Enhancement of Cabernet Sauvignon must extraction combining
ultrasound, mechanical stirring and enzymatic treatment
Luíza Dalagnol¹, Lucas Dal Magro¹, Vitória Silveira¹, Eliseu Rodrigues², Vitor Manfroi³ and Rafael Rodrigues¹,*
1
Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of
Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil.
2
Laboratory of Natural Antioxidants, Institute of Food Science and Technology, Federal University of Rio Grande do
Sul.
³ Institute of Food Science and Technology, Federal University of Rio Grande do Sul.
* Corresponding author:
E-mail address: [email protected] (R. C. Rodrigues) - www.ufrgs.br/bbb
Abstract. In this work the effects of ultrasound (US), mechanical stirring (MS) and a commercial enzyme
preparations (EP) were investigated on Vitis vinifera Cabernet Sauvignon must extraction (GME). Initially, the
enzyme preparation was characterized regarding six enzymatic activities and evaluated its application on GME
using US or MS, varying the extraction temperature (40, 50 and 60 °C) and enzyme concentration (0.01 to
2.0 U.g-1). Zimopec PX5® at the concentration of 1.0 U.g-1 of pectinase and 50 °C were chosen as the best
extraction conditions. Thus, it was tested the use of US and MS, individually or in combination, measuring
yield and quality parameters. The phenolic and anthocyanins compounds were identified and measured via
HPLC-DAD. Extraction yield, antioxidant activity and color, increased 7.1 %, 30.2 % and 9.6 %, respectively,
over the control, for the treatment with enzyme and combination of US and MS. Moreover, 33 phenolic
compounds were identified, and the total anthocyanin content was 40 % higher using the optimized process
compared to traditional extraction.
1 Introduction
Grape and its derivatives, such as wine and juice, are
renowned by the high content of bioactive compounds,
presenting nutritional value and some positive effects on
human health, due to their antioxidant and antiinflammatory properties [1-4]. These phenolic
compounds also contribute to the wine quality
parameters, as color, astringency and ageing ability.
However, their presence on the final product depends of
grape variety, maturity stage and the winemaking
techniques [5].
The extraction is one of the most important stages of
winemaking and juice process, since it affect the product
quality as well as the industry incomes [6]. Enzymatic
treatment has been employed to accelerate and improve
the extraction process, through the application of
pectinases, cellulases and hemicellulases that have the
ability of catalyzing the hydrolysis of structural
polysaccharides from plant cell wall, improving the must
extractability, and facilitating the release of bioactive
compounds [5,7,8].
Ultrasound (US) is an emerging and promising
technology that has been widely used to improve the
efficiency of various chemical, physical and
biotechnological processes on food industry [9-12],
minimizing processing time, enhancing quality and
ensuring the safety of food products [13]. The main effect
of US on liquid medium is the physical phenomenon of
acoustic cavitation [14-16], which consists on the
formation, growth and implosion collapse of
microbubbles dissolved in the liquid [9]. Cavitation could
provide potential benefits to the extraction process, as
intensification of mass transfer, improvement of solvent
penetration into plant tissue, and increasing on substrate
availability [17-19].
Ultrasound has been applied in combination with
enzymatic treatment (ET) to reduce maceration time
while increases the extraction yield [20-22].
Thus, in this work, the effects of ultrasound combined
with enzymatic treatment were studied on Vitis vinifera
cv. Cabernet Sauvignon must extraction (GME),
comparing to traditional mechanical stirring (MS)
process. Initially, the enzyme preparation Zimopec PX5®
were characterized regarding its enzymatic activities, and
evaluated the enzyme concentration and temperature for
GME on US and MS. Additionally, quality parameters
(total soluble solids, pH, yield, reducing sugars, titratable
acidity, color parameters, anthocyanin concentration and
antioxidant activity) of GME extracted by US and MS,
individually or in combination were analyzed. Finally, the
phenolic compounds and anthocyanins extracted were
identified by high performance liquid chromatograph
with diode array detector and mass spectrometry (HPLCDAD-MS).
Brazil), and ultrasound extraction was performed using
an ultrasonic bath (Unique Inc., model USC 2880A, 40
kHz, 300 W, Brazil).
2 Material and Methods
Extraction temperature (40, 50 and 60 °C) for GME was
evaluated on MS and US, and the enzyme concentration
(0.01 to 2.0 U.g-1 of PE activity) was evaluated on MS.
2.4.1 Extraction parameters
2.1 Chemicals
Zimopec PX5® was provided from Vêneto Mercantil
(Brazil). Ultra-pure water was obtained by the Milli-Q
water purifier system from Millipore (Bedford, MA,
USA). Gallic acid, galacturonic acid, polygalacturonic
acid, pectin from apple, xylan from beechwood, cyanidin,
Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), and Folin-Ciocalteu reagent were from
Sigma Aldrich (St. Louis, MO).
