Innovative Food Science and Emerging Technologies 45 (2018) 196–207

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Innovative Food Science and Emerging Technologies

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

Effect of citric acid and high pressure thermal processing on enzyme activity T
and related quality attributes of pear puree
Lei Zhoua, Wei Liua,⁎, Regine Stockmannb, Netsanet Shiferaw Terefeb,⁎
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
CSIRO Agriculture and Food, 671 Sneydes Rd., Werribee, Victoria 3030, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Pear puree was acidified with citric acid and was subjected to thermal (TP) and high pressure-thermal processing
Pear puree (HPTP). Citric acid inhibited peroxidase (POD) and polyphenol oxidase (PPO) and preserved the color of pear
High pressure thermal processing puree. The addition of citric acid (2%, w/w) increased total phenolic content (TPC) and oxygen radical absor-
Thermal processing bance capacity (ORAC) of pear puree by ~50%. POD was more sensitive than PPO to thermal and HPT in-
Citric acid
activation. Citric acid enhanced thermal and HPT inactivation and complete inactivation of POD was observed
Enzyme
after TP (90 °C, 7 min) and HPTP (600 MPa, 90 °C, 5 min) in acidified samples, while a maximum 60% in-
Quality
activation of PPO was observed under these conditions. TP resulted in a slightly better color retention of
acidified samples than HPTP under conditions of equivalent enzyme inactivation, whereas TPC and ORAC re-
mained stable irrespective of the process.
Industrial relevance: This study investigated the effect of thermal and high pressure thermal processing with and
without citric acid on the activity of oxidative enzymes and related quality attributes of pear puree. Citric acid
inhibited enzymatic browning, protected polyphenolic antioxidants against oxidative degradation and resulted
in 50% increase in antioxidant capacity when it was added at 2% (w/w). Acidified pear puree can possibly be
used as an ingredient in products such as yoghurts, jams and baby food after either thermal or high pressure
thermal processing due to its reduced oxidative enzyme activity, low pH, high antioxidant capacity and excellent
color retention.

1. Introduction enzymatic browning strongly affect consumer's purchasing decisions.

The popularity of commercially produced fruit purees for baby
weaning has increased in recent years, due to the nutritional benefits
and the convenience of these products (Álvarez, Cancela, Delgado-
Bastidas, & Maceiras, 2008). Fruit purees are also widely used in a
variety of products such as yoghurts, jams, smoothies and fruit ice
creams (Komes, Lovrić, & Kovačević Ganić, 2007; Patras, Brunton, Da
Pieve, & Butler, 2009). Pear is widely cultivated and it is a rich source of
dietary fiber, sugar, polyphenolic compounds and vitamins. Second
grade pears which are not suitable for the fresh fruit market can be used
for producing puree, creating value adding opportunities for farmers
and processers. Pear puree is one of the most widespread products in
baby food application (Tarea, Cuvelier, & Sieffermann, 2007). It is also
suitable for old people who have chewing and swallowing problems.
Besides, fruit purees in general are nutritionally superior to juices due
to their high fiber content and as such can be used as alternative snacks
by the general population. However, quality of pear puree deteriorates
rapidly due to enzymatic browning. Color changes induced by
Therefore, inactivation of browning related enzymes is crucial to
maintain the quality of pear puree.
Thermal processing is widely used in the food industry to achieve
the inactivation of enzymes and microorganisms in fruit and vegetable
products. However, thermal processing can also cause detrimental
changes in sensory and nutritional quality attributes (Terefe,
Buckow, & Versteeg, 2014). High pressure processing (HPP) is increas-
ingly being recognized as an alternative stabilization treatment to
thermal processing as it minimizes the degradation of quality attributes
and nutritional compounds (Terefe et al., 2013). Several studies have
reported that HPP inactivates the vegetative cells of spoilage micro-
organisms and some enzymes (Bayındırlı, Alpas, Bozoğlu, & Hızal,
2006; García-Parra, González-Cebrino, Delgado, Cava, & Ramírez,
2016). Enzymes such as lipoxygenase and polygalacturonase can be
inactivated by HPP at room temperature, while pectin methyl esterase,
peroxidase (POD) and polyphenol oxidase (PPO) resist inactivation
under this condition (Terefe et al., 2014). During the storage of pro-
cessed fruits and vegetables, the residual activity of PPO and POD leads

⁎
Corresponding authors.
E-mail addresses: liuwei@ncu.edu.cn (W. Liu), netsanet.shiferawterefe@csiro.au (N.S. Terefe).

