasingly lipids and therefore the
lipid fraction are seen as beneficial for health, provided they are
of specific composition and subscribe to nutritional
recommendations such as the proportion of saturated, mono
unsaturated, poly-unsaturated fatty acids and the ratio
omega-6/omega-3 [1, 2].
While lipid composition is important, the bio-availability of
the essential fatty acids is often determined by their
regio-distribution on the glycerol back bone. Current evidence
appears to suggest that the bio-availability of essential fatty
acids is enhanced when they occupy the sn2 position.
From these preliminary considerations, we have chosen to study
the lipid composition of seeds derived from three fruits: Kiwi
(actinidia chinensis, passion fruit (edulis passiflora) and guava
(psidium guajava). All three are marketed as either fresh or
processed products and are valued for their ascorbic acid contents.
As passion fruit, kiwi and guava contain on average 30
mg/100 g, 60 mg/100 g and 250 mg/100 g respectively.
In the case of kiwi and guava, this content can reach 300 and even
900 mg/100 g in certain varieties and according to the
stage of maturity [3, 4].
These fruits are processed into a range of products i.e. juices,
sorbets. However a considerable amount of the annual yield does not
meet the prescribed criteria both for the fresh and processed
markets thus leading to potential waste. In the case of kiwi this
can account for 10-15% of the harvest. Attempts have been made to
use these fruits in processing, but with little success, for
example kiwi juices from smaller fruits do not offer the same,
organoleptic quality and colour and cannot be stabilised. In this
context the extraction of the oil from the seeds may provide an
opportunity for adding value to a waste product. Therefore this
article reports the characterisation and nutritional quality of the
oils from these fruits.
Materials and methods
Material. Kiwis (Actinidia chinensis), Hayworth variety, were
provided by the Barniol Company (Perpignan, France). The guavas
(Psidium guajava), Beaumont variety came from Martinique (River
Crack, France) and the passion fruits (Passiflora edulis), Pourpre
variety of Kenya were provided by CIRAD Dept. FLHOR (Montpellier,
France).
Oil Extraction
The extraction of the seeds of kiwi was carried out after manual
peeling of the kiwis and pressing using an Auriol press. The
extract was subjected to simultaneous milling and sieving through a
6 mm mesh. The seeds are separated from the mixture by
decanting in water. The seeds of guavas and passion fruits were
obtained by manual extraction. Prior to extraction the seeds were
ground using a grinder supplied by Retsch (Germany). We used an
accelerated solvent extraction (ASE) system to extract oil witch
petroleum ether (purchased from VWR international SAS France). This
method is performed to optimize parameters such as temperature,
pressure, extraction time). It replaces soxlhet extractor in
several cases. Thanks to this method you can extract with higher
temperature than the solvent extraction boiling point. This system
is also interesting because it is shorter, it needs less solvent
and the results obtained are comparable to those of the reference
method [5, 6]). The oil was extracted from a 4g sample using a
semiautomatic ASE200 (Accelerated Solvent Extraction, Dionex, USA)
according to the following conditions: temperature 80 °C,
7 min static time, 5 min dynamic time and a total rinsing
volume of 11 ml. The cycle was repeated 5 times. This program
ensures complete extraction of the neutral lipids from the sample
at 80 °C under a pressure of 100 bars for 60 min. The
extract was collected in an inactinic glass bottle. The solvent was
driven off at 45 °C using a rotary evaporator. The last
solvent traces were removed by drying under nitrogen. The content
of oil was determined by gravimetry.
Transformation into fatty acids methyl esters (FAME) and GC
analysis for Fatty acids composition
In 25 mL round bottom flask, oil samples (10 mg) were
added to 3 mL sodium methylate solution with phenolphtalein.
