Effect of ozonation parameters on nutritional and microbiological quality of sugarcane juice

Abstract

Sugarcane juice is susceptible to rapid spoilage and quality deterioration owing to browning of the juice and fermentation. To address this issue, ozonation of the juice has been attempted in this work. For optimization of ozonation process, response surface methodology based on full factorial design with control variables of gas flow rates (3, 6.5, and 10 L/min); ozone concentration (10, 20, and 30% wt/wt); and exposure time (5, 12.5, and 20 min) were employed. Physicochemical and microbiological indices were affected significantly by the gas flow rate irrespective of other two independent variables. Regression analysis showed that the predicted quadratic model fitted well with the experimental data and was significant (p < .05) with coefficient of determination (R²) for all responses >.9. Significant reductions in nutritional parameters, such as polyphenol content (13.5%), flavonoid content (22.5%), ascorbic acid content (81.46%), and antioxidant capacity (30%) were observed after ozonation. However, the treatment resulted in 67.8% inactivation of polyphenol oxidase enzyme and 3.72 log reduction in total plate count. Sensory analysis revealed that the treated juice was acceptable in terms of sensory properties. Thus, the ozone treatment was proved effective in enzyme inactivation and microbial destruction that offered enhanced anti‐browning and antimicrobial properties to the sugarcane juice.

Practical applications
Sugarcane juice is susceptible to rapid spoilage and quality deterioration owing to the browning and fermentation resulting in its short shelf life. It is an extremely perishable product that loses its marketability because of processing problems. Considering the nutritional importance of this juice, it can be emerged as an energy drink if adequately treated. Consumer demands for minimally processed health foods have further encouraged the research related to techniques for processing of fruit juices. Ozone processing is one such potential nonthermal method which can enable extension of shelf life of sugarcane juice, yet retaining its freshness and organoleptic properties that in turn aid in the growth of juice processing industry.

Full text

ORIGINAL ARTICLE
Effect of ozonation parameters on nutritional and
microbiological quality of sugarcane juice
Chirasmita Panigrahi
1
| Hari Niwas Mishra
1
| Sirshendu De
2
1
Department of Agricultural and Food
Engineering, IIT Kharagpur, Kharagpur, India
2
Department of Chemical Engineering, IIT
Kharagpur, Kharagpur, India
Correspondence
Chirasmita Panigrahi, Department of
Agricultural and Food Engineering, IIT
Kharagpur, Kharagpur 721302, India.
Email: chirasmitapanigrahi8@gmail.com
Funding information
Ministry of Food Processing Industries, Govt.
of India, Grant/Award Number: IIT/SRIC/R/
TSJ/2019
Abstract
Sugarcane juice is susceptible to rapid spoilage and quality deterioration owing to
browning of the juice and fermentation. To address this issue, ozonation of the juice
has been attempted in this work. For optimization of ozonation process, response
surface methodology based on full factorial design with control variables of gas flow
rates (3, 6.5, and 10 L/min); ozone concentration (10, 20, and 30% wt/wt); and expo-
sure time (5, 12.5, and 20 min) were employed. Physicochemical and microbiological
indices were affected significantly by the gas flow rate irrespective of other two inde-
pendent variables. Regression analysis showed that the predicted quadratic model
fitted well with the experimental data and was significant (p< .05) with coefficient of
determination (R
2
) for all responses >.9. Significant reductions in nutritional parame-
ters, such as polyphenol content (13.5%), flavonoid content (22.5%), ascorbic acid
content (81.46%), and antioxidant capacity (30%) were observed after ozonation.
However, the treatment resulted in 67.8% inactivation of polyphenol oxidase enzyme
and 3.72 log reduction in total plate count. Sensory analysis revealed that the treated
juice was acceptable in terms of sensory properties. Thus, the ozone treatment was
proved effective in enzyme inactivation and microbial destruction that offered
enhanced anti-browning and antimicrobial properties to the sugarcane juice.
Practical applications
Sugarcane juice is susceptible to rapid spoilage and quality deterioration owing to the
browning and fermentation resulting in its short shelf life. It is an extremely perish-
able product that loses its marketability because of processing problems. Considering
the nutritional importance of this juice, it can be emerged as an energy drink if ade-
quately treated. Consumer demands for minimally processed health foods have fur-
ther encouraged the research related to techniques for processing of fruit juices.
Ozone processing is one such potential nonthermal method which can enable exten-
sion of shelf life of sugarcane juice, yet retaining its freshness and organoleptic prop-
erties that in turn aid in the growth of juice processing industry.
1|INTRODUCTION
Sugarcane juice is a delicious and pleasing beverage relished particu-
larly for its sweet taste, refreshing sensation and appealing flavor. This
juice is a proven health promoting drink known to prevent several
ailments, such as, jaundice, sore throat, constipation, heat stroke, and
many more owing to its enriched nutritional benefits (Khare, Lal,
Singh, & Singh, 2012). In view of consumer demands for nutritious
food products, sugarcane juice is a vital drink. The freshly prepared
juice is highly susceptible to contamination with a risk of infection
Received: 23 December 2019 Revised: 14 August 2020 Accepted: 17 August 2020
DOI: 10.1111/jfpe.13542
J Food Process Eng. 2020;e13542. wileyonlinelibrary.com/journal/jfpe © 2020 Wiley Periodicals LLC. 1of14
https://doi.org/10.1111/jfpe.13542

