Kinetic modelling of native apple polyphenol oxidases and

peroxidases inactivation under supercritical carbon

dioxide
Krystian Marszałeka,*, Bartosz Kruszewskia, Łukasz Woźniaka, Sylwia Skąpskaa
a
Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology, Department of Fruit and
Vegetable Product Technology, 36 Rakowiecka St., 02532 Warsaw, Poland
* krystian.marszalek@ibprs.pl

ABSTRACT
Supercritical carbon dioxide (SCCD) was applied for the native apple enzyme inactivation.
The polyphenol oxidases (PPO) and peroxidases (POD) inactivation kinetics significantly
depended on process parameters and kind of enzymes. An increase in the temperature and
pressure resulted in increasing of the k-value and decrease of D-value calculated for PPO and
POD. POD turned out to be more pressure and less temperature resistant compared to PPO.
The lowest decimal reduction time (D-value) and zT and zP- values were noted at 65°C and
60 MPa for PPO: 33.8 min, 90.3°C and 112.6 MPa, respectively. The activation energy (Ea)
increased for PPO and decreased for POD with increasing the pressure. Activation volume
(Va) generally increased with increasing the temperature (except for the harshest condition of
SCCD).
INTRODUCTION
Polyphenol oxidase (PPO, EC 1.14.18.1) and peroxidase (POD, EC 1.11.1.7) are the principal
enzymes involved in enzymatic browning of fruit and vegetable products. PPO is a copper-
containing enzyme responsible for hydroxylation of monophenols to o-diphenols which react
with endogenous amino acids and proteins to form complex brown pigments (Gong, Li, Liu,
Cheng, & Wang, 2015). POD carries a ‘b’-type haem as a prosthetic group, and as another
oxidoreductases, participates in several metabolic plant processes such as: catabolism of
auxins, lignification of the cell wall, oxidation of various electron donors with H2O2 (Elstner
& Heupel, 1976).
Supercritical carbon dioxide (SCCD) processing can be used as an alternative method to
thermal pasteurization. SCCD carried out at temperatures and pressures which are relatively
safe for heat-labile compounds, as well as sufficient for the inactivation of microorganisms
and tissue enzymes, enables to obtain juices of superior quality compared to pasteurized ones
(Bi, Wu, Zhang, Xu & Liao, 2011, Fabroni, Amenta, Timpanaro & Rapisarda, 2010,
Ferrentino & Spilimbergo, 2011, Gui, Wu, Chen, Liao, Hu, Zhang & Wang, 2007, Damar &
Balaban, 2006, Liu, Hu, Zhao & Song, 2012, Zhong, Black, Davidson & Golden 2008). A
limited number of reports have dealt with the influence of pressurized carbon dioxide on
enzymes, but our previous studies showed that SCCD gave satisfactory results of enzyme
inactivation in the fruit and vegetable matrix (Marszałek, Krzyżanowska, Woźniak &
Skąpska, 2016, Marszałek, Skąpska, Woźniak & Sokołowska, 2015). The mechanism of
enzyme inactivation by SCCD is hypothesized to be the result of lowering of local pH
(Chakraborty, Rao & Mishra, 2015). According to the other authors carbon dioxide under
pressure could cause changes in the conformation of the secondary structure of the enzyme
(Manzocco, Ignat, Valoppi, Burrafato, Lippe, Spilimbergo, Nicoli, 2016). Despite these
findings, the effect of SCCD treatment on the activity and structure of food enzymes is still
under study.

