Journal of Food Engineering 240 (2019) 105–113

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Pressurized liquid extraction of bioactive compounds from grape marc T

a b b
Débora Tamires Vitor Pereira , Adriana Gadioli Tarone , Cinthia Baú Betim Cazarin ,
Gerardo Fernández Barberoc, Julian Martíneza,∗
a
School of Food Engineering, Food Engineering Department, University of Campinas, UNICAMP, 13083-862, Campinas, SP, Brazil
b
School of Food Engineering, Food and Nutrition Department, University of Campinas, UNICAMP, 13083-862, Campinas, SP, Brazil
c
Departament of Analytical Chemistry, Faculty of Sciences, University of Cadiz, IVAGRO, República Saharaui Avenue s/n, C.P. 11510, Puerto Real, Cadiz, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: Extracts rich in monomeric anthocyanins (MAC) and total phenolic compounds (TPC) were obtained from grape
PLE marc by Pressurized Liquid Extraction (PLE). PLE was performed using ethanol and water mixtures (acidified or
Grape marc not) (50% w/w), pure ethanol and acidified water at temperatures from 40 to 100 °C. The best PLE conditions for
Anthocyanins MAC extraction (ethanol-water pH 2.0 [50% w/w]) resulted in 10.21 mg of malvidin-3-O-glucoside/g of dried
Phenolic compounds
grape marc (dr). Fifteen anthocyanins were identified and quantified in PLE extracts by UHPLC-UV-Vis. PLE with
Antioxidant capacity
ethanol-water (50% w/w) as solvent at 100 °C achieved the highest TPC content (65.68 mg GAE/g dr) and
Mathematical modeling
antioxidant capacity by ORAC (772.11 μmol TE/g dr) and FRAP (1452 mg TE/g dr) among the evaluated con-
ditions. Based on the results for extraction of monomeric anthocyanins and phenolics compounds, a sequential
PLE process was performed and proved to be viable for the recovery of two different extract fractions.

1. Introduction Bioactive compounds are found in vegetables and fruits.

The world production of grapes exceeded 75.8 million tons in 2016,
among which 35.3 million tons were destined to wine production, re-
sulting in 267 million hectoliters of wine for consumption and in-
dustrial purposes (OIV, 2016). Brazil currently ranks 20th in the world's
wine production, with approximately 1.6 million hectoliters in 2016
(OIV, 2016). Grape marc is the main solid waste from wine industries,
resulting from the fermentation process, containing seeds and skin as
main components. It is currently used as fertilizer, animal feed and as
substrate for extraction of tartaric acid (Negro et al., 2003; Silva et al.,
2000). However, only 30–40% of the anthocyanins contained in grapes
are extracted from the skins during winemaking. Therefore, grape marc,
which corresponds to 50% of the total grape mass, is rich in these
bioactive compounds and have functional properties, being capable to
act on human metabolism, preventing cardiovascular and inflammatory
diseases and certain cancers (Luque-Rodríguez et al., 2007; Polovka
et al., 2010; Teixeira et al., 2014). Anthocyanins also have applications
in food, cosmetics and pharmaceutical industries (Fontana et al., 2013).
Polyphenols stand out among these compounds, as secondary metabo-
lites found in cereals, fruits, wine and teas. They are classified in dif-
ferent groups, including flavonoids, such as anthocyanins (Bosscher
et al., 2009; Reis Giada, 2013). Anthocyanins are responsible for the
color of red fruits, being often used as natural pigments, and are anti-
oxidants that act in the prevention of several diseases (Duba et al.,
2015; Scalbert et al., 2005; Shrikhande, 2000; Yang et al., 1997).
Pressurized Liquid Extraction (PLE) uses liquid solvents in their
subcritical state with controlled temperature and pressure. At these
conditions, PLE is efficient in the extraction of phenolic compounds
from plants in short time using organic solvents and/or water (Otero-
Pareja et al., 2015; Teixeira et al., 2014). Once grape marc is a complex
mixture of bioactive compounds such as lignans, stilbenes, phenolic
acids and flavonoids, extracting individual compounds from this by-
product is a hard challenge (Teixeira et al., 2014). Emerging and more
efficient techniques, such as PLE, have been recommended to improve
the extraction of these compounds from plant matrices (Garcia-
Mendoza et al., 2017; Gonçalves et al., 2016; Machado et al., 2017,

Abbreviations: Del3Glu, delphinidin-3-O-glucoside; Cy3Glu, cyanidin-3-O-glucoside; Pet3Glu, petunidin-3-O-glucoside; Peo3Glu, peonidin-3-O-glucoside; Mal3Glu,
malvidin-3-O-glucoside; Del36AcG, delphinidin3-O-(6″-acetyl)-glucoside; Cy36AcG, cyanidin-3-O-(6″-acetyl)-glucoside; Pet36AcG, petunidin-3-O-(6″-acetyl)-glu-
coside; Peo36AcG, peonidin-3-O-(6″-acetyl)-glucoside; Mal36AcG, malvidin-3-O-(6″acetyl)-glucoside; Cy36CuG, cyanidin-3-O-(6″-p-coumaryl)-glucoside; Pet36CuG,
petunidin-3-O-(6″-p-coumaryl)-glucoside; cMa36CuG, malvidin-3-O-(6″-cis-p-cumaroyl)-glucoside; Peo36CuG, peonidin-3-O-(6″-p-coumaryl)-glucoside; tMa36CuG,
malvidin-3-O-(6″-trans-p-coumaroyl)-glucoside
∗
Corresponding author.
E-mail address: julian@unicamp.br (J. Martínez).

https://doi.org/10.1016/j.jfoodeng.2018.07.019
Received 16 February 2018; Received in revised form 17 July 2018; Accepted 19 July 2018
Available online 20 July 2018
0260-8774/ © 2018 Elsevier Ltd. All rights reserved.
D.T.V. Pereira et al. Journal of Food Engineering 240 (2019) 105–113

