Food Chemistry 307 (2020) 125549

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

High pressure processing of carrot juice: Effect of static and multi-pulsed T

pressure on the polyphenolic profile, oxidoreductases activity and colour
Justyna Szczepańskaa, , Francisco J. Barbab, Sylwia Skąpskaa, Krystian Marszałeka
⁎

a
Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology, Department of Fruit and Vegetable Product Technology, 36 Rakowiecka St., 02532 Warsaw,
Poland
b
Universitat de València, Faculty of Pharmacy, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Nutrition and Food
Science Area, Avda. Vicent Andrés Estellés, 46100 Burjassot, València, Spain

ARTICLE INFO ABSTRACT

Keywords: The aim of this study was to determine the influence of static and multi-pulsed hydrostatic pressure processing
Triple-TOF-LC-MS/MS analysis (HPP) treatments on the polyphenolic profile, oxidoreductase activity, colour, and browning index of carrot
Pathway of polyphenol compounds juice. Phenolic acids, flavonoids, lignans and other polyphenols were the predominant polyphenols detected
Polyphenoloxidase with Triple-TOF-LC-MS/MS. The highest concentration of ferulic acid, didymin, dihydro-p-coumaric acid, se-
Peroxidase
saminol and matairesinol isomers were found among all the compounds detected. After HPP treatment, irre-
Browning index
spective of the pressures applied, new simple polyphenols like oleuropein, 4-vinylsyringol, isocoumarin, and 4-
hydroxybenzaldehyde were detected. Both phenomena could be attributed to the release of bounded phenolic
compounds after applying HPP, as well as enzymatic degradation and/or condensation. The highest inactivation
of polyphenoloxidase (PPO) enzymes (57%) was obtained at 300 MPa × 3 pulses, and peroxidase (POD) en-
zymes (31%) at 600 MPa working in static mode. Significant changes in the colour parameters and browning
index were observed in all HPP-treated juices.

1. Introduction
Carrots (Daucus carota L.) are one of the most popular root crops
cultivated worldwide (Augšpole, Kince, & Cinkmanis, 2017). They are
usually consumed raw or processed in order to obtain various products,
such as juices and baby foods. Carrots are a valuable component of the
diet since they contain a wide variety of nutrients and bioactive com-
ponents such as carotenoids and polyphenols, among other things
(Augšpole et al., 2017; Priecina & Karklina, 2018).
Polyphenols are secondary metabolites of plants. They have an
important defensive function against free radicals (Gonçalves, Pinheiro,
Abreu, Brandão, & Silva, 2010). Most authors divide phenolic com-
pounds into five groups depending on the number of phenol rings they
contain and structural elements that bind them together. From this
perspective, the main groups of polyphenols are: flavonoids, phenolic
acids, lignans, stilbenes, and others not classified above (Augšpole
et al., 2017; Quirós-Sauceda et al., 2014).
During conventional thermal pasteurization of carrot juices, some
detrimental effects on the sensorial, nutritional and bioactive value of
the juice are discernible (Jabbar et al., 2014; Ma et al., 2013). Because
of this, researchers are seeking new solutions to increase the shelf-life of
fresh products while maintaining the highest quality. The sensory and
nutritional value of carrot juices can be affected by processing tech-
nologies. Proper thermal processing can entirely inactivate native tissue
enzymes, especially those in the oxidoreductases class. However, non-
thermal preservation techniques, such as high pressure processing
(HPP), are often not sufficiently effective in this aspect. High pressure
processing (HPP) is one of fastest growing innovative non-thermal food
processing methods that is used commercially. HPP enables the shelf-
life of food products to be increased by applying pressures of up to
600 MPa or lower pressure in a multi-pulse system, of even up to few
months under refrigeration (Barba et al., 2012; Jabbar et al., 2014;
Marszałek, Woźniak, & Skąpska, 2016; Putnik et al., 2019).
The most resistant plant enzymes, such as polyphenol oxidases
(PPO) and peroxidases (POD), are not properly inactivated with the use
of HPP (Butz, Koller, Tauscher, & Wolf, 1994). Tissue enzymes are
generally located in plastids, whereas most phenolic compounds can be
found in the vacuoles (Vaughn & Duke, 1984). PPO catalyzes two dis-
tinct types of reactions. The first is the hydroxylation of monophenols to
o-diphenols, and the second is the oxidation of o-diphenols to o-

Corresponding author.
⁎

E-mail addresses: justyna.szczepanska@ibprs.pl (J. Szczepańska), francisco.barba@uv.es (F.J. Barba), sylwia.skapska@ibprs.pl (S. Skąpska),
krystian.marszalek@ibprs.pl (K. Marszałek).