HPLC solvents, formic acid, acetonitrile and
methanol (Panreac Quimica SL, Barcelona, Spain) were
filtered through Millipore membranes of 0.45 µm.
2.2 Sample
Grapes from Vitis vinifera Cabernet Sauvignon variety
were kindly donated by Vitivinicola Jolimont (Canela,
RS, Brazil). The bunches were sanitized and stored at
−18 °C until their use.
2.4.2 Extraction protocol
The grape must extraction was performed under MS, US
and combination of both using Zimopec PX5 (1 U.g-1), at
50 °C for 30 min. Eight different protocols were tested
and the conditions are presented in Table 1. After
extraction, quality parameter were analyzed for each
condition.
Table 1. Extraction protocols for grape must extraction.
Experiment
Extraction protocol
Code
30 min under MS
MS-0
30 min under MS + enzyme
MS-E
30 min under US
US-0
30 min under US + enzyme
US-E
15 min under MS + 15 min under US
MSUS-0
15 min under MS + 15 min under US + enzyme
MSUS-E
15 min under US + 15 min under MS
USMS-0
15 min under US + 15 min under MS + enzyme
USMS-E
2.3 Enzymatic activities
Pectinase (PE), polygalacturonase (PG), pectin lyase
(PL), pectin methyl esterase (PME) and cellulase (CE)
activities were determined as described by Dal Magro et
al. [23]. Xylanase (XLN) activity was determined
following the methods proposed by Bailey & Biely et al.
[24] using xylan (1%) prepared in sodium acetate buffer
(50 mM, pH 5) as substrate. The reaction was carried out
for 5 min at 50 °C, under agitation, and the amount of
reducing groups formed was estimated by the 3,5dinitrosalicyclic acid (DNS) method proposed by Miller
[25]. One unit of enzyme was defined as the amount of
enzyme required to generate 1 μmol of reducing groups,
expressed as xylose, per minute under standard assay
conditions.
2.4 Grape Must Extraction
Firstly, grape bunches were destemmed and slightly
mixed to obtain a homogeneous sample. Then, 50 g of
grape berries were gently crushed and used to grape must
extraction (GME). To realize the enzymatic treatment,
0.5 mL of enzyme solution was added to grape samples
and the extraction was carried out by two techniques,
ultrasound and mechanical stirring. Controls without
enzyme were performed for each condition. At the end,
the grape must was pressed, analyzed and stored under
refrigeration.
Mechanical stirring extraction was carried out on an
agitated water bath (MA-093, Marconi, Piracicaba,
2.5 Analytical methods
2.5.1 Extraction yield
The extraction yield of each treatment was expressed as
percentage of mass of must per initial mass of grape as
described in the Eq. 1:
𝑌𝑖𝑒𝑙𝑑 % =
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑚𝑢𝑠𝑡
𝑥 100
𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑔𝑟𝑎𝑝𝑒𝑠
2.5.2 Reducing sugars, total soluble solids, acidity
and pH
Reducing sugars were determined by the DNS method as
proposed by Miller [25]. Titratable acidity was carried
out by titration with 0.1 N NaOH with phenolphthalein as
indicator and expressed in g.L-1 of tartaric acid. Total
soluble solids (°Brix) were measured with a refractometer
at 20.0±0.5 °C, and pH was measured by a digital pH
meter.
2.5.3 Antioxidant Capacity
Antioxidant capacity was measured by two methods:
Reducing capacity (RC), determined by Folin–Ciocalteu
method [26] and antioxidant activity (AA) by ABTS
method [27].
Color properties were determined using a Konica Minolta
Colorimeter (CR-400, Osaka Japan), and the obtained
data were express as L* (lightness/brightness), a*
(redness/greenness) and b* (yellowness/blueness). These
values were used to calculate; Chroma (C*), hue angle
(h*) and color difference (ΔE*ab).
2.5.5 Determination of phenolic compounds
The identification of phenolic compounds was carried out
in a high performance liquid chromatograph (HPLC)
(Shimadzu HPLC, Kyoto, Japan) equipped with two
pumps (model LC-20AD), on-line degasser (DGU-20A),
column oven (CTO-20A), automatic injection system
(SIL-20AHT), diode array detector (SPD-M20A) and,
connected in series to a mass spectrometer with a q-TOF
analyzer and electrospray ionization (ESI) source (Bruker
Daltonics, model micrOTOF-QIII, Bremen, Germany).
Before analysis, the grape must samples was centrifuged
at 10000 g for 5 min. The supernatant was filtrated in
0.22 μm cellulose acetate membrane filter (Millipore,
Massachusetts, USA), and injected (20 µL) into the
HPLC-DAD-MS or HPLC-DAD.