2015; Viganó et al., 2016a, 2016b). Moreover, the mathematical duplicate.

modeling of PLE kinetic curves can help comprehending the mass
transfer mechanisms of the process, thus allowing its optimization and
scale-up.
The investigation of the process operational parameters is necessary
to improve the PLE from grape marc and maximize the recovery of its
target compounds. Therefore, this study aimed to apply PLE to grape
marc to obtain extracts rich in anthocyanins and polyphenols.
2. Materials and methods
2.1. Materials
Grape (Vitis vinifera L. CV. Syrah) marc, composed by skins and
seeds, was donated by Adega Família Ferragut winery (Vinhedo, SP,
Brazil). This material was ground in a domestic blender to be homo-
genized and to reduce the resistance to mass transfer during extraction.
The ground raw material was stored in the dark at −18 °C for further
extractions.
2.2.1. Sequential PLE
The dynamic PLE method was applied in a sequential process, using
approximately 5.0 g of fresh grape marc, forming a fixed bed inside a
50 mL jacketed stainless steel column. Glass beads were used to com-
plete the cell volume. Pressure and solvent mass flow rate were kept
constant at 10.0 ± 0.5 MPa and 5.0 g/min, respectively. The total ex-
traction time was 220 min. Temperatures and solvents were chosen
according to the results obtained in the one step PLE, in which the best
conditions to recover anthocyanins and total phenolics were identified.
The PLE process was divided in two sequential steps to first recover
anthocyanins and then phenolic compounds. The first step was per-
formed at 40 °C with water-ethanol pH = 2.0 (50% w/w) as solvent,
and the second was performed at 100 °C with water-ethanol (50% w/
w). The fresh grape marc was kept inside the extraction cell after the
first step and used in the second step. The extracts were collected in
amber glass flasks and stored in the dark at −18 °C for further analyses.
The sequential PLE was performed in duplicate.

2.2. One-step pressurized liquid extraction (PLE) 2.3. Characterization of grape marc

PLE was performed in a high pressure extraction unit described by
Viganó et al. (2016a) using the dynamic method, which consists of the
continuous solvent flow through a fixed substrate bed. Approximately
5.0 g of fresh grape marc were used, forming a fixed bed inside a 50 mL
jacketed stainless steel column. Glass beads were used to complete the
cell volume. The sample was ground in a domestic blender to be
homogenized and to reduce the resistance to mass transfer during ex-
traction. The choice of temperatures and solvents was based on pub-
lished works, in which PLE was successfully applied in the extraction of
anthocyanins (Garcia-Mendoza et al., 2017; Machado et al., 2015;
Monrad et al., 2010; Paes et al., 2014). Temperatures of 40, 60, 80 and
100 °C were tested, using pure ethanol, water-ethanol (50% w/w),
water-ethanol pH = 2.0 (50% w/w) and acidified water (pH = 2.0) as
solvents, with pH adjusted with citric acid (Synth, São Paulo, Brazil).
Pressure and solvent mass flow rate were kept constant at
10.0 ± 0.5 MPa and 5.0 g/min, respectively. Extraction time was de-
fined by preliminary tests, when verified that the diffusion-controlled
extraction period had started. To assess the temperature effect on the
anthocyanin recovery, the best solvent for monomeric anthocyanins
was used at different temperatures (40, 55, 70 and 85 °C), keeping
pressure and solvent mass flow rate constant. In all extractions, the
Grape marc was subjected to chemical characterization by per-
forming the following analyses, according to AOAC (1997) methods:
moisture (method 931.04); lipids (method 963.15); proteins (method
970.22); ash (method 972.15); carbohydrates determined by difference;
direct determination of pH by phmeter (QUIMIS®, Mod.Q400AS,
Brazil).
2.4. Characterization of the extracts
2.4.1. Monomeric anthocyanin content
The monomeric anthocyanin content (MAC) of the extracts was
determined through the differential pH method (Giusti and Wrolstad,
2001) with some adaptations (Lee et al., 2005). The monomeric an-
thocyanin concentration was calculated considering the molar absorp-
tivity (ε) of 28,000 L/cm.mol and molecular mass of 493.2 g/mol,
which corresponds to malvidin-3-O-glucoside (Mv-3-glc), the major
anthocyanin found in grapes (Rockenbach et al., 2011), using Equation
(1). Results were expressed as mg Mv-3-glc/g dried grape marc (dr). All
measurements were performed in triplicates.
mg ⎛ mgMv3glc ⎞ A x MM x DF x 1000
MAC ⎛ ⎞⎜ ⎟ =

extracts were collected and evaluated at different times. For the
monomeric anthocyanins extraction, the beginning of the diffusional
extraction stage was verified at about 40 min. The extraction time for
total phenolics was 4 h, from when it was verified that the diffusion-
controlled extraction period had already begun. The total extraction
times are specified in Tables 1, 2 and 6. The extracts were collected in
glass flasks and stored in the dark at −18 °C until further analyses. For
the chromatographic analyses, the extracts were filtered through a
0.22 μm nylon syringe filter (Filter-Lab, Filtros Anoia, S.A., Sant Pere de
Riudebitlles, Barcelona, Spain). All extractions were performed in
⎝ L ⎠⎝ g dr ⎠ εx1 (1)
Where: MAC: Monomeric anthocyanins expressed as mg (Mv-3-glc)/g
dr; A = (Abs 520 nm–Abs 700 nm)pH 1,0 − (Abs 520 nm–Abs 700 nm)pH
4,5, sample absorbance; MM = molecular mass of malvidin-3-O-gluco-
side; DF = dilution factor; ɛ = molar absorptivity of malvidin-3-O-glu-
coside; 1 = correction optic path factor (1 cm).
2.4.2. Identification of anthocyanins by UHPLC-QToF-MS
The anthocyanins of the PLE extracts were identified by ultra high-
performance liquid chromatography (UHPLC) coupled to quadrupole-