Reaction medium was refluxed for 10 min. 3 mL chlorhydric
methanol were added to phenolphtalein discoloration and the mixture
was refluxed again for 10 min and then cooled to ambient
temperature. 8 mL hexane and 10 mL water were added and
the organic phase was recovered, dried over anhydrous sodium
sulfate and filtered for subsequent GC analysis: Agilent 6890 (Bios
Analytique, France) series using a Innowax capillary column (SGE,
Courtaboeuf, France) with the following characteristics: length,
30 m; internal diameter, 0.32 mm, film thickness
0.25 μm, Fatty acid methyl esters were directly injected into
the GC. Carrier gas: Helium debit 1.0 mL/min, splitting ratio:
1/80. Injector temp: 250 °C, FID Detector temp: 275 °C.
oven was heated from 185 °C to 225 °C at 5 °C/min
and held at 225 °C for 20 min. Quantitative data were
given by a D-2500 integrator (Merck, Darmstadt, Germany). Peak area
percentages obtained with the integrator were divided by molecular
weight of individual FAME (standards were purchased from
Sigma-Aldrich, France) to yield mole percents of fatty acids. Two
GC analyses of products from four experiments were made.
Regiodistribution analysis of triacylglycerols (TAG)
The regiodistribution analysis of TAG was performed after
degradation to partial acylglycerols with ethyl magnesium bromide
[7]. The resulting mixture of partial acylglycerols was separated
by preparative thin-layer chromatography. The plates were developed
with a chloroform/acetone/acetic acid solution (85:15:1 v/v/v). The
α-monoacylglycerols (α-MAG) band was visualized under ultraviolet
light (Rf = 0.26) and scrapped off. The α-MAG were converted to
fatty acid methyl esters and analyzed by GLC according to the
procedure described above.
Phytosterols analysis
The determination of phytosterols was carried out in accordance
with the corresponding AFNOR Method NF ISO 6799 [8]. Sterols were
extracted from 500 mg of lipid extract. The sample was
saponified using 5ml alcoholic Potassium carbonate (0.5N), under
reflux for 15 min. The unsaponifiable components were
separated from the soaps on a alumina column and washed with
diethyl ether. The solvent was evaporated off and the residue was
taken up in 1 mL of chloroform. The separation of sterols from
other unsaponifiable components was carried out by thin-layer
chromatography. The plates were developed with a hexane/diethyl
ether/acetic acid solution (80:20:1 v/v/v). The individual sterol
species were resolved using an Agilent 6890 coupled to a mass
spectrometer Agilent 5973N GC fitted with a SAC5 column (length:
30 m, internal diameter: 0.25 mm, film thickness
0.25 μm) that allowed the direct analysis of sterols without
derivatisation. The injector was set to 260 °C with a split
ratio of 1/50. The carrier gas was helium at a flow rate of
1.5ml/min and the oven temperature isotherm at 285 °C.The Mass
spectrometer was operated in the electron impact ionization mode,
the source temperature was programmed from 230 °C, the energy
of ionisation was 70 electrons volts and the parameters of
acquisition of the spectra are 40 to 400 uma for the range of mass.
Two GC analyses of products from three experiments were made.
Tocopherols analysis
Tocopherols are analysed by HPLC in accordance with the AFNOR
Method ISO 9936 [9]. The HPLC consists of modules provided by
Thermo-Finnigan (France): a quaternary pump (P1000XR), a sample
passor (AS1000) and a valve of injection to 6 ways provided with a
loop of 20 μL, a spectrofluorimetric detector (FL3000) and
software for data processing (PC 1000). The standards were
purchased from VWR international SAS France. The mobile phase
consists of a mixture of hexane/ dioxane (97:3 v/v), with a flow of
1 mL/min. The column was a silica hypersil of 5 μm
(0,4 cm by 25 cm). The wavelength of excitation was fixed
at 290nm and that of emission was regulated to 330 nm. Analysis
were run in triplicate.
Determination of free fatty acid content (FFA)
FFA content was determined by titration with NaOH 0.1 mol/L using
phenolphtalein as an indicator AOCS [10]. The amount of FFAs was
calculated as wt % oleic acid. Analyses were carried out in
triplicate.
Results and discussion
Oil Content and FFA content
The extraction of oil was carried out with accelerated extraction
apparatus system (ASE 200). Oil content was respectively 30.0, 18.6
and 12.6% for kiwi, passion fruit, and guava. The free fatty acid
content of the three oils extracted expressed as a percentage of
oleic acid was lower than 1% (respectively of 0.44%, 0.60%, and of
0.23% for kiwi, passion fruit and guava). This is quite acceptable
for a non-refined oil since, according to the codex Alimentarius
criteria of quality for a crude oil are a FFA content of maximum 2%
[11].