Phenolic compounds separation was performed
according to a method described by Rodrigues et al. [28]
in a C18 Phenomenex column (4 µm, 250 × 4.6 mm i.d.)
connected to a guard column (4 µm, 4 × 3 mm i.d.) using
a mixture composed of water:formic acid (99.5:0.5, v/v)
(A) and acetonitrile:formic acid (99.5:0.5, v/v) (B) as
mobile phase. The flow rate was 0.7 mL.min-1 and the
oven temperature was set at 40 °C. The UV–vis spectra
were obtained between 200 and 800 nm and the
chromatograms were processed at 280 nm (flavan-3-ols),
320 nm (hydroxycinnamic acids), 360 nm (flavonols),
and 520 nm (anthocyanins).
Phenolic compounds were tentatively identified
according to the elution order and retention time in the
reversed phase column, UV–vis, MS spectra features and
data available in the literature [29-34].
agitation methods, ultrasound and mechanical stirring,
measuring the extraction yield, °Brix and reducing
capacity. The results are showed in Table 2.
Table 2. Yield, total soluble solids (°Brix) and reducing
capacity (mg.L-1)of grape must treated with enzyme at
mechanical stirring (MS) and ultrasound extraction (US).
Temperature Method
US
40 °C
MS
US
50 °C
MS
US
60 °C
MS
3.2 Effects of temperature and enzyme
concentration on grape must extraction
Initially, the extraction temperature (40, 50 and 60 °C),
was tested on Cabernet Sauvignon must extraction at both
RC (mg.L-1)
8.81B
7.22b
15.4A
13.7a
14.2A
14.3a
a)
81
78
75
72
69
0.0
b)
0.5
1.0
1.5
2.0
-1
Enzyme concentration (U.g )
20.5
20.0
° Brix
Zimopec PX5 was characterized regarding its activities,
in U.mL-1: total pectinase, 9393.19; polygalacturonase,
1482.52; pectin lyase, 1482.52; cellulase, 1311.78; and
xylanase, 10831.14. The preparation did not present
pectin metyl esterase activity.
The knowledge of its exacts activities as well as the
amount of enzyme employed is essential to a correct
application. Therefore, GME was evaluated using
0.5 U.g−1 of total pectinase activity per gram of grape.
°Brix
19.1B
18.1c
19.8B
19.8b
20.0A
21.0a
Significant differences on physicochemical properties
were observed with temperature variation. °Brix and
reducing capacity increased at higher temperatures for
both agitation methods, and were statistically equal at
50 °C and 60 °C. The increase of the antioxidant capacity
at higher temperatures agrees with previous studies
[35,36], where it was observed an enhancement on
antioxidant capacity, possibly due to the higher extraction
of phenolic compounds by thermal treatments. On the
other hand, higher extraction yields were achieved at the
lowest temperature (40 °C), however, no statistical
differences were found to yield of extraction under US
between 40 and 50 °C.
3 Results and discussion
3.1 Enzymatic activity
Yield (%)
72.7A
73.1a
72.6A
68.5b
66.0B
65.3b
* Same lowercase letters indicate that response are equal for each
evaluated parameters on MS. Same uppercase letters indicate that
response are equal for each evaluated parameters on US.
Yield (%)
2.5.4 Determination of color properties
19.5
19.0
0.0
0.5
1.0
1.5
2.0
-1
Enzyme concentration (U.g )
Figure 1. Extraction yield (a) and °Brix (b) of grape must
extracted on MS with different enzyme concentrations.
The extractions performed using ultrasound obtained
higher values for yield, RC and ° Brix when compared to
MS. Ultrasound and enzyme treatment provided an
enhancement on extraction yield and grape must quality,
also allowing the use of milder processing conditions as
lower extraction temperatures. Therefore, for a
compromise between yield and quality, we selected 50 °C
for the subsequent analysis.
Subsequently, the enzyme concentration was
evaluated. The extraction yield (Figure 1.a) increased
with enzyme concentration reaching to the highest value
(79.3 %) at 1.0 U.g−1, remaining constant at higher
concentrations. However, the total soluble solids (Figure
1.b) were not affected by enzyme concentration.
3.3 Extraction protocols
Different treatments were carried out in order to verify
the effects of the agitation methods and the enzyme
activity on Cabernet Sauvignon must extraction. The
experiments were performed comparing both agitation
methods, with or without enzyme, as well as the
combination of both methods. The physicochemical
analysis and antioxidant capacity of the extracts obtained
are showed in Table 3 and Table 4, respectively.
Ultrasound improved the grape must extraction when
compared to MS, providing higher values for most of the
evaluated parameters, highlighting °Brix, reducing sugars
and reducing capacity, which increased 3.8 %, 11.7 %
and 19.2 %, respectively.
Table 3. Physicochemical analysis of GME with mechanical
stirring (MS) and ultrasound (US) via different extraction
methods.
MS-0
MS-E
US-0
US-E
USMS-0
USMS-E
MSUS-0
MSUS-E
Table 4. Analysis of antioxidant capacity for grape must
extracted with mechanical stirring (MS) and ultrasound (US)
techniques.