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 Apple juice is one of the most frequently consumed juices in the world. It is
commonly served as a clear juice, but there is a growing market for cloudy apple juices
(Braun, 2003). Apples and its preparations are recognized as promoters of health because of
their high content of bioactive compounds such as polyphenols, pectins and organic acids
(Włodarska, Pawlak-Lemańska, Górecki & Sikorska, 2016).
The majority of consumers prefer cloudy apple juice as a light, whitish yellow juice
with significant cloudiness and without sedimentation, harmonized in terms of sweet and sour
taste (Niu, Xu, Fang, Zhang, Yang, Liao & Hu, 2010). Unfortunately, during juice processing
and storage browning is initiated by enzymatic oxidation of polyphenols to coloured
quinones, which are then subjected to further reactions, leading to pigment formation (Lee,
Seo, Rhee & Kim, 2016). Browning is a major problem for many fruit and vegetable juices
due to the lowering of sensory quality and nutritional value. Fresh apple juice is highly
susceptible to enzymatic browning, because of considerable activity of oxidizing enzymes.
Therefore, proper control of the browning reaction in apple juice has gained growing attention
in the fruit processing industry (Lee, Seo, Rhee & Kim, 2016).
The present study was addressed to investigate the influence of SCCD treatment on the
PPO and POD inactivation kinetics of native enzymes present in apple juice. Fresh apple juice
were exposed to supercritical CO2 at different temperatures (35, 45, 65 °C), times (10, 20, 30
min) and pressures (10, 30, 60 MPa). Inactivation rate constants were used to estimate
decimal reduction time (D-value), 90% reduction of the D-value under different temperatures
and pressures (zt and zp -values), activation energy (Ea) and activation volume (Va) based on
the Eyring equation (Gui, Wu, Chen, Liao, Hu, Zhang & Wang, 2007, Manzocco, Ignat,
Valoppi, Burrafato, Lippe, Spilimbergo & Nicoli, 2016).

MATERIALS AND METHODS

Times New Roman, 12, single space
1.1.Reagents
The following chemicals were used in the study: polyvinylpyrrolidone (PVP) (~110 µm)
(Fluka, USA); catechol (>99%), hydrogen peroxide (30%), p-phenylenediamine and Triton
X–100 (Sigma-Aldrich, USA). Other reagents (analytical grade) like phosphate salts for
preparing the buffer were purchased in POCh (Warsaw, Poland). Ultrapure water was
obtained using a Direct-Q 3 UV system from Merck Milipore (Darmstadt, Germany).

1.2.Sample preparation
Fresh apple (Golden delicious cv.) were purchased from local market, washed with water, cut
in smaller pieces and squeezed to obtain juice (J 80 Ultra, Robot Coupe, France). One part of
this cloudy juicewas used as the control (unprocessed) sample and the second was preserved
with SCCD.

1.3.Supercritical carbon dioxide (SCCD) preservation

SCCD treatment was performed with a Spe-ed SFE 4 (Applied Separations, USA) system.
For each experiment, ca. 60 mL of apple juice was placed in a 160 mL glass jars without the
cap and then placed in a rinsed and sanitized (in the autoclave at 120 °C) pressure vessel (500
mL) which had been preheated to the experimental temperature 35, 45, or 65°C and then
exposed to a pressure of 10, 30, 60 MPa for 10, 20, 30 min. The pressure ramp was 60
MPa/min, and at the end of the SCCD treatment, the vessel was slowly depressurized over a
period of 5 min. Pressurization as well as depressurization time were not added to the process
time. After treatment, the jars with juices were immediately cooled. Experiments and
measurements were performed in duplicate.

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 1.4.PPO and POD activities
The activity of tissue and commercial enzymes was determined as described by Terefe, Yang,
Knoerzer, Buckow & Versteeg (2010). The extraction mixture comprised 0.2 M phosphate
buffer (pH= 6.5) containing 4% (w/v) polyvinylpyrrolidone (PVPP), 1% (v/v) Triton X–100
and 1M NaCl. The juices and the mixture (1 : 1, w/w) were homogenized (X120, CAT
Scientific, USA) for 3 minutes and centrifuged (380R, Hettich Instruments, Germany) at
14,000 g for 30 min at 4°C. The supernatant was used to determine PPO and POD activity.
For the PPO activity assay, 500 µL of the supernatant was introduced into 3 mL of
0.05 M phosphate buffer (pH 6.5) containing 0.07 M catechol, and the absorbance was
measured spectrophotometrically (6705 UV-VIS Spectrophotometer, Jenway, UK) at λ = 420
nm and 25°C for 10 minutes. A blank sample was prepared in the same way, by substituting
the supernatant with a phosphate buffer. The PPO activity was expressed as a ΔA/min/g of
fresh weight (FW) of the analysed sample.
For the POD activity assay, 1.5 mL of 0.05 M phosphate buffer (pH = 6.5) was added
to the mixture containing 200 µL of the supernatant, 200 µL of 0.05 M phosphate buffer
containing 1% p–phenylenediamine (w/v) and 200 µL of 1.5% (v/v) hydrogen peroxide.
Mixture absorbance was measured at λ = 485 nm, 25°C for 10 min. The POD activity was
expressed as a ΔA/min/g of FW of the analysed sample.