after consumption. Development of effective treatment which can
enhance the storability of sugarcane juice maintaining its inherent
quality, freshness, and nutrient content is of paramount importance
for improving its marketability.
Several preservation techniques have been followed to reduce
microbial counts and inhibit enzyme activity to retard the deteriora-
tion of sugarcane juice. The common techniques adopted include
thermal methods, such as, blanching, pasteurization, and so forth,
which assure microbiological safety (Kaavya, Pandiselvam, Kothakota,
Priya, & Prasath, 2019). However, these are accompanied by signifi-
cant loss of bioactive compounds which is not favorable in nutrient
enriched juice like sugarcane (Huang, Cheng, Hu, & Pan, 2015).
Among the various nonthermal or alternate thermal techniques that
are in vogue, ohmic heating (Abhilasha & Pal, 2018), pulsed electric
field (Kayalvizhi, Pushpa, Sangeetha, & Antony, 2016), microwave
combined with ultrasound treatment (Zia, Khan, Zeng, Shabbir, &
Aadil, 2019), high pressure processing (Sreedevi, Jayachandran, &
Rao, 2018), membrane processing (Panigrahi, Karmakar, Mondal,
Mishra, & De, 2018), and ozonation along with addition of lactic acid
(Garud, Priyanka, Rastogi, Prakash, & Negi, 2017) have reported sub-
stantial inactivation effects and are expected to facilitate the preser-
vation of inherent nutritional profile of juice. Garud et al. (2017) have
used the variation of one parameter at a time maintaining other oper-
ating parameters invariant (exposure time and lactic acid concentra-
tion were varied while fixing the ozone feed rate). However, an
appropriate optimization method needs to be adopted due to cross
effects of various important quality parameters. Furthermore, the
effects of processing on the antioxidants including polyphenols and
flavonoids as well as minerals were not considered by the relative
study which are essential parameters to be monitored in processed
juices.
Ozone is generated by splitting of oxygen atoms into nascent
oxygen and recombining with an oxygen molecule to become a tri-
oxygen molecule through the action of electric discharges. It pos-
sesses high oxidizing power that conveys a wider antimicrobial
spectrum attributing to its diffusion capacity. Either the ozone mole-
cules or the free radicals generated by the ozone decomposition
react with the organic compounds to cause their oxidation (Chiron,
Fernandez-Alba, Rodriguez, & Garcia-Calvo, 2000). Industrial guide-
lines are issued by Food and Drug Administration in 1997 for
granting ozone the “generally recommended as safe”status which
have triggered the application of ozone gas in juice processing. In
the food industry, ozone is used as disinfectant for the sanitization
of fruits and vegetables offering safe storage without altering their
physicochemical and organoleptic characteristics (de Souza
et al., 2018). The effectiveness of gaseous or dissolved ozone
applied is influenced by various factors, such as, concentration,
exposure time, pH, temperature, and food composition. Ozone inac-
tivates microorganisms through cell disruption due to oxidation
(Prabha, Barma, Singh, & Madan, 2015). The ozone left out after
reaction breaks down to nonhazardous products making it an
eco-friendly disinfecting agent (Asokapandian, Periasamy, & Swamy,
2018). Literature reports on use of ozone in fruit juice processing are
available, such as, apple juice (Torlak, 2014), citrus juices, like,
orange, lemon and lime (Shah, Sulaiman, Sidek, & Supian, 2019),
melon juice (Fundo et al., 2018), orange juice (Angelino, Golden, &
Mount, 2003; Patil, Bourke, Frias, Tiwari, & Cullen, 2009; Tiwari,
Muthukumarappan, O'Donnell, & Cullen, 2008), blackberry juice
(Tiwari, O'Donnell, Muthukumarappan, & Cullen, 2009), grape juice
(Tiwari, O'Donnell, Patras, Brunton, & Cullen, 2009a, 2009b), straw-
berry juice (Tiwari, et al., 2009a, 2009b), and peach juice (Jaramillo-
Sanchez, Garcia Loredo, Gomez, & Alzamora, 2018).
The process parameters of ozone treatment cannot be general-
ized and thus, the studies are critical for investigating the effective-
ness of ozonation to a specific juice. Therefore, the present study is
directed to optimize the ozone treatment parameters elucidating their
effects on nutritional, bioactive, and microbiological qualities of sugar-
cane juice.
2|MATERIALS AND METHODS
2.1 |Raw material
Sugarcane juice was extracted from the peeled canes of a local variety
Co87632 (“Sarayu”) and procured from the local market in IIT
Kharagpur campus. The juice was then filtered through cheese cloth
to discard the impurities. The clear filtrate was used for the
experiments.
2.2 |Ozone treatment
The cloth-filtered juice (100 ml) was used in each experiment of
ozone treatment. Gaseous ozone was generated in the ozonation
chamber by corona discharge effect from the compressed oxygen
feed gas as input using a laboratory scale ozone generator (OZ-AIR,
ISM 5, Creative OZ-AIR Pvt., Ltd., Noida, India). The photograph of
ozone generator is shown in Figure S1 (supporting information).
Ozonation was accomplished by injecting a gas mixture of oxygen
and ozone under constant agitation (100 rpm) into the juice through
a pipe fitted with a diffuser dipped in it creating bubbles and gas dis-
tribution. The ozone flow rate (L/min) is monitored using a regulator
fitted to a flow meter. The concentration of ozone (% wt/wt) in the
gas stream is recorded by a gas analyzer. To theoretically determine
the ozone concentration (g/m
3
), a conversion formula (1% wt/wt
ozone = 14.3 g ozone/m
3
oxygen) is used. The following formula
(Equation (1)) is used to calculate the amount of ozone required per
unit volume of juice.
Ozone dose g
ml

=
Ozone concentration g
m3

×Gasflow rate m3
min

×Treatment time minðÞ
Sample volume mlðÞ :
ð1Þ
There is provision of one exit pipe fitted in the sample bottle
for purging out the amount of unreacted gas into a water tank for
2of14 PANIGRAHI ET AL.

neutralization. Treatments were performed at room temperature
(25C). The schematic of the experimental set up is presented in
Figure 1.
2.3 |Experimental design and statistical analysis
Extrinsic process parameters, namely, gas flow rate, ozone concen-
tration, and exposure time are known to influence the ozone
processing. Preliminary experimental trials were conducted to ver-
ify the ranges (or levels) of variables. Gas flow rates of 3 and
10 L/min were the respective lowest and highest levels which
could be set by the instrument. From previous studies, it was found
that ozone concentration less than 10% (wt/wt) did not pose signif-
icanteffectoninactivation,yetrequiredmuchlongertimeforthe
same. Concentration beyond 30% (wt/wt) had insignificant effect
on enzymatic and microbial inactivation, rather resulted in destruc-
tion of nutritional compounds, like polyphenols, flavonoids,
vitamin C, antioxidants, and minerals. Exposure time of less than
5 min was insufficient to produce a significant effect while above
20mincausedahigherdestructionofnutritionalcompoundsand
FIGURE 1 Experimental set up for ozone treatment of sugarcane juice
TABLE 1 Full factorial experimental design matrix showing ozone treatment parameters and values of the responses
Run Independent variables Responses
Gas flow
rate (L/min)
Ozone concentration
(%wt/wt)
Exposure
time (min) BI
PhC
(mg GAE/ml)
PPO
activity (U/ml)
POD
activity (U/ml)
TPC
(log CFU/ml)
1 3 30 20 72.37 32.62 14.34 30.43 4.08
2 10 30 5 75.36 33.96 23.04 41.55 6.04
3 6.5 20 5 72.55 32.79 12.05 30.86 4.15
4 3 20 20 72.7 33.76 15.25 32.83 4.48
5 3 20 12.5 72.91 33.93 18.17 33.44 4.69
6 3 10 20 73.01 34.29 17.68 34.18 4.85
7 6.5 20 20 70.29 32.47 11.95 28.82 3.69
8 10 10 5 77.16 35.64 23.47 42.98 6.15
9 6.5 10 12.5 72.81 31.93 13.66 31.54 4.04
10 10 10 20 73.86 34.71 24.29 39.59 5.6
11 10 20 5 76.32 34.88 26.87 41.83 6.08
12 3 10 5 74.28 34.59 19.64 36.22 5.95
13 10 30 12.5 74.74 34.25 22.35 36.27 4.48
14 6.5 10 5 73.67 33.28 12.32 31.25 4.18
15 6.5 30 5 72.88 32.27 11.87 32.67 3.9
16 10 10 12.5 76.46 34.77 20.76 41.29 5.69
17 10 30 20 72.24 34.12 20.08 35.31 4.78
18 6.5 20 12.5 70.76 31.84 12.06 29.04 3.78
19 3 10 12.5 73.26 34.45 18.05 34.53 5.11
20 6.5 30 12.5 69.07 31.54 11.56 28.37 3.48
21 6.5 10 20 72.38 32.65 12.76 29.58 4.17
22 3 30 5 73.07 34.05 18.71 34.77 4.9
23 3 20 5 73.29 34.09 18.75 35.28 4.95
24 10 20 20 72.54 34.39 19.86 36.59 5.2
25 3 30 12.5 72.52 33.69 18.55 32.07 4.3
26 10 20 12.5 75.49 34.44 23.11 39.83 5.26
27 6.5 30 20 68.48 31.07 11.42 28.17 3.3
Abbreviations: BI, browning index; PhC, polyphenol content; TPC, total plate count.
PANIGRAHI ET AL.3of14