Extraction
method
MS-0
MS-E
US-0
US-E
USMS-0
USMS-E
MSUS-0
MSUS-E
Reducing capacity (mg.L-1)
ABTS (mM)
9843c
544.4c
609.1ab
567.9c
621.8a
586.2b
617.9ab
515.0d
616.3ab
10582bc
11001b
11933ab
10532bc
12792a
9511c
12042ab
*Same lowercase letters indicate that the extraction methods are equal
for each response. (p < 0.5). Standard deviations were lower than 5 %.
3.3.1 Physicochemical analysis and color attributes
Extraction
method
and obtained higher extraction of phenolic compounds,
which improved the antioxidant capacity.
pH
°Brix
Yield
(%)
3.66a
3.59bc
3.62ab
3.58bc
3.61b
3.54c
3.57bc
3.55c
18.0c
18.7a
18.8a
18.8a
17.8d
18.1c
18.2c
18.4b
71.7d
75.3b
72.3cd
74.4bc
74.2bc
75.8ab
73.6c
76.8a
Total
acidity
(g.L-1)
0.90b
0.97ab
0.94b
1.22a
0.88b
0.92b
0.96ab
1.13a
Reducing
sugars
(g.L-1)
219.2e
262.6ab
261.3b
262.8ab
220.3e
263.4a
257.6c
261.3b
*Same lowercase letters indicate that the extraction methods are equal
for each response. (p < 0.5). Standard deviations were lower than 5 %.
Ultrasound improved the grape must extraction when
compared to MS, providing higher values for most of the
evaluated parameters, highlighting °Brix, reducing sugars
and reducing capacity, which increased 3.8 %, 11.7 %
and 19.2 %, respectively. The use of US may ease the
extraction of phenolic compounds due to its ability to
degrade the plants cell wall through cavitation, enabling
the release of more bioactive compounds [19,22]. Abid et
al. [21] applied US treatment for apple juice extraction
Enzymatic treatment improved the physicochemical
parameters for both agitation methods, especially at MS,
where °Brix, yield, sugar content and antioxidant activity
increased 3.8 %, 5.0 %, 19.8 % and 11.9 %, respectively.
Under US treatment (US-E) only chroma and AA were
statistically different over the control (US-0), enhancing
6.7 % and 9.5 %, respectively.
Moreover, all studied parameters were improved by
US-E when compared to extraction at mechanical stirring
without enzyme (MS-0). The main differences between
those techniques were the enhancement of antioxidant
capacity (21.2 %) and reducing sugar (19.9 %) for grape
must treated using ultrasound and enzyme (US-E).
Furthermore, some authors observed an enhancement on
fruit extract composition by simultaneous treatment using
enzymes and ultrasound [22,37,38]. Tiwari et al. [39],
obtained a significant improvement of anthocyanin
content on grape juice sonicated, while, no differences
were found on pH, titratable acidity and °Brix.
As a final point, it was tested the combination of both
agitation methods. The experiments were carried out for
15 min in one method, and the remaining 15 min in the
other. Both combinations were analyzed: mechanical
stirring/ultrasound; ultrasound/mechanical stirring. When
MS was employed firstly (MSUS-0), °Brix and reducing
sugars were improved. On the other hand, when US was
used first (USMS-0), an enhancement of reducing
capacity (10.7 %) and antioxidant activity (19.6 %), was
achieved. The same behavior was observed when enzyme
was added.
The results of color parameters analysis from grape
must extraction are shown in Table 5. Brightness (L*)
significantly decreased with enzyme treatment for all the
agitation methods, showing an enhancement on color
attributes by the enzyme action. The parameters a*, b*,
C*, ΔE*ab and h* increased when US and enzyme
treatment were used, despite that, ultrasound was able to
enhance the color parameters even when applied without
enzyme. Regarding the color difference (ΔE*ab), the
extraction on MSUS-E presented the highest value (8.8)
comparing to the control (MS-0), indicating a great
improvement of color.
Table 5. Color parameters for grape must extracted with
mechanical stirring (MS) and ultrasound (US).
Extraction
method
MS-0
MS-E
US-0
US-E
USMS-0
USMS-E
MSUS-0
MSUS-E
L*
a*
b*
C*
∆E*ab
h*
49.7ab
46.1c
11.9b
47.6c
45.7bc
47.7b
12.3b
49.3bc
50.4a
44.3bc
47.7b
44.4bc
44.9bc
42.2c
47.5b
50.7ab
48.6b
50.5a
48.6b
50.2ab
12.2b
12.9b
12.8b
13.2ab
13.7ab
13.9a
49.0b
52.3a
50.39b
52.29a
50.5ab
52.18a
0.0
4.3d
1.5f
7.2b
3.3e
7.1b
5.7c
8.8a
0.25b
0.25b
0.25b
0.25b
0.26b
0.26b
0.27a
0.27a
*Same lowercase letters indicate that the extraction methods are equal
for each response. (p < 0.5) L* lightness, a* redness/greenness, b*
yellowness/blueness, C* chroma, h* hue angle, ∆E*ab color difference.
