a
ISSN 0101-2061 (Print)
ISSN 1678-457X (Online)
Food Science and Technology
DOI: http://dx.doi.org/10.1590/1678-457X.08317
Chemical composition of cold pressed Brazilian grape seed oil
Fernanda Branco SHINAGAWA1, Fernanda Carvalho de SANTANA1, Elias ARAUJO1, Eduardo PURGATTO1,
Jorge MANCINI-FILHO1*
Abstract
Grape seed oil (GSO) is an important by-product of the wine-making industry which has received attention as an alternative
source of vegetable oils; its chemical compounds can be influenced by agricultural practices and industrial processing. Knowledge
of the composition of Brazilian GSO is scarce; thus, this study aimed to analyze the chemical characteristics, as well as the
antioxidant activity of these oils. GSO samples were obtained from Brazilian markets and showed significantly high amounts
of phenolic, γ-tocotrienol and phytosterols as well as, the presence of several volatile compounds. Based on these results, is
possible to show that oils exhibited good antioxidant activity. Therefore, it can be inferred that Brazilian GSO had a considerable
content of phytochemical compounds with biological activity, which allows its association with other vegetable oils.
Keywords: seed oils; micronutrients; antioxidant activity.
Practical Application: In this study Brazilian grape seed oils were found to have potential to be used for some industrial sectors,
such as food ingredients and cosmetics industry. They showed high amount of polyunsaturated fatty acid and significant amount
of vitamin E, phenolics, phytosterols and volatile compounds. The knowledge regarding the composition of the products is
important once they are made from a sustainable way.
1 Introduction
Recent data from the Food and Agriculture Organization
of the United Nations (2014) shows that Brazil is the eleventh
highest grape producer in the world and its harvest corresponds
to approximately 1.5 million tons of grapes (Instituto Brasileiro
de Geografia e Estatística, 2012). The Rio Grande do Sul
state remains notable as the largest national producer, with
829.589 tons of production per year, representing approximately
55% of Brazil’s total cultivation within an increase of the wine
sector nationwide.
There is a worldwide trend to seek new sources of vegetable oil,
and a wide range of research has been conducted to identify new
oils from fruit, and especially fruit seeds (Madawala et al., 2012).
In this context, cold pressed grape seed oil is an environmentally
suitable vegetable oil as it is a value added by-product of wine
and grape juice-making process.
Lipid content in grape seed is around 7-20%. The importance
of grape seed use is mainly due to the fact that it is rich in lipids
and bioactive compounds, such as vitamin E, phytosterol and
phenolic compounds, among other components with biological
activity, which are important for food, pharmaceutical and
cosmetic industries (Kim et al., 2013; Rockenbach et al., 2010;
Nakamura et al., 2003).
Motivated by the lack of information on the chemical data
of grape seed oil (GSO) produced in the world and mainly by the
absence of information for Brazilian oils, the aim of this study
was to analyze the chemical composition of Brazilian GSO in
order to expand the knowledge of its characteristics and infer
its potential for human health.
2 Material and methods
2.1 Reagents and standards
All solvents were analytical grade, used within their expiration
dates and purchased from Merck (Darmstadt, Germany).
The hexane and isopropanol used in this study were high
performance liquid chromatography (HPLC) grade. Furthermore,
the boron trifluoride-methanol solution 14% (BF3 14%), fatty
acid methyl ester (FAME) mix (C4:0-C24:0), methyl tridecanoate
(C13:0), tocopherols (α-tocopherol, β-tocopherol, γ-tocopherol,
δ-tocopherol), δ-tocotrienol, campesterol, stigmasterol, sitosterol,
5-α-cholestane and β-carotene (type II, synthetic) were obtained
from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Oil samples
GSO was obtained from Brazilian markets between July
2012 and August 2013. The cold pressed samples were identified
as follows: UVB (Antônio Prado, RS, Brazil), URS (Bento
Gonçalves, RS, Brazil), OOV (Guaribalde, RS, Brazil) and CAC
(Estância Velha, RS, Brazil). After receiving the samples at the
Lipids Laboratory (Faculty of Pharmaceutical Science, University
of São Paulo, São Paulo, Brazil), they were fractionated into
amber glass bottles, nitrogen gas was added in the headspace
and the samples were maintained at –20 °C until further analysis.
Received 07 Mar., 2017
Accepted 25 July, 2017
1
Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Science, Universidade de São Paulo – USP, São Paulo, SP, Brazil
*Corresponding author: jmancini@usp.br
164 164/171
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
Shinagawa et al.
2.3 Color parameters
Color parameters were evaluated by colorimeter (ColorQuest
XE, Hunter Assoc. Laboratory, Reston, USA), with a field of
view of 10°, D65 type illuminant and slit diameter of 1 mm.
The following color coordinates were determined in the Cielab
system: lightness (L*), redness (a*, red to green axis) and
yellowness (b*, yellow to blue axis).
2.4 Fatty acid composition
Fatty acid methyl esters (FAME) were obtained using BF3
14% according to the Ce 2-66 method (American Oil Chemists’
Society, 2009). Separation of FAME was performed using a gas
chromatograph (Shimadzu Plus 2010, Kyoto, Japan), equipped with
a split injector system, an auto-sampler and fused silica capillary
column (SP-2560, 100 m × 0.25 mm × 0.2 µm). The column
temperature gradient was programmed between 140 and 220 °C
and detection was performed with a flame ionization detector
(FID) at 260 °C. Helium was used as a carrier gas (1 mL min-1).
