BIO Web of Conferences 15, 04006 (2019) https://doi.org/10.

1051/bioconf/20191504006
42nd World Congress of Vine and Wine

Antioxidant and antimicrobial effects of grape pomace extracts

C.E. Luchian1 , V.V. Cotea1 , L. Vlase2 , A.M. Toiu2 , L.C. Colibaba1 , I.E. Răschip3 , G. Nadăş4 , A.M. Gheldiu2 ,
C. Tuchiluş5 , and L. Rotaru1
1
“Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania
2
“Iuliu Hatieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Cluj-Napoca, Romania
3
Romanian Academy, “Petru Poni” Institute of Macromolecular Chemistry, Iaşi, Romania
4
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania
5
“Grigore T. Popa” University of Medicine and Pharmacy, Iaşi, Romania

Abstract. The use of antioxidants and antibacterials in food industry has become increasingly necessary
to ensure the high quality of food. Grape pomace is the main by-product of winemaking industry that
concentrates bioactive metabolites with more studied antioxidant activity and possible antibacterial activity.
The grape pomace contains fragmented skin, broken cells, pulp remains, stalks and seeds with high amount
of phenolic compounds due to their poor extraction during the winemaking process. Anthocyanins, catechins,
flavonol glycosides, phenolic acids, alcohols and stilbenes have been identified among the compounds
present in grape pomace. In this study, antibacterial activity against different pathogens (Escherichia coli
ATCC 25922, Candida albicans ATCC 90028, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus
aureus ATCC 25923) was evaluated and the relation with polyphenols content and antioxidant activity
of grape pomace from selected grapes from Iaşi vineyard was studied. The grape pomace samples were
obtained after the fermentation process from the 2017 harvest of Sauvignon Blanc, Traminer, Busuioacă de
Bohotin, Cabernet Sauvignon, Merlot, Fetească Neagră and Fetească Regală grape varieties. The antioxidant
properties were evaluated using the DPPH method, FRAP assay and Folin Ciocalteu method. The content
of resveratrol was quantified using an HPLC method. Samples with antioxidant activity showed the highest
phenolics content. This study reveals that grape pomace is a potential source of natural antioxidant agents.
The pomace extracts were tested to establish the effects on Gram-positive and Gram-negative bacteria. The
analysed samples exhibited insignificant antibacterial activity and the method requires optimization. Grape
marc represents an important source of resveratrol and other bioactive compounds that could be a valuable
source of antioxidants for further utilization in food and pharmaceutical industry.

1. Introduction naturally occurring phenolic compounds which include

anthoxanthins (flavones, flavonols, flavanones, flavanols,
The main organic waste produced in wine industries is chalcones and isoflavones), anthocyanins, leucoxanthins
grape pomace, representing 13% to 25% of the total weight and flavonoidal alkaloids [5, 6]. Grape seeds and skins are
of processed fruit [1]. often used as a dietary supplement, containing vitamins,
Wine-making by-products still contain significant minerals, and polyphenols. The polyhenols of grape
amounts of various bioactive compounds [2]. Grape seeds, such as catechins, epicatechin, procyanidin or some
pomace contain numerous phenolic acids and flavonoids dimmers and trimers, have been recognized for their health
which could possess antioxidant activity and antibacterial benefits. Catechins and their epimers serve as powerful
activities. antioxidants for directly eliminating superoxide anion
The antioxidant activity of phenolic compounds radicals. Proanthocyanidins are apparently responsible for
depends on the number of hydroxyl groups in the the action on the cardiovascular system [5].
molecule and the activity can be strengthened by steric Grape pomace represents a potential source of
hindrance. The electron withdrawing attribute of the resveratrol, one of the secondary plant metabolites [7].
carboxylate group in benzoic acids has a negative effect This compound has insulin sensitizing effects and
on the H-donating abilities of the hydroxy benzoates. maintains the blood glucose homeostasis, with numerous
Hydroxylated cinnamates are more effective than benzoate positive influences on health including anticancer action,
counterparts [3]. The antioxidant capacity of phenolic neuroprotective activity and antiaging properties [8, 9].
substances can perform as free radical scavengers, Since wine-making residues composition depends on
hydrogen donators, metal chelators and singlet oxygen grape variety, climate conditions, vineyard location, and
quenchers. winemaking process, it is relevant to study the antioxidant
Antibacterial properties of phenolic substances may be effects and antibacterial activity of each variety separately
due to iron deprivation or hydrogen bounding with vital and so to investigate the biotechnological applications of
proteins, such as microbial enzymes [3, 4]. Flavonoids are natural sources [4].
© The Authors, published by EDP Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 15, 04006 (2019) https://doi.org/10.1051/bioconf/20191504006
42nd World Congress of Vine and Wine

