Inactivation of peroxidase and polyphenoloxidase in coconut

water using pressure-assisted thermal processing

Angelica M. Chourio1, Fabiola Salais-Fierro1, Zahid Mehmood1, Sergio I.

Martinez-Monteagudo2, and Marleny D.A. Saldaña1,*

1
Department of Agricultural, Food and Nutritional Science, University of Alberta,

Edmonton AB, Canada, T6G 2P5

2
Dairy and Food Science Department, South Dakota State University, Brookings, SD,

United States of America, 57007

* Corresponding author

3-18A Department of Agricultural, Food and Nutritional Science,

Faculty of Agricultural, Life and Environmental Sciences,

University of Alberta, Edmonton, AB, Canada, T6G 2P5

Phone: (1) (780) 492-8018

Fax: (1) (780) 492-8914

E-mail: [email protected]

© 2018. This manuscript version is made available under the Elsevier user license
http://www.elsevier.com/open-access/userlicense/1.0/
Abstract

The effect of pressure assisted thermal processing (PATP) was evaluated on the inactivation

kinetics of polyphenol oxidase (PPO) and peroxidase (POD) and selected quality attributes of

coconut water. Coconut water from green young coconuts was treated at 200, 400 and 600

MPa, 40-90ºC, and 60-1800 s of holding time. The activities of PPO and POD were

determined using spectrophotometric methods. No enzymatic activity was detected for both

enzymes within 300 s at 90ºC/400-600 MPa. The combination of 400 MPa/90°C/300 s

yielded POD and PPO inactivation, and could be used in the industrial development of PATP

treated-coconut water. The POD showed to be more pressure-temperature resistant than the

PPO in coconut water. The pressure-temperature inactivation kinetics of PPO and POD in

coconut water were well described by the Weibull model. The activation energy for the

inactivation of POD and PPO were 107-192 and 41-191 kJ mol-1, respectively, while the

activation volume varied from -13.2 to 10.2 and -37 to 9.2 cm3 mol-1, respectively. Total

phenolic content extractability significantly increased after PATP treatments at all conditions

evaluated compared to the control. Low ∆E values of PATP treated coconut water were

obtained, indicating imperceptible change of color.

Keywords: Coconut water, enzyme inactivation, kinetics modeling, pressure-assisted

thermal processing.

2
Industrial relevance: Pressure Assisted Thermal Processing (PATP) is an emerging

technology that requires further research. The results of this study highlighted for the first

time the potential of PATP on polyphenoloxidase and peroxidase inactivation of coconut

water, maintaining color characteristics of coconut water. The pink color after PATP

treatment was not observed. In addition, the use of kinetic models helped to determine the

optimal conditions for enzyme inactivation. The outcomes of this study can be used for

further industrial development of PATP treated-coconut water.

3
1. Introduction

Coconut water is a clear and colorless liquid inside young green coconuts. This liquid is

mildly sweet when freshly extracted from young green coconuts (Purkayastha et al., 2012).

Due to its low-calorie content (17.4 kcal/100 g) and relative high concentration of minerals,

coconut water has been proposed as a natural beverage alternative to sport drinks. In addition,

coconut water is rich in many beneficial bioactive compounds, including vitamin C, vitamin

B, potassium, sodium, magnesium, calcium, arginine, alanine, lysine, and glutamic acid

(Cappelletti et al., 2015; Reddy, 2005).

Inside the hermetic cavity of the coconut, the liquid is sterile and its composition depends on

the maturity of the coconut (Luengwilai et al., 2014). However, when the coconut water is

removed from its cavity, it spoils within a day because of external contamination by

microorganisms during extraction, refrigeration, and packaging. Leite et al. (2000) found a

population of up to 5-log of Bacillus cereus in fresh coconut water stored under refrigeration.

Currently, there is a major challenge to assure microbiological safety while retaining

fresh-like quality attributes without using preservatives and additives. The activity of

peroxidase (POD) and polyphenoloxidase (PPO) present in coconut water also affects its

quality, including discoloration, off-flavor, turbidity, and short shelf-life (Ciou et al., 2011).

Coconut water is classified as a low-acid drink (pH=5.0-5.5) and therefore severe thermal

conditions are mandatory to warrant microbiological safety (Luengwilai et al., 2014). The

commercial production of coconut water employs ultra-high-temperature pasteurization

(UHT, 130-150˚C for at least 4 s). Unfortunately, such thermal treatment eliminates the

4
delicate flavor characteristics, which limits the marketability of the product (Jayanti et al.,

2010).