https://doi.org/10.1016/j.foodchem.2019.125549
Received 10 July 2019; Received in revised form 13 September 2019; Accepted 16 September 2019
Available online 30 September 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
J. Szczepańska, et al.
quinones (Mayer, 2006; Rodríguez-López et al., 2001; Yoruk &
Marshall, 2003). Quinones can be converted into brown pigments,
visible as a sediment, due to polymerization reactions (Mayer, 2006).
Our previous study indicated that the activity of POD in carrot juice was
almost 120 times higher compared to PPO, therefore indicating that
POD is the principal enzyme responsible for the browning of carrot
juice (Marszałek, Krzyżanowska, Woźniak, & Skąpska, 2016). More-
over, the activity of POD in carrot juice was approx. 3 times higher
compared to the same enzyme found in celery juice, and 10 times
higher compared to PODs found in strawberries (Marszałek,
Krzyżanowska, et al., 2016; Marszałek, Woźniak, et al., 2016). POD in
the presence of peroxides or oxygen as hydrogen acceptor catalyses the
oxidation reactions, leading to the formation of enzyme-hydrogen
donor complexes (Hemeda & Klein, 1991).
The application of multi-pulse HPP at low pressures can bring
economic benefits compared to classic HPP treatment. Unfortunately,
the high residual activity of tissue enzymes in HPP fruit and vegetable
products leads to the fast degradation of bioactive compounds during
storage, which results in the shortening of their shelf-life of up to
3–4 weeks (Marszałek et al., 2018). HPP-treated juices are available
worldwide for a long time and their market share is still increasing. In
case of carrot juice, mixtures are often used, carrot juice with the ad-
dition of e.g. apple, orange, beet or mango juices. For instance, there
are many reports about the application of multi-pulse HPP showing the
usefulness of this technique for effective microbial inactivation (Buzrul,
2014; Donsì, Ferrari, & Maresca, 2010; Pilavtepe-Çelik, Buzrul, Alpas,
Largeteau, & Demazeau, 2011), but there are no reports on the influ-
ence of multi-pulse HPP on the stability of tissue enzymes in cloudy
carrot juice and the phenolic profile changes. Therefore, the aim of the
present work is to evaluate the activity of cloudy carrot juice tissue
enzymes as well as the stability of its phenolic compounds after ap-
plying single- and multi-pulsed HPP.
2. Materials and methods
2.1. Reagents
The following standards and chemicals were used in the study: so-
dium dihydrogen phosphate monohydrate, sodium phosphate dibasic
heptahydrate, sodium chloride, formic acid, polyvinylpolypyrrolidone
(PVP) (~110 µm), Triton X-100, catechol (> 99%), hydrogen peroxide
industrial (30%) and p-phenylenediamine (Merck, Germany). Other
reagents, such as methanol (HPLC grade) and hydrochloric acid were
purchased from Avantor Performance Materials (Gliwice, Poland).
2.2. Preparation of the carrot juice
Fresh carrots (Daucus carota L.) were purchased from a local market,
hand-washed in tap water, cut into smaller pieces and pressed (J 80
Ultra, Robot Coupe, France). The juice was bottled in high density
polyethylene bottles (50 mL) and preserved immediately using HPP.
2.3. High pressure processing (HPP) treatments
The flexible bottles with carrot juice were placed in a 6-CAL70 HPP
chamber (Exdin Solutions, Poland). The volume of the chamber was
100 L, which corresponds to filling 132 bottles with a capacity of 0.5 L;
the maximum pressure being 600 MPa. The high-pressure chamber was
filled with tap water as the pressure-transmitting medium. The machine
was equipped with an automatic product feeder.
The process was carried out using various pressures: static pressure
of 450 MPa and 600 MPa for 5 min and three pulses of 300 MPa for
5 min (total 15 min) at ambient temperature (≈ 22 °C). The initial
temperatures of the pressure transmitting fluid were 13 °C, 11 °C and
4 °C, respectively for pressures of 300 MPa, 450 MPa and 600 MPa. The
initial temperature of the juices was 4 °C. The compression time was 30,
2
Food Chemistry 307 (2020) 125549
45 and 60 s, with a decompression time 4, 6 and 8 s, respectively for
pressures of 300 MPa, 450 MPa and 600 MPa. The HPP treatments were
performed in two replicates. The control sample consisted of fresh,
untreated juice. Samples for measuring tissue enzyme activity and
phenolic compounds were stored at −25 °C until analysis, whereas
colour changes were assessed immediately after the HPP process.
2.4. Determination of phenolic compounds using Triple-TOF-LC-MS/MS
2.4.1. Extraction procedure
Five milliliters of 80% (v/v) methanol containing 0.1% (v/v) of HCl
were added to 5 mL of carrot juice. To better extract the polyphenols,
the samples were treated with ultrasound for 5 min (45 kHz, 200 W,
25 °C, MKD Ultrasonic, Poland) and centrifuged (Rotina 380R, Hettich
Instruments, Germany) for 5 min at 4 °C with RCF 3670 × g. The su-
pernatant was transferred to a 25 mL flask; the extraction was repeated
four times. After filtration (pore size 0.45 µm, Macherey-Nagel,
Germany), the supernatant was used for further analysis.