FAME were identified by comparing the retention time of the
sample peaks with a standard mixture of 37 fatty acid methyl
esters, C4:0-C24:0 (Sigma Chemical Co St. Luis, MO, USA).
The results were expressed as direct area % for each peak identified.
2.5 Phytosterol composition
Phytosterol determination was followed by Duchateau et al.
(2002) with some modifications. Internal standard 5-α-colestane
(1.0 mg mL-1 hexane) was added to each sample before
saponification (methanolic KOH 3% at 50 ± 2 °C for 3 hours).
After three extractions with hexane, the organic phase was
collected, evaporated and ressuspended in 150 µL of hexane
before injection into a GC system. A gas chromatograph
(Shimadzu Plus GC 2010, Kyoto, Japan) equipped with a FID
detector and a fused silica capillary column LM 5 (5% phenyl
95% methylpolysiloxane 60 m × 0.25 mm internal diameter
with 0.25 µm particle size) was used. The GC program was as
follows: column temperature, 290 °C; detector temperature,
300 °C; helium (1 mL min-1); and split ratio 1/50. Phytosterols
were identified and quantified by comparing the relative retention
time of standard campesterol (C5157), stigmasterol (S 6126)
and β-sitosterol (S 9889) (Sigma-Aldrich Co., St. Louis, USA).
The results were expressed in mg 100 g-1 of oil.
2.6 Tocopherol/Tocotrienol profile
α-, β- and γ-tocopherol and γ-tocotrienol levels were
determined according to the Ce 8-89 method (American Oil
Chemists’ Society, 2009). The oil samples were diluted with hexane
(0.1 g mL-1) and filtered through a 0.22 µm PTFE membrane filter.
Then, samples were analyzed by an HPLC (Shimadzu CBM-20A,
Kyoto, Japan) consisting of an RF-10AXL fluorescence detector
(excitation = 295 nm and emission = 330 nm). A normal silica
phase column (Sim-pack CLC-SIL, 250 × 4.6 mm internal diameter
with 0.5 µm particle size) was used with hexane:isopropanol
(99:1 v/v) as a mobile phase. The system was operated isocratically
at a flow rate of 1 mL min-1. Identification of synthetic standards
tocopherols (α, β, γ, and δ-tocopherol and δ-tocotrienol) (Sigma
Chemical Co St. Louis, MO, USA) was conducted by comparing
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
the HPLC retention time with those of standard compounds
under the same operating conditions, and the quantification
was based on an external standard method. The results were
expressed in mg 100 g-1 of oil.
2.7 Total phenolics
The phenol extraction was carried out with a methanolic
extract obtained according to Bail et al. (2008) and quantification
was based on the colorimetric method by Hills & Swain
(1959) using Folin-Ciocalteau adapted to a microplate reader
(Biotek®, Synergy HT model, Winooski, VT, USA). Absorption was
measured at 720 nm. A standard curve was used, with gallic
acid (0.50 to 5.00 mg mL-1), obtaining a correlation coefficient
of 0.9987. The content of phenolic compounds in the oils was
expressed as mg of gallic acid equivalents per 100 grams of
sample (mg GAE 100 g-1).
2.8 Total carotenoids
Total amounts of carotenoids were determined using
Ranjith et al. (2006) following some modifications. GSO were
dissolved in hexane (0.1 g mL-1), vortexed for 30 s with NaCl
0.5%, and centrifuged for 10 min at 1500 g. Aliquots of 250 µL
of the upper phase were collected and measured at 460 nm with
a microplate reader. Carotenoid quantification was based on a
calibration curve with β-carotene standard, type II: synthetic
(Sigma-Aldrich Co., St. Louis, USA). The results were expressed
as mg of β-carotene equivalent per one hundred grams of oil
(mg bCE 100 g-1 of oil).
2.9 Total chlorophylls
Total chlorophyll analysis was performed by MinguezMosquera et al. (1991) based on a dilution of the sample in
cyclohexane PA, then read at 670 nm using spectrophotometric
equipment. Quantification was conducted by the following equation:
Total Chlorophyll = (Absorbance × 106) / (613 × 102 × oil density)
and the results were expressed as milligrams per kilogram of
chlorophyll oil (mg kg-1).
2.10 Volatile compounds
Volatile compounds were extracted by headspace solid-phase
microextraction and analyzed by gas chromatographic massspectrometric method (HS-SPME-CG/MS). SPME compatible
vials containing 1 g of the each oil were extracted isothermally
for 24 h to produce sufficient amounts of analytes at room
temperature (25 °C) and then the headspace was absorbed using a
pre-conditioned column (Supelco 57330-U, Melbourne, Australia)
for 30 minutes. After sampling had been carried out, the SPME
fiber was immediately exposed to the inlet temperature of the
GC–MS instrument. For the separation of volatile compounds,
a non-polar column (30 m × 0.25 mm × 0.25) (Hewlett-Packard
HP-6890 model, HP-5MS, California, USA) equipped with a mass
selective detector (Hewlett-Packard HP-5973, California, USA) was
used. The column temperature gradient was programmed between
40 and 160 °C and the injector temperature was 200 °C. After
4 min using the splitless mode of the expurgate GC–MS-system,
165/171 165
Brazilian grape seed oil composition
a constant flow of 1 mL min-1 was applied, carrying helium.
Mass spectra were recorded with a scan range of 10-300 amu.
