Journal of Biotechnology 164 (2013) 423–432

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Antimicrobial activity and composition profile of grape (Vitis vinifera)

pomace extracts obtained by supercritical fluids
Daniela A. Oliveira a , Ana Augusta Salvador a , Artur Smânia Jr. b , Elza F.A. Smânia b ,
Marcelo Maraschin c , Sandra R.S. Ferreira a,∗
a
Chemical and Food Engineering Department, Federal University of Santa Catarina, EQA/UFSC, C.P. 476, CEP 88040-900 Florianópolis, SC, Brazil
b
Microbiology and Parasitology Department, Federal University of Santa Catarina, CCB/UFSC, Brazil
c
Department of Plant Science, Federal University of Santa Catarina, CCA/UFSC, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The possibility of increasing the aggregated value of the huge amount of residues generated by winer-
Received 18 January 2012 ies around the world foment studies using the grape pomace – the residue from the wine production,
Received in revised form 1 August 2012 composed by seed, skin and stems – to obtain functional ingredients. Nowadays, consumers in general
Accepted 24 September 2012
prefer natural and safe products mainly for food and cosmetic fields, where the supercritical fluid extrac-
Available online 1 October 2012
tion is of great importance due to the purity of the extracts provided. Therefore, the objective of this
work is to evaluate the global extraction yield, the antimicrobial activity and the composition profile of
Keywords:
Merlot and Syrah grape pomace extracts obtained by supercritical CO2 (SC-CO2 ) and CO2 added with co-
Supercritical fluid extraction
Winery residue
solvent at pressures up to 300 bar and temperatures of 50 and 60 ◦ C. The results were compared with the
Antibacterial activity ones obtained by Soxhlet and by ultrasound-assisted leaching extraction methods. The main components
Antifungal activity from the extracts, identified by HPLC, were gallic acid, p-OH-benzoic acid, vanillic acid and epicatechin.
Phenolic profile The antibacterial and antifungal activities of the extracts were evaluated using four strains of bacte-
ria (Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa) and three fungi
strains (Candida albicans, Candida parapsilosis, Candida krusei). Despite lower extraction yield results, the
supercritical fluid extracts presented the highest antimicrobial effectiveness compared to the other grape
pomace extracts due to the presence of antimicrobial active compounds. Syrah extracts were less efficient
against the microorganisms tested and Merlot extracts were more active against Gram-positive bacteria.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction varieties against Gram-positive (Staphylococcus aureus and Bacil-

Functional properties such as antimicrobial and antioxidant
activity of plant extracts are the base of its application in food
preservation, pharmaceuticals, cosmetics, alternative medicine
and natural therapies (Bakkali et al., 2008). Therefore, in the last
several years, studies regarding extraction of natural compounds,
capable to avoid microorganisms development, present special
interest, especially related to microorganisms responsible for the
deterioration of foods and ones responsible for a great number of
diseases (Pinelo et al., 2007; Michielin et al., 2009).
Particular attention has been addressed to the use of grape
pomace, a winery industry residue formed around the world in
large amount and composed by skins, seeds, and stems, to obtain
high valued products (Pinelo et al., 2007). Katalinić et al. (2010)
presented good results studying the peel extract effect of 14 grape
∗ Corresponding author. Tel.: +55 48 3721 9448; fax: +55 48 3721 9687.
E-mail address: sandra@enq.ufsc.br (S.R.S. Ferreira).
lus cereus) and Gram-negative (Escherichia coli O157:H7, Salmonella
infantis and Campylobacter coli) bacteria. Papadopoulou et al. (2005)
studied the potential antimicrobial activity and the composition of
alcohol-free red and white wine extracts against pathogenic strains
of S. aureus, E. coli and Candida albicans. Han (2007) showed that a
grape seed extract alone inhibited growth of C. albicans yeast cells,
with a dose-dependent antifungal effect, and also presented a syn-
ergic effect with other antifungal (Amphorericin B) used to treat
fungal infections. Some other studies can be found in the litera-
ture evaluating the antimicrobial activity and/or the composition
of wines and grape pomace extracts against other strains of bacte-
ria and fungi (Daroch et al., 2001; Thimothe et al., 2007; Corrales
et al., 2010; Fikselová et al., 2010). Additional studies involve spe-
cific substances present in grapes. Most of these studies connect
the antimicrobial activity to the content of phenolic compounds
like resveratrol (Chan, 2002; Filip et al., 2003; Chatterjee et al.,
2005), but it can also be associated to pathogenesis-related pro-
teins (Monteiro et al., 2003), substances produced by the grapevine
under stress conditions (fungal attack or high UV exposure).