Table 1
Monomeric anthocyanins content (MAC), total phenolic content (TPC) and antioxidant capacity determined by ORAC and FRAP methods of the extracts obtained
from grape marc by PLE obtained in 40 min.
T(°C) Solvent S/F MAC (mg Mv-3-glc/g dr) TPC (mg GAE/g dr) ORAC (μmol TE/g dr) FRAP (mg TE/g dr)

100 Ethanol-Water (50% w/w) 253 1.90 ± 0.04D 65.68 ± 2.24A 772.11 ± 18.62A 1452.00 ± 129.62A
80 Ethanol 218 4.52 ± 0.10CD 36.74 ± 2.72B 503.88 ± 73.54BC 797.70 ± 161.48BC
60 Ethanol-Water (50% w/w) 218 8.76 ± 0.73AB 32.45 ± 6.58B 479.97 ± 83.97BC 897.30 ± 146.27B
40 Ethanol-Water (50% w/w) 217 6.23 ± 0.18BC 16.08 ± 0.74C 276.69 ± 13.16C 478.50 ± 31.32BC
40 Ethanol-Water pH 2.0 (50% w/w) 217 10.21 ± 0.53A 28.66 ± 0.29B 571.76 ± 22.73AB 478.40 ± 9.73BC
40 Water pH 2.0 220 7.42 ± 1.08AB 14.16 ± 0.85C 268.86 ± 30.95C 391.70 ± 32.38C

Results expressed as mean ± standard deviation. Equal capital letters in the same column indicate that there is no difference between the extraction methods.

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D.T.V. Pereira et al. Journal of Food Engineering 240 (2019) 105–113

(50% w/w). TA-UHPLC: total content of monomeric anthocyanins. Results are expressed as mean ± standard deviation. Equal capital letters in the same column indicate that there is no difference between the
Table 3
TA-UHPLC

10.573 ±

12.012 ±
1.823 ±

3.494 ±

6.434 ±

7.791 ±
0.402CD
1.507DE

1.245AB

0.718BC
Pearson's correlation matrix obtained between antioxidant capacity (ORAC and

0.764A
0.028E
FRAP) and extract composition (MAC and TPC) for the following PLE condi-
tions: Ethanol-watera (100 °C), Ethanol (80 °C), Ethanol-watera (60 °C), Ethanol-
watera (40 °C), Ethanol-watera pH 2.0 (40 °C), Water pH 2.0 (40 °C).
tMa36CuG

0.124ABC
0.269 ±

0.392 ±

1.049 ±

0.874 ±

1.496 ±

0.444 ±
0.226AB
0.013BC

0.274BC
0.243A
0.144C

ORAC FRAP

MAC r −0.418 −0.714

p 0.409 0.111
Peo36CuG

0.056 ±

0.087 ±

0.177 ±

0.176 ±

0.326 ±

0.111 ±
TPC r 0.940 0.958

0.038A
0.022B

0.006B

0.029B

0.020B

0.062B
p 0.005 0.003

a
(50% w/w).
cMa36CuG

0.038 ±

0.029 ±

0.102 ±

0.089 ±

0.279 ±

0.092 ±
0.013A
0.011B

0.040B

0.032B

0.023B

0.063B

time-of-flight mass spectrometry equipment (Q-ToF-MS) (Synapt G2,

Waters Corp., Milford, MA, USA). The injection volume was adjusted to
3 μL. The chromatographic separation was performed on a
Pet36CuG

0.045 ±

0.067 ±

0.212 ±

0.228 ±

0.421 ±

0.170 ±

2.1 mm × 100 mm reverse-phase C18 analytical column (Acquity UPLC

0.033A
0.008B

0.027B

0.057B

0.046B

0.079B

BEH C18, Waters, Milford, USA) and a particle size of 1.7 μm. For the
anthocyanins identification, water (2% formic acid) was used as solvent
A and methanol as solvent B, as mobile phases at a flow rate of 0.4 mL/
Cy36CuG

0.013 ±

0.008 ±

0.014 ±

0.013 ±

0.026 ±

0.011 ±
0.006AB

0.004AB

0.002AB

0.006AB
0.001A
0.001B

min. The gradient employed was: 0 min, 15% B; 3.30 min, 20% B;
3.86 min, 30% B; 5.05 min, 40% B; 5.35 min, 55% B; 5.64 min, 60% B;
5.94 min, 95% B; 7.50 min, 95% B. Total run time was 12 min, in-
Mal36AcG
Concentration of the anthocyanins (mg/g dr) identified by UHPLC-UV-vis in the PLE extracts obtained in 40 min under different conditions.

0.004ABC

cluding 4 min for re-equilibration. The anthocyanins determination was

0.171 ±

0.275 ±

0.826 ±

0.510 ±

0.690 ±

0.385 ±
0.069AB

0.002BC
0.096A
0.025C

0.208C

performed using an electrospray source operating in positive ionization

mode under the following conditions: desolvation gas flow
rate = 700 L/h, desolvation temperature = 500 °C, cone gas flow
Peo36AcG

0.004ABC
0.043 ±

0.080 ±

0.184 ±

0.121 ±

0.199 ±

0.109 ±
0.031AB

0.023BC

rate = 10 L/h, source temperature = 150 °C, capillary voltage = 700 V,

0.012A
0.004C

0.026C

cone voltage = 20 V and collision energy trap = 4 eV. The full-scan

mode (m/z = 100–1200) was used. Fifteen anthocyanins were identi-
Pet36AcG

fied (Fig. 1).