2.4.2. Triple-TOF-LC-MS/MS analysis
The determination of phenolic compounds in fresh and HPP-treated
carrot juices was carried out using a TripleTOF™ 5600 (AB SCIEX)
UPLC-MS/MS system equipped with Agilent 1260 Infinity (Agilent,
Waldbronn, Germany) and a 1.7 μm, 2,1 mm × 50 mm Acquity UPLC
C18 column (Waters, Cerdanyola del Vallès, Spain). An analysis of the
samples was performed within 22 min at a flow rate of 0.4 mL/min.
Samples were eluted using a gradient of 0.1% (v/v) formic acid, (sol-
vent A) and methanol with the addition of 0.1% (v/v) formic acid
(solvent B), as follows: from 0 to 13 min, 90% (A) and 10% (B); then
13–15 min, 100% (B) and finally 15–22 min, 90% (A) and 10% (B).
MS acquisition was conducted in negative mode, with a mass range
of 80–1200 m/z. The MS was using an IDA acquisition method with:
survey scan type (TOF-MS) and dependent scan type (product ion)
using −50 V of collision energy voltage. The MS parameters were as
follows: temperature 400 °C with curtain gas (CU) 25 psi, ion source gas
1 (GC1) 50 psi and ion source gas 2 (GS2) 50 psi, ion spray voltage
−4500 V, collision energy voltage (CE) −50 V and declustering po-
tential (DP) 90 V. IDA MS/MS was as follows: the collision energy was
fixed at 25 V, ion tolerance 50 mDa, ions that exceeded 100 CPS and the
dynamic background substraction activated. For analysis, PeakView
1.1. software with XIC Manager and Formula Finder was used, which
carried out data acquisition and processing. To quantify the phenolic
compounds, an external calibration curve was prepared with resvera-
trol and the results were expressed as mg of the resveratrol equivalents/
L of the juices.
2.5. Determination of polyphenoloxidase (PPO) and peroxidase (POD)
activities
The activities of polyphenoloxidase (PPO) and peroxidase (POD)
were determined spectrophotometrically according to methodology
previously reported by Terefe, Yang, Knoerzer, Buckow, & Versteeg,
2010. The extraction solution consisted of a 0.2 M sodium phosphate
buffer (pH = 6.5) containing 4% (w/v) polyvinylpolypyrrolidone
(PVPP), 1% (v/v) triton X-100 and 1 M NaCl. The carrot juice and ex-
traction mixture (4.2 mL : 4.2 mL, v/v) were shaken on a vortex (IKA,
Germany) for 1 min and centrifuged (Rotina 380R, Hettich Instruments,
Germany) at 11,000 × g for 30 min at 4 °C. The supernatant was cen-
trifuged once more under the same conditions and was used to de-
termine PPO and POD activity.
For the PPO assay, 300 µL of the supernatant was added to 3 mL of
0.05 M phosphate buffer (pH = 6.5) containing 0.07 M catechol. The
absorbance was measured at λ = 420 nm and 25 °C for 10 min using a
UV–visible spectrophotometer (6705 UV–vis Spectrophotometer,
Jenway, UK). The blank sample was prepared in the same way, but the
supernatant was replaced with a 0.05 M phosphate buffer (pH = 6.5).
J. Szczepańska, et al.
The activity of the PPO was expressed as a change in the absorbance/
min/mL of the analysed sample.
For the POD assay, 50 µL of the supernatant was added to 3 mL of
0.05 M phosphate buffer (pH = 6.5). To start the reaction 50 µL of 1%
p-phenylenediamine (w/v) in 0.05 M phosphate buffer (pH = 6.5) and
50 µL of 1.5% hydrogen peroxide (v/v) were added. The absorbance
was measured at λ = 485 nm and 25 °C for 10 min. A blank sample was
prepared in the same way, but the supernatant was replaced with a
0.05 M phosphate buffer (pH = 6.5). The activity of the POD was ex-
pressed as the change in the absorbance/min/mL of the analysed
sample. The analysis were carried out in triplicate.
The residual activity for both analysed enzymes (PPO and POD) was
calculated according to Eq. (1):
A O-glucoside were increased after applying HPP treatments by 75.0,
RA (%) = × 100
A0 (1)
where: A is the activity of the HPP-treated juice and A0 is the activity of
the fresh (untreated) juice.
2.6. Colour parameters and browning index
The colour of the carrot juices was measured using a Color Quest XE
colorimeter (HunterLab, USA) equipped with a xenon flashlamp.
Samples were placed into a 5 mL cuvette, 1 cm thick. The results were
expressed in accordance with the CIE L*a*b* system, using illuminant
D65 and 10° observer. The parameters were expressed as: L* (lightness/
darkness), a* (red/green), and b* (yellow/ blue). All of these measured
values were used to calculate the total colour difference (ΔE), which
showed a numerical difference in the colour of the samples after HPP
treatment compared to the control sample, according to Eq. (2)
(Maskan, 2001):
E= (L 0 L ) 2 + (a 0 a ) 2 + ( b0 b) 2 (2)
The browning index (BI) was calculated using Eq. (3) (Maskan,
2001):
100(x 0.31) a + 1.75L
BI = , where, x =
0.172 5.645L + a 3.012b (3)
2.7. Statistical analysis
All analyses were carried out in triplicate. The results were ex-
pressed as a mean value ± standard deviation (S.D.) and analysed
using STATISTICA 7.1 software (StatSoft, Tulsa, OK, USA) with one-
way analysis of the variance (ANOVA). Statistically significant differ-
ences between these values were assayed using Tukey’s test at a con-
fidence level of α = 0.05.
3. Results