Alternatively, hydrostatic pressure levels of 400–600 MPa at ambient temperature have been

effective in inactivating pathogenic and spoilage vegetative cells (Martinez-Monteagudo &

Balasubramaniam, 2016). Pressure pasteurized coconut water is already on the market in

North America with an estimated shelf-life of 12 d under refrigeration. There is industrial

interest to extend the shelf-life of coconut water longer than that of high pressure processing

to increase its marketability and further distribution (Gordon & Jackson, 2017). However,

pressure alone at moderate temperature (60°C) cannot inactivate spores. Pressure-assisted

thermal processing (PATP) is an emerging sterilization technology that consists in applying

high hydrostatic pressure (100-600 MPa) to a preheated sample (75-90°C) over certain time

(3-10 min) (Martinez-Monteagudo & Saldaña, 2014). Samples compressed hydrostatically

rise their temperature, allowing rapid and uniform heating at target process temperatures of

90-120°C. During PATP, the temperature of the food material increases due to physical

compression under pressure. Upon decompression, the product cools volumetrically to its

initial temperature. The PATP was first developed to inactivate bacterial spores and achieve

commercially sterility of low-acid foods (Sizer et al., 2002). In this study, it was

hypothesized that PATP can be used to obtain shelf stable coconut water with fresh like

attributes. Therefore, the main objective of this study was to investigate the effect of PATP

on the inactivation of PPO and POD enzymes, use different models to predict inactivation as

a function of pressure, temperature and holding time, and evaluate the change of total

phenolic content and color of coconut water treated by PATP.

5
2. Material and Methods

2.1. Coconut

Ten green coconuts imported from Vietnam were purchased from a local supermarket

(Sobeys, Edmonton AB, Canada). After sanitizing the monocarp with 10% ethanol solution,

a sterilized drill was used to perforate the coconut monocarp to remove the coconut water.

This water was filtered using a filter cloth, and manually swirly. The coconut water was then

stored at -18C for further PATP treatments.

2.2. Pressure-Assisted Thermal Processing

A four-vessel system (Apparatus U111 Unipress, Warszawa, Poland) was used for the

inactivation of PPO and POD in coconut water. Each vessel has a capacity of 8 mL. The

vessels were heated with a circulator thermostat (Lauda Proline RP 855 Low Temperature,

Lauda-Konigshofen, Germany) using propylene glycol as the pressure transmission fluid.

Polypropylene tubes (Cryogenic vial, Fisher Scientific, Pittsburgh, PA) of 3 mL were filled

with untreated coconut water. Samples were pressurized to 200, 400, and 600 MPa at

temperatures of 40, 60, 80, and 90C with holding times of 60, 120, 300, 600, 900 and 1800 s.

The samples were pressurized at a rate of 10 MPa s-1. At the end of the holding time, the

vessels were decompressed, and the samples were removed immediately from the

high-pressure vessels, cooled down with ice and stored at -18 C for further analysis. Earlier,

this experimental system was validated for kinetic studies (Martinez-Monteagudo & Saldaña,

2014, Martinez-Monteagudo & Saldaña, 2015). All experiments were performed at least in

triplicate.

2.3. Enzyme Activity

6
The POD activity was measured using a UV-VIS spectrophotometer (Jenway 6320D,

Standford, United Kingdom) according to the method described by Matsui et al. (2008), with

slight modifications. A test tube containing 3.5 mL of buffer (Na2HPO4·2H2O+KH2PO4, pH

6), 0.4 mL of ABTS (2.2 azino-bis 3– ethylbenzthiazoline-6-sulfonic acid) solution (0.02

mole/L) and 0.4 mL of hydrogen peroxide (0.1% v/v) was placed in a water bath at 25oC for

300 s. Then, 1 mL of coconut water was added to this solution and transferred to a cuvette

where the absorbance solution was measured at 405 nm and recorded every 10 s for 300 s.

The reference value of the POD was determined using a blank solution containing 3.5 mL of

buffer (Na2HPO4·2H2O+KH2PO4, pH 6.0), 0.4 mL of ABTS solution (0.02 mole/L), 0.4 mL

of hydrogen peroxide (0.1% v/v) and 1 mL of distilled water.

The PPO activity was measured using the same UV-VIS spectrophotometer according to the

method described by Matsui et al. (2008), with slight modifications. A test tube containing

2.25 mL of sodium phosphate buffer (0.2 mole/L, pH 6.0) and 0.75 mL of 0.2 mole/L

pyrocatechol solution was placed in a water bath at 25oC for 300 s. Coconut water sample (1

mL) was added to this solution and the absorbance was measured at 425 nm and recorded

every 10 s for 5 min. The reference value of PPO was determined using a blank solution

containing 0.75 mL of pyrocatechol, 2.25 mL of sodium phosphate buffer (0.2 mole/L, pH 6)

and 1 mL of distilled water. All POD and PPO analyses were carried out at least in triplicate.

For both enzymes, absorbance were plotted against time and the values of enzymatic activity

were calculated from the slope of the initial linear part of the curves following the method

reported by Matsui et al. (2008). The relative activity in coconut water samples were

determined using equation (1):

7
 Relative activity of enzyme = (1)

where, At is the mean of the enzyme activity after PATP treatment at specific processing

conditions, and Ao is the mean of the initial enzyme activity before the PATP treatment.

2.4. Quality Parameters

2.4.1. Color

Color was determined using a colorimeter (CR210, Minolta, Osaka, Japan), following the

methodology reported by Park et al. (2014). Briefly, the colorimeter was calibrated using a

white standard plate (L* = 97.83, a* = 0.43, b* = 1.98). The CIELAB L*, a* and b* values

represent the indicators of lightness, redness and yellowness, respectively. Total change in

color (∆E) was calculated using equation (2):

(2)

where, L*raw: lightness of coconut water before the PATP treatment, L*tret: lightness of

coconut water after the PATP treatment, a*raw: redness of coconut water before the PATP

treatment, a*tret: redness of coconut water after the PATP treatment, b*raw: yellowness of

coconut water before the PATP treatment, and b*tret: yellowness of coconut water after the

PATP treatment.