0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.09.014
424 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432
The studies above mentioned used liquid-solvent-based meth-
ods to obtain the extracts. These processes present limitations like:
high energy costs; elevated solvent use; high temperatures, inju-
rious for thermolabile substances; low selectivity; and vestiges of
solvent in the solute (Pourmortazavi and Hajimirsadegui, 2007). For
this reason, alternative methods to obtain natural bioactive sub-
stances have been studied, such as ultrasound-assisted leaching
and supercritical fluid extraction.
Supercritical fluid extraction (SFE) is based on the use of solvents
in conditions above the critical point, resulting in a liquid like den-
sity, gas like viscosity and the diffusivity values within two orders
of magnitude higher than that for typical liquids. This technology
presents several advantages over traditional liquid-solvent-based
methods including improved selectivity, expeditiousness, automa-
tion and environmental safety as the solvent can easily be removed
from the solutes by expansion to ambient pressure (Tena et al.,
1998; Brunner, 2005). Carbon dioxide (CO2 ) is commonly used in
SFE because, in contrast with organic solvents, it is non-toxic (clas-
sified as GRAS – generally recognized as safe – by the United States
Food and Drug Administration – US FDA), inexpensive and volatile,
with moderate critical conditions, thus no thermal or chemical
degradations of bioactive substances are expected (Pinelo et al.,
2007). Nevertheless, CO2 is a non-polar compound and presents
no dipole moment, which disqualifies its use as solvent for polar
compounds and limits its solvent power. To overcome this dis-
advantage, polar co-solvents can be used to increase the overall
solvent power of the fluid phase during SFE (Silva et al., 2008;
Michielin et al., 2009).
The quality and the composition of the extracts are strongly
dependant on the extraction technique, the solvent used, the origin
of the raw material, the part of the plant used (seeds, skin, leaves,
etc.), its storage condition and the pre-treatment applied (Moure
et al., 2001; Louli et al., 2004). The quality of the extract can be rep-
resented by its properties, such as its biological activities, which
can be determinant to compare different extraction methods.
Despite the recognized antioxidant capacity of substances
present in grapes, studies related to the antimicrobial activity of
grape pomace extracts, especially when obtained by supercritical
fluid extraction, are still needed. Therefore, the present work has
the objective to evaluate the use of different extraction methods,
such as low-pressure procedures using different solvents and SFE
with CO2 and with CO2 and co-solvent, to obtain grape pomace
extracts from Merlot and Syrah varietals. The extracting techniques
were compared in terms of global extraction yield and antibacterial
activity of the extracts, in an attempt to evaluate the potential of
the grape pomace to generate an alternative antimicrobial product.
2. Experimental procedures
2.1. Sample preparation
The raw material, pressed grape pomace derived from Merlot
and Syrah (Vitis vinifera) wine production, was kindly provided by
two wineries from Miolo Wine Group. The Syrah pomace, vintage
2008 from winery located in Vale do São Francisco – Bahia, Brazil,
vintage 2008, presented initial moisture content of 56 ± 2% (wet
base) and the Merlot pomace, vintage 2009, provided by the winery
from Vale dos Vinhedos – Rio Grande do Sul, Brazil that was received
with initial moisture content of 64 ± 1% (wet base). The raw mate-
rial (grape pomace) was dried at 32 ◦ C in a forced air circulation
oven (De Leo, Model A3 CARF, RS, Brazil) up to approximately 10%
moisture content (10.1 ± 0.3% for Syrah pomace, reached after 20 h
drying, and 10 ± 1% for Merlot pomace, reached after 21.5 h drying).
The total moisture content was assessed by maintaining the sam-
ples in a stove at 105 ◦ C until constant weight. Dried samples were
grounded in a knife mill (De Leo, Porto Alegre/RS, Brazil) and the
particle size was classified in sieve separator (Bertel Indústria Met-
alúrgica Ltda., Caieiras/SP, Brazil). The particle fraction of −20/+32
Mesh was selected to proceed the extractions.
2.2. Ultrasound-assisted leaching extraction (UE)
Ultrasound-assisted leaching extractions were conducted as
described by Benelli et al. (2010). Briefly, 3 g of dried and ground
grape pomace and 50 mL of solvent were used and placed inside
an evaporation flask connected to a condenser. The extraction was
performed in duplicate at room temperature for 30 min and using
the solvents water (H2 O), EtOH, EtOAc and Hx in decreasing order of
polarity index of the pure solvent using solvatochromic dyes indi-
cators as suggested by Reichardt (1994). The equipment used was
an ultrasonic cleaner bath (USC-700/55 kHz, Unique Ltda., Inda-
iatuba/SP, Brazil), which operates in a frequency of 55 kHz and
potency of 220 V.
2.3. Soxhlet extraction (SOX)
The organic solvents ethanol (EtOH), ethyl acetate (EtOAc) and
hexane (Hx) were applied for Soxhlet extraction. Samples (5 g) of
dried and grounded grape pomace were placed inside a thimble,
made by thick filter paper, and loaded into the main chamber of the
extractor which consisted of an extracting tube, a glass balloon and
a condenser. The assays were performed at solvent boiling temper-
ature during 8 h, with 150 mL of solvent continuously refluxing over
the sample. The solvents used in all procedures were 99% pure (CAQ
Ind. & Com., SP, Brazil). The extraction yield results were obtained
by the mean value of the duplicated experiments.
2.4. Supercritical fluid extraction (SFE)
The SFE laboratory apparatus is presented in Fig. 1 and
the experimental procedure for high-pressure extractions was
described by Michielin et al. (2009). The SFE assays used the 99.9%
pure CO2 (White Martins, Brazil) applied in a high pressure dynamic
unit with a constant CO2 flow rate of 13 ± 2 g/min during 4 h. The
fixed bed consisted of 25 g of dried and ground material, forming
a volume of 38.8 cm3 inside the extraction vessel. The extraction
yield values were determined for Merlot and Syrah pomaces. The
conditions of pressures (P) ranging from 150 to 300 bar and tem-
peratures (T) of 50 ◦ C and 60 ◦ C were applied for the Merlot pomace,
while the extraction with the Syrah pomace was conducted at
250 bar/60 ◦ C. The same condition was also used for Merlot pomace
to evaluate the influence of the addition of co-solvent (ethanol) to
the SFE in concentrations of 12.5%, 15% and 17.5%. All supercritical
assays were conducted at flow rate of 13 ± 2 g/min CO2 during 4 h
extraction process. In order to enable the co-solvent (CS) assays,
a CS pump (Constametric 3200, Thermo Separation Process, EUA)
was connected to the extraction line to supply the organic solvent
at high-pressure (modifier) at pre-established flow rate, to mixture
with CO2 flow before the extraction vessel, as described by Campos
et al. (2008). The solute was collected in amber flasks and the
mass quantified in an analytical balance (OHAUS, Model AS200S, NJ,
USA). After the extraction, the product from the co-solvent assays
was submitted to a solvent removal step, as described in Section
2.5.
2.5. Solvent–solute separation
The resulting mixtures solute/solvent from each extraction
technique (SOX, UE and SFE with CO2 + CS) were separated by
means of reduced pressure to remove the solvents in a rotary
 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432
Fig. 1. SFE Unit: E, extractor; V1, V2, V3, V4, VT and VR, control valves; PI1, PI2 and PI3, manometers; TI, temperature control; BC, co-solvent pump; BT1, BT2 and BT3,
thermostatic baths.
evaporator (Fisatom, model 802, São Paulo, Brazil) and obtain the
crude extracts free of solvents.
2.6. High performance liquid chromatography (HPLC)
Phenolic compounds in the grape pomace extracts were
detected by reversed-phase HPLC (RF-HPLC-UV–visible) (Shimadzu
LC-10, Kyoto, Japan) using a Shim-pack CLC-ODS C18 reverse-
phase column (250 mm length × 4.6 mm ID, 5 ␮m Ø), protected
by a 5 ␮m C18 reverse-phase guard column (Shim-Pack CLC-ODS,
4.6 mm ID) guard-column, with a UV–visible detector (Shimadzu
SPD 10A, 280 nm), as previously described by Morais et al. (2009).
For the determination of the phenolic compounds, approximately
10 mg of each extract were diluted in 1 mL EtOAc, centrifuged
(5000 rpm/10 min), and the supernatant filtered (0.22 ␮m). A
10 ␮L aliquot was injected into an HPLC column at 40 ◦ C. The
isocratic mobile phase was constituted of water/acetic acid/n-
butanol (350:1:10, v/v/v), at a flow rate of 0.8 mL/min. Quantitative
determination was based on the external standard method by com-
parison with the standard retention time and co-chromatography
of the pure phenolics of interest (0.1 mg/mL – Sigma–Aldrich, St.
Louis, MO, USA). Standard calibration curve was obtained by plot-
ting the area of peaks against different concentrations of gallic acid
(y = 3350x; r2 = 0.99), which was used as basis for calculation of the
extract composition. For each sample, the final concentration of the
compounds was determined by averaging the content after three
consecutive injections and the results are expressed in ␮g of gallic
acid equivalents (GAE) per g of dry extract.
The parameters of validation, such as detection and quantifi-
cation limits for the phenolic compounds in study (LOD and LOQ,
respectively), recovery (R, %), precision or relative standard devi-
ation (RSD, %), and accuracy (E, %), were established (ACS, 1980;
ICH Q2B, 1995) and previously reported (Andrade et al., 2012). The
LOD and LOQ were evaluated on the basis of the noise obtained with
analysis of non-spiked blank samples for phenolic acids (n = 8). LOD
and LOQ were defined as the concentration of the analyte that pro-
duced a signal-to-noise ratio of 3 and 10, respectively (ACS, 1980).
For the total phenols analysis, the LOD and LOQ were estimated by
425
the slope and mean standard deviation of gallic acid concentrations
used in the standard curve (ICH Q2B, 1995). Results of eight paral-
lel experiments indicated that recovery/accuracy ranged 80–100%,
RSD were <6%, and the LOD and LOQ were 0.36 and 1.08 lg/mL,
respectively for the phenolic acids of interest.
2.7. Antimicrobial activity
2.7.1. Microbial strains
The antimicrobial activity of grape pomace extracts obtained by
different extraction methods was evaluated against Gram-positive
bacteria S. aureus, ATCC 25923 (American Type Culture Collection)
and B. cereus ATCC 11778, Gram-negative bacteria E. coli ATCC
25922 and Pseudomonas aeruginosa ATCC 27853 and fungi strains
C. albicans ATCC 14053, Candida parapsilosis ATCC 22019, Candida
krusei ATCC 6258. Mueller-Hinton agar and broth media were used
for microbial growth. An overnight culture of each strain grown
in Mueller-Hinton broth was diluted in fresh medium to achieve
a final concentration of approximately 107 or 108 CFU/mL. All cul-
tures were incubated in aerobic conditions (Smânia et al., 1995;
Michielin et al., 2009).
Some aspects were considered for the selection of the microor-
ganisms for this study: S. aureus, B. cereus and E. coli are known for
causing foodborne diseases, P. aeruginosa is commonly resistant to
multiple antibiotics (Stover et al., 2000) and C. albicans, C. parap-
silosis and C. krusei are the main fungus responsible for invasive
bloodstream fungal infections, a significant cause of mortality in
immunocompromised patients (Selvarangan et al., 2003).
2.7.2. Agar diffusion method (ADM)
Each microbial suspension (108 CFU/mL) was inoculated onto
Mueller-Hinton agar plates, by means of spreading uniformly the
microbial suspensions on the agar surface using cotton swabs. Then
the agar surface was perforated with 7 mm diameter holes (four
holes), aseptically cut and filled with the different grape pomace
extracts, used at concentration of 20 mg extract/mL in a water
solution of DMSO (dimethyl sulfoxide) 10% because at this concen-
tration the DMSO is not an inhibitor to the microorganism growth,
426 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432