http://dx.doi.org/10.1016/j.ifset.2017.10.012
Received 19 July 2017; Received in revised form 12 October 2017; Accepted 12 October 2017
Available online 14 October 2017
1466-8564/ © 2017 Elsevier Ltd. All rights reserved.
L. Zhou et al.
to significant degradation of color and polyphenolic antioxidants
(Terefe, Tepper, Ullman, Knoerzer, & Juliano, 2016). Combined effects
of pressure and temperature during high pressure thermal (HPT) pro-
cessing were shown in some studies (Chakraborty, Rao, & Mishra, 2015;
García-Parra et al., 2016) to enhance the inactivation of oxidative en-
zymes. HPT processing (600 MPa, 100 °C, 3–5 min) of ‘Packham’ pear
slices resulted in effective inactivation of PPO and POD (Terefe et al.,
2016). However, PPO in Taylor's Gold pear puree showed very high
resistance to thermal and HPT processing (Sulaiman, Soo, Farid, and
Silva, 2015; Sulaiman, Soo, Yoon, Farid, and Silva, 2015).
Organic acids such as ascorbic acid and citric acid can also be used
to inhibit PPO and prevent the browning of fruit and vegetable products
via acidification and other mechanisms (Zhou et al., 2016). Citric acid
and citrate are widely used in the food industry for inhibition of
browning in canned fruits and other products (Altunkaya & Gokmen,
2008; Liu et al., 2013). However, limited information is available on the
impact of acidification of pear puree with citric acid on the activity of
oxidative enzymes and related quality attributes in pear puree sub-
jected HPT processing. The objective of this study was to evaluate the
combined effects of acidification by citric acid and HPTP or thermal
processing on the activities of PPO and POD and related quality attri-
butes of pear puree. Besides, this study compared the impact of thermal
processing and HPTP with equivalent degree of PPO inactivation, which
is often used as the measure of the effectiveness of pear retorting
(Kluter, Nattress, Dunne, & Popper, 1996), on the quality of pear puree
related to the activity of oxidative enzymes.
2. Materials and methods
2.1. Materials
Pears (cv. Packham) were purchased from local suppliers. Catechol,
p-phenylenediamine, Folin-Ciocalteu reagent and gallic acid were ob-
tained from Sigma-Aldrich (Castel Hill, NSW, Australia). Trolox and
2,2′-Azobis dihydrochloride (AAPH) were from Sapphire Bioscience
(Redfern, NSW, Australia). All other chemicals were purchased from
Merck (Kilsyth, VIC, Australia) or Sigma-Aldrich (Castle Hill, NSW,
Australia) and were of analytical or HPLC grade.
2.2. Pear puree samples preparation
The pears were ripened overnight at room temperature to obtain
approximate firmness of 6–8 kg. The firmness was measured by a hand
held penetrometer with an 8 mm probe (Fruit Pressure Tester, FT 327,
Italy). After washing, peeling and coring, the pear was sliced and
samples were prepared as follows (Chakraborty, Rao, & Mishra, 2014;
Terefe et al., 2016);
i. Non-acidified samples: pear slices were homogenized with a Bamix
mixer without any pretreatment.
ii. Samples acidified by dipping (AD): pear slices were dipped in 1.5%
citric acid solution for 30 min and homogenized.
iii. Samples acidified by acid addition (AA): pear slices were dipped in
citric acid and homogenized followed by addition of citric acid (2%
w/w) to attain pH 2.5.
Approximately 100 g puree were packed in a transparent retort
pouch (3 ply laminate: nylon (15 μm), clear polypropylene (70 μm),
adhesive (15 μm), oxygen transfer rate at 23 °C, 50% relative humidity
and 1 atm: ≤ 3 CC/m2/day, Amcor, Germany) and sealed with a va-
cuum packing system (Webomatic E50G, Warner Bonk, Bochum,
Germany) at − 0.9 bar for immediate processing by heat or HPT.
2.3. Thermal processing
Thermal processing was conducted in a thermostated water bath at
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Innovative Food Science and Emerging Technologies 45 (2018) 196–207
temperatures ranging from 40 °C to 100 °C for 10 min. Following the
thermal treatment, samples were rapidly immersed in ice water. In
order to compare the quality of pear puree with similar level of PPO
inactivation as HPT, further thermal treatments were conducted at 90
and 100 °C for 0, 1, 3, 5, 7 and 10 min. The core temperature of the
samples was continuously measured during the process and timing was
started after the samples attained the target experimental temperatures
(Terefe et al., 2013). An example temperature profile (for the experi-
ment at 90 °C for 3 min) is shown in Fig. 1 in the Supplementary ma-
terial. The color of the samples were measured immediately after pro-
cessing while the samples for enzyme assays were kept at −18 °C until
analysis (Terefe et al., 2016). Samples for TPC and antioxidant capacity
analyses were also frozen immediately and freeze dried prior to ana-
lyses (Terefe et al., 2013).
2.4. HPT processing
A 35 L high pressure vessel (Flow Pressure System QuINTUS® Food
Press Type 35 L-600 sterilisation machine, Avure Technologies, Kent,
WA, USA) was used in the HPT processing experiments. Packed pear
puree samples were processed at 600 MPa and temperatures of 20, 55
and 90 °C. The holding times were 1, 3 and 5 min. The packed samples
and processing water were kept at predetermined initial temperatures
of 5, 35 and 65 °C, respectively to attain the target processing tem-
perature of 20, 55 and 90 °C after compression to 600 MPa. The high
pressure vessel was maintained at the experimental temperatures. Thus,
the pear puree was processed at isothermal-isobaric conditions
(600 MPa, 20, 55 and 90 °C) for 1, 3 and 5 min. An example tempera-
ture profile (for experiments at 600 MPa/90 °C for 5 min) is shown in
Fig. 2 in the Supplementary material. The temperature in the high
pressure vessel was measured using a high pressure resistant tempera-
ture logger (HP-T process logger) described by Knoerzer et al. (2010).
The compression rate was 4.2 MPa/s and the decompression rate was
~40 MPa/s. After HPT processing, samples were immersed in an ice-
water for rapid cooling. The color of the samples were measured im-
mediately after processing while the samples for enzyme assays were
kept frozen at − 18 °C until analysis (Terefe et al., 2016; Terefe, Yang,
Knoerzer, Buckow, & Versteeg, 2010). Samples for TPC and antioxidant
capacity analyses were also frozen immediately and freeze dried prior
to analyses (Terefe et al., 2013).
2.5. Color measurement and browning index
A Minolta Chromameter (Minolta CR 300, Japan) was used to
measure the surface color (CIE L*, a* and b* values representing
lightness, green-red and yellow-blue color coordinates respectively) of
the samples. Each sample was measured three times at three points and
the average value was calculated. Browning index (BI) was calculated
from the colour indices using the following equation (Ergunes & Sefa,
2006):
100(x − 0.31)
BI =
0.172 (1)
where:
(a∗ + 1.75L∗)
x=
(5.645L∗ + a − 3.012b∗) (2)
2.6. PPO and POD activity assays
The extraction and activity assays of pear PPO and POD were con-
ducted according to Terefe et al. (2016). The enzyme extraction solu-
tion was 0.1 M phosphate buffer (pH 6.5) containing 1 M NaCl, 4% (w/
v) polyvinylpolypyrrolidone and 1% (v/v) Triton X-100. The pear puree
was defrosted at room temperature and 10 g samples were mixed with
20 mL enzyme extraction solution. After homogenizing for 3 min, the
L. Zhou et al. Innovative Food Science and Emerging Technologies 45 (2018) 196–207