0.049 ±

0.068 ±

0.240 ±

0.098 ±

0.286 ±

0.168 ±
0.026AB
0.048A

0.022A
0.007B

0.045B

0.011B

2.4.3. Separation and quantification of anthocyanins by UHPLC-UV-Vis

The separation and quantification of anthocyanins in PLE extracts
Cy36AcG

0.030 ±

0.056 ±

0.084 ±

0.054 ±

0.121 ±

0.071 ±
0.001AB

0.026AB

0.008AB

0.029AB
0.002A
0.010B

were performed on an Elite UPLC LaChrom Ultra system (VWR Hitachi,

Tokyo, Japan) consisting of an L-2200U Autosampler, an L2300
Column Oven, an L-2160U Pumps and an L-2420U UV–Vis Detector.
Del36AcG

The column oven was adjusted at 50 °C for the chromatography. UV–Vis

0.035 ±

0.043 ±

0.124 ±

0.038 ±

0.148 ±

0.090 ±
0.009CD

0,002CD
0.025AB

0.016BC
0.009D

0.006A

Detector was set at 520 nm for the analysis. Anthocyanins were ana-
lyzed on a Kinetex C18 100 Å column (100 × 2.1 mm I.D., particle size
2.6 μm, Phenomenex Inc., UK). A gradient method, using acidified
Mal3Glu

0.754 ±

1.795 ±

5.085 ±

3.247 ±

5.144 ±

3.837 ±
0.030AB

0.785AB
0.999BC

0.423A

0.480A
0.032C

water (5% formic acid, solvent A) and methanol (solvent B), working at
a flow rate of 0.7 mL/min, was used for the chromatographic separa-
tion. The gradient employed was as follows: 0 min, 0% B; 1.00 min, 5%
Peo3Glu

0.127 ±

0.328 ±

0.794 ±

0.521 ±

0.849 ±

0.655 ±
0.024AB

0.138AB
0.146BC

B; 3.30 min, 15% B; 4.80 min, 25% B; 6.00 min, 40% B; 7.00 min, 45%
0.058A

0.078A
0.005C

B; 9.00 min, 100% B; 12.00 min, 100% B; 12.70 min, 0% B. The UHPLC
method was used to obtain a calibration curve for malvidin chloride
(y = 232219.58x − 3574.06), which is the commercially available an-
0.114 ±

0.183 ±

1.066 ±

0.355 ±

1.250 ±

0.935 ±
Pet3Glu

0.090A

0.087A

0.230A
0.048B

0.215B

0.021B

thocyanidin standard for malvidin anthocyanins.

Simple linear regression (y = βx + α) has been performed with
concentration units of mg/L as independent variable “x”, and absor-
0.026 ±

0.051 ±

0.477 ±

0.055 ±

0.127 ±

0.124 ±
Cy3Glu

0.001A

0.022A

0.478A

0.003A

0.031A

0.033A

bance units (A.U.) as dependent variable “y”. Therefore, the slope

(β = 232219.58) has units of U.A. L/mg and the intercept
(α = 3574.06) has units of U.A. The regression equation and the de-
0.053 ± 0.025BC

termination coefficient (R2 = 0.9997) were calculated using Microsoft

Office Excel 2010. The detection and quantification limits for malvidin-
0.031 ±

0.463 ±

0.055 ±

0.652 ±

0.588 ±
Del3Glu

0.075AB

0.012BC

3-O-glucoside (0.199 mg/L and 0.663 mg/L) were calculated from 3x

0.156A

0.192A
0.044C

and 10x times the signal-to-noise ratio divided by the angular coeffi-
cients of the analytical curves. All the anthocyanins were quantified
Ethanol-Watera pH 2.0

using the calibration curve for malvidin chloride, considering each

extraction methods.

one's molecular mass. All analyses were performed in duplicate and the
Ethanol-Watera

Ethanol-Watera

Ethanol-Watera

results are expressed as mg of the respective anthocyanin/g dr.

Water pH 2.0
100 °C
Extraction

80 °C

60 °C

40 °C

40 °C

40 °C
Ethanol

2.4.4. Total phenolic content

Table 2

The total phenolic content (TPC) of the extracts was determined

a

through the Folin–Ciocalteu method (Singleton and Rossi, 1965), with

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D.T.V. Pereira et al. Journal of Food Engineering 240 (2019) 105–113

Table 4
Monomeric anthocyanin content (MAC), total phenolic content (TPC), antioxidant capacity (ORAC and FRAP) obtained in 40 min in grape marc extracts by PLE with
ethanol-water pH 2.0 (50% w/w).
T (°C) MAC (mg Mv-3-glc/g dr) TPC (mg GAE/g dr) ORAC (μmol TE/g dr) FRAP (mg TE/g dr)

25 6.20 ± 0.10B 19.41 ± 1.37D 311.96 ± 11.23D 245.47 ± 12.75C

40 10.21 ± 0.53A 39.08 ± 0.84C 571.76 ± 22.73BC 478.44 ± 9.73B
55 7.15 ± 0.11B 40.23 ± 2.41C 530.46 ± 36.80C 453.58 ± 24.49B
70 4.07 ± 0.31C 55.33 ± 0.76B 733.34 ± 62.15AB 557.96 ± 19.16B
85 4.12 ± 0.30C 76.38 ± 6.75A 876.03 ± 82.80AB 810.00 ± 74.74A

Results are expressed as mean ± standard deviation. Equal capital letters in the same column indicate that there is no significant difference between the extraction
methods.