FA composition and regiodistribution
The FA compositions are shown in table 1( Table
1 ). Kiwi seed oil was found to have a high percentage of
essential fatty acids, omega 6 linoleic acid C18:2 n-6 (16.1%) and
omega 3 linolenic acid C18:3 n-3 (62.3%) for a total of
polyunsaturated fatty acids of 78.4%. To the best of our knowledge,
this oil can be considered as one of the richest source of omega 3
linolenic acid with comparable amount as the one found in linseed
oil [12] and considerably higher than the one found in rapeseed or
soy oil. Saturated fatty acids (SFA) represented less than
10 % of the total fatty acids with palmitic C16:0 (5.4%) and
stearic acid C18:0 (2.7%) being the predominant ones in this
family. Finally, monounsaturated acids accounted for 13.2% of total
FA with a large majority of oleic acid C18:1 n-9 (12.6%). This
result and the others FA compositions of the tropical seed oils are
according to the previous studies [3,13,14].
Concerning the positional distribution of fatty acids within
TAG, the regiospecific analysis of kiwi seed oil showed that sn2
position was mainly occupied by unsaturated fatty acids with a
large majority of C18:3 n-3 (61.2%) followed by C18:2 n-6 (20.6%).
On the contrary, saturated fatty acids were almost exclusively
located on the two sn1 and sn3 external position. This results
underlines the nutritional importance of such regiodistribution of
essential fatty acids (C18:2 n-6 and C18:3 n-3) since the
localization of these latter at the central position of the TAG
backbone assure their optimal bioavailabilty during absorption of
kiwi seed oil [15]. Concerning passion fruit seed oil, a high ratio
of linoleic acid C18:3 was observed (73.4%) followed by oleic acid
C18:1 (13.7%) palmitic C16:0 (8.8%) and stearic C18:0 (2.4%) (table
2)( Table 2 ). Its regiodistribution
study showed that saturated fatty acids are absent from central sn2
position while mono unsaturated are distributed equally among the
three position. Finally, linoleic acid is preferentially located at
the sn2 position. The analysis of guava seed oil showed that this
is also a linoleic oil (C18:2, 77.0%). Its fatty acid composition
is comparable to the one of passion fruit with lesser amounts of
oleic acid C18:1 (7.6%). Regiodistribution illustrates that
linoleic occupies almost 90 % of the sn2 position in TAG while
SFA acid presents only 5%.
Phytosterols analysis. The determination of phytosterols was
carried out following the corresponding AFNOR Method [8] and GC-MS
analysis. Results showed that guava seed oil was the richest in
phytosterols with 329 mg/100 g whereas kiwi and passion fruit
oils contain respectively 269 and 209 mg/100 g (table 2). In
terms of different sterols proportion, stigmasterol was present in
a very large majority in guava (97%). Campesterol was detected at
9.9 mg/100 g accounting for 3.0% of total sterols. Others
phytosterols were present in traces amounts or not detected.
β-sitosterol is the more common phytosterol present in kiwi seed
oil (73.8%) while passion fruit oil contains equal amounts of
βsitosterol and stigmasterol. Finally, the presence of rare sterols
such as δ-7 stigmasterol (10.5 mg/100 g) and δ-7 avenasterol
(14.7 mg/100g) is worth noting. Phytosterols, especially
β-sitosterol, are been shown to exert protective effects against
cardiovascular diseases as well as many types of cancer, several
authors suggest. β-sitosterol can protect against oxidative stress
through modulation of antioxydant enzymes [16, 17]. These molecules
could be used against ageing skin cells.