Volatile compound identification was carried out using Wiley
275, NBS 75 K and in-house mass spectra libraries and partly
by the co-injection of reference compounds.
was used and the reaction was performed at 37 °C the reaction
was started by the thermal decomposition of AAPH (2,2’-azobis
[amidinopropane] dihydrochloride) at concentration of
135 mM. A Trolox calibration solution was used (6.25-100 µM).
Fluorescence was measured immediately after addition and
measurements were then taken every 5 min for one hour. ORAC
values were expressed as micromolar Trolox equivalents per
100 grams of sample (TE µM 100 g-1).
2.11 Oxidative established index (OEI)
The oxidative stability index was determined using Rancimat
equipament (model 743, Metrohm Ltd., Herisau, Switzerland)
according to the Cd 12b-92 method (American Oil Chemists’
Society, 2009). The induction period (IP) of oxidation at a
temperature of 120 °C and oxygen flow 20 L h-1 was determined
in hours (h).
2.13 Statistical analysis
All analyses were performed in triplicate and results
were expressed as mean values ± standard deviation (SD).
The Box-Cox transformation technique was used to normalize
non-normal data and significant differences were evaluated using
a variance analysis (ANOVA) test followed by a Tukey’s Test for
significance at the 5% level (p <0.05). For samples that could
not be normalized, the Kruskal Wallis Test was used followed
by Dunn’s Test (p <0.05). Correlation analyses were performed
using Pearson’s Test. A multivariate statistical analysis of selected
chemical data was performed using principal component analysis
(PCA). Statistical analyses were conducted using Prism 5 software
(GraphPad, California, USA) and Statistica 7 (Statsoft, Tulsa,
Oklahoma, USA).
2.12 Antioxidant capacity assays
ABTS+ (2,2 ‘azinobis [3-ethylbenzothiazoline-6-sulfonic acid])
radical scavenging activity was measured according to Re et al.
(1999) adapted for a microplate reader. TEAC measurements
were achieved by comparing decreased absorption after using
20 µL of GSO extract (Bail et al., 2008), reagent blank or Trolox
standard, respectively, with 200 µL of 7 mM ABTS+. Absorbance
was monitored at 734 nm 6 min after the addition of reactant
at 25 °C. The TEAC value is expressed as micromolar Trolox
equivalents per 100 grams of sample (TE µM 100 g-1).
3 Results
The ORAC method used was described by Prior et al. (2003)
the lipophilic and hydrophilic methods were carried out. For the
lipophilic method, samples were diluted in acetone:water (1:1)
with β-cyclodextrin. For the hydrophilic method, methanolic
extract was used according to Bail et al. (2008) and then diluted
in ethanol. An automated ORAC assay was carried out on a
microplate reader at 493nm (filter 485/20) and an emission of
515 nm (filter 528/20). For both methods, fluorescein (40 ηM)
3.1 Fatty acid composition
Lipid profiles are presented in Table 1; all analyzed samples,
without exception, showed higher linoleic acid (C18: 2 n-6)
concentrations ranging from 72.19 to 75.02%, followed by
monounsaturated oleic acid (C18: 1 n-9) between 14.80 to
17.34%, and saturated palmitic acid (C16:0) (9.72 to 10.22%).
Table 1. Fatty acid, vitamin E isomers and phytosterols content (g 100 g-1) of different grape seed oils obtained from Brazilian market.
OVB
Fatty Acid (g 100 g-1)
C16:0
C18:0
C18:1 c (n-9)*
C18:2 c (n-6)*
C18:3 (n-3)
ΣSFA
ΣMUFA
ΣPUFA*
Vitamin E isomers (mg 100 g-1)
α-T
γ-T
γ-T3
Phytosterols (mg 100 g-1)
Campesterol
Stigmasterol
β-sitosterol
URS
OOV
CAC
6.26 ± 0.07d
3.42 ± 0.08cd
15.83 ± 0.07e
74.15 ± 0.20ab
0.21 ± 0.01e
9.72 ± 0.14d
15.92 ± 0.07e
74.36 ± 0.20ab
6.52 ± 0.09c
3.36 ± 0.05cd
17.20 ± 0.05c
72.19 ± 0.06ab
0.49 ± 0.03a
9.99 ± 0.04cd
17.34 ± 0.05c
72.67 ± 0.05ab
6.61 ± 0.02bc
3.22 ± 0.23d
14.80 ± 0.07g
75.02 ± 0.31a
0.36 ± 0.03bcd
9.83 ± 0.22d
14.80 ± 0.07g
75.38 ± 0.29a
1.76 ± 0.01d
0.49 ± 0.02c
450.99 ± 10.34a
1.69 ± 0.02e
0.61 ± 0.02c
432.50 ± 14.47ab
1.33 ± 0.02f
0.51 ± 0.03c
453.48 ± 4.79a
1.34 ± 0.01f
0.47 ± 0.01c
417.03 ± 12.70b
12.78 ± 0.34b
31.98 ± 5.28a
83.50 ± 1.28a
13.79 ± 0.07a
32.63 ± 0.74a
88.86 ± 1.14a
12.90 ± 0.31b
30.66 ± 0.32a
84.17 ± 1.47a
13.51 ± 0.10ab
30.57 ± 0.19a
91.94 ± 0.32a
6.70 ± 0.01b
3.53 ± 0.04bc
15.31 ± 0.05f
74.12 ± 0.11ab
0.34 ± 0.01d
10.22 ± 0.05bc
15.31 ± 0.05f
74.47 ± 0.11ab
Data are mean ± SD (n=3). Means with different letter in a line are statistically significant at 5% level probability by Tukey Test. *Non-parametric data were obtained from Kruskal-Wallis
Test. Data n.d. (non-detectable). ƩSFA = sum of saturated fatty acids; ƩMUFA = sum of monounsaturated fatty acids; ƩPUFA = sum of polyunsaturated fatty acids. α-T: α tocopherol;
γ-T: γ tocopherol; γ-T3: γ tocotrienol.