Table 5 radical and it was determined using the method described by Ou et al.
Pearson's correlation matrix obtained between antioxidant capacity (ORAC and (2013). AC was expressed as μmol Trolox (μmol TE)/g dr and was used
FRAP) and extract composition (MAC and TPC) for PLE with ethanol-water pH to plot (5–125 μmol TE/L) the standard curve (y = 0.317x + 8.27) for
2.0 (50% w/w) at different temperatures (25, 40, 55, 70 and 85 °C). each assay. The regression equation and the determination coefficient
ORAC FRAP (R2 = 0.994) were calculated using Microsoft Office Excel 2010. The
linear regression has been performed with units of concentration in
MAC r −0.480 −0.437
μmol TE/L as dependent variable “y” and absorbance units (nm) as
p 0.413 0.461
TPC r 0.987 0.991
independent variable “x”. Therefore, the slope (β = 0.317) has units of
p 0.002 0.001 μmol TE/L.nm, and the intercept (α = 8.27) has units of μmol TE/L.
The linearity between the net area under the curve and the con-
centration was checked for the samples, and the fluorescences were
some modifications (Singleton et al., 1999). Gallic acid (GAE) was used used for the appropriate calculations. All measurements were per-
to plot the (0.005–0.07 mg GAE/mL) standard curve formed in triplicates.
(y = 0.0714x − 0.0023) and the regression equation and the determi- The antioxidant capacity FRAP was evaluated by the method de-
nation coefficient (R2 = 0.9902) were calculated using Microsoft Office scribed by Benzie and Strain (1996), performed with some modifica-
Excel 2010. The linear regression has been performed with concentra- tions. AC was expressed as Trolox equivalent units (mg TE/g dr) and
tion units of mg GAE/mL as dependent variable “y”, and absorbance was used to plot (0.0125–0.3 mg/mL) the standard curve
units (nm) as independent variable “x”. Therefore, the slope (y = 3.0736x + 0.0112). The regression equation and the determina-
(β = 0.0714) has units of mg GAE/mL.nm and the intercept tion coefficient (R2 = 0.9927) were calculated using Microsoft Office
(α = 0.0023) has units of mg GAE/mL. Results were expressed as mg Excel 2010. The linear regression has been performed with concentra-
gallic acid equivalent (GAE)/g dr. All measurements were performed in tion units of mg TE/mL as dependent variable “y” and absorbance units
triplicates. (nm) as independent variable “x”. Therefore, the slope (β = 3.0736)
has units of mg TE/mL.nm and the intercept (α = 0.0112) has units of
mg TE/mL. All measurements were performed in triplicates.
2.4.5. Antioxidant capacity
The antioxidant capacity (AC) of the extracts was determined
through the ferric reducing antioxidant power (FRAP) and the oxygen 2.5. PLE kinetic and modeling
radical absorbance capacity (ORAC) methods.
The ORAC method measures the absorption capacity of the oxygen The PLE kinetic behavior was investigated in terms of monomeric

Table 6
Parameters adjusted by the three-line Spline and two-site desorption kinetics models applied to PLE curves from grape marc in 4 h of extraction.
Solvent MAC (mg Mv-3-glc/g dr) TPC (mg GAE/g dr)

Ethanol-Water pH 2.0 (50% Water pH 2.0 Ethanol-Water (50% Ethanol-Water (50% Ethanol-Water (50% Ethanol-Water pH 2.0 (50% w/
w/w) w/w) w/w) w/w) w)

Temperature (°C) 40 40 60 100 60 40

Spline Model

tCER (min) 15.52 46.70 20.21 10.25 11.05 12.28

YCER 7.67 7.42 8.58 54.63 26.22 23.44
RecoveryCER (%) 75.12 100 100 62.89 52.92 59.35
tFER (min) 37.07 160.00 161.20 84.60 101.70 86.00
YFER 10.21 7.42 8.58 78.57 42.61 35.30
RecoveryFER (%) 100 0 0 90.44 86.00 89.38
R2 0.99 0.99 0.92 0.97 0.99 0.99

Two-site Model

y∞ 10.23 7.52 8.58 85.63 49.75 39.34

F 0.56 0.17 0.44 0.50 0.39 0.52
k1 (min−1) 0.10 0.24 0.56 0.41 0.36 0.02
k2 (min−1) 0.07 0.04 0.08 0.02 0.01 0.19
MRPD (%) 2.11 2.61 0.74 0.61 0.99 0.67
R2 0.99 0.99 0.99 0.99 0.99 0.99