Standard deviations were lower than 5 %.
3.3.2 Phenolic compounds and anthocyanin amount
Polyphenols chemical structure enables them to act as
antioxidants, scavenging and neutralizing free radicals
and collaborating to wine color stabilization [2].
Nevertheless, the extraction of these components depends
on grape maturity stage and extraction procedures
[40,41]. Hence, the grape must extracted by different
methods was analyzed for phenolic compounds
identification via HPLC-DAD-MS and the anthocyanin
concentration by HPLC-DAD.
The chromatograms processed at 280, 320, 360 and
520 nm (Fig. 2) showed the separation of 38 phenolic
compounds of Cabernet Sauvignon grape must. The
compounds identified by HPLC-DAD-MS were
summarized in Tables 6 and 7. A similar profile of
phenolic compounds with different peak intensity was
observed to all treatments. The main phenolic compound
was identified as Quercetin-hexoside (peak 23), which
has been reported as the most abundant phenolic
compound in grapes [7,31,34,41].
Furthermore, at the chromatogram processed at
280 nm, it was observed a peak (*) from a non-phenolic
compound, presenting molecular ion of m/z 366,
identified as 3-indolyl-(2R)-O-β-D-glucosyl-lactic acid,
as describe by Fabre et al. [33]. This compound did not
present any effect on the flavor of red wines because of
its nonvolatile nature, however, it has been associated to
ensure the wine quality contributing to wine astringency
taste due to strong structural homology with other
compounds [42].
Anthocyanins are important compounds of grapes
berries, responsible for color and antioxidant power.
These compounds were separated and identified in the
chromatogram processed at 520 nm and are described in
Table 7. The highest peaks (15 and 22) were identified
and are related to a co-elution of different anthocyanins.
Peak 15 was a mixture of peonidin 3-O-hexoside,
malvidin 3-O-hexoside and vitisin A-delphinidin-3hexoside, based on the ion molecular [M]+ at 463, 493
and 533, and mass fragments of MS2 at m/z 301, 331 and
371, corresponding to the loss of one hexose,
respectively. Moreover, peak 15 showed MS spectrum
and MS2 fragmentation patterns similar to data previously
reported in the literature [30,43]. Peak 22 was identified
as a mixture of malvidin 3-O-6-O-acetyl-hexoside and
peonidin 3-O-6-O-acetyl-hexoside, based on the ion
molecular [M]+ at 535 and 505, and mass fragments in
MS2 at m/z 331 and 301, corresponding to the loss of one
acetyl–hexoside (204 Da), respectively.
Despite of the large amount of phenolic compounds
identified, their quantification was not possible due the
low area and peak intensity. Nevertheless, their presence
has an important role in the stabilization of the wine
color, since they participate at the pigmentation reactions
with anthocyanins [26,44]. Therefore, only anthocyanins
could be quantified by HPLC-DAD, and the anthocyanin
concentration for each treatment is shown in Table 8. All
extraction methods showed similar profiles of phenolic
compounds, whereas differences on anthocyanin
concentration.
Table 6. Chromatographic, UV–vis, and mass spectroscopy characteristics of phenolic compounds in grape must, obtained by HPLCDAD-MS.
Peaks
1
2
3
4
5
6
7
8
9
11
14
23
25
26
29
30
31
Compound
Galloylhexoside I
Gallic acid
Not identified 1
Caffeic acid derivative I
p-Coumaric acid derivative I
2-S-Glutathionyl caffeoyl tartaric acid
Feruloyl hexoside
Caffeoyl hexoside pentoside
p-coumaric acid derivative II
p-coumaric acid derivative III
Proanthocyanidin dimer
Quercetin hexoside I
Quercetin hexoside II
Kaempferol hexoside
Rhamnetin hexoside
Caffeic acid derivative II
Caffeic acid derivative III
Caffeic acid derivative IV
tR (min)a
11.5
12.4
12.8
13.6
15.4
16.5
17.5
17.9
18.1
20.6
22.6
29.3
31.7
31.9
33.7
34.1
34.9
λmax (nm)b
281
269
290
288, 320
288, 300
325
328
282, 320
284, 306
281, 306
279
353
353
353
349
279/310
278/ 310
279/ 311
[M-H]331.0089
168.8989
359.0107
635.1317
457.0640
616.1261
355.0156
473.0346
361.0192
361.0192
577.1187
463.0592
463.0592
447.0798
477.0964
630.1352
630.1350
630.1353
MS2 (-) (m/z)c
168.8997, 124.8720
124.8740
124.8756
341.0316, 178.9198
162.9152
148.8837, 272.0256
192.9043
341.0304, 178.9206
162.9167, 118.9001
162.9153, 118.8962
289.8295 (source)
300.9593, 178.8838
300.9593
283.9746
477.0903, 313.9948, 283.9718
178.9429, 132.8700
178.9413, 132.7256
178.9209, 132.8879
Table 7. Chromatographic, UV–vis, and mass spectroscopy characteristics of anthocyanins content in grape must, obtained by HPLCDAD-MS.