Therefore, more antioxidant and antibacterial studies 2.4.2. DPPH assay

on grape pomace extracts are still needed in order
to identify the appropriate mechanisms to evaluate the The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was
maximum potential for each grape variety. performed to assess radical scavenging activity, by
The aim of the present study was to evaluate the bleaching of purple methanolic solution of the stable
composition of phenolic compounds in grape pomace from radical. Hydrophilic and lipophilic synthetic antioxidants,
seven different varieties from Iaşi Vineyard and assess quercetin and butylated hydroxytoluene (BHT) were used
their antioxidant and antibacterial activities to determine as standards reagents. The measure of antioxidant effect is
their potential as source of natural antioxidants and the disappearance of the DPPH absorbtion by the action of
antimicrobials. antioxidants. 20 µl of diluted extracts were added to 980 µl
DPPH solution (100 µM). The reduction in absorbance
was measured at 517 nm, using a UV-VIS JASCO
2. Materials and methods V-530 spectrophotometer after 30 minutes of incubation
period. After adding individual samples, the percentage
2.1. Grapes samples and winemaking inhibition of DPPH radical was calculated according to the
The necessary plant material for the investigation was formula: I = 100 (Ac -As )/Ac , where I – DPPH inhibition
represented by Merlot (V1), Cabernet Sauvignon (V2), (%), Ac – absorbance of control sample, As – absorbance
Busuioacă de Bohotin (V3), Muscat Ottonel (V4), of tested sample. Antioxidant activity was also expressed
Fetească Neagră (V5), Traminer (V6), Sauvignon Blanc as inhibitory concentration IC50 (calculated graphically)
(V7) grape varieties, harvested in autumn of 2017 at that refers to the concentration of the sample required to
full maturity from Iaşi vineyard – Bucium and Bohotin cause a 50% decrease in initial DPPH radical absorbance.
regions. The grapes were crushed and destemed, succeded Experiments were carried out in triplicate [11–13].
by maceration phase (24 hours for white varieties and
5 days for reds at 10 ◦ C) and then pressed. The marc 2.4.3. Ferric Reducing Antioxidant Power assay
resulted from pressing operation was dried and stored in
the freezer until analysis moment. At the moment of the The FRAP (ferric reducing antioxidant power) assay is
analysis, the marc samples were defrosted and dried in air based on the colour variation of a complex with Fe+3 ion
stream until constant weight, transformed to a powder and of the 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) radical
extracted with different solvents. due to the reduction of the ferric ion to the ferrous
iron (Fe+2 ) in this complex. Trolox solution was used
as reference. A curve absorbance was built according
2.2. Extraction phase to Trolox mass, the correlation coefficient (R2 ) for this
For the extraction, two different methods were compared. curve being 0.992. The final results were converted to µM
One extraction was realised using a 50% mixture of Trolox equivalents/100 mL extract [14]. All samples were
water and ethanol. Another extraction was performed with analysed in triplicate.
a 50% mixture of water and methanol. Two series of
extracts were prepared by mixing 2 g of solid samples with 2.4.4. Antimicrobial susceptibility tests
5 mL of aqueous mixture, followed by stirring for 24 h at
room temperature, and then centrifuged at 4500 rpm for The antimicrobial activity was studied using Gram
20 minutes at 4 ◦ C. The supernatants were recovered and positive bacteria (Staphylococcus aureus ATCC 25923),
used for analysis. Gram negative bacteria (Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853) and a pathogenic
yeasts (Candida albicans ATCC 90028).
2.3. Chemicals Antimicrobial tests of selected microorganisms were
Methanol, ethanol and acetonitrile of HPLC gradient, carried out using a disc-diffusion method (CLSI). A
ammonium acetate, formic acid, acetic acid of analytical- small amount of each microbial culture was diluted in
grade were purchased from Merck (Merck KgaA, sterile 0.9% NaCl until the turbidity was equivalent to
Darmstadt, Germany). Trans-resveratrol was purchased McFarland standard no. 0.5 (106 CFU/mL). Mueller
from Sigma (Sigma-Aldrich Chemie GmbH, Munich, Hinton agar (Oxoid) and Mueller-Hinton agar Fungi
Germany). Other chemicals used were of biochemistry (Biolab) were inoculated with the suspensions of the tested
grade. microorganisms. Sterile stainless steel cylinders (5 mm
internal diameter; 10 mm height) were applied on the
agar surface in Petri plates. Then, 100 µL of the tested
2.4. Methods of analyses compounds (1−7), with a concentration of 10 mg/mL each
2.4.1. Determination of total polyphenolic content were added into cylinders. The plates were left 10 minutes
at room temperature to ensure the equal diffusion of the
The total phenolic content (TPC) of the extracts was compound in the medium and then incubated at 35 ◦ C for
purchased using the Folin-Ciocalteu assay, with some 24 hrs.
revisions. The absorbance was measured at 760 nm, using As reference there were used commercial available
a JASCO UV-VIS spectrophotometer. Standard curve was discs with antimicrobial drugs containing Ciprofloxacin
prepared by using different concentrations of gallic acid. (5 µg/disk), Fluconazole (25 µg/disk) and Voriconazole
TPC was expressed as mg gallic acid/ mL extract (mg (1 µg/disk). After incubation, the diameters of inhibi-
GAE/mL extract) [10]. All samples were analysed in tion were measured. All assays were carried out in
triplicate. triplicate [15, 16].