3.1. Phenolic compounds profile by Triple-TOF-LC-MS/MS in fresh and
HPP cloudy carrot juice
Twenty-five polyphenol compounds were identified in the cloudy
carrot juice with the use of triple-TOF-LC-MS/MS analysis (Table 1). As
can be seen in Table 2 and Fig. 1, the sum of the phenolic compounds
identified in fresh carrot juice was 188.67 ± 6.27 mg/L (Fig. 1), ob-
serving an increased amount after applying HPP (300 MPa/3 pulses)
(219.80 ± 6.23 mg/L) or HPP (600 MPa) (246.11 ± 7.58 mg/L).
The identified polyphenols can be assigned mainly to 4 groups such
as: phenolic acids (nine compounds), flavonoids (four compounds),
lignans (three compounds) and other polyphenols (nine compounds).
Fig. 1 shows the contribution of these groups in fresh cloudy carrot
juice as well as after HPP treatment at different pressures. As shown in
the figure, the concentration of phenolic acids in fresh juice was
77.27 ± 2.67 mg/L. Among the phenolic acids, two simple acids and
3
Food Chemistry 307 (2020) 125549
their glycoside derivatives (ferulic acid, ferulic acid 4-O-glucoside, 4-
hydroxybenzoic acid and 4-hydroxybenzoic 4-O-glucoside) were de-
tected.
HPP treatment caused significant changes in the profile of the
phenolic acids (Table 2). As can be seen in the table, cinnamic acid
(5.67 ± 0.21 mg/L) was detected in the fresh carrot juice, whereas the
concentration of this acid was below the limit of detection after HPP
treatment. Ferulic acid was the most abundant compound in the phe-
nolic acids. The ferulic acid content (33.90 ± 0.44 mg/L) and dihydro-
p-coumaric acid content (16.15 ± 0.78 mg/L) were decreased by 11.8
and 77.8% after HPP treatment, respectively. The opposite was ob-
served for all the other phenolic acids. For instance, the concentration
of vanillic acid, ferulic acid 4-O-glucoside and 4-hydroxybenzoic acid 4-
48.4, 41.1%, respectively. Finally, the total phenolic acid content in-
creased from 77.27 ± 2.67 mg/L in fresh juice up to
62.81 ± 2.62 mg/L in HPP-treated juice (Fig. 1).
Four flavonoids (cirsimaritin, didymin, kaempferol 3-O-(6-acetyl-
galactoside) 7-O-rhamnoside and prodelphinidin dimer B3) were de-
tected and identified in the cloudy carrot juice before and after HPP
treatment. Their concentration ranged from 42.02 ± 1.84mg/L to
55.53 ± 1.40 mg/L in fresh juice and increased significantly after HPP
under each parameter used. The highest content of flavonoids was
noted for didymin and the concentration of this compound increased
significantly from 26.35 ± 0.78 mg/L to 42.13 ± 1.08 mg/L, after
HPP treatment. The opposite was observed for prodelphinidin dimer
B3. The highest pressure caused the degradation of this compound by
almost 35%. Cirsimaritin and kaempferol 3-O-(6-acetyl-galactoside) 7-
O-rhamnoside turned out to be most stable, but the changes were also
statistically significant. Multi-pulsed HPP caused a higher increase in
the flavonoid content than the highest applied static pressure.
Among the lignans, sesaminol 2-O-β-D-glucoside was the pre-
dominant phenolic compound found in fresh carrot juice with a con-
centration of 18.80 ± 0.26 mg/L. Isomers of matairesinol
(13.90 ± 0.17 mg/L) and sesaminol (11.50 ± 0.42 mg/L) were also
detected, promoting slight but significant increases of these compounds
under HPP treatment (Table 2, Fig. 1).
Four polyphenols, unclassified in the previously defined three
phenolic groups: pyrogallol, 4-vinylphenol, 4-vinylguaiacol and sir-
ingaldehyde were identified in untreated carrot juice. Moreover, five
new components were identified after the application of high pressure
in all samples (Table 2). This phenomenon was significant from the
point of view of the final total concentration of phenolic compounds.
The content of polyphenols classified to other polyphenols increased by
more than 3 times after applying pressure of 600 MPa (Fig. 1). Among
this group of polyphenols, the most interesting was oleuropein, not
detected in fresh juice, whereas its concentration increased in all HPP
treated juice, up to 45.30 ± 1.28 mg/L at 600 MPa. 3,4-DHPEA-AC
was not found in fresh and multi-pulsed HPP treated juices, but it was
detected after 450 MPa and 600 MPa treatment.
3.2. Polyphenol oxidase and peroxidase activity
The measured activity of polyphenol oxidase in fresh carrot juice
was 0.19 ± 0.01 ΔA/min/mL. The influence of HPP parameters on the
activity of oxidoreductive enzymes, expressed as residual activity, is
presented in Fig. 2. HPP treatment significantly decreased the activity
of PPO compared to fresh juice. A pressure of 450 MPa caused the in-
activation of this enzyme by 43.4%, whereas the application of higher
pressures (600 MPa) resulted in lower inactivation (30.0%). The best
results for PPO inactivation (a reduction of 57.9%) were obtained when
multi-pulsed HPP (3 × 300 MPa) was applied.
The activity of POD in fresh carrot juice was as high as
10.85 ± 0.14 ΔA/min/mL, therefore showing this enzyme was almost
60 times more active compared to PPO. The residual activity of POD
ranged from 68.9 to 88.2% after HPP treatment, and it was found that
J. Szczepańska, et al. Food Chemistry 307 (2020) 125549