2.4.2. Total Phenolic Content

The Folin-Ciocalteu method was used to determine total phenolic content (Singleton &

Rossi, 1965). Briefly, the sample aliquot (0.04 mL), distilled water (3.1 mL) and the

Folin-Ciocalteu reagent (0.2 mL) were mixed. After 6 min, sodium carbonate (20% w/v; 600

µL) was added and the mixture was incubated for 2 h in dark at room temperature. The

8
absorbance was measured at 765 nm. The final results were expressed as milligrams of gallic

acid equivalents per gram of coconut water (mg GAE g-1).

2.5. Modeling Enzyme Inactivation

The enzymatic inactivation models used for the experimental data are presented in Table 1.

The parameters of each model were calculated using Athena Visual Workbench, a powerful

software package for modeling and parameter estimation (www.athenavisual.com). The

predictive capability of the individual models was assessed by the coefficient of

determination (R2), the adjusted coefficient of determination (R2Adj), residual analysis, and

average absolute percentage of residuals (E).

Insert Table 1

The influence of temperature and pressure on the rate constant (ki) was expressed by

Arrhenius-type and Eyring-type models, respectively:

(7)

(8)

where, kT and kP are the reaction rate constants at a reference temperature (Tr) and pressure

(Pr), respectively; Ea is the apparent activation energy (kJ mol-1); ∆V# is the activation

volume (cm3 mol-1); T is the temperature (K); P is the pressure (MPa); and R is the universal

gas constant (8.314 J mol−1 K−1). The average values of the experimental temperatures and

pressures were used as Tr and Pr, respectively. In the case of the Weibull model, the effect of

temperature and pressure was evaluated using the mathematical model proposed by

Martinez-Monteagudo & Saldaña (2014, 2015).

9
 (9)

(10)

where, α is the scale parameter, αT and αP are the scale parameters at a reference temperature

and pressure, respectively.

3. Results and Discussion

3.1. Enzyme Inactivation of Coconut Water Treated by PATP

Fig 1a-c shows the remaining activity of POD in coconut water treated with PATP (200-600

MPa; 40-90°C; 0-1800 s). In general, the activity of POD gradually decreased over time, in

which the final activity depends on the combination of temperature and pressure. At 40°C

and 1800 s, the remaining activity was 0.52±0.02, 0.66±0.03, and 0.74±0.02 at 200, 400, and

600 MPa, respectively. A gradual decrease of the remaining activity of POD until reaching a

plateau after 600 s was observed at 60 and 80°C, independently of the pressure. The

exception of this generalization was observed at 90°C, where the remaining activity of POD

was not detected after 120-300 s, regardless of the pressure (200-600 MPa). Similar

observations have been reported in litchi pericarp, where POD was completely inactivated

after 90°C for 600 s and after 100°C for 60 s (Mizobutsi et al., 2010).

The remaining activity of PPO in coconut water treated with PATP is presented in Fig 1d-f.

At 40°C and 1800 s, the remaining activity of PPO was 0.21±0.01, 0.50±0.02, and 0.62±0.02

at 200, 400, and 600 MPa, respectively. These activity values are substantially lower than

those observed for POD at the same pressure and temperature conditions (Fig 1a-c),

demonstrating that POD was more pressure-temperature resistant than PPO in coconut water.

The remaining activity of PPO within the temperature range of 60-80°C and pressure of

200-600 MPa was between 0.13-0.33. Similar results were reported in the remaining POD

activity of strawberry puree and pineapple puree during thermal and high-pressure treatments

10
(Terefe et al., 2010; Chakraborty et al., 2015). The combination of 90°C and 200-600 MPa

yielded no detectable PPO activity after 300 s of treatment. A remarkable observation during

the inactivation of PPO is the existence of antagonist and synergist effects as a result of

different combinations of temperature and pressure. A noticeable antagonist effect was

observed at a combination of 40°C and 600 MPa, yielding an activity value of 0.60±0.02 after

1800 s, while an activity value significantly lower (0.21±0.02) was obtained at 40°C and 200

MPa. The behavior of higher remaining activity at a lower temperature (40oC) and high

pressure (200-600 MPa) for both PPO and POD could be attributed to the partial

rearrangements of the enzyme structures that enhanced the intramolecular interactions with

the substrate, resulting in a higher catalytic performance. In contrast, a synergistic effect can

be exemplified at a combination of 90°C and 600 MPa, where the remaining activity of PPO

was 0.08±0.01 after 120 s, while the combined effect of 90°C and 200 MPa, yielded an

activity value of 0.20±0.02. Inactivation of enzyme by combined action of pressure and

temperature is a complex phenomenon. The primary structure of the enzyme could be

minimally affected by pressure while the secondary structure suffers structural modifications

only at very high pressures. The tertiary structure is greatly affected by pressure because

pressure disrupts hydrophobic and electrostatic interactions (Ludikhuyze et al., 2003).

Consequently, water solvates the exposed charge groups, leading to a volume reduction that

inactivates the enzymes (Ludikhuyze et al., 2003).