as presented by Smânia et al. (1999). Finally, the plates were incu- 16

bated at 36 ◦ C for 24 h and then the inhibition effect was examined.
14
A positive result was defined as an inhibition zone (halo size) of
9 mm or more around the holes, indicating the presence of antimi- 12
crobial substance, or group of substances, in the extracts tested 10
(Smânia et al., 1999; Kitzberger et al., 2007; Michielin et al., 2009).

X0 (%)
The ADM was conducted for all strains of bacteria and for one 8
fungus strain, the C. albicans, as screening procedure for further 6
investigation by MIC method with other fungi strains.
4
2.7.3. Minimum inhibition concentration (MIC) 2
The extracts with positive inhibition zone (9 mm or more) in the
ADM assays, against at least one microorganism, were submitted to 0

15%
12.5%

17.5%

EtOH

EtOH
200 bar
250 bar
300 bar

250 bar
300 bar

EtOAc

EtOAc
150 bar

150 bar
200 bar

H2O
Hx

Hx
the determination of the minimum inhibition concentration (MIC)
by microdilution method in culture broth, as describe by Michielin
et al. (2009). The extracts were dissolved in DMSO 10% (20 mg of SFE CO2 50°C SFE CO2 60°C SFE SOX UE
extract/mL of DMSO) and, further, serial dilutions of the extracts CO2+EtOH
were performed to reach a final concentration range between 2000
Fig. 2. Global extraction yield results for Merlot grape pomace extractions using
and 3.9 ␮g/mL. One sample (10 ␮L) of each diluted solution (grape different techniques: SFE with pure SC-CO2 at 50 ◦ C and 60 ◦ C and pressures of 150,
pomace extracts), 85 ␮L of Mueller-Hinton broth and 5 ␮L inocu- 200, 250 and 300 bar; SFE (60 ◦ C/250 bar) with EtOH as co-solvent at concentrations
lum of the microbial suspension (107 CFU/mL) were added in each of 12.5%, 15% and 17.5%; SOX with Hx, EtOAc and EtOH; UE with Hx, EtOAc, EtOH
well of a microdilution plate with 96 wells. The sterility control and H2 O.