also hampered the juice quality in terms of flavor and taste. Thus,
the variable levels have been selected focusing on achieving a
desirable effect, that is, maximum inactivation of browning
enzymes (polyphenoloxidase [PPO] and peroxidase [POD]) and
microorganisms (bacteria and yeast and mold) with the least dam-
age to useful components (like polyphenols, antioxidants, etc.) and
organoleptic quality of juice. The independent variables were gas
flow rates (x
1
): 3, 6.5, and 10 L/min; ozone concentrations (x
2
):
10, 20, and 30% (wt/wt); and exposure time (x
3
): 5, 12.5, and
20 min. Total 27 (3
3
) experiments were conducted in this study
(Table 1). The dependent variables, browning index (BI) (Y
1
), poly-
phenol content (PhC) (Y
2
), PPO activity (Y
3
), POD activity (Y
4
), and
total plate count (TPC) (Y
5
) were selected as the response func-
tions. In Design Expert 7.0.0 software, the experimental design was
set and the statistical analysis was performed. Response surface
methodology based on a three-factor three-level full factorial
design was employed. The mean values of three readings for all
analyses were reported. These values were linked to the coded var-
iables by a second-order polynomial function using the least-square
regression method (Equation (2)):
Yn=β0+β1x1+β2x2+β3x3+β12x1x2+β13 x2x3+β23x1x3
+β11x2
1+β22x2
2+β33x2
3
ð2Þ
where Y
n
is the predicted response; β
0
is the model constant; β
1
,β
2
,
and β
3
are the linear coefficients; β
11
,β
22
, and β
33
are the quadratic
coefficients; and β
12
,β
13
, and β
23
are the cross product (interaction
term) coefficients.
The analysis of variance (ANOVA) tables were generated and
the statistical significance of all terms were arbitrated by computing
the F-value at a level of significance of 5, 1, 0.1, and 0.01%. The
regression coefficients of individual terms (linear, quadratic, and
interaction) were obtained which were then used to make statistical
calculation and to generate contour maps or three-dimensional
response surface plots. The regression models for the response vari-
ables were determined from the experimental design and the model
with non-significant lack of fit (p> .05) was only considered for
optimization.
2.4 |Process optimization and data validation
The predicted values (Y
n
) were transformed to a dimensionless
desirability function based on which the process optimization was
carried out. Optimized process conditions were found based on
the overall desirability D, which is the average of desirability of
the individual responses. The simulated result was validated by
experimentation with the optimized conditions. One-way ANOVA
trailed by Tukey post hoc test was performed to establish the
comparison between the control and ozone-treated sample by
evaluating the statistical variation in parameters at a probability
value of .05.
2.5 |Analysis of physicochemical and nutritional
parameters of sugarcane juice
2.5.1 |Total soluble solids and BI
Total soluble solids (TSS) were determined using a handheld refrac-
tometer (Erma, Tokyo, Japan).
BI, the degree of brown color development, is a common index of
browning in food products containing sugar. BI was determined using
Equation (3) (Sreedevi et al., 2018).
BI = 180:232 a+1:75b
ðÞ
5:645L+a−3:012b
ðÞ
ð3Þ
where L* denotes lightness, a* indicates red/green value, and b*
denotes yellow/blue value.
2.5.2 |Polyphenol content
PhC was determined by Folin–Ciocalteu's (FC) method against gal-
licacidasstandardusedforcalibration(Kamtekar,Keer,&
Patil, 2014). An aliquot of 0.25 ml of sample and 1.25 ml of FC
reagent were taken in a test tube, mixed, and shaken. After 5 min,
3.75 ml of 7.5% sodium carbonate was added and distilled water
wasusedtomakeuptototal12ml.Thereactionmixturewaskept
undisturbed for 2 hr in dark at room temperature (27 ± 2C) for the
development of deep blue color. The absorbance was noted at
760 nm in a UV–Vis spectrophotometer (M/s PerkinElmer, Shel-
ton, CT).
2.5.3 |Estimation of enzyme activity
PPO activity
PPO enzyme activity was estimated by carrying out enzyme assay as
described by Saxena, Makroo, Bhattacharya, and Srivastava (2018).
For reaction mixture, 1 ml of 0.2 M catechol solution was added to
0.2 ml of sample and 2 ml of 0.2 M sodium phosphate buffer (pH 6.5).
The absorbance was recorded at 420 nm at every 30 s interval by
spectrophotometer.
POD activity
POD activity was determined according to Kunitake, Ditchfield, Silva,
and Petrus (2014). The technique includes immersion of a test tube
containing 3.5 ml of 0.2 M phosphate buffer (pH 5.5) and 0.5 ml of
sample in a 35C water bath till the stabilization of the temperature. It
was followed by the addition of 0.75 ml of guaiacol (0.05%) and
0.25 ml of hydrogen peroxide (0.1%) and homogenization of the mix-
ture. The changes in absorbance were read at 470 nm in a spectro-
photometer at 1 min interval.
PPO and POD activity were calculated using Equation (4),
4of14 PANIGRAHI ET AL.

Activity U
ml

=Absample −Abblank

0:001 X tð4Þ
where Ab
sample
is the sample absorbance and Ab
blank
is the blank
absorbance and tis incubation time (min).
2.5.4 |Antioxidant capacity
Antioxidant capacity was estimated using free-radical scavenging
assay expressed as inhibitory concentration (IC
50
) (Kong, Yu, Zeng, &
Wu, 2015). Briefly, 0.1 ml of 10 mM 2.2-diphenyl-L-picry1 hydrazyl
methanolic solution was added to test tubes having sample volumes
in the range of 0.05–1 ml. The mixture was shaken properly and incu-
bated for 30 min. For control, sample was replaced with methanol.
The absorbance was noted at 517 nm and percentage scavenging
capacity was calculated using Equation (5).
Scavenging capacity %ðÞ=Abscontrol −Abssample
Abscontrol
×100 ð5Þ
where Abs
control
is the control absorbance and Abs
sample
is the sample
absorbance.
2.5.5 |Total flavonoid content
Total flavonoid content was determined by following the aluminum
chloride colorimetric assay using quercetin as standard (Kamtekar
et al., 2014). Sample (0.5 ml), distilled water (4 ml), and 5% sodium
nitrite solution (0.3 ml) were mixed. After 5 min, 0.3 ml of 10% alumi-
num chloride and 2 ml of 1 M sodium hydroxide were added. The
reaction mixture was made up to 10 ml with distilled water, shaken
well and finally, when yellowish orange color developed, the absor-
bance was read at 385 nm. The flavonoid content was expressed as
mg quercetin equivalent (QE)/ml juice.
2.5.6 |Vitamin C content
Vitamin C or ascorbic acid content was determined by
2,6-dichlorophenol indophenol titration method 967.21 according to
Association of Official Analytical Chemists (AOAC) (1998). Briefly,
20 ml of metaphosphoric acid extract of sample was taken in an Erlen-
meyer flask and titrated with 2,6-dichlorophenol indophenols dye.
The dye had earlier been standardized with vitamin C solution to find
an equivalence factor. Vitamin C content was calculated using the fol-
lowing formula (Equation (6)) and expressed as mg ascorbic
acid/100 ml.
Vitamin C = T×D×V
v×W×100 ð6Þ
where Tis the titer value, Dis the dye factor, Vis the volume made
up, vis the volume of extract taken for estimation, and Wis the
weight or volume of sample.
2.6 |Mineral profiling
Mineral composition of raw and ozone-treated sugarcane juice such
as calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), copper (Cu),
zinc (Zn), cobalt (Co), and manganese (Mn) were determined using
atomic absorption spectrometer (Analyst 700, PerkinElmer). The sam-
ple preparation includes acid digestion and filtration for removing the
digested residues. The filtrates were taken for mineral analysis. For all
elements, calibration was performed using individual standards.
2.7 |Microbiological evaluation
The microbial characteristics of sugarcane juice were evaluated in
terms of enumeration of TPCs and yeasts and mold count (YMC) by
the spread plate method according to American Public Health Associ-
ation (American Public Health Association, 1976). Nutrient agar and
yeast malt agar were taken as the growth media for TPC and YMC,
respectively, and corresponding incubation conditions were
37 ± 1C/24 hr and 25 ± 1C/72 hr. The colony counts were
expressed as log
10
colony forming unit (CFU)/ml (Equation (7)).
CFU
ml =CFU per plate ×Dilution factor
Volume of sample mlðÞ ð7Þ
2.8 |Sensory analysis
The sensory evaluation of the raw and ozone-treated sugarcane juice
was done by 10 trained members from Agricultural and Food Engi-
neering Department, Indian Institute of Technology, Kharagpur. The
attributes adjudged were color, taste, flavor, aroma, and overall
acceptability. The sensory testing was performed based on 9-point
Hedonic rating scale tests (1 = dislike extremely, 2 = dislike very much,
3 = dislike moderately, 4 = dislike slightly, 5 = like slightly, 6 = like
fairly well, 7 = like moderately, 8 = like very well, and 9 = like
extremely). Scores below 5 are regarded as “unacceptable”and above
6 are considered as “safe”(Pandraju & Rao, 2020).
3|RESULTS AND DISCUSSION
The effects of gas flow rate, ozone concentration, and exposure time
on BI, PhC, PPO activity, POD activity, and TPC are shown in Table 1.
PPO and POD inactivation are among the priority variables since
these enzymes initiate the browning process resulting in degradation
PANIGRAHI ET AL.5of14