3.2. Inactivation Models

Table 2 shows the performance of different models used to represent the inactivation of POD

and PPO in coconut water at PATP conditions. The first-order and fractional models failed to

adequate represent the experimental data, judging by low values of R2 (0.375-0.806 and

0.839-0.983, respectively) and high values of E (32.17-54.15% and 15.36-34.32%,

respectively). The R2Adj and E are parameters used for evaluating the fitting performance of

kinetics models. The R2 values indicate how well the model describes the experimental data,

11
R2Adj indicates the influence of the number of parameters on R2, and E compares the overall

error in terms of percentage. The Weibull and Isozyme models presented the best fitting

performance throughout the entire experimental domain (Table 2) with the highest R2 and

R2Adj and the lowest E values. The residual analysis (standardized residual against predicted

values) of Weibull and Isozyme models revealed random patterns (graph not shown), which

validates the estimation of parameters and prediction of new observations. The fitting

performance (Table 2) indicated almost no distinction between Weibull and Isozyme

models, with both models being statistically appropriate to describe the inactivation of POD

and PPO in coconut water treated with PATP. The isozyme model accounts for the presence

of isozymes with different stabilities, where the labile fraction is clearly identified from the

stable fraction (Gökmen, 2010). However, our experimental data cannot confirm the

existence of different fractions for both enzymes, which challenges the applicability of the

Isozyme model. On the other hand, the application of the Weibull model for enzyme

inactivation assumes that the inactivation followed an exponential probabilistic distribution

(Cunha et al., 1998). The information presented in Table 2 suggested that the inactivation of

POD and PPO can be seeing as a failure phenomenon, where a fraction of an intact enzyme is

reduced with time at isothermal and isobaric conditions, regardless of the inactivation

mechanism as the entire process is governed by probability laws. Weibull model has been

successfully used for modeling not only the temperature-pressure inactivation of spores

(Couvert et al., 2005; Van Boekel, 2002, Mafart et al., 2002, Fernandez et al., 2002) and

enzymes (Criado et al., 2015; Sampedro et al., 2014; Deylami et al., 2016) but also the

retention of bioactive lipids (Martinez-Monteagudo & Saldaña, 2015; Guo et al., 2017).

Insert Table 2
3.2.1. Modeling the Inactivation of POD and PPO

12
The temperature-pressure inactivation was evaluated by combining equation (7) with

equation (9), and equation (8) with equation (10). The resulting equations resemble that of

Arrhenius model (equation (11)) and Eyring model (equation (12)).

(11)

(12)

The influences of temperature and pressure on the fitting and kinetic parameters are provided

in Table 3 and Table 4, respectively. The fitting performance of both equations suggests that

both models and their associated parameters can be used to satisfactorily model the

inactivation of POD and PPO within the experimental domain. The scale parameter at a

reference temperature (αT) varied from 789 to 3640 for POD and from 54 to 229 for PPO. On

the other hand, the scale parameter at a reference pressure (αP) varied within the range of

35-2750 and 25-360 for POD and PPO, respectively. A general observation for both enzymes

was that αT increased with pressure while αP decreased with temperature. The αT and αP were

inversely proportional to the frequency or pre-exponential factor of Arrhenius and Eyring

equation. The pre-exponential factor can be seen as the reaction rate when no energetic

barrier is present (Martinez-Monteagudo & Saldaña, 2014).

Insert Table 3
The values for the shape parameter (β) were within the range of 0.18-0.53 and 0.16-0.47 for

POD and PPO, respectively, and no systematic influence of temperature and pressure was

observed. The estimated parameters for β were lower than 1, indicating deviation of the

first-order reaction. The main assumption of a first-order reaction is that the reactants

(enzymes) are unaffected by the food matrix, which is very unlikely in this case. Thus, the
13
inactivation of POD and PPO in coconut water was expected to deviate from the first-order

reaction. The parameters of activation energy (Ea) and activation volume ( ) are

oftentimes considered the most relevant kinetic information. The inactivation of POD and

PPO yields activation energies of 107-192 and 41-191 kJ mol-1, respectively. On the other

hand, the activation volume of POD and PPO varied from -13 to 13 and -38 to 9 cm3 mol-1.

Although a systematic relationship (Ea increasing with pressure and increasing with

temperature) can be observed, such relationship could not be statistically confirmed due to

the large variability of the activation parameters.

Insert Table 4
3.2.2. Activation Modes

The inactivation of enzymes is a very complex phenomenon involving a reversible partial

unfolding, followed by an irreversible modification that leads to the inactivation. The step of

unfolding-refolding is negligible due to its difficulty to measure experimentally. Thus, the

inactivation is governed by the irreversible step. This step is thought to be to follow the

Arrhenius and Eyring law when temperature and pressure is applied. Thus, the activation

parameters (Ea and ) are of great of importance since they provide essential information

for process development and optimization. However, a theory explaining the influence of

pressure and temperature on the Ea and , respectively, has not been developed. An

attempt was made to provide a meaningful interpretation of the activation modes during the

inactivation of POD and PPO under PATP conditions. It is convenient to express the Ea as a

dimensionless number (Arrhenius number, ) as a function of pressure (Fig 2a). Arrhenius

number is used for the analysis and optimization of complex reactions. The Arrhenius

number is the ratio of activation energy to the average kinetic energy (R∙Tr). Reactions having

small Arrhenius number are less sensitive to temperature and relative large increase in

temperature is needed to accelerate the reaction. On the other hand, large values of Arrhenius