was prepared with the culture broth and the DMSO (without grape
pomace and microorganism suspension). The growth control was
0.871 g/cm3 , according to Angus et al. (1976), resulting in an
prepared with the culture broth, the DMSO and 5 ␮L inoculum
enhancement in the solute solubility and on extraction rate. The
of the microbial suspension (107 CFU/mL) (without extracts). All
maximum X0 was achieved at 300 bar for both temperatures,
experiments were performed in duplicate and the plates incubated
5.0 ± 0.2% at 50 ◦ C and 5.2 ± 0.6% at 60 ◦ C. The SFE results were sta-
for 24 h at 36 ◦ C. Microbial growth was first detected by opti-
tistically similar at 95% level of significance (significant difference:
cal density (OD) (ELISA reader, CLX800 – Biotek Instruments). In
p < 0.05), according to Tukey test. Campos et al. (2007) obtained
case of turbidity or interference of the extracts color on the OD
a maximum extraction yield of 4.2% for the extract of Cabernet
detection, it was added 20 ␮L of an alcoholic solution (0.5 mg/mL)
Sauvignon pomace, using similar operational conditions and raw
of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chlo-
material pre-treatment as applied in the present work. The diver-
ride (Sigma) in order to expose the microbial growth. The MIC was
gence between the literature data (Campos et al., 2007) and the
considered the lower concentration of the extract that inhibited
present work (Fig. 2) was probably caused by the diversity in the
the microbial growth, after the incubation period. The results were
grape varietal and the lower CO2 flow rate used by the authors
expressed in ␮g/mL.
(3.3 g/min). Complementarily, Beveridge et al. (2005) presented
yields for SFE of Merlot and Syrah seed of 10.5 and 10.8% respec-
2.8. Statistical analysis
tively. The mentioned data (Beveridge et al., 2005) were higher than
the present work because the raw material used was formed only by
The results of the extraction yield (X0 ) were statistically evalu-
grape seed (meaning high lipophilic fraction) different than grape
ated by a one-way analysis of variance (ANOVA) by means of the
pomace that presents seeds, skin and stems.
software Statistica for Windows 7.0 (Statsoft Inc., USA) in order
The temperature effect, however, is more complex. At 150 bar,
to detect significant differences among the values. The significant
the enhancement in the temperature causes a reduction in the
differences (p < 0.05) were analyzed by Tukey test.
extraction yield due to the decrease in solvent density from
0.701 to 0.606 g/cm3 (Angus et al., 1976). On the other hand,
3. Results and discussion

3.1. Global extraction yield (X0 ) 16

14
The process efficiency is quantitatively related to the extraction
global yield (X0 ). The yield results (wet base) obtained by the dif- 12
ferent extraction procedures applied to grape pomace (SOX and 10
X0 (%)

UE methods with different solvents, and SFE with and without co-
8
solvent) are presented in Figs. 2 and 3 with their standard deviation
data. Fig. 2 shows the results provided by the pomace from Merlot 6
cultivar while Fig. 3 presents the data for Syrah varietal. 4
The results for SFE with CO2 for Merlot pomace show that
2
the extraction yield increases with operating pressure (from 150
to 300 bar) for both temperatures evaluated (50 and 60 ◦ C). This 0
250 bar