1.5.Kinetic data analysis

Inactivation of tissue enzymes was analyzed by using a conventional first-order reaction (Eq.
1) (Gui, Wu, Chen, Liao, Hu, Zhang & Wang, 2007):
(Eq. 1)
where A is a residual enzyme activity (%) at time t (min) and k is the inactivation rate
constant (min-1). The value of k was obtained from the regression of natural logarithm of A
versus time.
The decimal reduction time (D) was the treatment time needed for 90% decrease of
initial activity at a given pressure and temperature. D- value was computed as:
.
(Eq. 2)
The pressure and temperature increase needed for a 90% reduction of the D-value are
reflected by zP (MPa, Eq. 3) and zT (°C, Eq. 4), respectively:
(Eq. 3)
(Eq. 4)
The pressure and temperature dependence of k can be expressed by the activation
volume (Va, cm3/mol) and activation energy (Ea, kJ mol-1), as is shown in the Eyring (Eq. 5)
and Arrhenius (Eq. 6) equations, respectively (Gui, Wu, Chen, Liao, Hu, Zhang & Wang,
2007, Manzocco, Ignat, Valoppi, Burrafato, Lippe, Spilimbergo & Nicoli, 2016).
ln (Eq. 5)
ln (Eq. 6)
where P2 and P1, T2 and T1 are pressures and temperatures corresponding to the decimal
reduction times D1 and D2 or constant k1 and k2, respectively. R is the gas constant (8.31 cm3
MPa K-1 mol-1); T is the absolute temperature (K). The values of zP and zT are obtained as the
negative reciprocal slope of the regression line representing log D versus P and T,
respectively. Ea and Va are estimated from linear regression of ln k versus (1/T) and P,
respectively.

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 1.6.Statistical analysis
All analyses were conducted using Statistica 10 StatSoft® software. The significance of the
differences was determined based on an analysis of the variance with Fisher’s test (p-value ≤
0.05). The results were presented as a mean ± standard deviation of a minimum of two
replicates, performed on separate samples prepared according to the same procedure.

RESULTS
The initial PPO and POD activity in apple cloudy juices were 0.09 and 1.69 ΔA/min/mL
for PPO and POD, respectively (tab. 1). The kinetic rates of both enzymes were plotted in
logarithmic graphs (data not shown) as a function of various pressures and temperatures
during SCCD treatment for 10, 20 and 30 min. The linear relationships indicated the SCCD
inactivation of native enzymes in juices followed the first order reaction kinetic with high
determination coefficients. Both types of enzymes were sensitive for increasing the pressure
and temperature of SCCD treatment, but the inactivation kinetics were different and highly
dependent on the kind of enzymes . The kinetic rate constant (k-value) rised significantly with
the increase of temperature and pressure. According to this result it can be concluded that
POD was much more thermal and pressure resistant compared to PPO.
Table 1. Inactivation rate constants, activation energy and activation volume values of polyphenoloxidase (PPO)
and peroxidase (POD) in cloudy apple juice under supercritical carbon dioxide treatment

Pressure k-values (x 10-2 min-)1

Ea (kJ mol-1)
[MPa] 35°C 45°C 65°C
PPO 10 0.86cE 1.53cD 2.14dC 24.9e
30 1.27bD 2.07bC 3.50bB 28.5d
60 1.58aF 2.19aE 6.81aA 43.2c
Va (mL mol-1) -30.2D -18.0B -64.9E -
POD 10 0.32efD 0.80fC 2.85cdA 62.7a
30 0.39eF 0.91eD 3.01cA 58.2b
60 0.70dD 1.12dC 2.97cA 41.9cd
-1
Va (mL mol ) -42.0D -17.4C -2.0A -
Data: mean ± SD (n=3); k-value: kinetic rate constant Ea: activation energy ; Va: activation volume
Different small letters in columns and capital letters in rows indicate significantly different values (p≤ 0.05)