166 166/171
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
Shinagawa et al.
3.2 Phytosterol content
3.5 Total phenolic, carotenoid and chlorophyll compounds
Campesterol, stigmasterol and β-sitosterol concentrations ranged
between 12.78 to 13.79; 30.57 to 32.63 and 83.50 to 91.94 mg 100 g-1,
respectively (Table 1).
Total phenolic quantification showed a range from
13.92 to 27.87 mg 100 g-1 (Table 3). High values in cold pressed
GSO (13.92 to 27.87 mg 100 g-1) were observed and they were
statistically similar (p <0.001).
3.3 Tocopherols and Tocotrienol contents
Quantification of the tocopherol isomers (α- and γ-) and
γ-tocotrienol were conducted (Table 1). Low values were
found when the α-tocopherol isomer was quantified, ranging
between 1.33 to 1.76 mg 100 g-1. For all samples, γ-T3 was the
main isomer. Samples submitted to cold pressing showed levels
between 417.03 and 453.48 mg 100 g-1 of γ-T3 and only the CAC
sample differed significantly from the others.
Total carotenoids and chlorophyll, were also measured
(33.85 to 59.85 mg bCE 100 g-1 and 0.30 to 0.40 mg 100g-1,
respectively) (Table 3). For both pigments, the URS sample
showed a significantly higher value than other samples (both cases,
p <0.001). Carotenoid contents in oils is important, for instance,
as they provide color (strong correlation with component b*;
r=0.737, p <0.05) and have a relation the function of vitamin A
precursors (fat-soluble vitamin important to human metabolism).
3.4 Volatile compounds
3.6 Color parameters
Twenty five volatile compounds were identified: six alcohols, four
aldehydes, three carboxylic acids, four esters, three hydrocarbons,
two ketones and two terpenes (Table 2). Alcohol compounds
were more prevalent (%) for all analyzed samples.
Samples showed low luminance (L*), indicating high
turbidity, which ranged from 4.58 to 16.2; UVB was the
clearest (p <0.001) sample, while the URS stood out as
the most turbid. Positive value for the component b* was
Table 2. Volatiles compounds identification of different grape seed oil from Brazilian market based by HS-SPME-CG/MS.
Compounds
Alcohol
Ethyl alcohol
Isoamilic
Hexanol
n-Pentanol
1-octen-3-ol
Phenylethylalcohol
Aldehyde
Isopentanal
Pentanal
Hexanal
2-Heptenal
Carboxylic Acids
Acetic acid
Isovaleric acid
Hexanoic acid
Esters
Banana oil
Ethyl hexanoate
Ethanodiol, diacetate
Ethyl octanoate
Furan Compound
Furfural
Hydrocarbon
Hexane
Toluene
Styrene
Ketones
Acetoin
2,4-methyl-2-hexanone
Terpenes
α-Limonene
4-Carene
CAS number
Odor description
UVB
[64-17-5]
[123-51-3]
[111-27-3]
[71-41-0]
[3391-86-4]
[60-12-8]
Floral
Fruity
Citrus, eucalyptus
Fruity, banana-like
Mushroom, fruity
Honey, floral
[590-86-3]
[110-62-3]
[66-25-1]
[57266-86-1]
Fruity
Green grass
Rancid
+
[64-19-7]
[503-74-2]
[142-62-1]
Vinegar
Sweat smell, rotten
+
+
[123-92-2]
[123-66-0]
[542-10-9]
[106-23-1]
Sweet, fruity
Fruity, floral
+
+
+
+
[98-01-1]
Almond
[110-54-3]
[108-88-3]
[100-42-5]
Floral
Pungent, roasty
[513-86-0]
[105-42-0]
Buttery
Spicy, acetone
[5989-54-8]
[5208-50-4]
Fresh citrus, orange-like
Sweet, pungent
+
+
+
URS
OOV
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Fruity, floral
CAC
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ Positive
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
167/171 167
Brazilian grape seed oil composition
Table 3. Phytochemicals and pigments quantification from different grape seed oils obtained from Brazilian market and their antioxidant activity.