of phenolic compounds and formation of brown pigments. The values
for BI, PhC, PPO, POD, and TPC varied between 68.48 and 77.16,
31.07 and 35.64 mg GAE/100 ml, 11.42 and 26.87 U/ml, 28.17 and
42.98 U/ml and 3.3 and 6.15 log CFU/ml, respectively, depending on
the ozone concentration and contact time. The highest gas flow rate
(10 L/min) showed the least effect on PPO activity (48.86% reduction)
while with the same ozone concentration (30% wt/wt) and for same
exposure time (20 min), the decrease in PPO activity (71%) was
remarkable with 6.5 L/min of flow rate. Similar trends were observed
for other parameters.
The ANOVA of all process parameters of the predicted model
showing estimated regression coefficients, the significance level, and
correlation coefficient for each response variable is summarized in
Table 2. The coefficients of the polynomial (second-order) equations
(refer Equation (2)) were computed from the experimental data.
ANOVA was conducted to demonstrate the significant effects of pro-
cess variables on each response. Regression analysis showed that all
the responses were significantly affected by the process parameters.
Gas flow rate was the most crucial factor influencing the juice quality
parameters since the quadratic term associated with the flow rate was
significant for all the response variables evaluated (p< .0001). All
response variables demonstrated high correlation coefficients with
the mathematical model as determined by the adjusted R-square (adj
R
2
) values of .87, .91, .9, .96, and .92 for BI, PhC, PPO, POD, and TPC,
respectively. The 3D response surface graphs were generated to illus-
trate the effect of independent variables on the responses as shown
in Figures 2 and 3.
3.1 |Browning index
Anincreaseinlightnessandyellownessvaluesanddecreaseinred-
ness values were observed during ozonation. BI was found to be
affected significantly by flow rate, ozone concentration, and treat-
ment time. It was negatively associated with the linear effects of
gas flow rate as well as ozone concentration while positive correla-
tion was found with exposure time (p< .0001). The minimum BI
was found at flow rate 6.5 L/min and it increased when rate
increased to 10 L/min. The explanation of such behavior is pres-
ented in the subsequent section. Furthermore, the interaction of
gasflowratewithexposuretimehadasignificanteffecton
BI (p< .01).
TABLE 2 ANOVA for regression analysis of ozone treatment of sugarcane juice
Response
Terms
Intercept x
1
x
2
x
3
x2
1x2
2x2
3x
1
x
2
x
2
x
3
x
1
x
3
BI (R
2
= .92, adj R
2
= .87)
Coefficient 79.55 −2.01 −0.104 0.046 0.21 0.002 0.0004 −0.006 −0.003 −0.024
Fvalue —29.22 27.17 44.62 72.74 0.53 0.005 1.01 0.87 9.11
p-Value —<.0001*** <.0001*** <.0001*** <.0001*** .475 .94 .328 .364 .008**
PhC (R
2
= .94, adj R
2
= .91)
Coefficient 40.49 −2.13 −0.014 −0.14 0.167 −0.0007 0.004 0.0004 −0.0007 0.003
Fvalue —14.56 34.35 13.46 204.65 0.25 2.34 0.022 0.25 0.43
p-Value —.0014 ** <.0001*** .0019 ** <.0001*** .62 .14 .88 .62 .52
PPO activity (R
2
= .93, adj R
2
= .9)
Coefficient 35.94 −7.82 0.06 0.05 0.65 −0.0009 −0.002 0.002 0.002 −0.008
Fvalue —51.67 2.97 9.43 178.02 0.022 0.041 0.02 1.95 0.02
p-Value —<.0001*** .103 .0069 ** <.0001*** .884 .84 .889 .181 .895
POD activity (R
2
= .97, adj R
2
= .96)
Coefficient 49.91 −5.77 −0.04 −0.185 0.54 0.002 0.01 −0.007 −0.009 −0.019
Fvalue —189.74 33.24 72.87 334.36 0.36 2.46 1.01 6.84 3.92
p-Value —<.0001*** <.0001*** <.0001*** <.0001*** .572 .135 .328 .018* .064
TPC (R
2
= .95, adj R
2
= .92)
Coefficient 9.41 −1.28 −0.04 −0.134 0.11 0.0002 0.005 0.001 −0.001 −0.0009
Fvalue —34.86 41.07 36.99 175.62 0.063 7.48 0.35 1.53 0.13
p-Value —<.0001*** <.0001*** <.0001*** <.0001*** .805 .014* .56 .233 .72
Abbreviations: ANOVA, analysis of variance; BI, browning index; PhC, polyphenol content; POD, peroxidase; PPO, polyphenoloxidase; TPC, total plate
count.
*Significant with p< .05.
**Significant with p< .01.
***Significant with p< .0001.
6of14 PANIGRAHI ET AL.

3.2 |Polyphenol content
Regression analysis showed the negative effect of ozone concentra-
tion and treatment time on PhC (p< .001). PhC decreased with flow
rate up to a certain point, then it again increased. The explanation
related to such observation is discussed later. The PhC depends on
the flow rate of gas, where its linear (p< .01) effect was negative but
the quadratic (p< .0001) effect was positive. The interaction effects
were not significant on polyphenol concentration.
3.3 |PPO enzyme activity
PPO enzyme activity was found to be dependent on all three parame-
ters but mainly on the gas flow rate. The minimum enzyme activity
and maximum inactivation were obtained at 6.5 L/min flow rate of
gas. This observation is due to specific reasons which are explained
later in subsequent section. Increasing gas flow rate (p< .0001) and
decreasing exposure time (p< .01) decreased PPO activity. However,
ozone concentration did not show significant effect on PPO activity
(p> .05). The positive quadratic coefficient of gas flow rate indicated
that PPO activity significantly increased with this parameter.
3.4 |POD enzyme activity
POD activity has positive linear effects with gas flow rate, ozone con-
centration, and exposure time. It is strongly affected by exposure time
as the quadratic term linked to this independent variable is significant
(p< .0001). The activity becomes minimum at a particular value of gas
FIGURE 2 Response surface graphs showing the effect of gas flow rate and ozone concentration on (a) browning index, (b) polyphenol
content, (c) polyphenoloxidase (PPO) activity, (d) peroxidase (POD) activity, and (e) total plate count
PANIGRAHI ET AL.7of14

flow rate, that is, 6.5 L/min. The explanation of such behavior is given
in subsequent section. The interaction of ozone concentration with
exposure time negatively influenced the POD activity (p< .05). It
increases proportionally with both ozone concentration and
exposure time.
3.5 |Total plate count
The ozone treatment had a significant effect on microbial count of
sugarcane juice. After 20 min of processing, flow rate of 6.5 L/min
provided higher microbial destruction than 3 and 10 L/min at the
fixed ozone concentration. Also, the increase in exposure time
resulted in greater lethal effect. Statistical analysis showed that TPC
had negative linear effect with gas flow rate, ozone concentration,
and exposure time (p< .0001), but it had positive quadratic effect
with flow rate (p< .0001) and exposure time (p< .05).
It is noteworthy that, regardless of the ozone concentration and
exposure time, all the parameters showed a decreasing trend with gas
flow rate up to a certain range and increased thereafter. The desirable
effect of parameters studied herein was not achieved at either
extreme of flow rate rather at the middle range of values. This kind of
behavior needs explanation. The ozone solubility rate is affected by
factors, like pH, temperature, pressure, presence of impurities, ozone
bubble sizes, rate of gas flow, and time of contact which actually gov-
ern the efficacy of ozone treatment. Varied gas flow rates result in
production of different sizes of bubble. Bubble size is known to have
a great influence on ozone's solubilization rate, ozone mass transfer,
FIGURE 3 Response surface graphs showing the effect of ozone concentration and exposure time on (a) browning index, (b) polyphenol
content, (c) polyphenoloxidase (PPO) activity, (d) peroxidase (POD), and (e) total plate count
8of14 PANIGRAHI ET AL.