mixture was centrifuged (Sorvall RC 5C Plus) at 11,000g for 15 min at Table 1

4 °C. The supernatant was used as the crude enzyme extract for PPO and Physical and biochemical properties of pear puree.
POD activity assays.
Samples Non-acidified AD AA
For PPO assay, the reaction mixture consisted of 0.05 mL of enzyme
extract and 2 mL of 0.07 M catechol in sodium phosphate buffer pH 4.4 ± 0.1a 3.7 ± 0.1b 2.6 ± 0.1c
(0.05 M, pH 6.5) solution. The assay mixture was mixed and its ab- Brix 14.2 ± 0.1a 12.3 ± 0.1b 14.1 ± 0.2a
L⁎ value 47.9 ± 0.7c 52.0 ± 0.6b 55.0 ± 1.2a
sorbance was monitored at 420 nm for 1 min with a UV–visible spec-
PPO activity (OD/min/g FW) 6.9 ± 0.4a 6.7 ± 0.5a 3.4 ± 0.2b
trophotometer (UV-1700 Pharmaspec, Shimadzu) in a kinetic mode. POD activity (OD/min/g FW) 5.6 ± 0.1a 3.0 ± 01.b ND
For POD assay, the reaction mixture contained 0.04 mL of enzyme TPC (mg GAE/100 g FW) 20.7 ± 1.4b 16.5 ± 1.5c 31.2 ± 1.5a
extract, 0.1 mL of 1.0% (w/v) p-phenylenediamine, 0.1 mL of 1.5% (w/ ORAC (μmol TE/100 g FW) 300.3 ± 49.8b 246.3 ± 52.50b 447.7 ± 40
v) hydrogen peroxide and 2.7 mL sodium phosphate buffer (0.05 M,
Different letters in the same row represent significant difference (P < 0.05), FW: fresh
pH 6.5). The assay mixture was homogenized and its absorbance was
weight, GAE: Gallic acid equivalent, TE: Trolox equivalent, AD: acidified by dipping, AA:
monitored at 485 nm for 5 min with a UV–visible spectrophotometer in acidified by acid addition, ND: not detected.
a kinetic mode. The residual activities of the enzymes were calculated
in accordance with Eq. (3). 3. Results and discussion
Enzyme activity in processed sample
Residual activity (%) = × 100%. 3.1. Physical and biochemical properties of pear puree
Enzyme activity in untreated sample
(3) The pH of non-acidified pear puree was 4.4, decreasing to ~3.7
after dipping in 1.5% citric acid solution for 30 min. The pH of the AA
sample was much lower ~2.5 (Table 1). The soluble solid content
(Brix) of non-acidified and AD samples were 14.2 and 12.3, respec-
2.7. Total phenolic content (TPC) analysis
tively. During the dipping process, soluble solid from the pear slices
may have transferred to the low concentration dipping solution via
Extraction of phenolic compounds was carried out according to
osmosis resulting in reduced Brix of the AD samples. The SSC of AA
Ehala, Vaher, and Kaljurand (2005). Pear powder (40 mg) was dis-
samples was 14.1, which was due to the addition of critic acid after
persed in 2 mL of the extracting solution (70% methanol solution with
dipping. The browning of non-acidified pear puree occurred very fast
1% HCl). The mixture was sonicated for 40 min at 20 °C. Afterwards,
due to enhanced enzyme-substrate interaction during homogenization.
the extracts were centrifuged (Eppendorf 5417R) at 16400g for 15 min
Citric acid, both via dipping and direct addition, inhibited browning of
at 5 °C. The supernatant was used for TPC analysis. The TPC of the
pear puree (Fig. 1) and the L⁎ values of AD and AA samples were much
samples were analyzed in accordance with Folin-Ciocalteu method.
higher than the non-acidified samples (Table 1).
Accordingly, 20 μL of sample was diluted with 180 μL of water, and
Dipping treatment had little effect (P > 0.05) on PPO activity
then 1 mL of 0.2 N Folin-Ciocalteu reagent was added. After 3 min,
while about 50% decrease in PPO activity was observed in AA samples
800 μL of 75 g/L Na2CO3 was added and the samples were then in-
(Table. 1). The POD activity of non-acidified samples was 5.5 OD/min/
cubated for 45 min at 37 °C. The absorbance was measured at 765 nm
g FW. It decreased after dipping and there was no POD activity in the
with a UV–visible spectrophotometer and the TPC was expressed as
AA samples, indicating that pear POD was more susceptible to citric
milligram gallic acid equivalents per 100 g fresh weight based on a
acid than PPO. Similar effects were observed in an earlier study on pear
calibration curve developed using gallic acid as a standard.
slices where vacuum packing of sliced ‘Packham’ pears in syrups acid-
ified by citric acid resulted in 40% reduction of POD activity whereas
no inactivation of PPO was observed under the same condition (Terefe
2.8. Oxygen radical absorbance capacity (ORAC) analysis
et al., 2016). The TPC of AD samples was slightly lower (P < 0.05)
than the non-acidified, which might be due to the loss of water-soluble
Freeze-dried pear samples (40 mg) were mixed with 1.6 mL of the
polyphenols and other antioxidants during the dipping process. On the
extraction solvent (acetone-water mixture (80:20, v/v)). The mixture
other hand, the AA samples had ~50% higher TPC compared to the
was shaken for 60 min at 666 rpm and room temperature on a shaker
non-acidified samples. This could be due to the citric acid induced re-
(Heidolph multi reax). This was followed by centrifugation (Eppendorf
duction of the PPO and POD catalyzed oxidation of polyphenols in the
5417R) at 16400g for 15 min at 5 °C. The supernatant was used for
AA samples. The added citric acid might also contribute to the observed
subsequent ORAC analysis. The ORAC assay was carried out on a VIC-
TPC of the AA samples, since the TPC assay is based on the reduction of
TOR3 2030 microplate reader according to the method developed by
the Folin-Ciocalteu reagent where reducing agents other than poly-
Huang, Ou, Hampsch-Woodill, Flanagan, and Prior (2002). The ex-
phenols can contribute to the measured TPC value (Sánchez-Rangel,
citation wavelength and emission wavelength were 495 nm 515 nm,
Benavides, Heredia, Cisneros-Zevallos, & Jacobo-Velázquez, 2013). The
respectively. The experiment was conducted at pH 7.4 and 37 °C using a
ORAC antioxidant capacity showed the same trend as TPC with the
96-well plate. Fluorescein solution (150 μL), 25 μL of samples and 25 μL
highest ORAC value observed in the AA samples, which was ~50%
of buffer were added to each plate. The reaction was initiated by adding
higher than the non-acidified sample.
25 μL of AAPH and the decay of fluorescence was measured every 2 min
Citric acid is widely used to inhibit enzymatic browning of pro-
for 2 h and the area under the curve was calculated. Trolox was used as
cessed fruits. This is attributed to 1) the pH reduction of the matrix,
a standard.
which inhibits the activities of enzymes including PPO and POD (Terefe
et al., 2014; Zhou et al., 2016) and 2) chelating of metal ions (Ali, El-
Gizawy, El-Bassiouny, & Saleh, 2015; Terefe et al., 2014) essential for
2.9. Data analysis
the activity of the enzymes by citric acid i.e. copper ion in the active
sites of PPO (Mayer, 2006) and ferrous ion in the prosthetic heme group
All experiments were performed in triplicate. The values were ex-
of POD (Veitch, 2004). Some studies reported the effect of citric acid on
pressed as means ± standard deviation (SD). The analyses of variance
PPO and POD in tissue systems. Immersion of ‘Russet Burbank’ potato
(ANOVA) were performed for all experimental runs to determine sig-
slices in citric acid solution (1% and 2%) reduced PPO activity
nificance at 95% confidence. SPSS Version 17.0 was used for the sta-
(Tsouvaltzis & Brecht, 2017), while no inhibition of PPO activity was
tistical analysis.