14
number are indicative of the reaction sensitivity towards temperature and relative small raise

in temperature would result in substantial increase in the reaction rate. The Arrhenius number

varied from 37 to 67 for POD and from 14 to 65 for PPO. For both enzymes, the Arrhenius

number increased with pressure. A possible interpretation is that pressure levels of 600 MPa

may induce irreversible modifications of the enzyme structure that favor the inactivation by

temperature. Contrary, such modifications may occur to a lesser extend at pressure levels of

200 MPa. Consequently, the unfolding-refolding step becomes predominant and higher

temperatures are needed to overcome such physical barriers. Fig 2b shows the dimensionless

activation volume ( ) as a function of the temperature. The inactivation of POD yielded

values of -2.3 to 1.3, increasing with temperature. Similar tendency was observed for

the denaturation of PPO, where the values increase with temperature from -5.1 to 1.2.

In general, the for both enzymes revealed that the denaturation process (

is characterized by negative values. A possible interpretation is that at a given

temperature, for instance 40°C, the net effect of pressure is predominant over the thermal

effect. As the temperature increased (80-90°C), the dimensionless activation volume became

positive, meaning that the thermal effect is now the predominant force. These observations

exemplify the complexity of inactivation mechanisms of enzymes under pressure and

temperature. This indicates that the overall denaturation process is favored with pressure at

that given temperature. The forces affected by pressure in the denaturation process are

hydrogen-bondings, hydrophobic interactions, and electrostatic forces. Interestingly, the

extent of such forces increases with temperature. Indeed, increasing the temperature result in

positive values of the for both enzymes (Fig 2b), meaning that the denaturation

process is slightly inhibited by pressure. The influence of temperature on the is much

15
more complex than the relationship of Arrhenius number and pressure because the

may also represent inherit change in the volume as the enzyme unfolds. The is a

reflection of all molecular changes occurring during the progression of all steps involved

during the denaturation, folding-unfolding and irreversible inactivation. The enzyme

denaturation is a complex interplay process, where time, pressure, and temperature play a

significant role. We have postulated a denaturation process where both activation modes are

considered:

It is thought that the folding-unfolding step is mainly controlled by pressure. Hydrogen bonds

are related to solvent interactions, which may result in negative values (Weemaes et al.,

1998). Hydrophobic interactions involve the exposure of a non-polar component of the

structure with similar structural units, rather than to be exposed to solvation by water. The net

effect of such interactions results in positive values (Damodaran et al., 1996).

Electrostatic interactions include ion-pairing of ionizable groups, which ionic dissociation

greatly impact the conformational stability of the enzyme. Ionization volumes of synthetic

proteins (polylysine) are negative because the protein undergoes uncoiling of the helical

conformation, which results in a reduction of the molecular volume (Mozhaev et al., 1996).

Insert Fig 2

3.2.3. PATP Working Conditions for 90% PPO and POD Inactivation

The combined effect of pressure and temperature on the inactivation of POD and PPO is

illustrated through kinetic contour plot (Fig 3). The contour lines are the combinations of

temperature and time at a given pressure that lead to at least 90% of enzyme inactivation.

Such kinetic plot can be used for developing PATP process for coconut water. For instance,

the combination of 600 MPa and 85°C for 1000 s yields more than 90% of POD and PPO

16
inactivation. Designing a process that extends the shelf-life of coconut water can be derived

from the information presented in Fig 3 in combination with other quality parameters like

phenolic content and color change. It should be mentioned that the conditions illustrated in

Fig 3 cannot guarantee commercial sterilization of the coconut water since no information on

spore inactivation is available. In the absence of such information, the inactivation of

Bacillus amyloliquefaciens, a pressure-resistant spore, reported elsewhere (Ahn,

Balasubramaniam, & Yousef 2007; Martínez-Monteagudo, Gänzle, & Saldaña, 2014) was

used for interpretation of the kinetic data. In general, temperatures higher than 100°C in

combination with 400-600 MPa for 5 min (3000 s) yielded between 4-5 log reduction of

Bacillus amyloliquefaciens, achieving commercial sterilization. These processing conditions

would yield complete inactivation of PPO and POD in coconut water.

Insert Fig 3

3.4. The Effect of Other Quality Parameters on Coconut Water Treated by PATP

Color is one important parameter that influences product quality and purchase desire of the

consumers (Gökmen, 2010). The color related parameters of the coconut water, control and

samples after treatment at 40 and 80°C, and 400 and 600 MPa, are shown in Table 5 and

Table 6. The values of L* parameter ranged from 84.43±0.04 (coconut water treated at 80°C,

400 MPa for 300 s) to 87.14±0.04 (coconut water treated at 80°C, 600 MPa for 1800 s). No

significant difference was observed in L* values compared to control at 40°C/400-600 MPa

and 80oC/400 MPa. However, a slightly increase of L* value was observed after 600 s at

80°C/600 MPa compared to the control. Similarly, Garcia-Parra et al. (2016) reported a slight

increase of L* values of pumpkin puree, from 53.5 to 57.2 compared to the control, after
17
PATP treatment (600 MPa/80°C/300 s). Cappelletti et al. (2015) reported a slight decrease of

L* values of coconut water, from 99.59 to 98.35 compared to the control, employing high

pressure carbon dioxide (12 MPa, 45°C, 300-1800 s) and a slight decrease of L* values of

coconut water from 99.59 to 98.35 compared to the control, employing heat pasteurization

(90°C, 60 s).