and disinfection efficiency (Ahmad & Farooq, 1985; Prabha
et al., 2015). At high gas flow rate, lesser number of large sized bub-
bles are formed, which rise to the surface of the liquid quickly and
escape the medium allowing less time to contact. This results in poor
gas dissolution, leading to a lower rate of inactivation. At low gas flow
rate, a large number of small sized bubbles are produced and ozone
solubility increases owing to the larger contact surface area available
for mass transfer (Miller, Silva, & Brand~
ao, 2013). However, under
these conditions, inactivation is slow and takes a longer time to obtain
the same result (Cullen et al., 2010).
3.6 |Optimization of ozone treatment
The fitting of quadratic model with the experimental data can be well
predicted with R
2
for all responses >0.9 and hence, the analyzed
model for the response was adequate and satisfactory. The software
identified 14 solutions of the optimum conditions for the independent
variables but 6 solutions having similar desirability values, which are
shown in Table 3. Therefore, a range of treatment conditions can be
suggested, namely, gas flow rate 5.24–6.48 L/min, ozone concentra-
tion 26.41–27.92% (wt/wt), and exposure time 20 min. The flow rate
of 5.6 L/min, 26.4% concentration, and time 20 min were considered
optimum, which resulted in the highest desirability of 0.875. The
predicted responses under optimum treatment conditions were BI
69.7, PhC 31.7 mg GAE/100 ml, PPO activity 11.01 U/ml, POD activ-
ity 28.3 U/ml, and TPC 3.31 log CFU/ml. For these optimal results,
the amount of ozone needed per unit volume of juice was found to be
4.23 g/L.
3.7 |Reproducibility of the study
In order to determine and verify the reliability of the regression model
in predicting optimal responses, experiments were conducted at the
optimum process conditions for the purpose of validation. The experi-
mental and predicted values are shown in Table 4. It was observed
that the predicted values agreed well with experimental data. The
experimentation at the optimum conditions supported the result
showing residual error below 5%. Thus, the developed model can be
ascertained adequate and satisfactory to predict the responses of sug-
arcane juice ozonation.
3.8 |Comparisons between control and treated
samples
Figure 4 displays the pictures of fresh and ozone-treated sugarcane
juice. Increase in lightness and yellowness values were observed in
ozonized juice. Table 5 demonstrates the comparison between the
raw juice and ozone-treated juice. It was clearly observed that all the
parameters of ozone-treated sample differed statistically from the
control sample (p< .05).
Ozone treatment did not result in much reduction (only 6.3%
decrease) in TSS content. This is because sugarcane juice is a low acid
food and in neutral or acidic media, ozone reacts through the oxygen
radical that does not seem to degrade the sucrose molecule
(Davis, 2001). This was confirmed in other studies, such as, Davis,
Moodley, Singh, and Adendorff (1998) who found that destruction of
sugars under the applied treatment conditions (ozone = 0–3.0 kg/hr,
pH 6.0–7.0, and temperatures above 70C) was insignificant. Gomez,
Perez, and Ramos (1980) also showed no sugar degradation during
ozonation in neutral pH or slightly acidic conditions.
Ozone is decomposed to form hydroxyl radicals leading to oxida-
tion of endogenous phenols disintegrating the cellular membrane
structure. This finally leads to degradation of polyphenols (Cullen,
Tiwari, O'Donnell, & Muthukumarappan, 2009). The cleavage of aro-
matic rings during gallic acid oxidation also occurs simultaneously
(de Souza Sartori, Angolini, Eberlin, & de Aguiar, 2017). Although
ozonation of sugarcane juice has led to a 13.5% degradation of its
phenolic concentration, this can be considered less compared to other
related studies. Torres et al. (2011) found 49.7% degradation of total
phenol content during ozonation of apple juice with ozone concentra-
tion of 4.8% (wt/wt) for 10 min. This difference was observed due to
the variation in the structure of phenolic compounds and distribution
of their functional groups (Onopiuk, Półtorak, Moczkowska, Szpicer, &
Wierzbicka, 2017). As a result, some slow-to-oxidize organics remain
slightly less affected. This behavior is desirable for the retention of
the nutritional property of the juice. Apart from this, the reaction
medium for sugarcane juice is acidic and it is known that the
TABLE 3 Optimization of process conditions for ozone treatment of sugarcane juice
Flow
rate (L/min)
Ozone
concentration (%)
Exposure
time (min) BI
PhC
(mg GAE/100 ml)
PPO
(U/ml)
POD
(U/ml)
TPC
(log CFU/ml) Desirability
5.3 26.41 20 69.716 31.697 11.012 28.31 3.308 0.875
5.24 26.87 20 69.543 31.692 11.08 28.324 3.304 0.874
5.68 27.51 20 69.794 31.734 11.1 28.293 3.33 0.873
5.91 27.4 20 69.604 31.675 11.045 28.345 3.339 0.873
6.35 27.92 20 69.547 31.689 11.057 28.33 3.375 0.871
6.48 27.23 20 69.691 31.78 11.123 28.351 3.369 0.871
Abbreviations: BI, browning index; PhC, polyphenol content; POD, peroxidase; PPO, polyphenoloxidase; TPC, total plate count.
PANIGRAHI ET AL.9of14