16
17
18
19
20
21
22
24
26
27
28
Detector response at 280 nm (mAU)
tR (min)a λmax (nm)b
19.5
280/529
21.2
278/520
21.7
279/529
23.5
277/527
23.5
277/527
23.5
277/527
24.8
25.0
26.5
275/515
26.6
26.7
26.9
28.7
277/528
28.7
277/528
31.6
268/533
31.9
278/529
31.9
268/533
32.9
282/528
32.9
282/528
33.4
278/529
33.4
278/529
Compound
Delphinidin 3-O-hexoside
Cyanidin 3-O-glucoside
Petunidin 3-O-glucoside
Peonidin 3-O-glucoside
Malvidin 3-O-glucoside
Vitisin A - delphinidin -3-glucoside
Delphinidin 3-O-6-O-acetyl-glucoside
Malvidin 3-glucoside-pyruvate
Myricetin 3-hexoside
Cyanidin 3-O-6-O-acetyl-glucoside
Malvidin 3-6-O-acetylglucoside-pyruvate
Petunidin 3-O-6- acetyl-hexoside
Malvidin 3-O-6-O-acetyl-glucoside
Peonidin 3-O-6-O-acetyl-glucoside
Cyanidin 3-O-glucoside
Trans- Malvidin 3-O-6-O-coumaryl-glucoside
Petunidin 3-O-hexoside
Peonidin -3-6-O-coumaryl-glucoside
Cis –Malvidin 3-O-6-O-coumaryl-glucoside
Peonidin 3-caffeoyl-glucoside
Malvidin 3-caffeoyl-glucoside
Detector response at 320 nm (mAU)
Peaks
10
12
13
15
200
*
150
100
14
3
50
1
2
0
0
10
20
30
40
[M]+
465.1018
449.1099
479.1158
463.1215
493.1351
533.1241
507.1089
561.1282
481.1006
491.1111
603.135
521.1253
535.1441
505.1313
449.1095
639.1743
479.1212
609.1566
639.6232
625.1546
655.1589
50
40
30
20
6
10
7
8
11
9
4 5
0
10
20
Detector response at 520 nm (mAU)
Detector response at 360 nm (mAU)
23
40
30
25
26
10
0
10
20
30
Time (min)
30
40
50
Time (min)
50
0
29
30 31
0
50
Time (min)
20
MS2 (+) (m/z)c
303.0492
287.0557
317.0653
301.0712
331.0834
515.1123 /371.0711 /184.0310
303.0477
399.0739
319.0444
331.0711 /287.0526
399.0737
317.0642
331.0826
301.0701
287.0552
331.0833
317.0680
301.0695
331.0803
301.0691
331.0680
40
50
15
80
60
22
40
17
20
10
12
16
13
18
27
24
26 28
0
0
10
20
30
40
50
Time (min)
Figure 2. HPLC chromatograms of phenolic compounds in Cabernet Sauvignon must processed at 280 nm; 320 nm; 360 nm and
520 nm. Each peak numbered was identified and described in Tables 6 and 7.
Table 8. Anthocyanin concentration for grape must extracted
with mechanical stirring (MS) and ultrasound (US).
Extraction
method
Anthocyanin concentration (mg.L-1)
Acknowledgments
This work was supported by grants from CNPq (process
403505/2013-5) and scholarships (LMG Dalagnol, L Dal
Magro) from CAPES. The authors wish to thank Mr.
Ramiro Martínez (Novozymes, Spain) and LNF
Latinoamericana for kindly supplying the enzymes used
in this research, as well as Vitivinicola Jolimont (Canela,
Brazil) for providing the grapes.
peak 15
peak 22
Total
MS-0
30.2b
13.2c
43.4c
MS-E
30.7b
14.4c
45.1bc
US-0
28.4b
13.9c
42.3c
US-E
33.2b
16.7b
49.9bc
USMS-0
30.3b
16.7b
47.0bc
References
USMS-E
39.6a
19.2a
58.8ab
1.
MSUS-0
31.2b
14.8c
46.0bc
MSUS-E
42.1a
19.3a
61.4a
*Same lowercase letters indicate that the extraction methods are equal
for each response. (p < 0.5) Peak 15: mixture of peonidin 3-O-hexoside
+ malvidin 3-O-hexoside + vitisin A-delphinidin-3-hexoside. Peak 22:
mixture of malvidin 3-O-6-O-acetyl-hexoside and peonidin 3-O-6-Oacetyl-hexoside. Standard deviations were lower than 5 %.