The PATP treatment resulted in an increase of a* values in coconut water samples. The most

intense red color (a*) parameter was observed in coconut water samples treated at 80°C, 400

MPa for 600 and 1200 s (0.47±0.01), while the lowest a* values obtained was 0.13±0.01 at

40°C, 400 MPa for 600 s. Similarly, Cappelletti et al. (2015) reported an increase of a* values

in coconut water samples from 0.003 (control) to 0.07 and 0.11 after high pressure carbon

dioxide (12 MPa, 45°C, 300-3600 s) and heat pasteurization treatments (90°C, 60s),

respectively.

The blueness of processed coconut water ranged from -1.96±0.01 to -0.37±0.01. The PATP

caused increase in b* values of coconut water for all treatment conditions evaluated.

However, high temperature conditions, 80°C/600 MPa, resulted in higher increased in b*

values compared with the PTAP conditions, 40°C/600 MPa. Similarly, Terefe et al. (2009)

observed that the highest temperature evaluated (60°C) resulted in the highest increase in the

b* values of strawberries treated under PATP (300-600 MPa, 600 s). Cappelletti et al. (2015)

also reported an increase of b* of coconut water from 0.52 (control) to 1.02 after high

pressure carbon dioxide (12 MPa, 45°C, 300-1800 s) and from 0.52 (control) to 1.36 after

heat pasteurization treatments (90°C, 60 s). It is thought that the increase of b* values is

related to phenolic compounds oxidation of coconut water samples.

18
 Insert Table 5

Another important parameter is ∆E* which is used to denote difference between the colors of

two products. It is assumed that a consumer can distinguish the color of two samples when

∆E ≥ 5 units (Perez-Magarino et al., 2003). The values of ∆E* shown in Table 5 and Table 6

of coconut water ranged from 0.67±0.02 (coconut water treated at 40°C, 400 MPa for 120 s)

to 2.44±0.01 (coconut water treated at 80°C, 600 MPa for 1800 s). The PATP treatment of

coconut water resulted in ∆E* values ≤ 5 units. Cappelletti et al. (2015) reported ∆E* values

of 5.1 and 8.1 for coconut water after high pressure carbon dioxide (12 MPa, 45°C, 300-1800

s), ∆E* values of 5.1, and heat pasteurization treatments (90°C, 60s) respectively. However,

Liu et al. (2016) showed that cucumber juice treated under high pressure processing (HPP)

(500 MPa/300 s) and HTST (110°C/8.6 s) resulted in ∆E* values of 0.75 and 2.99,

respectively. Similarly, Barba et al. (2013) reported that blueberry juice treated under HPP

(200-600 MPa, 0-42ºC, 300-900 s) resulted in ∆E* values in the range of 0.83-1.44. The

PATP treated samples achieved low ∆E values resulting on imperceptible change of color of

coconut water samples.

Insert Table 6

3.4.1. Total Phenolic Content of Coconut Water

Total polyphenol content (TPC) of raw coconut water and treated coconut water samples at

40-120°C and 400-600 MPa are shown in Fig 4. The TPC in the raw coconut water was 0.07

mg GAE/g. The TPC values significantly increased up to 68% after PATP treatment at

120°C/600 MPa/120 s (0.15 mg GAE/g) and up to 69% (0.16 mg GAE/g) after 120°C/400

MPa/1800 s. The increase of TPC can be attributed to the disruption of cellular walls and
19
hydrophobic bonds during PATP treatment increasing extractability of antioxidant

compounds (Sharma et al., 2015). These results are in concordance with previous studies

reporting increase of TPC values after PATP. Garcia-Parra et al. (2016) found that the

combination of 600 MPa/70°C/60 s increased up to 65% TPC of pumpkin puree compared to

the control. Kaushik et al. (2016) also reported that total phenolic content of mango pulp

increased by 7-27% relative to the untreated sample after PATP (400 MPa/60°C/300 s).

Terefe et al. (2009) found no significant difference on TPC values of strawberries compared

with the control after PATP treatment (600 MPa/60°C/600 s). These results demonstrated the

potential of PATP to increase extractability of total phenolic content of coconut water.

Insert Fig 4

4. Conclusions

No enzymatic activity was detected for PPO and POD enzymes of green coconut water after

120 s at 90ºC/400-600 MPa. Both enzymes exhibited inactivation behavior as a function of

increasing temperature from 40 to 90°C. The POD enzyme of coconut water showed higher

resistance to PATP treatment than the PPO enzyme. The recommended conditions to ensure

more than 90% inactivation of POD and PPO in coconut water are 400 MPa/90oC/300 s. The

pressure assisted thermal inactivation kinetics of coconut water PPO and POD were well

described by the Weibull model, contributing to identify the PATP optimum conditions to

inactive PPO and POD of coconut water. The PATP treatment resulted in ∆E* values lower

than 5 units, indicating that original color of coconut water was maintained. The extraction of

total phenolic content of coconut water significantly increased after PATP treatment. These

20
results highlight the potential use of PATP for inactivation of PPO and POD enzymes of

coconut water to maintain its quality.