Table 1 Oil content, molar composition and central
position of kiwi, guavas and passion fruit seed oils. (Values in
brackets indicate for the considered fatty acid its centesimal
proportion in central position and external positions.) Two GC
analyses of products from four experiments were made for TAG
analysis, other values given are means of three determinations.
|
kiwi
|
Passion Fruit
|
Guava
|
|
Oil content (oil/seed w %)
|
30.0 ± 0.8
|
18.4 ± 0.6
|
12.6 ± 0.5
|
|
Mol%
|
Mol%
|
Mol%
|
|
Fatty acid
|
TAG
|
|
|
TAG
|
Fatty acid sn-2
|
Fatty acid sn1-3
|
TAG
|
Fatty acid sn-2
|
Fatty acid sn1-3
|
|
C14:0
|
-
|
-
|
-
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
|
C16 :0
|
5.4 ± 0.2
|
- (0)
|
8.1 (100) ± 0.3
|
8.8 ± 0.3
|
0.5 (1.9) ± 0.0
|
13.0 (98.1) ± 0.2
|
7.8 ± 0.2
|
3.6 (15.4) ± 0.2
|
9.9 (84.6) ± 0.3
|
|
C16 :1
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
0.2 ± 0.0
|
-
|
0.3 ± 0.0
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
|
C17 :0
|
0.1 ± 0.0
|
-
|
-
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
|
C18 :0
|
2.7 ± 0.1
|
0.2 (1.2) ± 0.0
|
4.0 (98.8) ± 0.1
|
2.4 ± 0.1
|
0.2 (2.8) ± 0.0
|
3.5 (97.2) ± 0.1
|
5.8 ± 0.2
|
1.4 (8.0) ± 0.1
|
8.0 (92.0) ± 0.3
|
|
C18:1n-9
|
12.6 ± 0.2
|
17.3 (45.8) ± 0.3
|
10.3 (54.2) ± 0.3
|
13.7 ± 0.3
|
14.6 (35.3) ± 0.3
|
13.6 (64.6) ± 0.4
|
7.6 ± 0.3
|
5.5 (24.1) ± 0.2
|
8.7 (75.9) ± 0.3
|
|
C18:1n-7
|
0.3 ± 0.0
|
0.1 ± 0.0
|
0.4 ± 0.0
|
0.6 ± 0.0
|
0.2 ± 0.0
|
0.8 ± 0.1
|
0.6 ± 0.0
|
0.1 ± 0.0
|
0.9 ± 0.1
|
|
C18:2n-6
|
16.1 ± 0.3
|
20.6 (41.4) ± 0.4
|
13.9 (58.6) ± 0.3
|
73.4 ± 1.3
|
84.6 (38.4) ± 1.6
|
67.8 (61.6) ± 1.3
|
77.0 ± 1.6
|
89.5 (38.7) ± 1.6
|
70.8 (61.3) ± 1.5
|
|
C18:3n-3
|
62.3 ± 0.5
|
61.2 (32.4) ± 0.5
|
62.9 (67.6) ± 1.4
|
0.4 ± 0.0
|
-
|
0.6 (100) ± 0.0
|
0.3 ± 0.0
|
-
|
0.5 ± 0.0
|
|
C20 :0
|
0.2 ± 0.0
|
-
|
0.3 ± 0.0
|
0.2 ± 0.0
|
-
|
0.3 ± 0.0
|
0.5 ± 0.0
|
-
|
0.8 ± 0.1
|
|
C20 :1
|
0.2 ± 0.0
|
-
|
0.3 ± 0.0
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
0.1 ± 0.0
|
-
|
0.2 ± 0.0
|
|
Σ saturated FA
|
8.4 ± 0.4
|
0.2 (0.8) ± 0.0
|
12.4 (99.2) ± 0.3
|
11.6 ± 0.5
|
0.7 (2.0)
|
17.1 (98.0) ± 0.5
|
14.3 ± 0.5
|
5.0 (11.7) ± 0.3
|
19.0 (88.3) ± 0.5
|
|
Σ mono unsaturated FA
|
13.2 ± 0.3
|
17.4 (43.7) ± 0.3
|
11.2 (56.3) ± 0.3
|
14.6 ± 0.4
|
14.8 (33.8) ± 0.3
|
14.5 (66.2) ± 0.3
|
8.4 ± 0.4
|
5.6 (22.2) ± 0.3
|
9.8 (77.8) ± 0.3
|
|
Σ poly unsaturated FA
|
78.4 ± 1.1
|
81.8 (35.0) ± 1.3
|
76.0 (65.0) ± 1.4
|
73.8 ± 1.4
|
84.6 (38.2) ± 1.6
|
68.4 (61.8) ± 1.3
|
77.3 ± 1.6
|
89.5 (38.6) ± 1.6
|
71.2 (61.4) ± 1.3
|
Table 2 Phytosterols content and proportions in kiwi,
passion fruit and guava seed oils. (Values in brackets indicating
the centesimal proportion of each phytosterols.) Tr = traces lower
than 0.1 mg/l00 g. Nd = Not detected. All values given are means of
three determinations.