UVB
Cielab parameters
L*
16.23 ± 0.89a
a*
-0.28 ± 0.05c
b*
10.24 ± 0.46a
Total Minorities (mg 100 g-1)
Phenolics
27.87 ± 3.69a
Carotenoids
51.67 ± 2.08b
Chlorophylls
0.35 ± 0.09b
Oxidative Stability Index (h)
Induction Time
3.09 ± 0.55a
Antioxidant Activity (µM TE 100 g-1)
ORAC lipophilic
224.00 ± 22.28c
ORAC hidrophilic
625.14 ± 35.44b
TEAC
180.50 ± 15.82a
URS
OOV
CAC
4.58 ± 0.45d
0.86 ± 0.22bc
4.58 ± 0.70cd
7.95 ± 0.36c
-1.11 ± 0.44b
8.40 ± 0.74b
7.50 ± 0.09c
-1.05 ± 0.18b
8.22 ± 0.18b
13.92 ± 3.60b
59.85 ± 3.06a
0.40 ± 0.05a
18.21 ± 1.56b
33.94 ± 4.93c
0.30 ± 0.03c
16.22 ± 0.56b
33.85 ± 4.16c
0.36 ± 0.14b
2.94 ± 0.26ab
2.36 ± 0.18bc
1.37 ± 0.09d
432.10 ± 17.75a
728.21 ± 17.01a
53.41 ± 3.97bc
362.92 ± 29.75b
588.84 ± 5.73bc
205.52 ± 9.76a
370.32 ± 4.20b
563.83 ± 14.81c
192.67 ± 6.99a
Data are mean ± SD (n=3). Means with different letter in a line are statistically significant at 5% level probability by Tukey Test. L*: luminosity; a*: green/red; b*: blue/yellow. Oxidative
stability index evaluation was compared with soy oil (3.67 ± 0.05 h) as a standard in the same conditions. ORAC: Oxygen radical absorbance capacity; TEAC: Trolox equivalent
antioxidant capacity.
obtained, with the UVB sample having the highest intensity
of yellow (10.24), and the URS sample had the lowest (4.58).
Negative values of component a* were found, ranging from
–0.28 to -1.11 (Table 3).
3.7 Oxidative Stability Index (OSI)
Induction times are showed in Table 3. The degree of
unsaturated fatty acids and the presence of minor components
in oils are important during the evaluation of the oxidation
stability of oils. An average induction time of 2.44 h was noted.
UVB showed statistically higher oxidative stability under the
accelerated conditions used in this study (120 °C for 20 h L-1).
URS and UVB samples showed higher induction times compared
to the others (3.09 and 2.94 h, respectively). In addition, both
samples also exhibited higher concentrations of minor components
with antioxidant activity, which may have contributed to their
increased stability.
3.8 Antioxidant capacity assays
The TEAC assay was based on the ABTS+ radical scavenging.
A range from 53.41 to 205.52 µM TE 100 g-1 activity is shown
in Table 3. Cold pressed samples such as OOV and CAC had
the highest values of antioxidant activity by this method, with
a significant difference (p <0.001) when compared to UVB and
URS. The antioxidant activity of Brazilian GSO is indicated in
Table 3. The URS sample showed a higher value statistically
(432.10 µM TE 100 g-1) when compared to the other samples
in the lipophilic ORAC methodology. On the other hand, the
OOV (362.92 µM TE 100 g-1) and CAC (370.32 µM TE 100 g-1)
samples showed statistically lower values compared to the
URS sample; however, the results did not differ between them.
UVB showed a significantly lower value (224.00 µM TE 100 g-1).
The hydrophilic ORAC assay showed the highest activity for URS
(728.21 µM TE 100 g-1) followed by UVB and OOV (625.14 and
588.84 µM TE 100 g-1, respectively).
168 168/171
4 Discussion
It is widely known that vegetable oils consumption rather
than of solid fats is vital to maintaining normal metabolism, in
this context GSO consumption plays an important role in human
health once the proportions of fatty acids identified increased in
the order of AGS < MUFA < PUFA, within an average of 9.94,
15.84 and 74.22%, respectively; this is in agreement with the
ranges recommended by international legislation (from 58 to
78% of LA, 12 to 28% oleic acid and 5.5 to 11% palmitic acid)
(Food and Agriculture Organization of the United Nations, 2001)
and data reported by Sabir et al. (2012) and Crews et al. (2006).
High content of PUFAs such as linoleic acid is highly correlated
with functional properties, such as a decrease of human total
serum cholesterol and LDL-c (Dhvamani et al., 2014).
Seed oils are not only a source of fatty acid, but also a valuable
source of micronutrients, such as sterols, carotenoids and tocols.
β-sitosterol was found to be a major phytosterol compound
in Brazilian GSO. No statistically significant result was found
between cold pressed samples, with a mean of 77.72 mg 100 g-1,
i.e. over 70% of total phytosterols. This value is in agreement with
those found for Rubio et al. (2009). The significant presence of
phytosterols in GSO reinforces the potential health benefits of
its consumption, since they have a similar chemical structure to
cholesterol, so compete for intestinal absorption sites, reducing
body cholesterol absorption capacity (Laakso, 2005).
Tocols are the major primary antioxidant group present in
vegetable oils (Fernandes et al., 2013). The γ-tocotrienol isomer
was observed as a major GSO constituent. In relation to this
content, it can be seen that cold pressed oils had a concentration
that was 2 times higher when compared to the legislation
(Food and Agriculture Organization of the United Nations,
2001). This correspond to more than 96% of total isomers, with
concentrations in the range between 57.22 and 453.48 mg 100 g-1.
In contrast, tocopherol concentrations (α- and γ-T) represented
less than 1% of the total from cold pressed samples (Table 1).
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
Shinagawa et al.
Our finds showed close values from Brazilian GSO to other
grape seed oils (Fernandes et al., 2013; Madawala et al., 2012;
Crews et al., 2006). In this context, γ-tocotrienol activity has
received great attention due to its anti-inflammatory effects, acting
on the NF-kB signaling pathway, confirming the functional effect
of these compounds (Kaileh & Sen, 2010) and the inhibition of
oxidative stress in HepG2 cells (Choi et al., 2010).