Table 1
Phenolic compounds identified by triple-TOF-LC-MS/MS in fresh and all HPP-treated carrot juice.
Phenolic compound Formula Retention time (min) Expected mass (m/z) Found mass (m/z) Mass error (ppm)

Phenolic acids
4-Hydroxybenzoic acid C7H6O3 8.49 137.0244 137.0245 0.3
Vanillic acid C8H8O4 4.82 167.0350 167.0351 0.7
Cinnamic acid C9H8O2 11.47 147.0452 147.0453 1.3
p-Coumaric acid C9H8O3 11.27 163.0401 163.0402 0.7
Dihydro-p-coumaric acid C9H10O3 15.69 207.0663 207.0667 2.2
Ferulic acid C10H10O4 13.49 193.0506 193.0509 1.3
4-Hydroxybenzoic acid 4-O-glucoside C13H16O8 8.49 299.0772 299.0773 0.2
Protocatechuic acid 4-O-glucoside C13H16O9 5.67 315.0722 315.0721 −0.3
Ferulic acid 4-O-glucoside C16H20O9 8.85 355.1035 355.1030 −1.2

Flavonoids
Cirsimaritin C17H14O6 17.48 313.0718 313.0724 2.0
Didymin C28H34O14 12.82 593.1876 593.1900 4.0
Kaempferol 3-O-(6-acetyl-galactoside) 7-O-rhamnoside C29H32O16 12.15 635.1618 635.1611 −1.0
Prodelphinidin dimer B3 C30H26O13 18.23 593.1301 593.1318 3.0

Lignans
Matairesinol isomers C20H22O7 11.92 373.1293 373.1287 −1.4
Sesaminol 2-O-β-D-glucoside C26H28O12 13.67 531.1508 531.1513 0.9
Sesaminol isomers C32H38O17 13.04 693.2036 693.2039 0.4

Other polyphenols
Pyrogallol C6H6O3 10.80 125.0244 125.0245 1.0
4-Hydroxybenzaldehyde C7H6O2 11.38 121.0295 121.0297 1.4
4-Vinylphenol C8H8O 12.50 119.0502 119.0503 0.6
4-Vinylguaiacol C9H10O2 12.96 149.0608 149.0609 0.6
Syringaldehyde C9H10O4 7.02 181.0506 181.0511 2.5
4-Vinylsyringol C10H12O3 10.79 179.0714 179.0715 0.8
3,4-DHPEA-AC C10H12O4 10.57 195.0663 195.0665 0.9
Isocoumarin C11H12O4 13.54 207.0663 207.0664 0.5
Oleuropein C25H32O13 13.26 539.1770 539.1768 −0.4

Table 2
Qualitative and quantitative profile of polyphenols identified in fresh carrot juice and under HPP treatment at pressures 300 MPa × 3, 450 MPa and 600 MPa and
room temperature for 5 min (mg/L).
Phenolic compounds fresh juice 300 MPa (3 pulses) 450 MPa 600 MPa

Phenolic acids
4-Hydroxybenzoic acid 3.40 ± 0.10 a 3.70 ± 0.10 ab 3.93 ± 0.21 bc 4.30 ± 0.20c
Vanillic acid 2.40 ± 0.35 a 3.87 ± 0.21 b 4.03 ± 0.23 b 4.23 ± 0.21 b
Cinnamic acid 5.67 ± 0.21 a N.D. N.D. N.D.
p-Coumaric acid 1.55 ± 0.07 a 1.85 ± 0.21 b 1.73 ± 0.06 ab 1.85 ± 0.07 b
Dihydro-p-coumaric acid 16.15 ± 0.78 b 3.60 ± 0.28 a 3.77 ± 0.21 a 3.75 ± 0.21 a
Ferulic acid 33.90 ± 0.44 b 30.93 ± 0.57 a 29.97 ± 0.58 a 29.93 ± 0.70 a
4-Hydroxybenzoic acid 4-O-glucoside 5.57 ± 0.32 a 7.27 ± 0.55 b 7.20 ± 0.10 b 7.90 ± 0.62 b
Protocatechuic acid 4-O-glucoside 5.53 ± 0.23 a 6.47 ± 0.25 b 6.40 ± 0.26 b 6.30 ± 0.26 b
Ferulic acid 4-O-glucoside 3.10 ± 0.17 a 4.27 ± 0.06 bc 4.10 ± 0.01 b 4.55 ± 0.35 c

Flavonoids
Cirsimaritin 3.67 ± 0.9 a 4.40 ± 0.24 b 4.40 ± 0.11 b 3.60 ± 0.23 a
Didymin 26.35 ± 0.78 a 36.20 ± 0.78 b 42.13 ± 1.08 c 34.43 ± 0.38 b
Kaempferol 3-O-(6-acetyl-galactoside) 1.40 ± 0.02 a 2.10 ± 0.13 b 1.80 ± 0.09 ab 1.70 ± 0.11 ab
7-O-rhamnoside
Prodelphinidin dimer B3 10.60 ± 0.14 b 7.80 ± 0.22 a 7.20 ± 0.12 a 6.81 ± 0.34 a

Lignans
Matairesinol isomers 13.90 ± 0.17 a 15.53 ± 0.15 b 16.83 ± 0.31 c 18.80 ± 0.17 d
Sesaminol 2-O-β-D-glucoside 18.80 ± 0.26 a 18.33 ± 0.65 a 19.47 ± 1.06 a 19.43 ± 0.31 a
Sesaminol isomers 11.50 ± 0.42 a 13.47 ± 0.51 b 12.50 ± 0.56ab 11.70 ± 0.17 a

Other polyphenols
Pyrogallol 9.13 ± 0.49 a 9.87 ± 0.12 ab 10.87 ± 0.26 b 10.17 ± 0.15 b
4-Hydroxybenzaldehyde N.D. 1.97 ± 0.21 a 2.13 ± 0.12 ab 2.43 ± 0.21 b
4-Vinylphenol 11.97 ± 0.29c N.D. 2.90 ± 0.13 b 2.05 ± 0.42 a
4-Vinylguaiacol 2.73 ± 0.06 a 2.67 ± 0.15 a 2.60 ± 0.06 a 2.70 ± 0.05 a
Syringaldehyde 1.35 ± 0.07 a N.D. N.D. N.D.
4-Vinylsyringol N.D. 10.23 ± 0.55 a 10.73 ± 0.15 a 14.25 ± 0.64 b
3,4-DHPEA-AC N.D. N.D. 4.40 ± 0.10 a 4.63 ± 0.15 a
Isocoumarin N.D. 4.77 ± 0.06 a 4.80 ± 0.42 a 5.30 ± 0.35 a
Oleuropein N.D. 30.50 ± 0.23 a 39.60 ± 0.38 b 45.30 ± 1.28 c

N.D.: not detected. Data are presented as the mean ± S.D. from three independent experiments. Different letters in the same row means statistically significant
differences (α = 0.05).