Acknowledgements

The authors thank to the Natural Sciences and Engineering Research Council of Canada

(NSERC, #05356-2014) for funding this project. ZM thanks his PhD Pakistan scholarship

from the International Research Support Initiative Program (IRSIP

1-8/HEC/HRD/2015/3673) during his stay at University of Alberta. SIMM thanks his salary

support of USDA National Institute for Food and Agriculture.

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27
Figure legends

Fig 1. Relative enzymatic activity during pressure-assisted thermal processing: peroxidase

activity at: (a) 200, (b) 400, and (c) 600 MPa; and polyphenoloxidase activity at: (d) 200, (e)

400, and (f) 600 MPa. ● 40, ○ 60, ▼80, and ∆ 90°C. Curves represent data obtained with

Equation (11).
Fig 2. Dimensionless activation parameters for the inactivation of peroxidase and

polyphenoloxidase: (a) Influence of pressure on the dimensionless activation energy, and (b)

influence of temperature on the dimensionless activation volume. The error bars were

calculated from the 95% confidence interval.

Fig 3. Inactivation of peroxidase (POD) and polyphenoloxidase (PPO) in coconut water

treated with pressure assisted thermal processing. The contour lines represent the require

conditions to inactivate 90% of the enzyme activity.

Fig 4. Total phenolic content of coconut water after PATP treatment.

28
Fig 1

29
Fig 2

30
Fig 3

31
 0.20
0.18 Control
Total phenolics (mg GA/g)

0.16
40 °C/400 MPa
0.14
0.12 40 °C/600 MPa
0.10
0.08 80 °C/400 MPa
0.06 80 °C/600 MPa
0.04
0.02 120 °C/400 MPa
0.00
120 °C/600 MPa
120 300 600 1800
Holding time (s)

Fig 4

32
Table legends

Table 1. Kinetic equations used to represent the inactivation of peroxidase and

polyphenoloxidase in coconut water treated with pressure-assisted thermal processing.

Table 2. Fitting performance of inactivation models for peroxidase and polyphenoloxidase in

coconut water treated at pressure-assisted thermal processing conditions.

Table 3. Fitting parameters of Arrhenius-type equation (11) for the inactivation of

peroxidase and polyphenoloxidase in coconut water treated with pressure-assisted thermal

processing.

Table 4. Fitting parameters of Eyring-type equation (12) for the inactivation of peroxidase

and polyphenoloxidase in coconut water treated with pressure-assisted thermal processing.

Table 5. Color parameters of coconut water treated by PATP at 400 MPa.

Table 6. Color parameters of coconut water treated by PATP at 600 MPa.

33
Table 1

Model Mathematical expression Equation number

First-order 3

Fractional 4

Weibull 5

Isozyme 6

At – enzyme activity at a given time; Ao – initial enzyme activity; ki – inactivation rate constants for equations (3)

and (4); t – holding time; A∞ - enzyme activity after prolonged processing; α, β – scale and shape parameters,

respectively; Al – enzyme activity of the labile fraction; As – enzyme activity of the stable fraction; kl –

inactivation rate constant of the labile fraction; and ks – inactivation rate constant of the stable fraction.

34
Table 2

Parameters First-order model, Equation (3)

Peroxidase (POD) Polyphenoloxidase (PPO)
200 MPa 400 MPa 600 MPa 200 MPa 400 MPa 600 MPa
R2 0.539 0.375 0.801 0.806 0.584 0.684
R2Adj 0.529 0.361 0.796 0.802 0.544 0.677
E (%) 35.55 36.54 32.17 54.15 45.32 41.84
Parameters Fractional model, Equation (4)
Peroxidase (POD) Polyphenoloxidase (PPO)
200 MPa 400 MPa 600 MPa 200 MPa 400 MPa 600 MPa
R2 0.901 0.844 0.925 0.983 0.839 0.907
R2Adj 0.893 0.833 0.919 0.982 0.828 0.901
E (%) 16.81 20.54 26.65 15.36 34.32 27.17
Parameters Weibull model, Equation (5)
Peroxidase (POD) Peroxidase (POD)
200 MPa 400 MPa 600 MPa 200 MPa 400 MPa 600 MPa
2
R 0.949 0.916 0.977 0.967 0.962 0.973
R2Adj 0.946 0.912 0.976 0.965 0.961 0.972
E (%) 12.27 15.36 13.61 19.33 15.48 13.32
Parameters Isozyme model, Equation (6)
Peroxidase (POD) Peroxidase (POD)
200 MPa 400 MPa 600 MPa 200 MPa 400 MPa 600 MPa
2
R 0.963 0.873 0.953 0.987 0.921 0.948
2
R Adj 0.959 0.858 0.947 0.986 0.912 0.921
E (%) 10.23 19.09 21.26 12.32 24.47 13.39
R – coefficient of determination; R Adj – adjusted coefficient of determination; E – residual
2 2

analysis, and average absolute percentage of residuals.