There are several minor compounds present in vegetable
oils, including fat-soluble vitamins, phytosterols, pigments and
phenolic compounds. Some of them had significant antioxidant
capacity, which means greater potential to inhibit lipid oxidation
reactions to which oils are more susceptible, i.e. the presence of
minor constituents contributes to product quality and nutritional
value improvements. There are numerous studies reporting minor
compound concentrations in GSO produced in many countries,
such as Portugal, Spain, Italy and France (Fernandes et al., 2013;
Navas, 2009; Rubio et al., 2009; Crews et al., 2006). Similarly, it
was important to characterize GSO from the Brazilian market
as no data were found in the literature.
Our results showed a great variability of minor compound
concentrations, which may be related to the different production
area to obtain the Brazilian GSO products. As the cold pressing
process does not involve chemical or heat treatment, this stands
out as an interesting method once consumers prefer, currently,
natural and healthy products, and especially those which maintain,
integrally or otherwise, the bioactive compounds present in
seeds (Passos et al., 2010).
There is a significant loss of many compounds, particularly
phenolic acids, during oil extraction due to the low solubility
in the lipophilic phase, however some has potential to migrate
during extraction; as a result, turbidity can be found. In this
sense, Table 3 shows low luminance value (L*) for the samples,
which indicate high turbidity. The total phenolics quantification
in the present study was in agreement with the results of different
studies in the literature (Siger et al., 2008).
Color evaluation in commercial products is important, not
only as an attribute that contributes to consumer acceptance, but
also because of its relationship with the bioactive compounds
present. Data from CieLab coordinates showed a predominance
of component b* (yellow) and component a* (green). These results
indicate a yellowish color, which is characteristic of vegetable
oils. No data were found regarding GSO color in the literature;
nevertheless the results obtained from palm, soybean, sunflower,
olive, corn and pumpkin cold pressed oils ranged from 44.8 to 69.5
for the parameter L*, with negative values for parameter a*
(range from 0.2 to 4.4) and parameter b* (range from 9.2 to 28.8)
(Rezig et al., 2012).
Volatile compounds were obtained, suggesting that compounds
such as hexanol and isoamyl alcohol resulted from the seed
fermentation process, indicating an herbaceous and fruity note,
respectively. Aldehydes such as pentanal, hexanal and furfural,
fruity and/or floral notes, as well as ethyl hexanoate and ethyl
octanoate esters were presented in the samples. On the other
hand, it should be noted that during the oxidation process, some
volatile compounds are formed which impart an unpleasant
flavor, as observed by the identification of isovaleric acid, styrene
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
and 2-heptenal from cold pressed URS, OOV and CAC samples.
Cavalli et al. (2004) demonstrated that during the extraction
process, maturation stage and cultivation practices can deeply
influence the volatile compounds profile of oils.
In order to characterize the potential antioxidant activity
present in commercial GSO and due to the complexity of
interactions and diverse mechanisms found in many antioxidant
compounds present in vegetable oils, three different methods
were used. As TEAC method is based on electron transfer
from an antioxidant compound to an oxidant, this method
do not show significant correlation with ORAC, hydrophilic
and lipophilic assays, whereas for these other two methods
they are based on hydrogen atom transfer of an antioxidant
compound to block the peroxyl radical. On the other hand,
because of the similarity between the TEAC methodology and
the determination of total phenolic compounds, we found a
strong and significant correlation between the two methods
(r = 0.791; p <0.05). Meanwhile, antioxidant capacity identified
by the hydrophilic and lipophilic assays were compared with the
ORAC method and the hydrophilic method gave results that were
higher than the lipophilic method, showing that after extraction,
antioxidant compound selection occurs. Our suggestion is that
in the hydrophilic method, from the hydrophilic GSO extract,
the selection of water-soluble compounds occurs, such as
phenolic derivatives of benzoic acid (gallic acid) and/or alcoholic
derivatives (flavonoids, secoiroids, lignans). On the other hand,
in the lipophilic GSO extract, major contributors to antioxidant
activity are namely carotenoids (β-carotene, lutein), tocopherols
and tocotrienols, chlorophylls, polymeric proanthocyanidines
and high molecular weight tannins (Leão et al., 2014).
In general, high antioxidant activity was found; this outstanding
activity could be attributed to the high phytochemical content,
as they are rich in phenolics, carotenoids and tocopherols
(see Tables 1 and 2). However, during the extraction process,
regardless of the possibility of the significant loss of many of these
minor compounds, due to their low solubility in oil, a strong
positive correlation was found between the lipophilic ORAC
method values and the content of compounds which have a greater
affinity to non-polar components such as carotenoids (r = 0.792,
p < 0.05), vitamin E activity (r = 0.860, p < 0.01), γ-T3 (r = 0.81;
p < 0.05) and total phytosterols (r = 0.910, p < 0.01), as well as
with their fractions campesterol, β-sitosterol and stigmasterol
(r = 0.909, r = 0.908 and r = 0.887, p < 0.01, respectively).
Antioxidant activity determination by the TEAC method showed
that samples had activities ranging from 53.41 to 205.52 µM TE 100 g-1.
Bail et al. (2008) investigated Austrian GSO antioxidant capacity
(from 9.0 to 116.0 µM TE 100 g-1 of oil) and Fernandes et al.
(2013) worked with ten different oils from many varieties of
Portuguese grapes, founding a range from 33.4 to 48.9 mmol
TE 100 mL-1 of oil.