198
L. Zhou et al.
observed in fresh-cut Fuji apples dipped in 0.5% citric acid (Chen, Hu,
He, Jiang, & Zhang, 2016). Similarly, POD activity in fresh-cut Chinese
water chestnut (Pen, Jiang, & Xu, 2004) and ‘Packham’ pear slices
(Terefe et al., 2016) decreased after dipping in citric acid solution as
observed in this study.
3.2. Thermal inactivation of PPO and POD in pear puree
The thermostability of PPO and POD in pear puree was investigated
first at temperatures between 25 and 100 °C. The thermal treatment
included the preheating step until the core of the sample reached the
target temperature and the subsequent 10 min isothermal treatment.
The PPO activity of non-acidified puree samples did not undergo sub-
stantial changes at temperature between 25 and 70 °C with a slight
increase (P < 0.05) of about 10% observed after 10 min at 60 °C
(Fig. 2A). At temperatures higher than 70 °C, the PPO activity of non-
acidified samples drastically decreased (P < 0.05) and the residual
activity was 37.8% after 10 min treatment at 90 °C. Similar thermo-
stability of PPO was observed in AD samples, with the activity re-
maining the same (P > 0.05) after treatments at temperatures between
25 and 60 °C (Fig. 2A). However, processing at 70 °C and higher for
10 min induced a significant drop (P < 0.05) in PPO activity of AD
samples. This indicates the sensitizing effect of citric acid on pear PPO
to thermal inactivation. The maximum inactivation of 59.0% was ob-
served after 10 min treatment at 80 °C with no further increase
(P > 0.05) in the level of inactivation with increase in temperature to
90 and 100 °C. With regard to AA samples, the added citric acid induced
50.1% inactivation of PPO. The activity decreased to 38.8% as tem-
perature increased to 80 °C, with no further decrease (P > 0.05) with
increase in treatment temperature, indicating the existence of a ther-
mostable PPO isoenzyme fraction, which is also resistant to inactivation
by citric acid or low pH.
Further studies on the isothermal inactivation kinetics of PPO were
conducted at 90 °C (Fig. 2C) and 100 °C (Fig. 2D). At 90 °C, the PPO
activity in non-acidified and AD samples decreased continuously and
the residual activity was about 40% after 10 min at 90 °C. On the other
hand, the PPO activity of AA samples decreased to about 40% during
the first 1 min and no significant inactivation (P > 0.05) was observed
with increase in treatment time (Fig. 2C). At 100 °C, substantial in-
activation of PPO was observed in all samples during temperature
equilibration (t = 0) (Fig. 2D). The PPO activity in non-acidified and
AD samples decreased further albeit slowly with no further inactivation
during longer treatment. On the other hand, no significant decrease
(P > 0.05) in PPO activity was observed during the thermal treatment
of AA samples after temperature equilibration. The residual PPO ac-
tivity in all samples was about 40%, further confirming the existence of
a thermostable PPO isoenzyme in pear which is also resistant to in-
activation by citric acid or low pH.
For PPO in Taylor's Gold pear puree, no thermal inactivation was
observed between 32 and 71 °C (Sulaiman, Soo, Farid, and Silva, 2015),
which is similar with the result in this study. However, the enzyme was
less resistant to thermal treatment at 80 °C and about 85% inactivation
was achieved after 10 min (Sulaiman, Soo, Yoon, et al., 2015). Likewise
PPO in Rabdosia serra (Maxim.) Hara was totally inactivated by thermal
treatment at 90 °C for 6 min (Lin et al., 2012). Similar results were
observed in apple (Pink lady; Granny smith; Jonagold) juice (Yi et al.,
2017). On the other hand, PPO from some plants was found to be highly
thermostable. For example, PPO in strawberry puree (cv. Aroma) was
not inactivated even after 30 min at 100 °C (Terefe et al., 2010). Si-
milarly, Deylami, Rahman, Tan, Bakar, and Olusegun (2016) reported
that complete inactivation of PPO from mangosteen pericarp (Garcinia
mangostana L.) was not possible even after 12 min at 100 °C. Clearly,
there is a wide variability in the thermostability of PPO depending on
the source, cultivar and matrix composition.
Similar percentage residual PPO activity (~ 40%) remained in all
pear puree samples (non-acidified, AD and AA) after 10 min treatment
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Innovative Food Science and Emerging Technologies 45 (2018) 196–207
at temperatures as high as 100 °C, which indicates the presence of a
thermostable PPO isoenzyme in pear which is also resistant to in-
activation by citric acid or low pH. The existence of PPO isoenzymes
have been reported in several plants such as Ataulfo mango
(Cheema & Sommerhalter, 2015), lettuce (Lactuca sativa L. var. capitata)
(Zlotek & Gawlik-Dziki, 2015) and ‘Yali’ pear (Yan, Li, He, Liang, & Li,
2013). Yan et al. (2013) reported that six PPO isoenzymes were de-
tected in ‘Yali’ pear (Pyrus bretschneideri Rehd).
In contrast to PPO, POD in pear puree displayed very high sensi-
tivity to thermal inactivation (Fig. 2B). The activity of POD in non-
acidified samples remained stable after 10 min treatments at tempera-
ture between 25 and 50 °C. Substantial inactivation of the enzyme was
observed after treatments at temperatures higher than 50 °C with
complete inactivation after 10 min treatment at 80 °C. Moreover, higher
thermosensitivity of POD was observed in AD samples. Thermal treat-
ment at 50 °C for 10 min significantly decreased POD activity and the
enzyme was completely inactivated after treatments at 70 °C and
higher. With regard to AA samples, POD activity was below detection
limits indicating the more pronounced effect of citric acid and reduced
pH on the activity of POD compared to PPO in pear puree. Likewise, pH
reduction induced by ascorbic acid resulted in a higher inactivation of
POD than PPO in lettuce (Lactuca sativa L. var. capitata) (Landi,
Degl'Innocenti, Guglielminetti, & Guidi, 2013). Although, POD as the
most thermoresistant enzyme in vegetables, being the established in-
dicator enzyme for the adequacy of blanching processes, PPO is more
thermoresistant compared to POD in many fruits (Vamos-
Vigyazo & Haard, 1981). There are no studies in literature that com-
pared the thermostability of pear PPO with POD.
3.3. Effect of HPT processing on the activity of PPO and POD
As shown in Fig. 3A, 600 MPa high pressure at 20 °C had a limited
effect on the activity of PPO in pear puree regardless of the degree of
acidification. No increase in the level of inactivation was observed with
increase in treatment time. Likewise, no inactivation of PPO was ob-
served in non-acidified puree samples during HPT treatment at 55 °C.
Rather, slight increases (P < 0.05) in the activity of PPO were ob-
served after 3 min treatment at 20 °C and 1 min treatment at 55 °C,
which is most probably due to increase in enzyme extraction as a
consequence of high pressure induced tissue disruption. Such an in-
crease in enzyme activity following high pressure processing at mild
temperature condition is commonly observed in tissue systems (Terefe
et al., 2010; Terefe et al., 2016).
HPT processing at 55 °C resulted in a significant inactivation
(P < 0.05) of PPO in AD samples with 36.2% inactivation after 5 min
treatment, indicating the sensitizing effect of citric acid and low pH on
pear PPO towards HPT inactivation as in the case of thermal inactiva-
tion (Fig. 3B). Interestingly, no significant inactivation (P > 0.05) of
PPO was observed in the AA samples under this condition. Never-
theless, the residual activity of PPO in the AD samples after 5 min
treatment was similar to that of the AA samples (P > 0.05). It is pos-
sible that the PPO fraction that is sensitive to combined citric acid and
HPT treatment at 55 °C had already been inactivated in the AA samples
during acidification. That may explain the apparent stability of the
remaining PPO towards HPT treatment at 55 °C in AA samples. In-
creasing the processing temperature to 90 °C led to significant in-
activation (P < 0.05) of PPO in all samples (Fig. 3C). The inactivation
of PPO in non-acidified samples increased with increase in processing
time to a maximum of 41% after 5 min treatment at 90 °C. The in-
activation effect under the same condition was higher (P < 0.05) in
AD samples with 58.7% inactivation observed after 5 min HPT treat-
ment at 90 °C. The PPO activity in AA samples decreased from 53.8% to
43.0% after 1 min treatment at 90 °C and no further inactivation was
observed during longer processing times (Fig. 3C). The maximum in-
activation under the most intense HPT processing condition in-
vestigated (600 MPa, 90 °C, 5 min) was about 60% in AD and AA puree
L. Zhou et al.
samples, with a residual activity of about 40% as in the case of thermal