2.
3.
The total anthocyanin concentration varied from 42.3
mg.L−1 to 61.4 mg.L−1, and MSUS-E provided an increase
of 41.5 % on anthocyanins contents over the control (MS0), followed by USMS-E (35.5 %) and US-E treatments
(17.5 %). The results showed that the combination of MS,
US and ET was more effective than individual
application, possibly because the use of MS alone was
not enough to break the grape cell wall and release the
phenolic compounds, and the use of US for 30 min could
be excessive, promoting the degradation of anthocyanins
by the chemical effect. Thus, the combination of 15 min
of US plus 15 min of MS improved the results, being the
enzyme treatment essential to obtaining higher
anthocyanin values. Carrera et al. and González-Caetano
et al. [45,46] also found higher amounts of anthocyanin
for extraction combining US and enzyme application.
Data from the analyses of antioxidant capacity (Table
4) revealed a positive correlation between these methods
with the total anthocyanin contents and phenolic
compounds. This is in agreement with previously studies,
which indicates that treatments with richer content of
phenolic compounds also have higher antioxidant
activities [39,47].
4.
4 Conclusion
12.
Considering the evaluated parameters, temperature
promoted changes on quality components of Cabernet
Sauvignon must. High temperatures had a positive effect
on antioxidant compounds extraction. Ultrasound
required lower operating temperatures to obtain extracts
with similar yield and antioxidant characteristics than
those resulted from MS extraction. Our results showed a
synergistic effect between ultrasound, mechanical stirring
and enzyme treatment on Cabernet Sauvignon must
extraction. The simultaneous treatment of grape must by
those techniques enhanced the extraction yield and
improved the quality of grape must, comparing to
individual application.
13.
5.
6.
7.
8.
9.
10.
11.
14.
15.
16.
17.
18.
B. Lochab, S. Shukla and I. K. Varma, RSC
Advances, 4, 21712-21752 (2014)
M. De Nisco, M. Manfra, A. Bolognese, A. Sofo, A.
Scopa, G. C. Tenore, F. Pagano, C. Milite and M. T.
Russo, Food chemistry, 140, 623-629 (2013)
J. Lachman, M. Šulc, K. Faitová and V. Pivec,
International Journal of Wine Research, 1, 101-121
(2009)
I. M. Toaldo, F. A. Cruz, T. d. L. Alves, J. S. d.
Gois, D. L. G. Borges, H. P. Cunha, E. L. d. Silva
and M. T. Bordignon-Luiz, Food Chem., 173, 527535 (2015)
M. Ortega-Heras, S. Pérez-Magariño and M. L.
González-Sanjosé, LWT - Food Sci. Technol., 48,
1-8 (2012)
L. Wang and C. L. Weller, Trends in Food Science
& Technology, 17, 300-312 (2006)
S. R. Segade, C. Pace, F. Torchio, S. Giacosa, V.
Gerbi and L. Rolle, Food Research International, 71,
50-57 (2015)
A. B. Bautista-Ortín, A. Martínez-Cutillas, J. M.
Ros-García, J. M. López-Roca and E. Gómez-Plaza,
Int. J. Food Sci. Technol., 40, 867-878 (2005)
T. J. Mason, E. Riera, A. Vercet and P. LopezBuesa, Application of ultrasound, in: Emerging
technologies for food processing, Eds: D.-W. Sun,
p. 323-351 (Elsevier Academic Press, San Diego,
USA, 2005)
H. Feng, G. V. Barbosa-Cánovas and J. Weiss,
Ultrasound
Technologies
for
Food
and
Bioprocessing (Springer, New York, 2011)
Y. Liu, H. Zhang and S. Wei, RSC Advances, 5,
46598-46607 (2015)
A. Singh, S. Ahmad and A. Ahmad, RSC Advances,
5, 62358-62393 (2015)
D. Knorr, A. Froehling, H. Jaeger, K. Reineke, O.
Schlueter and K. Schoessler, Annual review of food
science and technology, 2, 203-235 (2011)
T. J. Mason, L. Paniwnyk and J. P. Lorimer,
Ultrasonics sonochemistry, 3, S253-S260 (1996)
A. C. Soria and M. Villamiel, Trends in Food
Science & Technology, 21, 323-331 (2010)
N. Abdullah and N. L. Chin, Agriculture and
Agricultural Science Procedia, 2, 320-327 (2014)
T. J. Mason, A. J. Cobley, J. E. Graves and D.
Morgan, Ultrasonics Sonochemistry, 18, 226-230
(2011)
D. Pingret, A.-S. Fabiano-Tixier and F. Chemat,
Food Control, 31, 593-606 (2013)