Hx

EtOAc

EtOH

Hx

EtOAc

EtOH

H2O

behavior is explained by the fact that the solvent power of the

supercritical solvent (SC-CO2 ) is related to its density, which
enhances with pressure at constant temperature and decreases
with the elevation of the temperature at constant pressure. There- SFE CO2 60°C SOX UE
fore, the extraction yield increased in 25% at 50 ◦ C by changing
Fig. 3. Global extraction yield results for Syrah grape pomace extractions using dif-
pressure from 150 to 300 bar (from 4.0 ± 0.6% to 5.0 ± 0.2%, w/w), ferent techniques: SFE with pure SC-CO2 at 60 ◦ C and 250 bar; SOX with Hx, EtOAc
which is equivalent to a solvent density increase from 0.606 to and EtOH; UE with Hx, EtOAc, EtOH and H2 O.
 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432
at higher pressures, the increase in temperature enhances the
extraction yield because of the raise in solute vapor pressure with
temperature. The competition of these opposite effects provides
the inversion of the extraction yield isotherms, i.e., the crossover
point, as expressed by Michielin et al. (2005). Furthermore, from
SC-CO2 results in Fig. 2 we observe the slope of the yield isotherms
with pressure, i.e., at 50 ◦ C the yield enhances 2.5% by increasing
pressure from 150 to 200 bar, while at 60 ◦ C the yield increases
17.6% for the same pressure change. This variation in yield slope
suggests a crossover pattern of the yield isotherms which lays
between 175 and 180 bar. This suggestion is only a proposal of
behavior since no significant difference at the level of 95% was
detected for the extraction yield values.
Then, for practical purposes, the crossover point limits the dom-
inant effect regions: below the crossover pressure the density is
the dominant effect while above this condition the solute vapor
pressure is the leading mechanism affecting the extraction process.
Campos et al. (2007), studying the SFE from Cabernet Sauvignon
grape pomace with pressure conditions of 150, 200 and 300 bar
and temperatures of 40, 50 and 60 ◦ C, presented a crossover pres-
sure near 190 bar. A similar behavior was also detected by several
authors related to different solid matrixes (Michielin et al., 2005,
2009; Danielski et al., 2007; Kitzberger et al., 2007; Mezzomo et al.,
2010).
For the low pressure techniques (SOX and UE) the results from
Figs. 2 and 3 show an increase in the global extraction yield with
the solvent polarity. The best global yields were obtained by Soxhlet
extractions using ethanol, with no significant difference between
varietals (Merlot: 14.6 ± 0.2%, Syrah: 14 ± 1%). The highest extrac-
tion yields obtained by UE assays were achieved with water as
solvent, 9.1 ± 0.1% for Merlot and 12 ± 1% for Syrah. The lowest yield
value was provided by the UE with hexane (3.4 ± 0.1% for Mer-
lot extract), a non-polar solvent. Similarly, the data obtained by
SFE with the non polar CO2 was also low compared to other sol-
vents, indicating that grape pomace contains high amount of polar
components, extracted preferably by increasing solvent polarity.
Comparing the results obtained by SOX and UE for all solvents
used (low pressure methods), the best performance achieved by
SOX is probably due to characteristics inherent to this extrac-
tion method: higher temperatures reached, solvent recirculation
(that enlarges contact time between phases) and solute–solvent
interactions (solvent viscosity and superficial tension reduction
on boiling point). These features can promote also the solubili-
zation of sugar and pectin compounds from the raw material (grape
pomace), which increases the global yield, although not necessarily
improving the extract bioactivity. Campos et al. (2008) presented
slightly lower yield results for Soxhlet extractions of Cabernet Sauvi-
gnon pomace with hexane (5.64%), ethyl acetate (8.9%) and ethanol
(13.20%). This difference can be related to the varietal and also to
the lower extraction time applied (6 h), compared to the present
work time (8 h).
Finally, the statistical analysis show no significant difference
between varietals at level of 95% for the results presented in
Figs. 2 and 3, except for the data obtained by UE with ethanol and
with water, which showed highest extraction yields for the Syrah
varietal.
The co-solvent for the SFE of Merlot pomace was selected
considering the yield results from SOX, which indicated EtOH as
the solvent that provided the higher extraction yield values. Then,
EtOH was applied as co-solvent at 60 ◦ C, 250 bar and concentra-
tions between 12.5 and 17.5% (w/w). This concentration range
was defined to narrow the range from Campos et al. (2008) which
used EtOH as co-solvent in SFE of Cabernet sauvignon pomace
at concentration between 10 and 20% (w/w). Results show an
increase in global yield up to 171% for extraction using 15% of
EtOH, compared with SC-CO2 , reaching the value of 13.31 ± 0.04%
427
(w/w). This behavior is due to the increase in the number of soluble
components in the mixture, suggesting the extraction of polar and
non-polar components from grape pomace material, enhancing
the global yield but reducing the selectivity. It is also relevant that
EtOH is a GRAS (generally recognized as safe by US-FDA) solvent,
representing a positive contribution for food applications. Similar
to the work by Campos et al. (2008), the addition of co-solvent
enhances the global extraction yield with increasing EtOH concen-
tration from 12.5% to 15% (w/w) and then, raising EtOH content up
to 17.5% (w/w) the global yield decreases. The initial augment in
extraction yield (changing EtOH from 12.5 to 15%) is due to rupture
in solute/solid matrix interactions and consequent substitution
with co-solvent molecules in solid active sites (Hollender et al.,
1997). Above 15% co-solvent content, the increase in ethanol
concentration may induce the saturation of CO2 with EtOH, with
consequent formation of two phases, for the specific conditions of
temperature and pressure of the system. The statistical analysis of
the global yield results for co-solvent assays indicates significant
difference between 12.5% and 15% (w/w) concentrations. This
behavior was also discussed by Berna et al. (2001) for resveratrol
solubility in CO2 and ethanol mixtures and by Campos et al. (2008)
for extraction of Cabernet Sauvignon grape pomace, which also
identified the highest extraction yield with 15% EtOH.
The extraction yield values obtained by SFE with CO2 are closer
to the ones obtained by UE method, while the addition of EtOH
to SC-CO2 produced results close to the performance of the SOX
operation, regardless the use of lower temperature in SFE com-
pared to SOX. The similarity detected by yield values from SFE and
UE was also reported by Palma et al. (1999). The results presented
in terms of extraction yield estimate quantitatively the efficiency
of the processes; nevertheless it is also important to evaluate the
quality of the extraction methods. Therefore, in order to provide
a qualitative assessment, it is important to observe the product
characteristics such as composition, discussed in Section 3.2, and
biological activity, discussed in Section 3.3.
3.2. Phenolic profile
The extracts obtained by SFE and by low pressure methods
(LPM) for Merlot and Syrah grape pomaces were analyzed. The
results are presented in Table 1 and show the quantification of
the phenolic compounds, mainly phenolic acids identified in each
extract, with the concentration expressed in gallic acid equivalents
(GAE) resulted from a correlation with a standard calibration curve.
The compounds identified in most samples were gallic and p-OH-
benzoic acids which are usually found in major quantities in grape
seeds and skin, respectively (Kammerer et al., 2004).
Unlike the data reported by Tsuda et al. (1995) no increase in the
amount of extracted epicatechin was observed with the elevation of
pressure and temperature for the SFE with CO2 . However, the addi-
tion of ethanol as co-solvent in general enhances the epicatechin
extraction, for example, 2008 ± 3 GAE ␮g/g extract was determined
for the SFE at 250 bar and 60 ◦ C with 17.5% EtOH, compared to
non epicatechin detected at the same condition without ethanol
(Table 1). Similar behavior was reported by Tsuda et al. (1995) and
by Adil et al. (2007). Pinelo et al. (2007) obtained higher gallic acid
and epicatechin concentrations for the extract obtained by SFE with
EtOH, when compared to low pressure extractions. The same per-
formance was observed in the present work regarding mainly to
the gallic acid concentration for Merlot extracts which ranged from
zero to 1624 ± 4 GAE ␮g/g extract for SOX and SFE with 17.5% EtOH
respectively (Table 1).
The phenolic profile presented in Table 1 can be affected
by factors such as: (1) because phenolic compounds are highly
reactive, the extraction conditions (like temperature, time, solvent
applied) can deeply modify the phenolic profile of the extracts by
428 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432

Table 1
Phenolic profile of grape pomace extracts determined by HPLC, expressed in ␮g of gallic acid equivalents (GAE) per g of extract.

Extraction method Grape varietal Process conditions Epicatechin Gallic acid Tannic acid p-OH-benzoic acid Vanillic acid Protocatecuic acid

Merlot 50◦ C/150 bar 355 ± 1 108.2 ± 0.9 ND 498 ± 7 315 ± 5 ND

Merlot 50◦ C/200 bar ND 1159 ± 8 ND 116 ± 1 189 ± 4 ND
Merlot 50◦ C/250 bar 54 ± 1 3969 ± 6 3849 ± 5 1221 ± 7 460.0 ± 0.3 ND
Merlot 50◦ C/300 bar 67 ± 7 613 ± 1 501 ± 5 2332 ± 10 146 ± 9 ND
SFE SC-CO2 Merlot 60◦ C/150 bar ND 1216 ± 7 ND ND 337 ± 1 ND
Merlot 60◦ C/200 bar ND 436 ± 10 ND 46 ± 1 106 ± 1 ND
Merlot 60◦ C/250 bar ND 142.0 ± 0.1 303 ± 7 888.1 ± 0.1 505 ± 3 ND
Merlot 60◦ C/300 bar 95.5 ± 0.1 445 ± 7 384 ± 1 714 ± 3 588 ± 5 ND
Syrah 60◦ C/250 bar ND 643 ± 9 ND 263 ± 1 ND ND