treatment indicating that the thermoresistant PPO fraction is also
pressure resistant. As in the case of thermal processing, the presence of
citric acid enhanced the inactivation effect of HPT on pear PPO. High
pressure slightly inhibited the inactivation of pear PPO in non-acidified
samples at 90 °C, which is opposite to the commonly observed sy-
nergistic effect of high pressure and elevated temperature on the in-
activation of enzymes (Terefe et al., 2014). On the contrary, the ap-
plication of high pressure increased the inactivation of PPO in AD
samples both at 55 °C and 90 °C. No inactivation of PPO was observed
in AD samples after 10 min treatment at 60 °C under atmospheric
condition whereas 36.2% inactivation was observed after 5 min treat-
ment at 55 °C and 600 MPa (Figs. 2A and 3B). It appears that effects of
temperature and pressure on PPO become synergistic with acidifica-
tion. Similar result was observed in high-pressure processed pineapple
(Chakraborty et al., 2014).
Unlike PPO, high pressure processing at 20 °C for 3 and 5 min had
significant inactivation effects (P < 0.05) on POD in non-acidified
samples (Fig. 3D). Processing at 20 °C for 5 min resulted in 25.0% de-
crease in POD activity. For AD samples, POD activity decreased from
61.7% to 48.4% after processing at 20 °C for 5 min. POD activity was
negligible in AA samples, i.e. it was below the detection limit. The in-
activation of POD increased with increase in HPT temperature to 55 °C
both in the non-acidified and AD samples (Fig. 3E). After HPT proces-
sing at 55 °C for 5 min, the residual activity of POD was 62.5% and
24.2% for non-acidified and AD samples, respectively. HPT at 90 °C
induced rapid decrease in POD activity (Fig. 3F). The residual activity
of POD in non-acidified sample was only 12.3% after HPT processing at
90 °C for 5 min, whereas, POD in AD samples was completely in-
activated after processing for 1 min. About 75% inactivation of pear
POD in non-acidified samples occurred after 1 min treatment under the
same condition. These results indicate that pear POD is relatively more
sensitive to HPT inactivation compared to PPO and HPT at high tem-
perature completely inactivate the enzyme in acidified pear puree
within a very short time.
The effect of HPP or HPT on PPO in plant tissues is widely studied
and results vary. In an earlier study on ‘Packham’ pear slices packed in
syrup acidified with citric acid (Terefe et al., 2016), a significantly
higher inactivation was observed during HPT compared to this study at
all the studied condition with 80% inactivation of PPO after 3 min
treatment at 600 MPa and 80 °C compared to a maximum of 60% in-
activation after 5 min treatment at 600 MPa and 90 °C in this study.
HPP at room temperature also induced inactivation of PPO in ‘Tongzi I’
strawberry pulps (Cao et al., 2011) and Amasya apple juice (Bayındırlı
et al., 2006). On the other hand, PPO in some plants tissues was re-
ported to be highly resistant to HPT. For instance, high pressure pro-
cessing at temperature ranging from 24–90 °C, pressure ranging from
100 to 690 MPa and treatment times between 5 and 10 min did not
have significant effect on PPO in ‘Aroma’ strawberry puree (Terefe
et al., 2010). PPO in pumpkin puree was found to be resistant to HPT
with only 40% to 50% inactivation after 1 min treatment at 900 MPa
and 80 °C (García-Parra et al., 2016). A number of studies have shown
that acidic media can increase the susceptibility of PPO to HPP in-
activation, in agreement with our observation in this study. For in-
stance, Chakraborty et al. (2014) observed increased inactivation of
PPO in pineapple puree at lower pH. Weemaes, Ludikhuyze, Broeck,
and Hendrickx (1998) reported that the threshold pressure for in-
activation of avocado PPO at room temperature decreased from
850 MPa at pH 8.0 to 450 MPa at pH 4.0.
Significant inactivation of POD by HPP at room temperature has
been reported in many plants such as ‘Aroma’ (Terefe et al., 2010) and
‘Tongzi I’ (Cao et al., 2011) strawberry, pineapple (Chakraborty et al.,
2014) and ‘Packham’ pear (Terefe et al., 2016) as in this study. On the
other hand, POD in Reineta apple slices was found to be highly resistant
to HPP inactivation with no inactivation observed at pressure lower
than 800 MPa (Prestamo, Arabas, Fonberg-Broczek, & Arroyo, 2001).
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Innovative Food Science and Emerging Technologies 45 (2018) 196–207
Mild or high temperatures have been investigated to enhance the sen-
sitivity of POD to high pressure inactivation. Pressure and temperature
acted in synergistic manner on POD inactivation in pineapple at pres-
sure from 400 to 600 MPa and temperatures from 30 to 70 °C
(Chakraborty et al., 2015). POD in ‘Aroma’ strawberry puree was al-
most completely inactivated after 5 min treatment at 90 °C regardless of
the pressure level (Terefe et al., 2010). A study on ‘Packham’ pear slices
packed with citric acidified syrup reported similar sensitivity of POD in
pear slices towards HPP at 20 °C and HPT at high temperature (Terefe
et al., 2016). However, HPT at 40 and 60 °C resulted in activity increase
of POD, which might be attributed to higher release and extraction of
bound POD (Terefe et al., 2016).
Our results showed that compared to PPO, pear POD was more
sensitive towards HPP and HPT as well as heat inactivation. Similar
higher sensitivity of POD towards HPP or HPT induced inactivation
compared to PPO has been reported for ‘Aroma’ strawberry (Terefe
et al., 2010), pineapple (Chakraborty et al., 2014) and ‘Hass’ avocado
(Woolf et al., 2013). In contrast, POD in ‘Packham’ pear slices packed in
acidified syrup was found to be more resistant to HPT inactivation at
temperatures up to 60 °C compared to PPO (Terefe et al., 2016). The
sugar in the syrup may have affected the sensitivity of PPO and POD
differently rendering PPO more susceptible to HPT inactivation com-
pared to POD.
Organic acids such as citric acid and ascorbic acid are commonly
used in the food industry for controlling enzymatic browning in pro-
cessed fruits and vegetables. The effects of organic acids are not limited
to their effect on the matrix pH but other chemical mechanisms are also
involved. With respect to citric acid, both its metal chelating effect and
pH reduction contribute to its inhibitory effect on PPO and enzymatic
browning (Ali et al., 2015; McEvily, Iyengar, & Otwell, 1992). A
number of studies have shown that organic acids and other anti-
browning agents augment the effect of HPP or HPT on oxidative en-
zymes and reduce browning via pH reduction and other mechanisms
(Chakraborty et al., 2014; Guerrero-Beltran, Swanson, & Barbosa-
Canovas, 2005; Kingsly, Balasubramaniam, & Rastogi, 2009). The con-
formation of enzymes is generally maintained by covalent bonding,
hydrogen bonding, hydrophilic and hydrophobic interactions
(Tauscher, 1995). HPP disrupt the natural balance of the hydrophobic
interaction and change the conformation of enzymes, leading to in-
activation (Yi et al., 2012). Ionita, Stanciuc, Aprodu, Rapeanu, and
Bahrim (2014) stated that in acidic pH the dissociation followed by the
unfolding of the native tetrameric PPO state predominate, causing ex-
posure of the hydrophobic residues. The exposure of hydrophobic
groups and the disruption of enzyme conformation are generally more
pronounced with the combination of low pH and high pressure
(Chakraborty et al., 2014) and may explain the synergistic inactivation
effect of acidic pH and HPT observed in this study.
3.4. Effect of processing on the color of pear puree
Substantial change in color occurred in the non-acidified samples
during pureeing due to enzymatic browning whereas the AD and the AA
samples retained the initial color of the pear slices (Fig. 1). The effect of
HPT processing on color was dependent on the type of sample and the
processing condition (temperature and time). Data on the effect of HPT
processing on the lightness of pear puree are shown in Fig. 4A–C.
Overall, high pressure processing at 20 and 55 °C had minimal effect on
L⁎ value of non-acidified samples (Fig. 4A and B). HPT at 90 °C resulted
in a significant increase in L⁎ values with increase in processing time
(Fig. 4C). The L⁎ value increased from the initial low value of 47.4 to
51.3 and 51.6 respectively after 3 and 5 min processing at 90 °C. This
was accompanied by visually noticeable lightening of the samples that
were brown prior to processing. This was unexpected since non-enzy-
matic browning occurs at such high processing temperature, which
would have the opposite effect on the color of the samples. It is possible
that HPT at 90 °C might have induced the leakage of pigments from the
L. Zhou et al. Innovative Food Science and Emerging Technologies 45 (2018) 196–207