35
Table 3

Parameter Arrhenius-type Equation (11)

200 MPa 400 MPa 600 MPa
POD PPO POD PPO POD PPO
αT 788.9±214.6 54.4±9.5 3169.8±262.9 144.1±53.9 3640.4±81.1 228.5±66.5
β 0.25±0.05 0.23±0.04 0.18±0.06 0.16±0.04 0.26±0.05 0.18±0.04
Ea 107.2±20.4 40.8±12.1 172.9±25.6 144.5±37.3 192.2±34.6 190.5±43.1
R2 0.949 0.967 0.916 0.965 0.977 0.973
R2Adj 0.945 0.965 0.912 0.961 0.976 0.971
E (%) 12.27 19.33 15.56 15.48 13.61 13.32
The error was calculated from the 95% confidence interval. POD – peroxidase; PPO – polyphenoloxidase; αT –

scale parameter at a reference temperature; β – shape parameter; Ea – apparent activation energy (kJ mol-1); R2

— coefficient of determination; R2Adj — adjusted coefficient of determination; E - average absolute percentage

of residuals.

36
 Table 4

Parameter Eyring-type Equation (12)

40°C 60°C 80°C 90°C
POD PPO POD PPO POD PPO POD PPO
αP 2750±230 360±20 298±15 258±45 176±35 31±11 35±9 25±9
-13.2±4.6 -37.7±9.4 4.1±2.1 -16.2±4.2 13.3±3.1 -4.1±1.7 10.2±3.6 9.2±3.2
β 0.28±0.06 0.21±0.05 0.24±0.05 0.18±0.04 0.24±0.07 0.19±0.04 0.53±0.09 0.47±0.01
R2 0.879 0.951 0.895 0.946 0.967 0.967 0.988 0.993
R2Adj 0.873 0.948 0.881 0.944 0.961 0.965 0.987 0.991
E (%) 4.44 8.91 9.27 14.15 18.5 18.51 16.69 14.25

The error was calculated from the 95% confidence interval. POD – peroxidase; PPO – polyphenoloxidase; αP –

scale parameter at a reference pressure; β – shape parameter; – activation volume (cm3 mol-1); R2 —

coefficient of determination; R2Adj — adjusted coefficient of determination; E - average absolute percentage of

residuals.

37
 Table 5

Color parameters
Time 40°C/400 MPa 80°C/400 MPa

(s) L *
a*
b *
 L*
a* b* 
C 85.52±0.01a 0.07±0.01c -2.28±0.01e - 85.52±0.01a 0.07±0.01f -2.28±0.01g -
a a b a a d f
60 85.18±0.01 0.20±0.01 -1.67±0.01 0.71±0.01 84.76±0.01 0.23±0.05 -1.88±0.01 0.87±0.01f
120 85.18±0.05a 0.19±0.02a -1.71±0.01c 0.67±0.02d 86.35±0.05a 0.35±0.01b -0.82±0.01c 1.70±0.0Sc
300 85.26±0.02a 0.22±0.01a -1.56±0.01a 0.78±0.01b 84.43±0.04a 0.21±0.05e -1.78±0.01e 1.21±0.01e
600 85.26±0.01a 0.13±0.01b -1.57±0.01a 0.76±0.01b 86.85±0.01a 0.47±0.01a -0.43±0.05b 2.31±0.01a
1200 84.48±0.03a 0.20±0.03a -1.74±0.01c 1.18±0.01a 86.09±0.03a 0.47±0.01a -0.37±0.05a 2.03±0.02b
1800 85.37±0.01a 0.22±0.01a -1.68±0.01bc 0.64±0.01d 85.31±0.01a 0.29±0.05c -0.85±0.01d 1.46±0.01d

Values are means of at least two replications. C: Control; L*: lightness; a*: red for positive values and green for

negative values; b*: yellow for positive values and blue for negative values.

38
Table 6

Color parameters
Time 40°C/600 MPa 80°C/600 MPa

(s) L *
a*
b *
 L*
a* b* 
C 85.52±0.01a 0.07±0.01e -2.28±0.01f - 85.52±0.01c 0.07±0.01d -2.28±0.01e -
a b b c c c b
60 85.21±0.01 0.25±0.02 -1.37±0.01 0.98±0.01 85.75±0.01 0.31±0.01 -1.03±0.01 1.29±0.01c
120 84.83±0.02a 0.20±0.02c -1.96±0.01e 0.77±0.01f 85.60±0.01c 0.34±0.01b -1.03±0.01c 1.28±0.01c
300 85.28±0.03a 0.20±0.01c -1.51±0.05c 0.82±0.02e 85.40±0.01c 0.31±0.01c -1.11±0.02d 1.20±0.05d
600 84.85±0.04a 0.23±0.01b -1.68±0.01d 0.91±0.01d 86.48±0.03a 0.37±0.01a -0.67±0.02b 1.90±0.05b
1800 85.98±0.01a 0.29±0.02d -1.00±0.02d 1.15±0.01b 87.14±0.04a 0.38±0.01a -0.48±0.01a 2.44±0.01a

Values are means of at least two replications. C: Control; L*: lightness; a*: red for positive values and green for

negative values; b*: yellow for positive values and blue for negative values.

39