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D.T.V. Pereira et al.
Fig. 1. Chromatogram of the anthocyanins analyzed by ultra high-performance liquid chromatography-UV-Vis (520 nm) in the PLE extracts from grape marc.
anthocyanins and total phenolic anthocyanins, as described in Section
2.3, collecting extract fractions at predetermined times of 5, 10, 20, 30,
40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 min. Each col-
lected volume was measured and their MAC and TPC were determined.
Experimental curves of MAC and TPC against time were plotted. PLE
kinetics was performed in duplicate and the extracts were analyzed in
triplicate. Two mathematical models, Spline Model and Two-site kinetic
desorption model, described in Sections 2.5.1 and 2.5.2, were adjusted
to the experimental PLE curves obtained at the extraction conditions
that provided the highest MAC and TPC.
2.5.1. Spline model
The three-line Spline model, presented in Equations (2)–(4), was
fitted to the experimental data using the PROC NLIN procedure of the
SAS University Edition software, as described by Rodrigues et al.
(2003). Each fitted line represents an extraction stage related to a mass
transfer mechanism: constant extraction rate (CER), falling extraction
rate (FER) and diffusion-controlled periods (DC), as described by
Meireles (2008).
y = b0 + b1 t for t≤ t CER
y = b0 − tCER b2 + (b1 + b2) t for t CER < t≤ tFER
y = b0 − tCER b2 − tFER b3 + (b1 + b2 + b3) t for tFER < t
Where: y = response variable (MAC = mg malvidin-3-O-glucoside (Mv-
3-glc)/g dried grape marc (dr) or TPC = mg gallic acid equivalent
(GAE)/g dr); bi (i = 0, 1, 2, 3; MAC = mg Mv-3-glc/g dr.min or
TPC = mg GAE)/g dr.min) linear coefficients of lines; t = time (min);
tCER = CER time (min); tFER = FER time (min).
Using the adjusted parameters, the MAC and TPC achieved in tCER
and tFER were calculated using Equation (4) (yCER and yFER). Then,
using Equation (5), MAC and TPC recoveries were calculated at each
time.
yt ∗100
Recovery (%) =
y240
Journal of Food Engineering 240 (2019) 105–113
Where: yt = response (MAC = mg Mv-3-glc/g dr or TPC = mg GAE)/g
dr) at 240 min, tCER (min) or tFER (min), as appropriate.
2.5.2. Two-site kinetic desorption model
The two site desorption model (Kubátová et al., 2002) was adjusted
to the MAC and TPC curves. This model considers that a fraction of the
extractable compounds is easily accessible to the solvent, being located
near the particle surface and being desorbed at a fast rate (washing
process). The remaining fraction is located within the substrate parti-
cles; therefore it is not easily accessed by the solvent, being, thus,
desorbed at a slower rate (slow diffusion). Equation (6) describes the
model:
yt = y∞ ∗ [1 − f ∗e−k1 t − (1 − f ) ∗e−k2 t ] (6)
Where yt = response variable (MAC = mg malvidin-3-O-glucoside [Mv-
3-glc]/g dried grape marc [dr] or TPC = mg gallic acid equivalent
[GAE]/g dr) at the time t (min); y∞ = response variable at saturation
(MAC = mg Mv-3-glc/g dr or TPC = mg GAE)/g dr); f = fraction of
(2) easily accessible extract; t = extraction time (min); k1 = first-order rate
constant describing easily accessible material desorption (min−1);
(3)
k2 = first-order rate constant describing the slow diffusion (min−1).
(4) This model was fitted to the experimental data using the Microsoft
EXCEL Solver, minimizing the mean relative percent deviation.
2.6. Statistical analyses
The results were statistically evaluated by the One-way analysis of
variance (ANOVA), and significant differences at the 5% level were
analyzed by the Tukey's test. The Pearson test was performed to obtain
correlation (r) values between MAC, TPC and AC (ORAC and FRAP).
The tests were performed using MINITAB® software (Release 16.1.0,
Minitab Inc., State College, PA, USA). MAC, TPC, FRAP and ORAC were
expressed as mean ± standard deviation.
(5)
109
D.T.V. Pereira et al.
3. Results and discussion
3.1. Physical and chemical characteristics of grape marc
The ground grape marc presented the following composition:
moisture: 81.8 ± 0.5 g/100 g wet basis (w.b.); lipids: 3.55 ± 0.07 g/
100 g dry basis (d.b.); proteins: 12 ± 1 g/100 g (d.b.); ash:
0.99 ± 0.07 g/100 g (d.b.); carbohydrates: 83.2 ± 0.4 g/100 g (d.b.);
pH: 3.6 ± 0.4.
3.2. Pressurized liquid extraction
Table 1 presents the monomeric anthocyanins content, total phe-
nolic content and antioxidant capacities evaluated by FRAP and ORAC
in the PLE extracts obtained from grape marc in 40 min. The antho-
cyanin quantification by UHPLC is reported in Table 2.
3.2.1. Monomeric anthocyanin content
As shown in Table 1, the highest MAC was achieved in the extrac-
tion with ethanol-water (50% w/w) with pH 2.0 at 40 °C. The lowest
MAC was obtained with ethanol-water (50% w/w) at 100 °C. Similar
results were found in PLE from juçara (Euterpe edulis Mart.) residues for
water-ethanol pH 2.0 (50% w/w) at 40 °C, with 7.7 mg cyanidin-3-ru-
tinoside (Cy-3-rut)/g dr and using water-ethanol (50% w/w) at 80 °C,
reaching 1.80 (Cy-3-rut)/g dr (Garcia-Mendoza et al., 2017). High
temperatures may reduce the anthocyanins recovery, since these com-
pounds are thermally sensible and thus can be easily degraded. The
anthocyanins degradation can be caused primarily by oxidation, clea-
vage of covalent bonds or increased oxidation reactions due to thermal
processing. However, the anthocyanins recovery in acid medium is
increased due to their stability in pH from 1.0 to 3.0 and also to the cell
wall disruption by acid hydrolysis, releasing the phenolics bound in the
cellular matrix, thus enhancing mass transfer and increasing the ex-
traction yield of these compounds (Khoo et al., 2017; Peron et al., 2017;
Santos et al., 2012; Türker and Erdogˇdu, 2006). Several studies also
extracted anthocyanins from grape marc using different techniques.
Melo et al. (2015) obtained 1.41 and 0.99 mg of Mv-3-glc/g lyophilized
marc from Syrah and Petit Verdot grapes, respectively, by ultrasonic
assisted extraction. Rockenbach et al. (2011) reported different mono-
meric anthocyanins concentrations (1.84, 3.94, 7.02, 11.22 mg Mv-3-
glc/g dried grape marc) in four different grape varieties, respectively
(Isabel, Merlot, Cabernet Sauvignon and Bordeaux), obtained by ex-
traction with mechanical agitation. In addition to the extraction
method, the processing technology, cultivar, genetic and climate have
great influence on the recovery of compounds from vegetables (Doshi
et al., 2006; Patras et al., 2010; Tseng and Zhao, 2013).
Adams (1973) and Markaris et al. (1957) reported two mechanisms