Other authors confirmed that the inactivation rate of tissue enzymes highly depended on
the kind of enzyme, initial activity as well as process conditions (Paciulli, Medina-Meza,
Chiavaro & Barbosa-Canovas, 2016, Chakraborty, Baier, Knorr & Mishra, 2015). Gui, Wu,
Chen, Liao, Hu, Zhang and Wang (2007) in the experiment on cloudy apple juice treated by
SCCD at 8 – 30 MPa and 35 – 55 °C obtained slightly lower k-values for PPO compared with
presented in this paper. It can be explained by the use of different apple cultivar as well as a
higher initial activity of PPO.
The k-values at fixed pressures were significantly temperature dependent as reflected by
the activation energy (Ea) values for PPO and POD. Calculated Ea (eq. 6) increased for PPO
and decreased for POD with increasing the pressure. Activation energy ranged between 24.0 –
43.2 and 41.9 – 62.7 kJ mol-1 for PPO and POD, respectively. The discrepancy in the trends
shown by Ea values for PPO and POD might be attributed to the presence of several
izoenzyme fractions in the case of both enzymes as well as a different initial concentration
and source of enzyme (Chakraborty, Baier, Knorr & Mishra, 2015).

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 The pressure dependency of k-value for both PPO and POD was significantly affected by
temperature. The value of the activation volume (Va) indicates that high pressure favoured the
inactivation process, resulting in a negative volume change. The values of Va decreased from -
18.0 to -64.9 mL mol-1 for PPO and from -2.0 to -42.0 mL mol-1 for POD. The lowest Va
value was noted for PPO at 65°C, amounting for -64.9 mL mol-1.
The obtained Va values for PPO and POD were significantly lower but followed the main
trends, noted by other authors in the high pressure preservation technique (Chakraborty,
Baier, Knorr & Mishra, 2015, Chakraborty, Rao, & Mishra, 2015). Increase of activation
volume at higher temperature has been also reported in the case of actinidin in kiwifruit
(Katsaros, Katapodis & Taoukis, 2009). The different trends in changes of Ea and Va with T
and P between PPO and POD might be explained by variant thermal and pressure expansion
in their structures during rearrangement through compaction caused by high pressure as well
as by a decrease of pH under SCCD treatment.
Table 2. Decimal reduction time and z-value of polyphenoloxidase (PPO) and peroxidase (POD) in cloudy apple
juice under supercritical carbon dioxide treatment
Pressure D-values ( min) for PPO
(MPa) ZT (°C)
35°C 45°C 65°C

10 267.8aC 150.5aD 107.6aE 145.4a

30 181.3bC 111.3bD 65.8bF 121.9b

60 145.8cC 105.2cD 33.8dF 90.3d

ZP (MPa) 280.4D 394.7C 112.6F -

D-values ( min) for POD
ZT (°C)
35°C 45°C 65°C

10 730.2aA 286.9aB 80.7cF 93.9e

30 591.4cA 251.7bB 76.4dE 94.8e

60 327.0eA 206.4cB 77.5dE 108.1b

ZP (MPa) 275.1E 512.6B 2962.0A -

Data: mean ± SD (n=3); D-value: decimal reduction time; zp - value: 90% reduction of D-value under different
pressures; zT - value: 90% reduction of D-value under different temperatures
Different small letters in columns and capital letters in rows indicate significantly different values ≤ 0.05