Merlot 12.5% EtOH 1194 ± 10 1230.9 ± 0.1 ND 5005 ± 5 38.5 ± 0.7 ND

SFE CO2 + CS
Merlot 15.0% EtOH ND ND ND ND ND 5371 ± 70
250 bar/60 ◦ C
Merlot 17.5% EtOH 2008 ± 3 1624 ± 4 263 ± 2 6587 ± 3 152.1 ± 0.6 ND

Syrah EtOH 53 ± 1 106 ± 3 203 ± 2 228 ± 5 ND ND

SOX
Merlot EtOH ND ND ND ND ND 98647 ± 42

Syrah EtOH – – – – – –
UE
Merlot EtOH 1219 ± 6 510 ± 5 642 ± 1 5066.1 ± 0.6 ND ND

favoring the formation of polymers, as reported by Pinelo et al.
(2005); (2) the grape pressing operation for wine production allow
the contact between monomeric procyanidins, like epicatechin,
and polyphenoloxidase enzyme, inducing their oxidation and
polymerization interfering on their extraction (Monrad et al.,
2010); (3) degradation caused by light exposure as some phenolics
are photosensitive, reducing their concentration to non detectable
levels (Maier et al., 2009). Besides that, the storage conditions of
the raw material as well of the extracts also affect the product
composition and its biological properties. These factors may have
influenced the composition differences among the extracts tested.
The Merlot extracts obtained by SOX and by SFE with 15% EtOH
were the only samples containing a single identified component,
the protocatechuic acid, which was not detected in other extracts.
This behavior could be explained by the high reactivity of the other
phenolic components, probably induced by the solvent EtOH and
the extraction conditions. Besides, the high concentration of proto-
catechuic acid may be related to the high polarity of that compound
(Murga et al., 2002), suggesting the need of lower polar solvents for
the extraction of other phenolics, especially considering the usual
occurrence of glycosylated or methylated derivatives of the phe-
nolic acids in plant materials (Harborne, 1991). In general, higher
EtOH contents in the extractant solution allow higher recoveries
of phenolics, but it seems to be the case when those secondary
metabolites occur predominantly in their aglycone form, explain-
ing the higher phenolic content were presented in the extract
with 17.5% EtOH. Less polar forms of those secondary metabolites,
e.g., glycosylated, methylated, and acetylated are preferentially
extracted upon lower EtOH/CO2 ratio.
The interest in bioactive substances from grape and its prod-
ucts such as wine was first motivated by the “French paradox”.
Hence, studies revealed phenolic compounds like gallic acid, cate-
chin and several other phenolics as inhibitors of human low density
lipoprotein (LDL) oxidation, consequently reducing the risk of car-
diovascular diseases (Frankel et al., 1993, 1995; Teissedre et al.,
1996). Several other studies related grape substances to health
benefits, such as epicatechin to antimutagenic and antiviral activ-
ities (Saito et al., 1998), tannic acid to hepatic lipids decrease
(Do et al., 2011) and gallic acid with the prostate cancer inhi-
bition (Veluri et al., 2006). Vanillic acid is the main metabolic
product of the aldehyde vanillin which has shown antimicrobial
(Ravichandran et al., 2011), anti-mutagenic and anti-carcinogenic
effects (Lirdprapamongkol et al., 2005). The esterified p-OH-
benzoic acid shows antimicrobial activity and is widely used as
preservatives in food, cosmetics, and pharmaceutical products
(Soni et al., 2005). The gallic acid was also identified as an antibacte-
rial component and structure–activity correlation assays revealed
that its three hydroxyl groups were effective for antibacterial activ-
ity against E. coli and Salmonella enteritidis and all of the substituents
of the benzene ring were effective against S. aureus (Shoko et al.,
1999). Therefore the compounds identified in the present study
confirm the importance of the reuse of grape pomace to obtain
bioactive compounds.
The main focus of the present evaluation was related to the
determination of the phenolic composition from the various grape
pomace extracts, even though these extracts are very complex mix-
tures, containing several classes of substances that could present
various biological activities, as already reported in the literature
(Ul’chenko et al., 2009; Tenore et al., 2011). Other phenolic com-
pounds might be tightly bound to the lignocellulosic backbone of
the biomass in study and the extraction conditions were not able
in recovering them to the organosolvent fraction. Furthermore, the
quality of the samples was determined by the antimicrobial activity
representing one of the several biological potentials of the pomace
extracts, as described in Section 3.3.
3.3. Antimicrobial activity
3.3.1. Agar diffusion method (ADM)
Although the agar diffusion method is sensitive to detect micro-
bial growth, it is a qualitative test and should not be recommended
to quantify the antimicrobial activity of a substance based on the
size of the inhibition zone formed during the analysis (Rios et al.,
1988). Anyhow, all grape pomace extracts were tested in ADM in
order to provide indication for further evaluation by the method
minimum inhibition concentration (MIC).
Samples of all extracts obtained by the different methods (SFE,
SOX and UE) and solvents were tested against S. aureus, B. cereus,
E. coli, P. aeruginosa and C. albicans by means of ADM. The results
achieved for the extracts tested against the above mentioned
microorganisms are presented in Table 2 in terms of size of inhibi-
tion zone (mm). The extracts obtained by LPM (except with hexane)
and by SFE with EtOH are not listed in Table 2 because no antimicro-
bial activity against any of the microorganisms tested was observed.
The results from Table 2 show that the extracts were mostly
more effective against the Gram-positive bacteria (S. aureus and
B. cereus), compared to the results for the Gram-negative ones
(E. coli and P. aeruginosa). The same behavior was observed by
Smith-Palmer et al. (1998), Boussaada et al. (2008) and Michielin
et al. (2009) studying extracts from other raw materials. The higher
 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432
Table 2
Antimicrobial activity of grape pomace extracts, evaluated by agar diffusion method.
Extraction Method Grape varietal Process conditions
Merlot 50 ◦ C /150 bar
Merlot 50 ◦ C/200 bar
Merlot 50 ◦ C/250 bar
Merlot 50 ◦ C/300 bar
SFE SC-CO2 Merlot 60 ◦ C/150 bar
Merlot 60 ◦ C/200 bar
Merlot 60 ◦ C/250 bar
Merlot 60 ◦ C/300 bar
Syrah 60 ◦ C/250 bar
SOX Syrah Hx
resistance of the Gram-negative bacteria is probably related to its
two layer cell membrane and to the strong hydrophilicity of the