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BIO Web of Conferences 15, 04006 (2019) https://doi.org/10.1051/bioconf/20191504006
42nd World Congress of Vine and Wine

2.4.5. Determination of resveratrol using HPLC Table 1. TPC in analysed extracts (±SD).
A methanolic solution (10 mg/mL) of trans-resveratrol was Sample TPC (mg GAE/mL) ±SD TPC (mg GAE/mL) ±SD.
prepared and stored at 4 ◦ C and protected from light. Extract with ethanol Extract with methanol
Before analysis, aliquots of the prepared solution were V1 2.7075 ± 0.002 2.421 ± 0.09
adequately diluted with bidistilled water. Cis-resveratrol
V2 2.08 ± 0.002 0.642 ± 0.03
was prepared from a standard solution of trans-resveratrol
after its irradiation for 10 minutes using an UV lamp V3 2.1375 ± 0.001 0.404 ± 0.02
(254 nm). V4 2.76 ± 0.001 0.771 ± 0.05
Sample preparation. The samples were diluted ten V5 2.7775 ± 0.002 1.452 ± 0.07
folds with bidistilled water, then centrifuged at 10,000 rpm V6 2.5725 ± 0.003 1.242 ± 0.05
for 5 minutes. An aliquot was then injected into the
V7 2.03 ± 0,003 0.884 ± 0.06
chromatographic system.
The experiment was performed using an Agilent 1100 ±SD standard deviation; GAE – gallic acid.

HPLC Series system (Agilent, SUA) equipped with an

autosampler G1311A. For the separation, a reversed-phase
Zorbax SB-C18 analytical column (100 × 3.0 mm i.d., Table 2. Antioxidant activity of extracts using DPPH method.
3.5 µm particles) was used. The column was operated
Sample IC50 (µg/mL) IC50 (µg/mL)
at 40 ◦ C in a G1316A oven. For the elution a degasser
(G1322A), and a binary gradient pump (G1311A) were Extract with ethanol Extract with methanol
used. The isocratic elution was achieved using a mixture V1 24.21 ± 0.82 98.142 ± 4.31
of 1 mM ammonium acetate/acetonitrile (73/27, v/v). The V2 34.60 ± 1.02 134.48 ± 5.27
flow rate was 1 mL/min and the injection volume 5 µL. V3 33.42 ± 0.91 144.9 ± 6.41
All solvents were filtered using 0.5 mm (Sartorius) filters V4 20.93 ± 0.79 132.17 ± 5.11
and degassed in an ultrasonic bath. The detection of trans-
and cis-resveratrol was carried out using an Agilent Ion V5 20.59 ± 0.75 115.78 ± 4.29
Trap VL mass spectrometer (Agilent, SUA). The mass V6 30.71 ± 0.96 119.86 ± 4.35
spectrometer was operated using an atmospheric pressure V7 44.04 ± 1.24 127.96 ± 4.74
chemical ionisation (APCI) ion source in negative mode. Quercetin 5.59 ± 0.13 5.59 ± 0.13
The nitrogen was used as nebulising and dry gas. The BHT 15.88 ± 1.06 15.88 ± 1.06
APCI heater was set at 350 ◦ C, the nebulizer pressure
Note: Values are the mean ± SD (n = 3).
60 psi, dry gas flow was 5 L/min and was heated at 250 ◦ C.
The mass spectrometer operated in multiple reactions
monitoring mode and was set to monitor the transition m/z
227 → m/z 185. Table 3. Antioxidant activity of extracts using FRAP method.
Chromatographic and mass spectrometric data acquisi- Sample µM Trolox /100 mL µM Trolox /100 mL
tion were performed using Chemstation software (Agilent
Extract with ethanol Extract with methanol
Technologies, Palo Alto, CA, USA), version B.01.03
and LC/MSD Trap Control (Bruker Daltonik, GmbH, V1 1432.37 ± 9.68 1164.22 ± 12.46
Bremen, Germany), version 5.3, while data processing V2 957.40 ± 8.25 359.75 ± 6.84
was performed using LC/MSD Data Analysis and Quant V3 1003.39 ± 9.01 186.09 ± 5.58
Analysis software (Bruker Daltonik, GmbH, Bremen, V4 1435.27 ± 10.37 406.17 ± 7.53
Germany), version 1.7. In order to find the optimum
V5 1688.51 ± 11.43 782.09 ± 9.31
ion source (MS interface) and mobile phase composition
the signal to noise (S/N) ratios were calculated for each V6 1252.91 ± 10.05 473.73 ± 8.27
injection [17]. V7 937.09 ± 9.29 434.35 ± 8.01
Trolox 2073.91 ± 26.08 2073.91 ± 26.08
3. Results and discussion Note: Values are the mean ± SD (n = 3).