Fig. 1. Photographs of non-acidified pear puree, pear puree

samples acidified by dipping (AD) and acid addition (AA) in
transparent retort pouches immediately after sample pre-
paration.

Fig. 2. The effect of thermal treatment for 10 min on the activity of PPO (A) and POD (B) in pear puree samples. The thermal inactivation of PPO in pear puree at 90 °C (C) and 100 °C (D).
Control: non-acidified, AD or AA, samples which were not thermally processed. Time 0 samples are samples that were pre-heated to the target experimental temperatures and cooled
immediately after that. Timing in all cases started after the samples attained the target experimental temperatures and the thermal treatment includes the preheating step to the target
temperature.

outermost cell layers into the intracellular space or the exudation of
pigments with the tissue fluid (Krebbers, Matser, Koets, & Van den Berg,
2002; Terefe, Matthies, Simons, & Versteeg, 2009), counteracting the
effect of non-enzymatic browning. The intense process may also have
induced the degradation of the brown pigments formed by enzymatic
oxidation. Moreover, HPT at 90 °C caused substantial inactivation of
PPO and POD in non-acidified samples and may have inhibited further
enzymatic browning.
Interestingly, high pressure processing at all temperatures caused a
slight but significant decrease in the lightness of AD and AA samples.
The effect was not visually noticeable since the initial L⁎ values of these
samples were relatively high. The decrease in lightness after processing
at 55 and 90 °C might be due to non-enzymatic browning. Several
studies have reported non-enzymatic browning and a decrease in L⁎
value induced by thermal or HPT processing (Deylami et al., 2016;
Terefe et al., 2016). The color change during processing at 20 °C could
be due to enzymatic browning facilitated by increased enzyme-sub-
strate interaction as a consequence of high pressure induced cell dis-
ruption.
The effect of HPT on the browning index of pear puree is shown in
Table 1 in the Supplementary material. The highest browning index of
9.27 was observed in non-acidified pear puree. Compared to non-