outer membrane acting as a strong barrier, compared to the sin-
gle membrane of the Gram-positive bacteria which facilitates the
penetration of lipophilic compounds (Smith-Palmer et al., 1998).
Nevertheless, the study presented by Katalinić et al. (2010)
related to grape skin extracts from 14 different varietals applied
as antimicrobial agents show no significant differences in suscep-
tibility of Gram-positive and Gram-negative bacteria. Otherwise,
extracts obtained from different berries such as blueberry, rasp-
berry, cranberry, strawberry, among others, presented strong
antimicrobial activity against Gram-negative bacteria (Puupponen-
Pimiä et al., 2001).
The results from Table 2 show that all extracts obtained by
means of SC-CO2 were effective against S. aureus (inhibition zone
>9 mm). Among the microorganisms studied in this work, the P.
aeruginosa was the most resistant one because only one extract pro-
vided a positive result (halo size >9 mm), i.e., the SC-CO2 extract at
250 bar and 60 ◦ C. According to the literature, the Gram-negative
bacterium P. aeruginosa appeared to be less sensitive to the action
of several plant essential oils such as rosemary (Pintore et al.,
2002), geranium (Dorman and Deans, 2000) and Rhaponticum
acaule DC (Boussaada et al., 2008). Considering this aspect and the
other results from Table 2 we observe that the SC-CO2 extract at
250 bar and 60 ◦ C was the only extract effective against all bacte-
ria tested (Gram-positive and Gram-negative). Besides that, the
extract obtained by SC-CO2 at 150 bar and 60 ◦ C was also very effec-
tive against the bacteria tested, except P. aeruginosa (7 mm halo),
and effective as well against the fungus C. albicans with halo size of
21 mm.
The Syrah pomace extract obtained by SOX–Hx was the only
LPM sample that presented a positive result on ADM assays, and
just against one microorganism, the bacterium B. cereus. Accord-
ing to Palma et al. (1999), the lipid fraction from grape pomace,
easier extracted by CO2 and hexane, may present antimicrobial
behavior due to the presence of sterol components (lipophilic). This
characteristic explains the performance of the extracts obtained by
SC-CO2 and SOX–Hx listed in Table 2. Additionally, Baydar et al.
(2004) observed that grape skin extracts showed no antimicro-
bial activity against some food spoilage and pathogenic bacteria,
including S. aureus, B. cereus, E. coli and P. aeruginosa, while the
grape seed extracts, rich in fatty acids, provided high antimicro-
bial activity against the same microorganisms. Therefore, the best
accomplishment presented by SC-CO2 samples magnifies the SFE
to obtain grape pomace extracts with antimicrobial activity against
different microorganisms. Besides that, the supercritical process
also provides extraction in the absence of air and light, which pro-
tects the bioactivity of the extracts (Palma et al., 1999). The best
performance of SC-CO2 was already reported by Kitzberger et al.
(2007) for shiitake extracts and by Palma et al. (1999) for grape
429
Inhibition Zone (mm)
S. aureus B. cereus E. coli P. aeruginosa C. albicans
12 11 7 0 0
9 10 0 0 17
11 10 7 0 0
12 10 7 0 0
10 9 8 7 21
10 8 9 7 0
10 9 10 10 0
10 7 9 0 0
10 10 0 0 0
0 14 0 0 0
seed extracts, when comparing the results with the ones obtained
by other methods including SFE with co-solvent.
Related to the antifungal activity, the results from Table 2 show
that only two extracts were active against C. albicans: SC-CO2 at
200 bar/50 ◦ C and at 150 bar/60 ◦ C (halo sizes of 17 and 21 mm
respectively). Because the ADM is not a quantitative method, there
is no direct relation between halo size and antimicrobial poten-
tial. Therefore, further evaluation of the extracts behavior was
conducted to detect the minimum inhibitory concentration (MIC)
values for the microorganisms strains studied in this work.
3.3.2. Minimum inhibition concentration (MIC)
The grape pomace extracts that showed suitable results in ADM
(above 9 mm halo size) were submitted to the test of microdilution
in culture broth to determine the minimum concentration capable
to inhibit the growth of certain microorganism, i.e., the minimum
inhibition concentration (MIC). The MIC results for Gram-positive
and Gram-negative bacteria are presented in Table 3 while the data
obtained by all fungi strains tested (C. albicans, C. parapsilosis and
C. krusei) are listed in Table 4. Standard deviation values are also
listed in Tables 3 and 4 when the results between duplicates were
not coincident.
According to Michielin et al. (2009), plant materials can be
classified as antimicrobial agents based on the MIC values of its
extracts. Duarte et al. (2007) and Wang et al. (2008) classified
the extracts as: strong inhibitors for MIC value below 500 ␮g/mL;
moderate inhibitors for MIC between 600 and 1500 ␮g/mL; weak
inhibitors for MIC above 1600 ␮g/mL. This classification is very use-
ful to detect the potential of various plant materials with biological
activity.
The supercritical extracts obtained from Merlot pomace by SC-
CO2 at 300 bar/50 ◦ C and at 150 bar/50 ◦ C showed the lowest MIC
values against S. aureus, 625 ± 375 and 750 ± 250 ␮g/mL, respec-
tively. Again, the extracts were more effective (lowest MIC values)
against Gram-positive bacteria, mainly S. aureus, comparing to
Gram-negative ones (E. coli and P. aeruginosa). Only the supercrit-
ical extract obtained for Merlot by SC-CO2 (50 ◦ C, 300 bar) showed
moderate activity against E. coli and against P. aeruginosa, inhibi-
ting their growth with a MIC value of 1000 ␮g/mL, while all other
extracts behaved as weak inhibitors against the Gram-negative
bacteria (MIC above 1600 ␮g/mL). Thus, in general, supercriti-
cal extracts from grape pomace could be classified as moderate
inhibitors against Gram-positive bacteria (S. aureus and B. cereus).
Jayaprakasha et al. (2003) presented MIC values from grape seed
extracts similar to the results obtained in the present work. Palma
et al. (1999) and Katalinić et al. (2010) indicate a slight tendency of
red cultivars to be more effective against Gram-positive bacteria,
while Gram-negative bacteria seems to be more affected by white
cultivars. But further tests are needed to confirm results in other
in vitro and in vivo assays, e.g., in selected food model systems.
430 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432

Table 3
MIC (␮g/mL) values of grape pomace extracts determined by the microdilution method against bacteria.