3.1. Polyphenols analysis

Total phenolic content varied considering the analysed V5 (1.45 GAE/mL) and V6 (1.24 GAE/mL), followed by
extracts. Different values of polyphenols concentration V7 (1.24 GAE/mL) and V4 extracts (0.88 GAE/mL).
were obtained depending on the solvent used for the The extraction method in which ethanol was used was
extraction. more efficient in determining the amount of phenolic.
The obtained concentrations of total polyphenols from
analysed samples are presented in Table 1. 3.2. Antioxidant activity assay by DPPH method
In the case of the extract obtained with ethanol, the
higher concentration in total polyphenols was registered In order to evaluate the ability of extracts and synthetic
in V5 extract (2.77 mg GAE/mL), V4 (2.76 mg GAE/mL), antioxidants quercetin and BHT to donate the hydrogen
and V1 (2.70 mg GAE/mL), followed by V6 extract atom, the stable free radical DPPH was used. The results
(2.57 mg GAE/mL). Smaller contents of polyphenols were obtained for the evaluation of the antioxidant activity using
found in samples V3, V2 and V7. On the other hand, in the DPPH bleaching assay are presented in Table 2. All
the case of the methanol extract, the highest values for the extracts were able to reduce DPPH radical with different
analysed parameters were recorded at V1 (2.42 GAE/mL), degrees of scavenging activity.

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42nd World Congress of Vine and Wine

Table 4. Antibacterial and Antifungal Activities of the Tested Compounds.

Diameter of inhibition zones (mm)
Compounds
S. aureus E. coli Pseudomonas aeruginosa C. albicans
ATCC 25923 ATCC 25922 ATCC 27853 ATCC 90028
1 – – – 12.0 ± 0.00
2 – – – –
3 – – – –
4 – – – –
5 – – – –
6 – – – –
7 – – – 13.0 ± 0.00
Ciprofloxacin 5 µg/disc) 27.7 ± 0.06 30.0 ± 0.00 30.0 ± 0.00 *NT
Fluconazol (25 µg/disc) NT* NT* NT* 30.0 ± 0.00
Voriconazol (1 µg/disc) NT* NT* NT* 29.5 ± 0.50
*NT-not tested.