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L. Zhou et al.
Fig. 3. Effect of high pressure-thermal processing (600 MPa) on the activity of oxidative enzymes in pear puree samples. (A) (B) (C): PPO; (D) (E) (F): POD. Control: non-acidified, AD or
AA, samples which were not processed by high pressure thermal processing.
acidified pear puree, the browning index of AD and AA samples were
much lower. For non-acidified pear puree, only HPT at 90 °C for 3 and
5 min resulted in significant decreases in browning index, which was in
accordance with the increase in lightness. High pressure processing at
20 °C caused significant increases in the browning index of AD samples.
High pressure processing at all temperatures had minimal effect in the
browning index of AA samples; only HPT at 55 °C for 3 and 5 min re-
sulted in significant increases in browning index. Overall, the changes
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Innovative Food Science and Emerging Technologies 45 (2018) 196–207
in browning index of pear puree were related to the changes in light-
ness. The reductions in browning index corresponded with increases in
lightness.
The data on the effects of thermal processing on the lightness of pear
puree samples are presented in Fig. 4D–F. As in the case of HPT, the L⁎
value of non-acidified samples increased after thermal processing at 90
and 100 °C. The L⁎ value increased with processing time up to 3 min
with no further increase observed during longer processing (Fig. 4D).
L. Zhou et al. Innovative Food Science and Emerging Technologies 45 (2018) 196–207

Fig. 4. Effect of high pressure-thermal (600 MPa) and thermal treatment processing on lightness (L⁎ value) of pear puree samples. (A) (B) (C): HPT; (D) (E) (F): thermal treatment.
Different letters on the bars within the same group represent significant difference (P < 0.05). Control: non-acidified, AD or AA, samples which were not processed by thermal or high
pressure processing. Time 0 samples are samples that were pre-conditioned and pressurized to the target experimental temperature and pressure and immediately depressurized with zero
hold time.

203
L. Zhou et al. Innovative Food Science and Emerging Technologies 45 (2018) 196–207

Fig. 5. Effect of high pressure-thermal processing (600 MPa) on TPC and ORAC antioxidant capacity of pear puree samples. (A) (B) (C): TPC; (D) (E) (F): ORAC. Different letters on the
bars within the same group represent significant differences (P < 0.05). Control: non-acidified, AD or AA samples which were not processed by high pressure thermal processing.

Similar mechanisms as in HPT may explain the observed increase in
lightness after thermal processing. In the case of AD samples, L⁎ value
increased after 0 to 5 min thermal processing at 90 °C with the highest
lightness of 56.6 after 5 min processing. However, further thermal
treatment induced significant decrease of L⁎ value. At 100 °C, similar
changes were observed and the highest lightness was observed after
1 min processing (Fig. 4E). These results might be due to initial exu-
dation of pigments with the tissue fluid dominating the effects of non-
enzymatic browning during shorter processing times. With respect to
AA samples, a significant decrease in L⁎ values was observed after 7 and
10 min at 90 °C. At 100 °C, the decrease in L⁎ values was higher and
occurred even after the shortest processing time of 1 min (Fig. 4F). It
seems that the effect of non-enzymatic browning dominated during
processing at 100 °C and counteracted the possible effect of exudation
of pigments with the tissue fluid.

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3.5. Effect of HPT processing on the TPC and ORAC of pear puree
The TPC values of HPT processed pear puree ranged from 16.2 to
37.7 mg GAE per 100 g fresh weight. The observed values are in the
range (109–261 mg/100 g dry weight) reported for different pear cul-
tivars (Yim & Nam, 2016). The added citric acid induced significant
changes in pear puree and the TPC in AA samples was the highest. High
pressure processing at 20 °C had no significant effect on the TPC of pear
puree (Fig. 5A). At 55 °C, a significant increase in TPC values was ob-
served in AD samples after HPT processing for 5 min (Fig. 5B). Sig-
nificant increase in TPC values was also observed in AD and AA samples
treated at 600 MPa and 90 °C (Fig. 5C). These changes in TPC might be
due to increased extractability of polyphenols (Patras et al., 2009) and
other antioxidants due to tissue disruption coupled with reduced en-
zymatic degradation resulting from substantially reduced activity of
PPO and POD at these conditions (Terefe et al., 2013). In the non-
acidified samples and during processing at 20 °C, enzymatic degrada-
tion may have counteracted the effect of tissue disruption on extraction.
Enzymatic oxidation played a major role in the degradation of poly-
phenolic compounds in high pressure processed samples (Terefe et al.,
2013). PPO catalyzes the hydroxylation of monophenols to o-diphenols
and the oxidation of o-diphenols to o-quinones in the presence of
oxygen (Espín, Jolivet, & Wichers, 1998). POD also catalyzes the oxi-
dation of polyphenolic compounds in the presence of hydrogen per-
oxides (Terefe et al., 2013).
With respect to ORAC antioxidant capacity, citric acid treatment
induced a similar change as TPC. The ORAC antioxidant capacity in AA
samples was the highest. High pressure processing had little effect on
the ORAC antioxidant capacity of pear puree samples regardless of
sample formulation or processing condition, the exception being the
slight but significant increase in ORAC antioxidant capacity in AD
samples after HPT processing at 90 °C for 3 min (Fig. 5F). A different
trend was observed to that of TPC, which might be due to the assays
being based on different chemical principles. The Folin-Ciocalteu
method that is used to measure TPC is based on an electron transfer
reaction (Huang, Ou, & Prior, 2005), while the ORAC assay is based on
a hydrogen atom transfer reaction mechanism (Prior, Wu, & Schaich,
2005).
Some studies reported no significant change in TPC following high
pressure or HPT processing of fruit puree (Cao et al., 2011; Terefe et al.,
2009). On the other hand, Patras et al. (2009) and Corrales, Toepfl,
Butz, Knorr, and Tauscher (2008) reported a slight increase in TPC of
blackberry puree (Rubus fruticosus cv, Loughness) and Dornfelder grape
by-products following high pressure processing. The increase in TPC
might be related to an increased extractability due to tissue disruption
induced by high pressure (Patras et al., 2009). However, there are a
number of studies that reported a decrease in TPC induced by thermal
treatment or HPT processing at high temperature (Chen, Hu, et al.,
2016; Chen, Pan, et al., 2016; Terefe et al., 2013). Nevertheless, the
decrease in TPC may not be necessarily due to degradation of poly-
phenols but also other antioxidant compounds such as vitamin C, which
contribute to the antioxidant capacity of the material. With respect to
ORAC antioxidant capacity of fruit products, Terefe et al. (2013) re-
ported that both thermal and high pressure processing (600 MPa, 25 °C)
induced slight but significant decreases in ORAC antioxidant capacity of
strawberry (Camarosa, Festival, and Rubygem). In contrast, significant
increase in ORAC of gooseberry pulp was observed after 1, 3 and 5 min
treatment at 500 MPa and room temperature (Vega-Gálvez et al.,
2014). Hence, the effect of high pressure on ORAC antioxidant capacity
of fruit products could be dependent on on the type of fruits as well as
the processing condition.
3.6. Comparison of thermally and HPT processed pear puree with
equivalent enzyme inactivation
Conventional thermal processing is the most common technology
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Innovative Food Science and Emerging Technologies 45 (2018) 196–207
used in the preservation of food products. In order to determine whe-
ther high pressure results in better preservation of pear puree, the
quality attributes of samples produced by the two processes under
equivalent level of PPO and POD inactivation were compared (Table 2).
For the non-acidified samples, 40.8% inactivation of PPO and 87.7%
inactivation of POD were attained after HPT at 90 °C for 5 min. Thermal
treatment at 90 °C for 3 min and 100 °C for 0 min resulted in a similar
effect. As can be seen in Table 2, the three processes resulted in similar
quality retention of non-acidified pear puree with no significant dif-
ferences in color (L⁎ value), TPC and ORAC antioxidant capacity. For
the AD samples, HPT at 90 °C for 5 min resulted in 58.7% inactivation
of PPO and complete inactivation of POD. The same inactivation effect
was attained with thermal treatment at 90 °C for 5 min and 100 °C for
3 min. There was no significant difference in TPC and ORAC anti-
oxidant capacity of pear puree after HPT and thermal treatments.
However, better color i.e. a higher L⁎ value was observed after thermal
treatment compared to HPT. Similar results were found in AA samples.
HPT at 90 °C for 1 min, thermal treatment at 90 °C for 3 min and 100 °C
for 0 min resulted in the same inactivation effect on PPO and POD,
while the L⁎ value of AA samples after thermal treatment was much
higher than that after HPT. Compared with HPT, thermally processed
AA samples showed slightly higher TPC and ORAC antioxidant capacity
although the difference was not statistically significant. It has to be
noted that this study compared only quality attributes related to the
activity of oxidative enzymes under similar inactivation condition. It is
possible that HPT may result in better retention of flavor, rheological
properties and other quality attributes, which need further investiga-
tion.
Although many studies reported high pressure processing had lim-
ited effects on color and nutritional values of products making it an
interesting alternative to thermal treatment (Marszałek,
Mitek, & Skąpska, 2015; Oey, Lille, Van Loey, & Hendrickx, 2008), some
studies found that HPP did not offer advantages in regards to some
quality attributes of food products. Terefe et al. (2013) reported that no
significant difference was observed in ORAC antioxidant capacity re-
tention of strawberry after high pressure and thermal pasteurization,
and high pressure did not have an advantage in the preservation of
phenolic phytochemicals. Polydera, Stoforos, and Taoukis (2003) ob-
served that color changes were not significantly different between
thermal and pressure treated orange juice.
4. Conclusion
This study showed that pear PPO is a thermostable and barostable
enzyme with no significant inactivation during thermal processing at
temperatures as high as 70 °C and high pressure treatment at 600 MPa
and 20 and 55 °C. On the other hand, POD was found to be relatively
more thermolabile and pressure labile. The addition of citric acid in the
puree enhanced the susceptibility of both PPO and POD to thermal and
combined high pressure-thermal inactivation. Acidification alone to
pH 2.5 resulted in complete inactivation of POD and partial inactivation
(50%) of PPO. The maximum PPO inactivation was ~60% regardless of
the degree of acidification and the intensity of the thermal and high
pressure processes, indicating that pear PPO has a stable isoform which
is resistant to temperature, pressure and citric acid induced inactiva-
tion.
Both dipping and acid addition treatment inhibited enzymatic
browning and preserved the color of pear puree. Acidification by direct
addition of citric acid also enhanced the antioxidant capacity of pear
puree. Processing on the other hand did not have significant effects on
TPC and ORAC antioxidant capacity of pear puree regardless of the
degree of acidification. Quality retention of pear puree after equivalent
(on the basis of equivalent PPO and POD inactivation) thermal and high
pressure processing with respect to color, TPC and ORAC antioxidant
capacity was largely similar with no advantage of high pressure pro-
cessing over thermal processing. The study however was limited to
L. Zhou et al. Innovative Food Science and Emerging Technologies 45 (2018) 196–207