The linear relationships indicated that the inactivation of both enzymes in juice under
SCCD followed the first order reaction kinetic. The baro- and thermal- resistivity of PPO was
very much reflected by the D-value which ranged from 33.8 to 267.8 minutes, whereas for
POD from 77.5 to 730.2 min (tab 2). Within this zone, PPO was more sensitive towards
SCCD than POD.
Similar trends were obtained by Gui, Wu, Chen, Liao, Hu, Zhang and Wang (2007) for
apple PPO after SCCD treatment at 30 MPa and 35 – 55 °C and by Manzocco, Ignat, Valoppi,
Burrafato, Lippe, Spilimbergo and Nicoli (2016) for mushroom polyphenoloxidase in model
solutions. According to the previous study, inactivation of native PPO and POD present in
strawberry juice was easier (Marszałek, Woźniak & Skąpska, 2015, Marszałek, Skąpska,
Woźniak & Sokołowska, 2015), whereas inactivation of these enzymes in vegetable juices
treated by SCCD was insufficient (Marszałek, Krzyżanowska, Woźniak & Skąpska, 2016,
Marszałek, Krzyżanowska, Woźniak & Skąpska, 2017). This phenomenon could be attributed
to the different matrix effect, as well as much higher initial pH noted in vegetable juices.
Some authors explain this effect with the occurrence of the natural self defence mechanism of

5 
 
vegetable plant cells to various biotic and abiotic stress (Spilimbergo, Komes, Vojvodic,
Levaj, & Ferrentino, 2013).
The zt and zp values for inactivation of enzymes were shown in Table 2. The pressure and
temperature dependency of z-value for both PPO and POD was significantly changed under
the parameters used in the current study. The zT-value of POD increased with increasing the
pressure, whereas zT-value of PPO followed the opposite trend. The zT-value ranged from 90.3
to 145.4 °C for and from 93.9 to 108.1 for POD. The zP-value generally increased with
increasing the temperature of the process (the exception was PPO at 65°C). The zP values
ranged from 112.6 to 280.4 MPa for PPO and from 275.1 to 2962.0 MPa for POD.
Other authors reported zT-value as high as 108°C for PPO from apple juice preserved by
SCCD, what is in line with results of the current study (Gui, Wu, Chen, Liao, Hu, Zhang &
Wang 2007). However, Rayan, Gab-Alla, Shatta and El-Shamei (2011) noted a much lower
value of zT, from 11.8 to 15.3 °C for PPO and POD, respectively in heated cauliflower.
Significantly lower zP -values were noted by Zhou, Wang, Hu, Wu, and Liao (2009) and Gui,
Wu, Chen, Liao, Hu, Zhang and Wang (2007) in their study on the inactivation of pectin
methylesterase from carrot and peach and polyphenoloxidase from apple with SCCD,
respectively.

CONCLUSION
The polyphenol oxidases (PPO) and peroxidases (POD) inactivation kinetics
significantly depended on process parameters. An increase of the temperature and pressure
resulted in an increase of the k-value and a decrease of the D-value for both type of enzymes.
The POD turned out to be more thermal and pressure resistant compared to the PPO. The
lowest decimal reduction time (D-value), zT and zP- value was noted for PPO. The highest
influence of temperature on zT- value and pressure on zP- value was noted at the lowest
pressure and temperature, respectively. The activation energy (Ea) of PPO decreased and of
POD increased with increasing the pressure, whereas activation volumes (Va) were inversely
related. In conclusion, SCCD treatment is a promising technique to obtain high quality apple
juices with low enzyme activity and future research are needed for determination of changes
in polyphenol profile as a results of enzyme activity during long time storage.

ACKNOWLEDGEMENTS
This research was supported by the Project number 2015/17/D/NZ9/02079 of the National
Science Centre, Poland.

REFERENCES
[1] BI, X., WU, J., ZHANG, Y., XU, Z. & LIAO, X. Innovative Food Science and Emerging Technologies, Vol.
12, 2011, p. 298- 304.
[2] BRAUN, G. Fruit Processing, Vol. 13, 2003, p. 422-425.
[3] CHAKRABORTY, S., BAIER, D., KNORR, D., MISHRA, H.N. Food and Bioproducts Processing, Vol.
95, 2015, p. 281-291.
[4] CHAKRABORTY, S., RAO P.S. & MISHRA, H.N. Innovative Food Science and Emerging Technologies,
Vol. 27, 2015, p. 57-68.
[5] DAMAR, S. & BALABAN, O. Journal of Food Science, Vol. 71(1), 2006, p. 1-11.
[6] ELSTNER, E.F. & HEUPEL, A. Planta, Vol. 130, 1976, 175-180.
[7] FABRONI, S., AMENTA, M., TIMPANARO, N. & RAPISARDA, P. Innovative Food Science and
Emerging Technologies, Vol. 11, 2010, p. 477- 484.
[8] FERRENTINO, G. & SPILIMBERGO, S. Trend in Food Science and Technology, Vol. 22, 2011, p. 427-
441.
[9] GONG, Z., LI, D., LIU, C., CHENG, A. & WANG, W. LWT- Food Science and Technology, Vol. 60,
2015, p. 1095-1099.