Total phenolic content showed high, positive and significant
correlation with carotenoids content (r = 0.756, p < 0.05),
component b* (r = 0.934, p < 0.01), tocopherol content (r = 0.822,
p < 0.05) and hydrophilic antioxidant activity when using the
ORAC method (r = 0.831, p < 0.05).
169/171 169
Brazilian grape seed oil composition
In summary, our results indicate GSO obtained from Brazilian
markets showed presence of important minor components
(such as phenolics, carotenoids, γ-tocotrienol and β-sitosterol)
and linoleic essential fatty acid, which leads GSO to be recognized
as an interesting ingredient for human consumption.
It is widely known that vegetable oils consumption in
place of solid fats is vital important to maintaining for health
maintenance. In particular, this context, grape seed oil stands
out as a suitable alternative to other commonly used vegetable
oils because of its once contains high amounts of n-6 fatty
acid and bioactive compounds and equally importantly, it is
an environmentally friendly oil as it is a sustainable option for
agro-industrial to obtain a value added a by-product of wine
and grape juice-making processes.
5 Conclusions
Agro-industrial waste is an excellent way of adding value to
crop production. In this context, the wine processing industry
produces tons of seeds as by-products. According to our analysis,
grape seed oils are rich in essential and other health-benefitting
fatty acids. In particular, Brazilian grape seed oil contains a
higher number of volatiles, and from the nutritional aspect,
high amount of total antioxidant capacity and total phenols.
Our results demonstrate the viability of developing nutraceuticals
or functional food ingredients from these commercial cold
pressed grape seed oils for optimal human health.
Acknowledgements
The authors are grateful for financial support of CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)
for the Doctoral scholarship to Fernanda B. Shinagawa and
Fernanda C. de Santana and CNPq (559768/2010-9) for the
project funding.
References
American Oil Chemists’ Society – AOCS. (2009). Official methods and
recommended practices (6th ed.). Champaign: AOCS.
Bail, S., Stuebiger, G., Krist, S., Unterweger, H., & Buchbauer, G. (2008).
Characterization of various grape seed oils by volatile compounds,
triacylglycerol composition, total phenols and antioxidant capacity.
Food Chemistry, 108, 1122-1132. PMid:26065780. http://dx.doi.
org/10.1016/j.foodchem.2007.11.063.
Cavalli, J. F., Fernandez, X., Lizzani-Cuvelier, L., & Loiseau, A. M.
(2004). Characterization of volatile compounds of French and
Spanish virgin olive oils by HS-SPME: Identification of qualityfreshness markers. Food Chemistry, 88(1), 151-157. http://dx.doi.
org/10.1016/j.foodchem.2004.04.003.
Choi, Y., Lee, J., Kim, Y., Yoon, J., Jeong, H. S., & Lee, J. (2010). A
Tocotrienol-Rich Fraction from grape seeds inhibits oxidative stress
induced by tert-Butyl Hydroperoxide in HepG2 cells. Journal of
Medicinal Food, 13(5), 1240-1246. PMid:20726785. http://dx.doi.
org/10.1089/jmf.2009.1342.
Crews, C., Hough, P., Godward, J., Brereton, P., Lees, M., Guiet, S., &
Winkelmann, W. (2006). Quantitation of the main constituents
of some authentic grape-seed oils of different origin. Journal of
Agricultural and Food Chemistry, 54(17), 6261-6265. PMid:16910717.
http://dx.doi.org/10.1021/jf060338y.
170 170/171
Dhvamani, S., Rao, Y. P. C., & Lokesh, B. R. (2014). Total antioxidant
activity of selected vegetable oils and their influence on total
antioxidant values in vivo: a photochemiluminescence based
analysis. Food Chemistry, 164, 551-555. PMid:24996369. http://
dx.doi.org/10.1016/j.foodchem.2014.05.064.
Duchateau, G. S. M. J. E., Bauer-Plank, C. G., Louter, A. J. H., van der
Ham, M., Boerma, J. A., van Rooijen, J. J. M., & Zandbelt, P. A. (2002).
Fast and accurate method for total 4-desmethyl sterol(s) content
in spreads, fat-blends, and raw materials. Journal of the American
Oil Chemists’ Society, 79(3), 273-278. http://dx.doi.org/10.1007/
s11746-002-0473-y.
Fernandes, L., Casal, S., Cruz, R., Pereira, J. A., & Ramalhosa, E. (2013).
Seed oils of ten traditional Portuguese grape varieties with interesting
chemical and antioxidant properties. Food Research International,
50(1), 161-166. http://dx.doi.org/10.1016/j.foodres.2012.09.039.
Food and Agriculture Organization of the United Nations – FAO.
(2001). Codex standard for named vegetable oils: Codex STAN 2101999. Rome: FAO.
Food and Agriculture Organization of the United Nations – FAO.
(2014). FAOSTAT. Retrieved from http://faostat3.fao.org.
Hills, W. E., & Swain, T. (1959). The phenolic constituents of Punnus
domestica. The quantitative analysis of phenolic constituents. Journal
of the Science of Food and Agriculture, 19, 63-68.
Instituto Brasileiro de Geografia e Estatística – IBGE. (2012).
Produção Agrícola Municipal. Retrieved from ftp://ftp.ibge.gov.br/
Producao_Agricola
Kaileh, M., & Sen, R. (2010). Role of NF-κB in the anti-inflammatory
effects of tocotrienols. Journal of the American College of Nutrition,
29(3, Suppl), 334S-339S. PMid:20823493. http://dx.doi.org/10.108
0/07315724.2010.10719848.