Table 2
Comparison of the quality of thermally and HPT processed pear puree with equivalent enzyme inactivation.

Sample Treatment Time (min) Residual PPO (%) Residual POD (%) L⁎ value TPC (mg GAE/100 g FW) ORAC (μmol TE/100 g FW)

Non-acidified 90 °C/600 MPa 5 59.2 ± 2.0a 12.3 ± 1.5a 51.6 ± 0.50a 18.8 ± 1.00a 245.9 ± 18.4a
90 °C 3 62.6 ± 3.2a 10.1 ± 2.1a 52.4 ± 1.3a 18.3 ± 1.5a 257.4 ± 20.9a
100 °C 0 53.3 ± 1.7b 6.2 ± 0.6b 51.5 ± 1.1a 18.6 ± 0.8a 274.4 ± 16.8a
AD 90 °C/600 MPa 5 41.3 ± 1.7a ND 52.30 ± 1.20a 20.9 ± 1.00a 282.5 ± 28.7a
90 °C 5 45.0 ± 1.9a ND 56.6 ± 0.6b 22.218 ± 0.3a 287.4 ± 40.1a
100 °C 3 41.1 ± 1.1a ND 55.1 ± 0.6b 20.4 ± 0.9a 322.0 ± 23.1a
AA 90 °C/600 MPa 1 45.4 ± 5.0a ND 51.3 ± 0.4a 35.2 ± 1.3a 415.3 ± 49.8a
90 °C 3 45.2 ± 2.8a ND 56.6 ± 0.3b 36.6 ± 1.3a 467.1 ± 17.3a
100 °C 0 43.9 ± 0.7a ND 56 ± 0.4b 37.7 ± 1.1a 463.9 ± 24.0a

Different letters in the same group represent significant difference (P < 0.05). Time 0 samples are samples that were heated or pressurized to the target experimental temperatures or
pressure after preprocessing and immediately cooled or depressurized. FW: fresh weight, GAE: gallic acid equivalent, TE: trolox equivalent, AD: acidified by dipping, AA: acidified by acid
addition, ND: not detected.

quality attributes related to the activity of oxidative enzymes and did
not include flavor, rheology and other quality parameters, where high
pressure processing may have distinct advantage. Future work will in-
vestigate the comparative effects of thermal and high pressure proces-
sing on other quality attributes and the impact of the residual PPO
activity on color and antioxidant capacity of pear puree during storage.
Acknowledgments
We gratefully acknowledge the support provided by Rod Smith and
Daryl Unthank during the high pressure processing. We also thank
China Scholarship Council (No. 201606820014) for providing funding
for Lei Zhou. We are also grateful for the financial support of this study
by the National Natural Science Foundation of China (No. 31460435).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ifset.2017.10.012.
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