6 
 
 [10] GUI, F., WU, J., CHEN, F., LIAO, X., HU, X., ZHANG, Z. & WANG, Z. Food Chemistry, Vol. 100,
2007, p. 1678- 1685.
[11] KATSAROS, G.I., KATAPODIS, P. & TAOUKIS, P.S. Journal of Food Engineering, Vol. 94, (1), 2009, p.
40-45.
[12] LEE, B., SEO, J.D., RHEE, J. & KIM, C.Y. Food Chemistry, Vol. 201, 2016, p. 315-319.
[13] LIU, Y., HU, X., ZHAO, X. & SONG, H. Innovative Food Science and Emerging Technologies, Vol. 13,
2012, p. 112- 119
[14] MANZOCCO L., IGNAT A., VALOPPI F., BURRAFATO K. R., LIPPE G., SPILIMBERGO S., NICOLI
M. C. The Journal of Supercritical Fluids, Vol. 107, 2016, p. 669-675.
[15] MARSZAŁEK, K, SKĄPSKA, S, WOŹNIAK, Ł, SOKOŁOWSKA, B. Innovative Food Science and
Emerging Technologies, Vol. 32, 2015, p. 101-109
[16] MARSZAŁEK, K., WOŹNIAK, Ł., SKĄPSKA, S. Żywność- Nauka Technologia Jakość, Vol. 2 (99),
2015, p. 114-123.
[17] MARSZAŁEK, K., KRZYŻANOWSKA, J., WOŹNIAK, Ł., SKĄPSKA, S. The Journal of Supercritical
Fluids, Vol. 117, 2016, p. 26-32.
[18] MARSZAŁEK, K., KRZYŻANOWSKA, J., WOŹNIAK, Ł., SKĄPSKA, S. LWT- Food Science and
Technology, 2016, DOI: 10.1016/j.lwt.2016.11.01
[19] NIU, S., XU, Z., FANG, Y., ZHANG, L., YANG, Y., LIAO, X & HU, X. Innovative Food Science and
Emerging Technologies, Vol. 11, 2010, p. 91-97
[20] PACIULLI, M., MEDINA-MEZA, I.G., CHIAVARO, E. & BARBOSA-CANOVAS, G.V. LWT-Food
Science and Technology, Vol. 68, 2016, p. 98-104.
[21] RAYAN, A.M.M., GAB-ALLA, A.A., SHATTA, A.A. & EL-SHAMEI, A.S. European Food Research and
Technology, Vol. 232, 2011, p. 319-326.
[22] SPILIMBERGO, S., KOMES, D., VOJVODIC, A., LEVAJ, B. & FERRENTINO, G. The Journal of
Supercritical Fluids, Vol. 79, 2013, p. 92- 100.
[23] TEREFE, N.S., YANG, Y., KNOERZER, K., BUCKOW, R. & VERSTEEG, C. Innovative Food Science
and Emerging Technologies, Vol. 11, 2010, p. 52-60.
[24] WŁODARSKA, K., PAWLA-LEMAŃSKA, K., GÓRECKI, T. & SIKORSKA, E. Journal of Food
Quality, 2016, DOI: 10.1111/jfq.12208
[25] ZHONG, Q., BLACK, D., DAVIDSON, P., M., & GOLDEN, D.A. Journal of Food Protection, Vol. 71,
2008, p. 1015-1017.
[26] ZHOU, L., WANG, Y., HU, X.S., WU, J.H., & LIAO, X.J. Innovative Food Science and Emerging
Technologies, Vol. 10, 2009, p. 321- 327.

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