19. F. Chemat, Zill-e-Huma and M. K. Khan, Ultrason.
Sonochem., 18, 813-835 (2011)
20. L. N. Lieu and V. V. M. Le, Ultrason. Sonochem.,
17, 273-279 (2010)
21. M. Abid, S. Jabbar, T. Wu, M. M. Hashim, B. Hu,
S. Lei, X. Zhang and X. Zeng, Ultrasonics
Sonochemistry, 20, 1182-1187 (2013)
22. W. Tchabo, Y. Ma, F. N. Engmann and H. Zhang,
Industrial Crops and Products, 63, 214-225 (2015)
23. L. Dal Magro, D. Goetze, C. T. Ribeiro, N. Paludo,
E. Rodrigues, P. F. Hertz, M. P. Klein and R. C.
Rodrigues, Food and Bioprocess Technology, 9,
365-377 (2016)
24. M. J. Bailey, P. Biely and K. Poutanen, Journal of
biotechnology, 23, 257-270 (1992)
25. G. L. Miller, Anal. Chem., 31, 426-428 (1959)
26. V. L. Singleton and P. Esau, Adv. Food Res. Suppl.,
1, 1-261 (1969)
27. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M.
Yang and C. Rice-Evans, Free radical biology and
medicine, 26, 1231-1237 (1999)
28. E. Rodrigues, L. R. B. Mariutti and A. Z.
Mercadante, J. Agric. Food Chem., 61, 3022-3029
(2013)
29. B. Bai, F. He, L. Yang, F. Chen, M. J. Reeves and J.
Li, Food chemistry, 141, 3984-3992 (2013)
30. L. Dal Magro, L. M. G. Dalagnol, V. Manfroi, P. F.
Hertz, M. P. Klein and R. C. Rodrigues, LWT Food Sci. Technol., (2016)
31. R. Flamini and P. Traldi, Mass spectrometry in
grape and wine chemistry (John Wiley & Sons,
Hoboken, USA, 2009)
32. E. Puértolas, G. Saldaña, S. Condón, I. Álvarez and
J. Raso, Food chemistry, 119, 1063-1070 (2010)
33. S. Fabre, C. Absalon, N. Pinaud, C. Venencie, P.-L.
Teissedre, E. Fouquet and I. Pianet, Analytical and
bioanalytical chemistry, 406, 1201-1208 (2014)
34. A. Vallverdú-Queralt, N. Boix, E. Piqué, J. GómezCatalan, A. Medina-Remon, G. Sasot, M. MercaderMartí, J. M. Llobet and R. M. Lamuela-Raventos,
Food chemistry, 181, 146-151 (2015)
35. S. G. Cabrera, J. H. Jang, S. T. Kim, Y. R. Lee, H. J.
Lee, H. S. Chung and K. D. Moon, J. Food Process.
Preserv., 33, 347-360 (2009)
36. M. d. S. Lima, I. d. S. V. Silani, I. M. Toaldo, L. C.
Corrêa, A. C. T. Biasoto, G. E. Pereira, M. T.
Bordignon-Luiz and J. L. Ninow, Food Chem., 161,
94-103 (2014)
37. B. Dang, T. Huynh and V. Le, International Food
Research Journal, 19, 509-513 (2012)
38. V. Nguyen, T. T. Le and V. V. M. Le, Int. Food.
Res. J., 20, 377-381 (2012)
39. B. K. Tiwari, A. Patras, N. Brunton, P. J. Cullen and
C. P. O’Donnell, Ultrason. Sonochem., 17, 598-604
(2010)
40. D. Villano, M. S. Fernández-Pachón, A. M.
Troncoso and M. C. García-Parrilla, Food
chemistry, 95, 394-404 (2006)
41. E. S. Vázquez, S. R. Segade and I. O. Fernández,
European Food Research and Technology, 231, 789802 (2010)
42. O. Cala, E. J. Dufourc, E. Fouquet, C. Manigand,
M. Laguerre and I. Pianet, Langmuir, 28, 1741017418 (2012)
43. M. Stefova and V. Ivanova, Analytical methodology
for characterization of grape and wine phenolic
bioactives, in: Fruit and cereal bioactives: sources,
chemistry, and applications, Eds: Ö. Tokuşoğlu and
C. A. Hall III, p. 409-427 (CRC Press, Boca Raton,
USA, 2011)
44. Z. Liang, B. Wu, P. Fan, C. Yang, W. Duan, X.
Zheng, C. Liu and S. Li, Food chemistry, 111, 837844 (2008)
45. C. Carrera, A. Ruiz-Rodríguez, M. Palma and C. G.
Barroso, Analytica chimica acta, 732, 100-104
(2012)
46. M. González-Centeno, F. Comas-Serra, A. Femenia,
C. Rosselló and S. Simal, Ultrasonics
Sonochemistry, 22, 506-514 (2015)
47. A. Golmohamadi, G. Möller, J. Powers and C.
Nindo, Ultrasonics Sonochemistry, 20, 1316-1323
(2013)