Extraction Method Grape varietal Process conditions Inhibition Zone (mm)a

S. aureus B. cereus E. coli P. aeruginosa C. albicans

Merlot 50 ◦ C/150 bar 12 11 7 0 0

Merlot 50 ◦ C/200 bar 9 10 0 0 17
Merlot 50 ◦ C/250 bar 11 10 7 0 0
Merlot 50 ◦ C/300 bar 12 10 7 0 0
SFE SC-CO2 Merlot 60 ◦ C/150 bar 10 9 8 7 21
Merlot 60 ◦ C/200 bar 10 8 9 7 0
Merlot 60 ◦ C/250 bar 10 9 10 10 0
Merlot 60 ◦ C/300 bar 10 7 9 0 0
Syrah 60 ◦ C/250 bar 10 10 0 0 0

SOX Syrah Hx 0 14 0 0 0
a
MIC, minimum inhibitory concentration (␮g/mL). The values with standard deviation (SD) are the average concentration from duplicates and represent adjacent dilutions.
For the other results (without SD) the MIC for the duplicates were obtained for the same dilution tested (coincident results).

Table 4
MIC (␮g/mL) values of grape pomace extracts determined by the microdilution method against fungi.

Extraction method Grape varietal Process conditions MIC (␮g/mL)a

C. albicans C. parapsilosis C. krusei

50 ◦ C/200 bar 500 1000 500

SFE SC-CO2 Merlot
60 ◦ C/150 bar > 2000 2000 1500 ± 500
a
MIC, minimum inhibitory concentration (␮g/mL). The values with standard deviation (SD) are the average concentration from duplicates and represent adjacent dilutions.
For the other results (without SD) the MIC for the duplicates were obtained for the same dilution tested (coincident results).

Although the size of the inhibition zone from ADM indicates the
strain sensibility to the antimicrobial substance, the halo size values
(Table 2) are not directly related to the samples potency by means
of MIC results (Rios et al., 1988; Kitzberger et al., 2007; Michielin
et al., 2009). For instance, the Syrah extract by SOX–Hx presented
high halo size (ADM data) against B. cereus while its MIC values
indicate weak inhibition. The same behavior was also detected by
other extracts.
For the extracts that presented positive result against C. albicans
in ADM it was applied the microdilution test also against C. parap-
silosis and C. krusei. The results for the Merlot varietal are shown in
Table 4. Similar to the results presented by the bacteria tests, the
size of halo formed on ADM test is not directly related to the potency
of the extract detected by the microdilution test. The best anti-
fungal activity was presented by the SC-CO2 /200 bar/50 ◦ C extract
presenting a moderate inhibition against all three fungi tested.
Finally, evaluating the resistance of different microorganisms,
Papadopoulou et al. (2005) reported that S. aureus was more sen-
sitive than E. coli and C. albicans when submitted to the action of
alcohol-free red and white wine extracts, a behavior also detected
in the present study.
Besides the high number of studies concerning the use of grape
products as antimicrobial agents, there is still a lack of literature
information involving similar combination of raw material type
and preparation, extraction method and microbiological assays
methodology applied to determine the antimicrobial activity, in
order to compare the results. Despite this fact and considering the
results obtained, two grape pomace extracts were considered rel-
evant in terms of quality aspects: the Merlot extract by SC-CO2
at 200 bar and 50 ◦ C, which presented medium inhibition power
against all three fungi and the two Gram-positive bacteria tested;
and the Merlot extract by SC-CO2 at 300 bar and 50 ◦ C a moderate
inhibitor against all bacteria tested.
The antimicrobial activity could be related to the plant cul-
tivation conditions since the active components are usually
synthesized as a response to stress such as microorganisms attack
or strong UV radiation (Filip et al., 2003; Berna et al., 2001). Then,
the better antimicrobial results provided by the selected Merlot
SC-CO2 extracts (50 ◦ C at 200 bar and 300 bar) can be attributed
to the phenolic substances identified such as gallic, p-OH-benzoic
and vanillic acids, well known as antimicrobial compounds (Shoko
et al., 1999; Soni et al., 2005). Besides the above mentioned com-
ponents, other bioactive substances may also have participated as
antimicrobial agents. According to Borchers et al. (2004) natural
extracts may be more beneficial than isolated constituents, as the
synergic positive interaction of compounds may change the prop-
erties of bioactive individual components. The literature reports the
presence of other antimicrobial active substances in grape pomace
extracts such as phytosterols, triterpenes (Ul’chenko et al., 2009),
flavonols (Thimothe et al., 2007) and anthocyanins (Duman et al.,
2009; Tenore et al., 2011) although they were not the focus of the
analytical procedure used in the present work.
4. Conclusions
The present study show that supercritical fluid extraction is
effective to obtain grape pomace extracts with good recovery of
antimicrobial active compounds. Also, the use of grape pomace as
raw material for different extraction methods is promising due
to the high quality of the substances that remain in this indus-
trial residue. SOX and UE presented highest global process yield,
notably when using ethanol and water as solvents respectively,
although those extracts show no antimicrobial activity. However,
results from classical solvent extraction were useful to indicate a
suitable co-solvent for the SFE, in order to improve the global yield
of the extraction. In SFE, the ethanol showed higher efficiency as
co-solvent at 15% (w/w) concentration, providing results compara-
ble to SOX. Despite lower global yield results of SC-CO2 extractions,
these extracts inhibited the growth of the microorganisms tested.
Some SFE conditions produced extracts with moderate activity
against all fungi and bacteria tested. In general, SC-CO2 extracts
were more effective against Gram-positive bacteria (S. aureus and
B. cereus) comparing to Gram-negative (E. coli and P. aeruginosa).
These results denote the SFE as the most efficient method to obtain
valuable antimicrobial extracts from grape pomace, contributing to
increase the aggregate value of this important by-product from the
wine industry.
 D.A. Oliveira et al. / Journal of Biotechnology 164 (2013) 423–432
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