Kim, Y., Choi, Y., Ham, H., Jeong, H. S., & Lee, J. (2013). Protective effects
of oligomeric and polymeric procyanidin fractions from defatted
grape seed on tert-butyl hydroperoxide-induced oxidative damage
in HepG2 cells. Food Chemistry, 137(1-4), 136-141. PMid:23200001.
http://dx.doi.org/10.1016/j.foodchem.2012.10.006.
Laakso, P. (2005). Analysis of sterol from various food matrices.
European Journal of Lipid Science and Technology, 107(6), 402-410.
http://dx.doi.org/10.1002/ejlt.200501134.
Leão, K. M. M., Sampaio, K. L., Pagani, A. A. C., & da Silva, M. A. A.
P. (2014). Odor potency, aroma profile and volatiles composition
of cold pressedoil from industrial passion fruit residues. Industrial
Crops and Products, 58, 280-286. http://dx.doi.org/10.1016/j.
indcrop.2014.04.032.
Madawala, S. R. P., Kochhar, S. P., & Dutta, P. C. (2012). Lipid components
and oxidative status of selected specialty oils. Grasas y Aceites, 63,
143-151. http://dx.doi.org/10.3989/gya.083811.
Minguez-Mosquera, M. I., Rejano-Navarro, L., Gandul-Rojas, B.,
Gomez, A. H. S., & Garrido-Fernandez, J. (1991). Color-pigment
correlation in virgin olive oil. Journal of the American Oil Chemists’
Society, 68(5), 332-336. http://dx.doi.org/10.1007/BF02657688.
Nakamura, Y., Tsuji, S., & Tonogai, Y. (2003). Analysis of proanthocyanidins
in grape seed extracts, health foods and grape seed oils. Journal of
Health Science, 49(1), 45-54. http://dx.doi.org/10.1248/jhs.49.45.
Navas, P. B. (2009). Chemical composition of the virgin oil obtained by
mechanical pressing form several grape seed varieties (Vitis vinífera
L.) with emphasis on minor constituents. Archivos Latinoamericanos
de Nutricion, 59(2), 214-219. PMid:19719020.
Passos, C. P., Silva, R. M., da Silva, F. A., Coimbra, M. A., & Silva, C.
M. (2010). Supercritical fluid extraction of grape seed (Vitis vinifera
L.) oil. Effect of the operating conditions upon oil composition and
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
Shinagawa et al.
antioxidant capacity. Chemical Engineering Journal, 160(2), 634-640.
http://dx.doi.org/10.1016/j.cej.2010.03.087.
Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L.,
Hampsch-Woodill, M., Huang, D., Ou, B., & Jacob, R. (2003). Assays
for hydrophilic and lipophilic antioxidant capacity (oxygen radical
absorbance capacity (ORACFL)) of plasma and other biological and
food samples. Journal of Agricultural and Food Chemistry, 51(11),
3273-3279. PMid:12744654. http://dx.doi.org/10.1021/jf0262256.
Ranjith, A., Sarin-Kumar, K., Venugopalan, V. V., Arumughan, C.,
Sawhney, R. C., & Singh, V. (2006). Fatty acids, tocols, and carotenoids
in pulp oil of three sea buckthorn species (Hippophae rhamnoides, H.
salicifolia, and H. tibetana) grown in the Indian Himalayas. Journal
of the American Oil Chemists’ Society, 83(4), 359-364. http://dx.doi.
org/10.1007/s11746-006-1213-z.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & RiceEvans, C. (1999). Antioxidant Activity Applying an Improved ABTS
radical cation decolorization assay. Free Radical Biology & Medicine,
26(9-10), 1231-1237. PMid:10381194. http://dx.doi.org/10.1016/
S0891-5849(98)00315-3.
Food Sci. Technol, Campinas, 38(1): 164-171, Jan.-Mar. 2018
Rezig, L., Chouaibi, M., Msaada, K., & Hamdi, S. (2012). Chemical
composition and profile characterisation of pumpkin (Cucurbita
maxima) seed oil. Industrial Crops and Products, 37(1), 82-87. http://
dx.doi.org/10.1016/j.indcrop.2011.12.004.
Rockenbach, I. I., Rodrigues, E., Gonzaga, L. V. & Fett, R. (2010). Fatty
acid composition of grape (Vitis vinifera L. and Vitis labrusca L.)
seed oil. Brazilian Journal of Food Technology, III SSA, 23-26.
Rubio, M., Alvarez-Ortí, M., Alvarruiz, A., Fernandéz, E., & Pardo,
J. E. (2009). Characterization of oil obtained from grape seeds
collected during Berry development. Journal of Agricultural and
Food Chemistry, 57(7), 2812-2815. PMid:19256538. http://dx.doi.
org/10.1021/jf803627t.
Sabir, A., Unver, A., & Kara, Z. (2012). The fatty acid and tocopherol
constituents of the seed oil extracted from 21 grape varieties (Vitis
spp.). Journal of the Science of Food and Agriculture, 92(9), 1982-1987.
PMid:22271548. http://dx.doi.org/10.1002/jsfa.5571.
Siger, A., Nogala-Kalucka, M., & Lampart-Szczapa, E. (2008). The
content and antioxidant activity of phenolic compounds in coldpressed plant oils. Journal of Food Lipids, 15(2), 137-149. http://
dx.doi.org/10.1111/j.1745-4522.2007.00107.x.
171/171 171