|
Kiwi
|
Passion Fruit
|
Guava
|
|
Total amount (mg/100 g)
|
269 ± 8.0
|
209 ± 7.4
|
329 ± 9.3
|
|
Campesterol
|
7.2 ± 0.8 (2.1)
|
28.2 ± 1.9 (13.5)
|
9.9 ± 0.9 (3.0)
|
|
Stigmasterol
|
7.8 ± 0.8 (2.3)
|
87.1 ± 3.2 (41.7)
|
319.1 ± 8.2 (97.0)
|
|
β-Sitosterol
|
251.3 ± 6.2 (73.8)
|
87.2 ± 3.2 (41.5)
|
Tr
|
|
δ-5 avenasterol
|
2.7 ± 0.4 (0.8)
|
6.9 ± 0.9 (3.3)
|
Tr
|
|
δ-7 Stigmasterol
|
10.5 ± 0.9 (3.1)
|
Nd
|
Nd
|
|
δ-7 avénasterol
|
14.7 ± 1.4 (4.3)
|
Nd
|
Nd
|
Tocopherols analysis
Guava seed oil was found to be the richest in tocopherols with 665
ppm in the crude oil (table 3)( Table 3
). Passion fruit and kiwi showed lower quantities with respectively
465 and 312 ppm In guava seed oil, γ-tocopherol was the predominant
with 550 ppm accounting for 82.7% of total tocopherols. A
comparable repartition was observed with kiwi seed oil, which
contains a majority of γ-tocopherol (84.3%) while α-tocopherol
accounted for about 15.4%. β-tocopherols and δ-tocopherols were
only present in traces quantities in this oil. Finally, passion
fruit oil contain both γ-tocopherols and δ-tocopherols in
comparable amounts (217 and 243 ppm respectively). The quantity and
nature of tocopherols naturally present in such unsaturated oils is
of crucial importance regarding their oxidative stability. Indeed,
tocopherols are natural antioxidants and depending of their nature,
they can differ in their antioxidant capacity. Indeed,
γ-tocopherols and δ-tocopherols are believed to be better
antioxidant than α-and β-tocopherols [18]. Therefore, one can
expect passion fruit and guava seed oils to be better protected
against oxidation than kiwi seed oil owing to their higher amounts
of γ-and δ-tocopherols.
Table 3 Tocopherols weight contents in kiwi, passion
fruits and guava seed oils (Values in brackets indicate the
centesimal proportion of each tocopherol). Tr = traces lower than
0.1 mg/100 g. All values given are means of three determinations.
|
Kiwi
|
Passion Fruit
|
Guava
|
|
312 ± 7.5
|
465 ± 8.4
|
665 ± 7.5
|
|
α-tocopherol
|
49 ± 0.6 (15.4)
|
5.0 ± 0.5 (1.1)
|
107 ± 1.9 (16.1)
|
|
β-tocopherol
|
Tr
|
Tr
|
3.0 ± 0.4 (0.5)
|
|
γ-tocopherol
|
263 ± 5.5 (84.3)
|
217 ± 5.8 (46.5)
|
550 ± 6.4 (82.7)
|
|
δ-tocopherol
|
Tr
|
243 ± 5.5 (52.4)
|
5.0 ± 0.4 (0.7)
|
Conclusion
In summary, the results of this study illustrate the nutritional
potential of the oils derived from the seeds of kiwi, passion fruit
and Guava. Such oils were shown to contain significant amounts of
polyunsaturated essential fatty acids with excellent
bioavailability due to their preferential localisation at the sn2
position of triacylglycerols backbone. Moreover, these oils contain
also significant amounts of sterols and tocopherols which justify
their use in nutritional and cosmetic industry. The valorization of
these waste materials becomes an attractive alternative because of
the promising commercial application for its seed oil in cosmetics.
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