A lower IC50 value represents a higher bleaching A positive correlation between scavenging activity
effect, thus a better antioxidant activity. Differences can be determined by FRAP method and total phenolic content
observed depending on the solvent used in the extraction. was observed. The results are varying in correlation to the
All analysed extracts showed lower DPPH scavenging used method.
activity than standards quercetin and BHT. In the case The highest antioxidant activity for the extrac-
of extract obtained with ethanol, the highest radical tions with ethanol was determined for V5 sample
scavenging activity was determined for extract V5 and and V4 (1688.51 µM Trolox/100 mL, and 1435.27 µM
V4 (20.59 µg/mL, and 20.93 µg/mL respectively), with Trolox/100 mL respectively). Using the FRAP assay,
positive correlation between scavenging activity on DPPH the following order in antioxidant activity was found:
and total phenolic content. The extracts V2 and V3 showed V7 <V2 <V3 <V6 <V1 <V4 <V5 <Trolox.
comparable antioxidant activity by DPPH method, and the In the second situation (extract with methanol), the
results could also be correlated with the content in total highest scavenging activity was noted for V1 and V5
polyphenols. The lower antioxidant effect was observed extracts (1164.22 µM Trolox /100 mL, and 782.09 µM
for extracts V3, V2, V7 (IC50 value = 33.42 µg/mL, Trolox /100 mL respectively). Using the FRAP assay, the
34.60 µg/mL, 44.04 µg/mL and respectively), and this following order in antioxidant activity was found: V3 <V2
could be due to the smaller concentration of polyphenols <V4 <V7 <V6 <V5 <V1 <Trolox.
(2.03−2.13 mg GAE/mL). Considering the obtained The antioxidant activity using the FRAP assay could
results by DPPH method, the following order in be correlated with the results obtained by DPPH assay.
antioxidant activity could be established: V7 <V2 <
V3 <V6 <V1 <V4 <V5 <BHT<quercetin. According to
this method, all analysed extracts showed high antioxidant 3.4. Antimicrobial activity
effects (IC50 < 50 µg/mL) [12].For the second extract The diameters of the inhibition zones (in mm) corre-
(with methanol), the strongest antioxidant activity was sponding to the tested compounds are shown in Table 1.
noted in V1 sample, (with IC50 = 98.142 µg/mL) and V5 All assays were carried out in triplicate. Results are
(115.78 µ g/mL) extract, with positive correlation between expressed as means ±SD. Against Gram negative and
scavenging activity on DPPH and total phenolic content. Gram negative bacteria all the tested samples have
Considering the obtained results, the following order in insignificant activity. The V1 and V7 extracts registered a
antioxidant activity could be established: V3 <V 2 < reduced antifungal activity. The influence of grape pomace
V4 <V7 <V6 <V5 <V1 <BHT<quercetin. According extract on the growth of fungi and bacteria is expressed in
to this method, the analysed extracts showed weak Fig. 1.
antioxidant effects [11].
The results could be related to the presence of
higher amounts of phenolic compounds and indicates that 3.5. Determination of resveratrol using HPLC
different active principles can contribute to antioxidant
properties of natural products. The various antioxidant Resveratrol is a very polar compound and it exist as
properties of these extracts may be due to the variability trans- and cis- isomers, both present in grapes [18]. For
of composition and content in phytochemicals, and the the extraction of resveratrol, the yield was determinate
method used for extraction. Ethanol solvent may be by using ethanol. The resveratrol content identified
preferable for the extraction preparation. using a chromatographic determination is presented in
Table 5.
Figure 2 express a chromatogram of trans- and
3.3. Ferric Reducing Antioxidant Power
cis-resveratrol standard solution at LOQ (limit of
(FRAP) assay
quantification level). The concentration of trans-resveratrol
Table 3 express the evaluation of the antioxidant activity in grape pomace extract varied considerably, depending on
using the FRAP assay. the grape variety.

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42nd World Congress of Vine and Wine

A B

C D
Figure 1. The influence of grape pomace extract on the growth of Candida albicans ATCC 90028 (A), Escherichia coli ATCC 25922
(B) and Pseudomonas aeruginosa ATCC 27853 (C), Staphylococcus aureus ATCC 25923 (D).

4. Conclusions
The results showed a close relationship between content of
phenolic compounds and antioxidant capacity. All extracts
were able to reduce DPPH radical with different degrees of
antioxidant activity.
A positive correlation between scavenging activity
determined by FRAP method and total phenolic content
being observed. Extract with ethanol showed better
results for determination of phenolic compounds from
grape pomace. The analysed grape varieties showed a
good potential as source of bioactive compounds that
Figure 2. Chromatogram corresponding to a standard mixture of could be applied as natural antioxidants in food and
trans- and cis-resveratrol at the limit of quantification level (peak cosmetic products, in order the increase their shelf life.
1 = trans-resveratrol; peak 2 = cis-resveratrol). Against Gram negative and Gram positive bacteria all
the tested samples have an insignificant activity. The V1
Table 5. The Resveratrol content of pomace extracts. and V7 extracts registered a reduced antifungal activity.
Grape marc represents an important source of trans-
µg/mL conc in extract trans+cis trans/cis resveratrol, with great potential for application in food and
Sample
conc trans conc cis µg/mL µg/mL pharmaceutical industry.
V1 23.54 ± 0.12 2.01 ± 0.06 25.55 ± 0.09 11.72 ± 0.12
The research was funded by the PN-III-P1-1.1-TE-2016-2038
V2 13.21 ± 0.15 3.27 ± 0.11 16.48 ± 0.08 4.04 ± 0.11
project, no. 77/8.05.2018 and FDI project - final registration code
V3 10.27 ± 0.13 0 10.27 ± 0.11 0 CNFIS-FDI-2019-0267.
V4 22.68 ± 0.08 0 22.68 ± 0.13 0
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42nd World Congress of Vine and Wine

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