for the anthocyanins degradation: 1) Hydrolysis followed by the pyr-
ilium ring opening in the 1–2 position, forming a chalcone, and further
degradation that leads to a brown precipitate, the final product of
pigment degradation; 2) Cleavage of the sugar fraction and formation of
aglycone, a highly labile and unstable compound. These mechanisms
may explain the brownish extracts obtained at 80 and 100 °C.
3.2.2. Identification and quantification of anthocyanins by UHPLC
Fig. 1 shows the chromatogram of the anthocyanins separated from
the extracts from grape marc by UHPLC–UV–Vis and identified by
UHPLC-QToF-MS. The detection and complete separation of the fifteen
identified anthocyanins were obtained in an 8-min run time. Table 2
shows the concentration (mg/g dried grape marc) of the fifteen an-
thocyanins identified and quantified in the extract. Among these an-
thocyanins, petunidin-3-O-glucoside, peonidin-3-O-glucoside, mal-
vidin-3-O-glucoside, malvidin-3-O-(6″-acetyl) glucoside and malvidin-
3-O-(6″-trans-p-coumaroyl) glucoside, highlighted with shadings in
Table 2, corresponded to over 78% of the identified pigments. The
concentrations obtained by UHPLC are very similar to those found by
110
Journal of Food Engineering 240 (2019) 105–113
the differential pH method and followed the same trend of MAC, ac-
cording to the extraction condition, as discussed in Section 3.2.1.
Temperature, binary solvents and acidified medium were decisive to
intensify anthocyanins recovery. Extractions at mild temperatures
minimize their thermal degradation. The use of binary mixtures may
have a synergistic effect: whereas a solvent increases the solubility of
target compounds, the other may favor their desorption, increasing the
extraction yields. Finally, acid media can improve the anthocyanins
extraction by releasing and solubilizing these compounds from plant
matrices due to the denaturation of cell membranes and by stabilizing
the anthocyanins through flavilium cation formation (Jackman et al.,
1987; Ju and Howard, 2003; Mustafa and Turner, 2011). Fontana et al.
(2013) extracted 1.356 mg total anthocyanins/g dr from White Zin-
fandel grape marc by maceration with methanol/formic acid/water
(60:37:3, v/v), and 1.195 mg/g dr using subcritical water at 140 °C.
Monrad et al. (2010), using a subcritical solvent composed of 50%
ethanol in water at 100 °C, achieved 4.974 mg total anthocyanins/g dr
from Sunbelt grape marc (V. labrusca L.). Melo et al. (2015) applied
ultrasonic assisted extraction to Syrah grape marc, the same variety
used in this work, and identified and quantified the anthocyanins del-
phinidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-O-glu-
coside by HPLC, obtaining, respectively, 0.05, 0.44 and 1.71 mg/g dr.
The highest anthocyanin yield was achieved using water-ethanol at pH
2.0 and 40 °C. Therefore, it can be stated that PLE at mild temperatures
with acidified binary solvents improves the solvent transport into the
matrix and promotes greater anthocyanins dissolution, thus increasing
the extraction yield of these compounds. From the presented results,
PLE can be considered an efficient technique to extract bioactive
compounds from grape marc, achieving better results than conventional
and other emerging methods.
3.2.3. Total phenolic content
Table 1 shows that PLE at 100 °C favored the extraction of phenolic
compounds with ethanol-water (50% w/w). This is mainly due to the
increased solubility and diffusivity of phenolics in this solvent at high
temperatures, besides the decrease in viscosity, surface tension and
breakdown of Van der Waals, hydrogen and dipole-dipole bonds, re-
sulting in higher extraction yield (Kim et al., 2009; Mustafa and Turner,
2011).
3.2.4. Antioxidant capacity
The antioxidant capacities (AC) of the extracts obtained from grape
marc, evaluated by FRAP and ORAC, are presented in Table 1. High PLE
temperatures affected both antioxidant capacities. The highest ORAC
and FRAP results were found in the extract obtained at 100 °C with
ethanol-water (50% w/w), coinciding with the best condition for TPC,
ethanol-water (50% w/w) at 100 °C. This indicates that the antioxidant
capacity of the extracts is mainly due to their phenolic compounds. The
Pearson correlations between TPC and AC (ORAC and FRAP) are shown
in Table 3. The correlation coefficients (r) are high and positive for
ORAC and FRAP, indicating that both antioxidant capacities are
strongly related to TPC. However, correlations between MAC vs FRAP
and ORAC showed p-values above 0.05, and r far from −1 and 1, which
means that MAC and AC are not correlated. Similar behaviors for TPC
and AC were also reported by Viganó et al. (2016a, 2016b), Machado
et al. (2017, 2015), Monrad et al. (2010), Martins et al. (2016), Dai
et al. (2009) and Ju and Howard (2003). Comparing the methods for
determining AC, the results obtained by FRAP are higher than those of
ORAC. In the FRAP method, the antioxidant capacity is measured by
the reduction of Fe3+ to Fe2+, due to the electrons transfer, whereas in
ORAC the determination is based on the absorption capacity of oxygen
radicals.
3.3. Temperature effect on the PLE of bioactive compounds
Table 4 shows MAC, TPC and AC (FRAP and ORAC) of the PLE
D.T.V. Pereira et al.
Fig. 2. PLE curves of monomeric anthocyanin content (MAC) of grape marc,
experimental and adjusted by Spline and two-site kinetic desorption models.
extracts obtained with ethanol-water (50% w/w, pH 2.0) at different
temperatures. The highest MAC was again achieved at 40 °C, differing
statistically from the other temperatures. At 70 and 85 °C, lower
monomeric anthocyanin contents were obtained. Therefore, the nega-
tive effect of high temperatures on the monomeric anthocyanins ex-
traction was confirmed. Temperature of 40 °C combined with the binary
mixture of ethanol-water pH 2.0 (50% w/w) was decisive to achieve
high monomeric anthocyanin concentrations in PLE, as discussed in
Sections 3.2.1 and 3.2.2. Higher phenolic content and antioxidant ca-
pacity were obtained as the extraction temperature increased, resulting
in positive Pearson correlations for TPC vs ORAC and FRAP, and ne-
gative for MAC vs FRAP and ORAC, as presented in Table 5. Some
authors have reported that extraction at high temperatures may favor
the increase of the antioxidant capacity due to the formation of anti-
oxidant compounds derived from the Maillard reaction (García-Marino
et al., 2006; Plaza et al., 2010; Vergara-Salinas et al., 2013).
3.4. PLE kinetics
PLE kinetics was performed in the three conditions that obtained the