degradation by ozone is more pronounced in basic pH because of the
superior role of hydroxyl radical having a higher oxidation potential of
2.80 V at elevated pH as demonstrated by Esplugas, Gimenez, and
Contreras (2002). The reduction in flavonoid content (22.5%) after
ozonation is expected due to the sensitivity of flavonoids toward oxi-
dative stress. Polyphenols and flavonoids are associated with the
property of inhibiting oxidation by neutralizing generated free radicals
and contribute to the antioxidant capacity that was reduced by 30%
during ozonation of sugarcane juice. Ascorbic acid in ozone-treated
sugarcane juice was reduced significantly by 81.46%. The reason that
justifies this observation is the interaction of ozone or secondary oxi-
dants with the enzyme ascorbate oxidase which is known to promote
the oxidation (or degradation) of ascorbic acid into dehydroascorbic
acid. This enzyme gets activated under stress conditions in ozone
environment (Yeoh, Ali, & Forney, 2014). The findings of the current
study are in line with that of few past research studies. Tiwari
et al. (2009a, 2009b) described the decrease in content of some poly-
phenols in grape juice caused by ozonation. The results of a study
conducted by Alothman, Kaur, Fazilah, Bhat, and Karim (2010) illus-
trated the negative effects of ozone treatment (8 ml/s for 30 min) on
nutritional value of guava fruit. A decrease of 23.5 and 45.9% in total
PhC was observed for banana and guava, respectively. Flavonoid con-
tents of ozone-treated pineapple, banana, and guava were reduced by
50, 25.5, and 47.2%, respectively, for these fruits. Vitamin C content
was decreased by 46.44, 12.2, and 67.13%, respectively. Shah, Supian,
and Hussein (2019) found 34.33% loss in ascorbic acid of pummelo
juice when ozonized at 600 mg/hr for 50 min. According to Angelino
et al. (2003), ascorbic acid of orange juice was almost fully degraded
after 90 min exposure to 0.9 g/hr gaseous ozone. Tiwari, O'Donnell,
Muthukumarappan, and Cullen (2009) reported 69.5% reduction in
ascorbic acid in orange juice after 10 min of ozone treatment at a flow
rate of 0.0625 L/min. Tiwari, O'Donnell, Brunton, and Cullen (2009)
TABLE 4 Validation of regression
model developed for ozone treatment of
sugarcane juice
Response parameters Predicted values Experimental values
a
Residual
BI 69.716 67.35 ± 0.09 2.37
PhC (mg GAE/100 ml) 31.697 32.54 ± 0.03 0.85
PPO activity (U/ml) 11.012 12.66 ± 0.12 1.65
POD activity (U/ml) 28.31 31.41 ± 0.28 3.11
TPC (log CFU/ml) 3.308 4.02 ± 0.68 0.9
Abbreviations: BI, browning index; PhC, polyphenol content; POD, peroxidase; PPO, polyphenoloxidase;
TPC, total plate count.
a
Values are presented as mean ± SD of three replicates.
FIGURE 4 Pictures of fresh sugarcane juice and ozone-treated
sugarcane juice
TABLE 5 Comparison of nutritional, biochemical, microbiological,
and sensory parameters between control and treated samples
Parameters
Control (raw)
juice
Ozone-treated
juice
TSS (Brix) 14.73 ± 0.06
a
13.8 ± 0.0
b
BI 75.21 ± 0.04
a
67.35 ± 0.09
b
PhC (mg GAE/100 ml) 37.62 ± 0.03
a
32.54 ± 0.03
b
Total flavonoid content
(μg QE/ml)
5.38 ± 0.02
a
4.17 ± 0.03
b
Vitamin C content (mg ascorbic
acid/100 ml)
2.3 ± 0.22
a
0.42 ± 0.06
b
Antioxidant activity (IC
50
) 0.47 ± 0.006
a
0.61 ± 0.014
b
PPO activity (U/ml) 39.27 ± 0.04
a
12.66 ± 0.12
b
POD activity (U/ml) 126.28 ± 0.07
a
31.41 ± 0.28
b
TPC (log CFU/ml) 7.74 ± 0.46
a
4.02 ± 0.68
b
Yeast mold count (log CFU/ml) 5.27 ± 0.54
a
2.84 ± 0.18
b
Mineral (mg/100 ml)
Calcium 18.28 ± 1.19
a
16.13 ± 1.53
a
Magnesium 13.09 ± 0.61
a
12.1 ± 0.25
a
Sodium 1.75 ± 0.03
a
1.65 ± 0.04
b
Iron 1.39 ± 0.26
a
1.34 ± 0.13
a
Copper 0.095 ± 0.01
a
0.077 ± 0.006
a
Zinc 0.216 ± 0.02
a
0.188 ± 0.02
a
Cobalt 0.018 ± 0.005
a
0.016 ± 0.002
a
Manganese 0.237 ± 0.05
a
0.183 ± 0.02
a
Sensory parameter scores
Color 8.22 ± 0.11
a
8.44 ± 0.08
b
Flavor 8.48 ± 0.08
a
8.14 ± 0.12
b
Aroma 8.61 ± 0.08
a
7.98 ± 0.11
b
Taste 8.48 ± 0.18
a
8.26 ± 0.09
a
Overall acceptability 8.46 ± 0.15
a
7.82 ± 0.14
b
Note: Values with different letters indicate significant differences (p< .05)
according to the Tukey test.
Abbreviations: BI, browning index; PhC, polyphenol content; POD, peroxi-
dase; PPO, polyphenoloxidase; QE, quercetin equivalent; TPC, total plate
count; TSS, total soluble solid.
10 of 14 PANIGRAHI ET AL.

and Tiwari et al. (2009a, 2009b) observed that the nutritional qualities
of tomato and strawberry juice were significantly affected by ozone
processing. The treatment has resulted in 96 and 85.8% reductions of
ascorbic acid in respective juices at an ozone concentration of 7.8%
(wt/wt) and time of 10 min.
Significant inactivations of PPO (67.8%) and POD (75.3%)
enzymes were obtained after ozonation of sugarcane juice (p< .05).
Thus, the residual activities (RAs) for PPO and POD were found to be
32.2 and 24.8%, respectively. When compared with the result findings
of Garud et al. (2017) in ozone-treated sugarcane juice, it was
observed to be similar for POD inactivation (25% RA) but higher in
terms of PPO inactivation (58% RA). The reason for enzyme inactiva-
tion is the alteration of the active site of sulfhydryl ( SH) groups in
cysteine residue as a result of oxidation (Chakraborty, Kaushik, Rao, &
Mishra, 2014). The active free radical produced by ozone, attack sus-
ceptible functional groups in solution (Rosenfeld et al., 2015). The
cleavage of NH CO bond and blocking of enzyme active sites are
also responsible for the inactivation of endogenous enzymes by
ozone. The enzyme activity is controlled by its active sites. Alteration
in the active sites leading to the protein denaturation result in the
changes in functionality or the losses in the enzymatic activity (Huang,
Chang, & Wang, 2015). The contribution of PPO in enzymatic discol-
oration can be accentuated by the observation that BI of the ozonized
juice was less than that of the fresh juice by 10.5%. Jaramillo-Sanchez
et al. (2018) achieved 97.3 and 99.8% PPO and POD inactivation in
peach juice after exposing it to 0.2 mg O
3
min
−1
ml
−1
ozone for
12 min. Rico, Martín-Diana, Frías, Henehan, and Barry-Ryan (2006)
stated that the oxidizing potential of ozone enables its greater efficacy
in reduction of activities of PPO and POD enzymes which was
observed while treating fresh-cut lettuce with ozone.
The ozone treatment caused a significant reduction in microbial
count (3.72 log in TPC and 2.43 log in YMC). Garud et al. (2017)
reported the reduction of total bacterial count by 3.9 log after ozona-
tion. However, they reported the absence of yeast and mold in
ozone-treated sample that differed from the result observed in the
present study. This so happened since the susceptibility of microor-
ganisms to ozone not only depends on the amount of ozone applied
but is extremely affected by nature of product, characteristics, initial
number, and physiological conditions of microbial cells as well as pro-
tection caused by organic matter. Several other researchers have also
elucidated the reduction of the microbial load by treatment with
ozone. Farajzadeh, Qorbanpoor, Rafati, and Isfeedvajani (2013)
showed that ozonation of date with 10 g/hr ozone gas for 3 hr led to
76 and 53% reduction in bacterial and yeast and mold load, respec-
tively. Agnihotri, Borse, Bhandarkar, Subramaniam, and
Bhardwaj (2018) found 6.7 log decrease in total viable count and 4.2
log decline in YMC after application of 1 ppm ozone for 3 min to raw
onion. The mechanism involving microcidal effect of ozone is very
complex. Ozone oxidizes vital cellular constituents such as proteins,
nucleic acids in cytoplasm and disrupts cell bacterial attachment by
destroying the membrane barrier (Miller et al., 2013). It causes irre-
versible damage to unsaturated lipids, oxidizes sulfhydryl groups
abundant in respiratory enzymes present in cell membranes and
attacks the peptidoglycans in cell envelops, spore coats and virus cap-
sids (Prabha et al., 2015; Tzortzakis, 2016). The surface active proper-
ties of free suspended bacteria cause their migration toward the
ozone bubbles and they are likely to be inactivated by ozone of com-
paratively high concentrations prevailing at the interface of the liquid
and gas bubble (Hill & Spencer, 1974). Singlet oxygen is a preferential
intermediate species that plays a part in the biochemical damage
occurred by ozonation (Agnez-Lima et al., 2012). Past studies reveal
that both molecular ozone and the free radicals resulted out of ozone
break down are equally important as reactive species in this inactiva-
tion mechanism.
Ozone treatment did not appear to affect Ca, Mg, Fe, Cu, Zn,
Co, and Mn significantly (refer Table 5). A slight decrease of 6% in
Na concentration was observed (p< .05). The minerals and organic
matter present in the medium interfere in catalyzing ozone decom-
position that in turn decreases the ozone solubility and hence its
efficiency. The organic constituents which are readily available com-
pete with microorganisms for applied ozone (Priyanka, Rastogi, &
Tiwari, 2014). Therefore, a higher concentration of ozone is required
for disinfection in complex solutions, like, juices owing to the ozone
requirement of organic nutrients present in the solution. However,
in the current study, increasing the ozone concentration beyond
30% (wt/wt) showed no significant effect on inactivation (data not
shown). This is probably because much higher ozone concentration
makes condition of saturation that causes the incorporation of fur-
ther ozone ineffective, due to which longer times need to be
devoted for achieving the same level of log reduction (Cullen
et al., 2010). Koseki and Isobe (2006) also showed no further bacte-
rial reduction above 5 ppm ozone while washing lettuce in ozonized
water for 2.5 min. Longer exposure time too does not work well
because sensitive cells get destroyed within the initial phase of treat-
ment but the cells that further survive will become resilient to the
ozone action causing reactivation later. Williams, Sumner, and
Golden (2004) also had a similar observation that the presence of
organic components posed a hindrance for the disinfection in case
of orange juice and apple cider ozonation.
The changes in scores of sensory parameters are reported in
Table 5. It was observed that the color of juice was slightly improved
after ozonation as specified by the corresponding scores. There was
no significant change in taste of juice after treatment (p< .05). The
scores for flavor and aroma of the treated sample were closer to that
of the untreated one. The overall acceptability score of the ozonized
juice was 7.82, indicating its acceptable quality and safety.
4|CONCLUSIONS
The current study dealt with establishing the efficacy of ozone
processing of sugarcane juice in achieving PPO and POD inactivation
and microbial destruction ensuring minimal enzymatic spoilage as well
as microbiological safety. All the variables markedly affect the
PANIGRAHI ET AL.11 of 14