4
J. Szczepańska, et al. Food Chemistry 307 (2020) 125549

purity of the brown colour.

4. Discussion

4.1. Changes in the polyphenol profile of carrot juice

The qualitative as well as quantitative polyphenol profile in carrot

Fig. 1. Concentration of main polyphenol groups in fresh and HPP-treated
carrot juice.
Fig. 2. Residual activity of polyphenoloxidase (PPO) and peroxidase (POD) in
fresh and HPP-treated carrot juice.
the higher the pressure applied, the higher the inactivation of the POD.
Moreover, it was also observed that the highest pressure (600 MPa) was
the most effective to inactivate POD, observing a strong negative cor-
relation between the POD activity and the level of pressure applied
(r = −0.970).
3.3. Colour parameters and browning index
All measured colour parameters of fresh and HPP-treated carrot
juice are shown in Table 3. HPP led to a significant darkening of all the
preserved samples compared to fresh juice (p < 0.05), the darkest
colour being observed after multi-pulsed HPP treatment (300 MPa × 3
pulses). Moreover, HPP juices were less red and yellow compared to
fresh carrot juice. The calculated coefficient, known as total colour
difference (ΔE), exceed the value of 5, which means that the changes
were significant even for inexperienced observers. These changes were
confirmed by the browning degree (BI) coefficient, which represents the
juice has still not been fully investigated, and probably depends on
many factors. For instance, the concentration of polyphenols in carrot
roots strongly depends on the variety, climate, soil, cultivation and even
technological processes applied in juice production (Ma et al., 2013;
Surjadinata & Cisneros-Zevallos, 2012). In a previous study, it was
proved that the concentration of phenolic compounds increased from
the interior (xylem) to the exterior (peel) (Gonçalves et al., 2010). The
same authors confirmed the results obtained in our study showing that
phenolic acids were the most abundant group of polyphenols found in
carrots. Except for ferulic acid, p-coumaric, cinnamic and vanillic acid
which have already been identified by several research groups
(Augšpole et al., 2017; Surjadinata & Cisneros-Zevallos, 2012) a few
other phenolic acids such as 4-hydroxybenzoic acid and protocatechuic
acid derivatives, were found in the present work.
On the other hand, the presence of chlorogenic acid, 5-caffeoyl-
quinic acid and caffeic acid identified in carrot tissue by other authors
(Ma et al., 2013; Neacsu et al., 2015; Surjadinata & Cisneros-Zevallos,
2012) was not confirmed in our research. The phenolic profile included,
among others, three glucosylated derivatives of hydroxybenzoic (4),
ferulic (12), protocatechuic (13) acids (Table 1, Fig. 3). Smeriglio et al.,
2018 studied the anthocyanin profile in black carrot and found that the
2″ position was the binding site of glucose. This site of glucose binding
could be the same in our study.
HPP significantly influenced the polyphenol profile. The increase in
the concentration of selected phenolic compounds such as 4-hydro-
xybenzoic acid, vanillic acid, didymin, pyrogallol and glucoside deri-
vatives of 4-hydroxybenzoic, protocatechuic and ferulic acids, can be
justified by the better extraction of bonded acids from tissue treated
using HPP or the enzymatic hydrolysis of higher polyphenols into
simple phenols. It is known that the use of ultrasound pre-treatment
promotes the extraction of valuable compounds, e.g. polyphenols (Zhu
et al., 2016; Zhu et al., 2017). However, the decrease observed for other
phenolic compounds such as cinnamic acid, dihydro-p-coumaric acid,
prodelphinidin dimer B3, 4-vinylphenol and syringaldehyde can be
explained by the lower baro-stability of these compounds, enzymatic
degradation as well as by the polymerization induced by enzymatic
reactions or high pressure. For instance, Patras, Brunton, Da Pieve,
Butler, and Downey (2009) reported an increase (up to 5%) in the total
phenolics content in carrot purées after high pressure treatment of
400–600 MPa/15 min/20 °C, whereas after traditional pasteurization,
the authors noted a decrease (≈10%) in total polyphenols. In another
study, Augšpole et al. (2017) observed a significant decrease in the
concentration of cinnamic acid after the six-month storage of carrot
roots. The authors attributed this decrease to the oxidation of the main
polyphenols or other biochemical processes.
In our study, cinnamic acid was only detected in fresh juice, which
could indicate that this compound is very sensitive to high pressure
and/or enzymatic degradation, or it could be used as a substrate in

Table 3
Color parameters of fresh and HPP-treated carrot juice pressures 300 MPa × 3, 450 MPa and 600 MPa and room temperature for 5 min.
Color parameter fresh juice 300 MPa (3 pulses) 450 MPa 600 MPa

L* 44.93 ± 0.04d 40.94 ± 0.03a 41.38 ± 0.01c 41.19 ± 0.01b

a* 27.27 ± 0.01d 24.24 ± 0.02a 24.61 ± 0.01c 24.39 ± 0.01b
b* 42.61 ± 0.03d 39.52 ± 0.06a 39.83 ± 0.02b 40.01 ± 0.03c
ΔE – 5.89 ± 0.03c 5.24 ± 0.01a 5.39 ± 0.01b
BI 223.31 ± 0.24a 228.73 ± 0.37c 227.88 ± 0.23b 230.95 ± 0.29d

Data are presented as the mean ± S.D. from three independent experiments. Different letters in the same row means statistically significant differences (p > 0.05).