highest MAC and in TPC contents. Fig. 2 and Fig. 3 show the PLE ki-
netics for MAC and TPC, respectively, and Table 6 shows the para-
meters of the PLE curves adjusted by the three-line Spline and two-site
kinetic desorption models.
Fig. 3. PLE curves of total phenolics content (TPC) from grape marc, experi-
mental and adjusted by the Spline and two-site kinetic desorption models.
111
Journal of Food Engineering 240 (2019) 105–113
3.4.1. Spline model
Based on the determination coefficients (R2), all curves were better
fitted with the three-line Spline model. Table 6 shows that most
monomeric anthocyanins, from 75 to 100%, were extracted during the
CER period, from 15 to 47 min, at all conditions. This result reinforces
Meireles (2008), who states that about 70–90% of the soluble material
can be extracted during the CER period. This stage has an important
contribution to the plant compounds extraction, since it is characterized
by the convective extraction of the solute present on the matrix surface,
which is easily accessible. For TPC, the recovery in the CER period
ranged from 53% to 63%, being more significant at 100 °C with
ethanol-water (50% w/w) as solvent. The FER period allowed in-
creasing the phenolic recovery from 27 to 33%, depending on the
condition. For MAC, the FER period had an impact of 24.88% at 40 °C
with ethanol-water pH 2.0 (50% w/w). At the other conditions, the
anthocyanins were completely extracted in the CER period. The results
presented in Table 6 show that the best PLE condition to obtain MAC
was 40 °C with ethanol-water pH 2.0 (50% w/w) in 37 min. For TPC,
the best condition was 100 °C with ethanol-water (50% w/w) in ap-
proximately 85 min. These extraction conditions were efficient for the
convective mass transfer in the CER period and convection-diffusion in
the FER stage, as observed in Figs. 2 and 3. These results confirm that
PLE promotes high extraction rate of the target compounds in short
time. To improve PLE yield, it would be interesting to investigate the
influence of ethanol concentration on both extractions, since solvent
mixtures may improve solubility and increase the interaction between
the target compounds and the solvent (Mustafa and Turner, 2011).
3.4.2. Two-site kinetic desorption model
The curves adjusted by the two-site desorption model fitted well to
the experimental data, as shown in Figs. 2 and 3, proving the accuracy
of this model to describe PLE of anthocyanins and phenolic compounds.
Solvents and temperatures affected the solubility of anthocyanins and
phenolics, influencing the values of y∞, f and the external (k1) and
internal (k2) mass transfer coefficients, as shown in Table 6. For MAC,
the acidified hydroethanolic mixture achieved greater fraction (f) of
easily accessible anthocyanins, resulting in greater y∞. At the same
extraction condition, both mass transfer coefficients contributed to the
extraction of anthocyanins. Therefore, it can be stated that the solvent
mixture increases the solvation power, and acid media contribute to
break cell matrix bonds and stabilize anthocyanins, promoting high
yields of these compounds. On the other hand, for TPC, ethanol-water
(50% w/w) at high temperatures (100 °C) resulted in high y∞, and
mass transfer was mainly convective, since k1 is approximately 22 times
greater than k2. The washing step in this case was almost instantaneous,
followed by a long diffusive period, as shown in Fig. 3.
3.4.3. Sequential PLE
In the PLE kinetics of MAC and TPC, the optimal conditions and
extraction times differ according to the compound of interest. This
observation is important if one intends to extract two or more com-
pounds from the same matrix. Based on the highest MAC and TPC, a
sequential PLE process was carried out to obtain two different extracts:
one rich in anthocyanins and the other in phenolic compounds. The
anthocyanin and phenolic yields as functions of time in the sequential
PLE process are shown in Fig. 4. The first PLE step achieved MAC of
9.96 mg Mv-3-glc/g dr, and TPC of 31.20 mg GAE/g dr. The mild
temperature of this step prevented the anthocyanins thermal degrada-
tion before the second step, and low pH helped increasing their ex-
traction yield. In the second step, an efficient extraction of phenolic
compounds, 86.00 mg GAE/g dr, was observed, since high temperatures
intensified the extraction of heat-resistant phenolics, as discussed in
Section 3.2.3. Therefore, the sequential PLE process allows recovering
anthocyanins and phenolic compounds in high yields using suitable
extraction conditions for each group of compounds.
Fruit by-products have a high concentration of nutraceutical
D.T.V. Pereira et al.
Fig. 4. Monomeric anthocyanin contents (MAC) and total phenolic contents
(TPC) obtained with time by sequential PLE extraction.
compounds, especially grape marc, and may also have antioxidant
properties. This makes the extraction of these compounds important
and attractive for application as food ingredients, pharmaceuticals and
cosmetic formulations.
4. Conclusions
PLE presented great potential for the recovery of bioactive com-
pounds from grape marc, evidenced by high yields of anthocyanins and
phenolic compounds. Solvent type and temperature had influence on
the recovery of phenolics and anthocyanins. For anthocyanins, the best
PLE condition was ethanol-water pH 2.0 (50% w/w) at 40 °C. Fifteen
anthocyanins were identified and quantified in the PLE extracts by
UHPLC, with five of them accounting for more than 78% of the iden-
tified pigments. For the recovery of phenolic compounds, the solvent
ethanol-water (50% w/w) at 100 °C was the most efficient and, in these
extracts, obtained by PLE, presented high antioxidant capacities. The
Spline model allowed identifying three PLE stages, and the two-site
kinetic desorption model provided an interpretation of the solutes mass
transfer in the external and internal parts of the matrix. The sequential
PLE process proved to be a viable strategy to obtain two extract frac-
tions through different solvents and temperatures combinations,
leading to extracts with specific compositions. Investigations must be
carried out to concentrate the target compounds of the extracts and
evaluate the economic viability of the process.
Acknowledgments
The authors wish to thank CAPES (AUXPE-PROEX 087/2016), CNPq
(13241/2016-2) for support in form of a scholarship and FAPESP
(2015/11932-7) for the financial support. The authors thank Espaço da
Escrita- Pró-Reitoria de Pesquisa – UNICAMP – for the language services
provided.
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