response parameters. Increasing the ozone concentration and expo-
sure time, the inactivation effect increased. However, under the range
studied herein, the middle flow rate (6.5 L/min) achieved higher disin-
fection effect than the extreme levels (3 and 10 L/min). The treatment
had a significant impact on the chemical and nutritional composition.
Simultaneous optimization allowed proposing an optimal solution,
namely, gas flow rate 5.6 L/min, 26.4% (wt/wt) ozone concentration,
and exposure time 20 min with maximum desirability 0.875. The vali-
dation of the results demonstrated that the experimentally deter-
mined values were in close agreement with the predicted ones. The
sugarcane juice with enhanced anti-browning and antimicrobial prop-
erties was obtained when treated under optimal ozonation conditions.
The results illustrated the effectiveness of applied ozone dose of
4.23 g/L in suppression of enzyme activity (67.8% PPO and 75.3%
POD) and reduction in cell viability (3.72 log TPC reduction and 2.43
log YMC reduction).
ACKNOWLEDGMENT
This work is partially supported by a grant from the Ministry of Food
Processing Industries, Govt. of India, under the scheme No. IIT/SRIC/
R/TSJ/2019, dated March 28, 2019.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Chirasmita Panigrahi: Conceptualization; data curation; formal analy-
sis; investigation; methodology; software; validation; writing-original
draft. Hari Niwas Mishra: Funding acquisition; resources; supervision;
writing-review and editing. Sirshendu De: Supervision; writing-review
and editing.
DATA AVAILABILITY STATEMENT
Research data are not shared.
ORCID
Chirasmita Panigrahi https://orcid.org/0000-0002-1135-7289
REFERENCES
Abhilasha, P., & Pal, U. S. (2018). Effect of ohmic heating on quality and
storability of sugarcane juice. International Journal of Current Microbiol-
ogy & Applied Sciences,7(1), 2856–2868. https://doi.org/10.20546/
ijcmas.2018.701.340
Agnez-Lima, L. F., Melo, J. T., Silva, A. E., Oliveira, A. H. S.,
Timoteo, A. R. S., Lima-Bessa, K. M., …Menck, C. F. (2012). DNA dam-
age by singlet oxygen and cellular protective mechanisms. Mutation
Research/Reviews in Mutation Research,751(1), 15–28. https://doi.org/
10.1016/j.mrrev.2011.12.005
Agnihotri, V., Borse, K., Bhandarkar, H., Subramaniam, V., & Bhardwaj, S.
(2018). Efficacy of ozone to reduce total viable count, yeast and mould
count, coliform count and enterobacteriaceae count in raw onion and
dehydrated onion products. Plant Archives,18(2), 2811–2821.
Ahmad, M., & Farooq, S. (1985). Influence of bubble sizes on ozone solubil-
ity utilization and disinfection. Water Science and Technology,17,
1081–1090. https://doi.org/10.2166/wst.1985.0203
Alothman, M., Kaur, B., Fazilah, A., Bhat, R., & Karim, A. A. (2010). Ozone-
induced changes of antioxidant capacity of fresh-cut tropical fruits.
Innovative Food Science & Emerging Technologies,11(4), 666–671.
https://doi.org/10.1016/j.ifset.2010.08.008
American Public Health Association. (1976). Compendium of methods for
the microbiological examination of foods. Washington, DC: APHA.
Angelino, P. D., Golden, A., & Mount, J. R. (2003). Effect of ozone treat-
ment on quality of orange juice. IFT Annual Meeting Book of Abstract,
2003, Abstract No. 76C-2 Chicago, IL: Institute of Food Technologists.
Asokapandian, S., Periasamy, S., & Swamy, G. J. (2018). Ozone for fruit
juice preservation. In Fruit Juices (pp. 511–527). Kidlington, UK: Aca-
demic Press. https://doi.org/10.1016/B978-0-12-802230-6.00025-4
Association of Official Analytical Chemists (AOAC). (1998). Official
methods of analysis (16th ed.). Gaithersburg, MD: AOAC International.
Chakraborty, S., Kaushik, N., Rao, P. S., & Mishra, H. N. (2014). High-
pressure inactivation of enzymes: A review on its recent applications on
fruit purees and juices. Comprehensive Reviews in Food Science and Food
Safety,13(4), 578–596. https://doi.org/10.1111/1541-4337.12071
Chiron, S., Fernandez-Alba, A. R., Rodriguez, A., & Garcia-Calvo, E. (2000).
Pesticide chemical oxidation: State of the art. Water Research,34,
366–377. https://doi.org/10.1016/S0043-1354(99)00173-6
Cullen, P. J., Tiwari, B. K., O'Donnell, C. P., & Muthukumarappan, K.
(2009). Modelling approaches to ozone processing of liquid foods.
Trends in Food Science and Technology,20(3–4), 125–136. https://doi.
org/10.1016/j.tifs.2009.01.049
Cullen, P. J., Valdramidis, V. P., Tiwari, B. K., Patil, S., Bourke, P., &
O'Donnell, C. P. (2010). Ozone processing for food preservation: An
overview on fruit juice treatments. Ozone: Science & Engineering,32(3),
166–179. https://doi.org/10.1080/01919511003785361
Davis, S. B. (2001). The chemistry of colour removal: A processing per-
spective. South African Sugar Technologists Association,75, 328–336.
Davis, S. B., Moodley, M., Singh, I., & Adendorff, M. W. (1998). The use of
ozone for colour removal at the Malelane refinery. South African Sugar
Technologists Association,72, 255–260.
de Souza, L. P., D'Antonino Faroni, L. R., Heleno, F. F., Cecon, P. R.,
Gonçalves, T. D. C., da Silva, G. J., & Prates, L. H. F. (2018). Effects of
ozone treatment on post harvest carrot quality. LWT - Food Science
and Technology,90,53–60. https://doi.org/10.1016/j.lwt.2017.11.057
de Souza Sartori, J. A., Angolini, C. F. F., Eberlin, M. N., & de Aguiar, C. L.
(2017). Criegee mechanism as a safe pathway of color reduction in
sugarcane juice by ozonation. Food Chemistry,225, 181–187. https://
doi.org/10.1016/j.foodchem.2017.01.028
Esplugas, S., Gimenez, J. F., & Contreras, S. (2002). Comparison of different
advanced oxidation processes for phenol degradation. Water Research,
36, 1034–1042. https://doi.org/10.1016/S0043-1354(01)00301-3
Farajzadeh, D., Qorbanpoor, A., Rafati, H., & Isfeedvajani, M. S. (2013).
Reduction of date microbial load with ozone. Journal of Research in
Medical Sciences,18(4), 330.
Fundo, J. F., Miller, F. A., Tremarin, A., Garcia, E., Brand~
ao, T. R. S., &
Silva, C. L. M. (2018). Quality assessment of cantaloupe melon juice
under ozone processing. Innovative Food Science & Emerging Technolo-
gies,47, 461–466. https://doi.org/10.1016/j.ifset.2018.04.016
Garud, S. R., Priyanka, B. S., Rastogi, N. K., Prakash, M., & Negi, P. S.
(2017). Efficacy of ozone and lactic acid as non thermal hurdles for
preservation of sugarcane juice. Ozone: Science & Engineering,40(3),
198–208. https://doi.org/10.1080/01919512.2017.1415802
Gomez, M., Perez, P., & Ramos, R. (1980). Method of sugar refining with
ozone. Proceedings - International Society of Sugar Cane Technologists,
17(3), 2066–2071.
Hill, A. G., & Spencer, H. T. (1974). Mass transfer in a gas sparged ozone
contactor. Paper presented the First International Symposium on Ozone
for Water and Wastewater Treatment. Washington, DC.
Huang, H. W., Chang, Y. H., & Wang, C. Y. (2015). High pressure pasteuri-
zation of sugarcane juice: Evaluation of microbiological shelf life and
12 of 14 PANIGRAHI ET AL.