5
J. Szczepańska, et al.
Fig. 3. The proposed pathway of polyphenol compounds.
certain reactions, such as polymerization. According to the results
presented by Priecina and Karklina (2018) the increase in the 4-hy-
droxybenzoic acid concentration could occur due to the degradation of
anthocyanins to benzoic acid derivatives during thermal processing.
HPP treatment applied at ambient temperature could lead to the bio-
synthesis of phenolic compounds induced by high pressure or a chain of
enzymatic reactions (proposed in Fig. 3). For instance, the cinnamic
acid (1) could be hydroxylated to p-coumaric acid (5) by cinnamate-4-
hydroxylase (Lehka et al., 2017). It is one of many steps occurring in the
flavonoids biosynthetic pathway in plants (Converti, Aliakbarian,
Domínguez, Vázquez, & Perego, 2010; Sircar & Mitra, 2009).
There are three possible ways to further transform p-coumaric acid.
According to Sircar and Mitra (2009), p-coumaric acid (5) can be the
substrate in 4-hydroxybenzaldehyde (2) synthesis, which in turn is
converted into 4-hydroxybenzoic acid (3). They studied the accumu-
lation of the latter compound in the hairy roots of carrots and con-
firmed this biosynthetic pathway. A similar trend, regarding the in-
crease of 4-hydroxybenzoic acid concentration was observed in our
study under high pressure conditions. Another possible way of trans-
forming p-coumaric acid (5) to 4-vinylphenol (11) is associated with
cinnamate decarboxylase activity. This transformation was observed in
6
Food Chemistry 307 (2020) 125549
wine during fermentation. The authors observed that the yeasts Bret-
tanomyces were responsible for 4-ethylphennol and 4-vinylphenol
synthesis. The same enzyme caused the formation of 4-vinylguaiacol (8)
from ferulic acid (7) (Wedral, Shewfelt, & Frank, 2010).
The third pathway of p-coumaric acid's (5) transformation could be
due to its hydroxylation into caffeic acid (6) (Gao, Yu, Xu, Cheng, &
Lou, 2015). Subsequently, caffeic acid (6) in further enzymatic reac-
tions can be transformed to ferulic acid (7) by caffeic acid O-methyl
transferase (Gao et al., 2015). Later, ferulic acid (7), can be transformed
into 4-vinylguaiacol (8). The next step is the conversion of 4-vi-
nylguaiacol (8) to vanillic acid (9), followed by the formation of pro-
tocatechuic acid (10). Two of the three described pathways could occur
in HPP-treated juices, which could explain why cinnamic acid (1) was
only detected in fresh juice, whereas 4-hydroxybenzoic acid (3) and
vanillic acid (9) concentrations increased after pressurization. On the
other hand, as mentioned earlier, some higher polyphenols such as
prodelphinidin dimer B3, in HPP-treated juice with an active enzymatic
system, can be decomposed to smaller molecules resulting in the in-
crease in the concentration of pyrogallol. However, the pressure can
contribute to the formation of macromolecules, such as oleuropein.
Surjadinata and Cisneros-Zevallos (2012), in their studies
J. Szczepańska, et al.
evaluating the differences in the polyphenol profile of different parts
(slices, pieces, and shreds) of carrots, noted that intensive disintegra-
tion induced an increase in the concentration of isocoumarin. Our re-
search confirmed these findings. Isocoumarin was only detected in
carrot juice after HPP treatment of the juices. Furthermore, the same
authors proved that p-hydroxybenzoic acid was present in shredded
carrot, whereas it was not detected in carrot roots. It is evident that
tissue disintegration is connected with the release of enzymes re-
sponsible for several reactions, thus resulting in the release of phenolic
compounds from tissue or their synthesis from other polyphenols. As
mentioned earlier, HPP can accelerate this reaction due to the better
extraction of polyphenols from the tissue, because pressurization causes
disruption of the salt bridges and also the hydrophobic bonds found in
membranes of cells and changes in the membrane's permeability which
induces the release of phenolic compounds from tissues. Furthermore,
the concentration of polyphenol compounds is directly related to the
residual activity of oxydoreductive enzymes, especially PPO. The in-
crease in the content of some compounds was observed and confirmed
by other authors, who noticed that stress conditions may involve the
synthesis and accumulation of e.g. caffeoylquinic acid in carrot pro-
ducts (Heredia & Cisneros-Zevallos, 2009; Jacobo-Velázquez, Martínez-
Hernández, Del C. Rodríguez, Cao, & Cisneros-Zevallos, 2011).
4.2. Polyphenol oxidase and peroxidase activity
There are many current studies on the possibility of PPO and POD
inactivation under HPP in different fruit and vegetable matrices. The
conclusions from these works are ambiguous because some of them
suggest that HPP may decrease the activity of tissue enzymes (Garcia-
Palazon, Suthanthangjai, Kajda, & Zabetakis, 2004; Jabbar et al., 2014;
Jung, Lee, Kim, & Ahn, 2013; Marszałek, Woźniak, et al., 2016),
whereas other confirmed that the activity of tissue enzymes may be
increased (Butz et al., 1994; Garcia-Palazon et al., 2004; Huang et al.,
2013; Liu et al., 2016). There are also reports indicating that the degree
of inactivation of enzymes is proportional to the value of the pressure
applied, while other suggest that mild pressurization ensures optimal
results. Marszałek, Woźniak, et al. (2016) studied enzyme activity in
strawberry purée under HPP and they noted only 17.4% POD activity
after treatment at 600 MPa. The efficiency of POD inactivation was
almost four times higher than in the current study. On the other hand,
the lowest pressure of 300 MPa induced a similar degree of inactivation
of PPO and POD compared to the present study, even though we used
multi-pulsed HPP. Despite the fact that the initial activity of both en-
zymes was much higher in carrot juice (especially POD activity) com-
pared to the results found for strawberries, these findings indicate that
the enzymes in carrots are more resistant to pressure than strawberry
enzymes. Our study confirms the findings of other authors showing that
PPO is easier to inactivate under HPP than POD (Huang et al., 2013; Liu
et al., 2016). Jabbar et al. (2014) reported that oxidoreductive enzyme
activity in carrot juice gradually decreased with increasing pressure
level. However, they obtained only 55% and 51% reduction in the PPO
and POD activity, respectively, in carrot juice pressurized at 450 MPa
for 10 min. Similarly to our study, the POD was more resistant than
PPO, which was also confirmed by Jung et al. (2013). On the contrary,
(Ortuño, Duong, Balaban, & Benedito, 2013) noted that the PPO and
POD activities in feijoa purée increased by 2% and 40%, respectively
after processing at 300 MPa for 5 min at room temperature. They ob-
tained a reduction of 62% in the PPO activity and 78% in the POD
activity at 600 MPa for 5 min. This study reported more than 2 times
higher inactivation of oxidoreductive enzymes than in our work, which
may result, among other things, from the different matrices used for the
studies.
7
Food Chemistry 307 (2020) 125549
4.3. Colour parameters and browning index
Jabbar et al. (2014) observed similar changes in the colour para-
meters in carrot juice subjected to HPP as those found in our study. The
L*, a* and b* values in HPP-treated samples decreased significantly,
which was probably caused by PPO and POD activity. These oxidor-
eductive enzymes are responsible for browning reactions as they use
phenolic compounds as a substrate. Different results were obtained by
Patras et al. (2009) in their study on tomato and carrot purées. The a*
parameter significantly increased when the carrot purée was subjected
to HPP treatment. Furthermore, it was confirmed that carotenoids could
be better extracted due to the disintegration of chromoplast under high
pressure. In the study of Ortuño et al. (2013), after pressurization of
feijoa purée, parameters L* and b* decreased, which are consistent with
our study. Marszałek, Woźniak, et al. (2016) reported that the colour
changes could also be caused by enzymatic and non-enzymatic
browning reactions, occurring at an increased temperature and the low
pH of the HPP-treated fruit samples.
5. Conclusions
From the results obtained in the present study, it can be concluded
that HPP generally enhanced the total polyphenol content. The con-
centrations of 4-hydroxybenzoic acid, vanillic acid, didymin, pyrogallol
and glucoside derivatives of 4-hydroxybenzoic, protocatechuic and
perulic acids were increased after HPP treatment. However, the cin-
namic acid, dihydro-p-coumaric acid, prodelphinidin dimer B3, 4-vi-
nylphenol and syringaldehyde content turned out to be sensitive to HPP
treatment under the process parameters applied in our experiment.
Oleuropein, 4-vinylsyringol, isocoumarin and 4-hydroxybenzaldehyde
were found only in HPP-treated juices, the first two compounds were
detected in carrot products for the first time. The observed changes in
the polyphenol profile during pressurization can be justified by enzy-
matic reactions well known as enzymatic browning. In turn, the re-
duction in the content of some phenolic compounds after the applica-
tion of HPP may be the result of the oxidation of these compounds,
simple pressure degradation or polymerization into the higher poly-
phenols. The possible pathways of polyphenol changes have been pro-
posed.
The HPP treatments led to a reduction in the activity of oxidor-
eductive enzymes. POD was more resistant than PPO under HPP
treatment in all pressure levels. The highest pressure (600 MPa) caused
the highest inactivation of POD (31%) and the lowest inactivation of
PPO (30%). Multi-pulsed HPP (300 MPa × 3 pulses) led to a reduction
in the PPO activity of 57%. Pressurization also caused significant
changes in the colour of carrot juice. Changes expressed as the total
colour difference (ΔE) exceed 5 which means that the changes were
visible to the inexperienced observer.
Considering the increase in the total polyphenol content in carrot
juice, preservation under HPP (450 MPa, 600 MPa) is more favorable
than lower pressure with pulses. None of the pressure levels applied
gave a satisfactory reduction in the activity of oxidoreductive enzymes,
which may result in the darkening of the juice during storage. Obtained
results are very promising for further research under optimization of
HPP treatment of carrot juice focused on the inactivation of tissue en-
zymes with limitation of polyphenol changes.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
J. Szczepańska, et al.
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