quality evolution during refrigerated storage. Food and Bioprocess
Technology,8(12), 2483–2494. https://doi.org/10.1007/s11947-015-
1600-2
Huang, N., Cheng, X., Hu, W., & Pan, S. (2015). Inactivation, aggregation,
secondary and tertiary structural changes of germin-like protein in Sat-
suma mandarine with high polyphenol oxidase activity induced by
ultrasonic processing. Biophysical Chemistry,197,18–24. https://doi.
org/10.1016/j.bpc.2014.12.001
Jaramillo-Sanchez, G. M., Garcia Loredo, A. B., Gomez, P. L., &
Alzamora, S. M. (2018). Ozone processing of peach juice: Impact on
physicochemical parameters, color, and viscosity. Ozone: Science &
Engineering,40(4), 305–312. https://doi.org/10.1080/01919512.
2017.1417111
Kaavya, R., Pandiselvam, R., Kothakota, A., Priya, E. B., & Prasath, V. A.
(2019). Sugarcane juice preservation: A critical review of the state of
the art and way forward. Sugar Technology,21(1), 9–19. https://doi.
org/10.1007/s12355-018-0622-2
Kamtekar, S., Keer, V., & Patil, V. (2014). Estimation of phenolic content,
flavonoid content, antioxidant and alpha amylase inhibitory activity of
marketed polyherbal formulation. Journal of Applied Pharmaceutical Sci-
ence,4(9), 61–65. https://doi.org/10.7324/JAPS.2014.40911
Kayalvizhi, V., Pushpa, A. J. S., Sangeetha, G., & Antony, U. (2016). Effect
of pulsed electric field (PEF) treatment on sugarcane juice. Journal of
Food Science and Technology,53(3), 1371–1379. https://doi.org/10.
1007/s13197-016-2172-5
Khare, A., Lal, A. B., Singh, A., & Singh, A. P. (2012). Shelf life enhancement
of sugarcane juice. Croatian Journal of Food Technology, Biotechnology
and Nutrition,7(3–4), 179–183.
Kong, F., Yu, S., Zeng, F., & Wu, X. (2015). Preparation of antioxidant and
evaluation of the antioxidant activities of antioxidants extracted from
sugarcane products. Journal of Food and Nutrition Research,3(7),
458–463. https://doi.org/10.12691/jfnr-3-7-7
Koseki, S., & Isobe, S. (2006). Effect of ozonated water treatment on
microbial control and on browning of iceberg lettuce (Lactuca sativa
L.). Journal of Food Protection,69(1), 154–160. https://doi.org/10.
4315/0362-028X-69.1.154
Kunitake, M., Ditchfield, C., Silva, C., & Petrus, R. (2014). Effect of pas-
teurization temperature on stability of an acidified sugarcane juice
beverage. Ciência e Agrotecnologia,38(6), 554–561.
Miller, F. A., Silva, C. L., & Brand~
ao, T. R. (2013). A review on ozone-based
treatments for fruit and vegetables preservation. Food Engineering
Reviews,5(2), 77–106. https://doi.org/10.1007/s12393-013-9064-5
Onopiuk, A., Półtorak, A., Moczkowska, M., Szpicer, A., & Wierzbicka, A.
(2017). The impact of ozone on health-promoting, microbiological, and
colour properties of Rubus ideaus raspberries. CyTA-Journal of Food,
15(4), 563–573. https://doi.org/10.1080/19476337.2017.1317669
Pandraju, S., & Rao, P. S. (2020). High-pressure processing of sugarcane
juice (Saccharum officinarum) for shelf-life extension during ambient
storage. Sugar Technology,22(2), 340–353. https://doi.org/10.1007/
s12355-019-00769-y
Panigrahi, C., Karmakar, S., Mondal, M., Mishra, H. N., & De, S. (2018).
Modeling of permeate flux decline and permeation of sucrose during
microfiltration of sugarcane juice using a hollow-fiber membrane mod-
ule. Innovative Food Science & Emerging Technologies,49,92–105.
https://doi.org/10.1016/j.ifset.2018.07.012
Patil, S., Bourke, P., Frias, J. M., Tiwari, B. K., & Cullen, P. J. (2009). Inacti-
vation of Escherichia coli in orange juice using ozone. Innovative Food
Science & Emerging Technologies,10(4), 551–557. https://doi.org/10.
1016/j.ifset.2009.05.011
Prabha, V., Barma, R. D., Singh, R., & Madan, A. (2015). Ozone technol-
ogyinfoodprocessing:Areview.Trends in Biosciences,8(16),
4031–4047.
Priyanka,B.S.,Rastogi,N.K.,&Tiwari,B.K.(2014).Opportunitiesand
challenges in the application of ozone in food processing. In
Emerging technologies for food processing (pp. 335–358). San Diego:
Academic Press. https://doi.org/10.1016/B978-0-12-411479-1.
00019-X
Rico, D., Martín-Diana, A. B., Frías, J. M., Henehan, G. T., & Barry-Ryan, C.
(2006). Effect of ozone and calcium lactate treatments on browning
and texture properties of fresh-cut lettuce. Journal of the Science of
Food and Agriculture,86(13), 2179–2188. https://doi.org/10.1002/
jsfa.2594
Rosenfeld, M. A., Razumovskii, S. D., Shchegolikhin, A. N.,
Konstantinova, M. L., Sultimova, N. B., Kozachenko, A. I., …
Leonova, V. B. (2015). Nature of active intermediate particles formed
during ozone-induced oxidation. Doklady Biochemistry and Biophysics,
461, 139–141. https://doi.org/10.1134/S1607672915020180
Saxena, J., Makroo, H. A., Bhattacharya, S., & Srivastava, B. (2018). Kinetics
of the inactivation of polyphenol oxidase and formation of reducing
sugars in sugarcane juice during Ohmic and conventional heating. Jour-
nal of Food Process Engineering,41(3), e12671. https://doi.org/10.
1111/jfpe.12671
Shah, N. N. A. K., Sulaiman, A., Sidek, N. S. M., & Supian, N. A. M. (2019).
Quality assessment of ozone-treated citrus fruit juices. International
Food Research Journal,26(5), 1405–1415.
Shah, N. N. A. K., Supian, N. A. M., & Hussein, N. A. (2019). Disinfectant of
pummelo (Citrus grandis L. Osbeck) fruit juice using gaseous ozone.
Journal of Food Science and Technology,56(1), 262–272. https://doi.
org/10.1007/s13197-018-3486-2
Sreedevi, P., Jayachandran, L. E., & Rao, P. S. (2018). Browning and bioac-
tive composition of sugarcane juice (Saccharum officinarum) as affected
by high hydrostatic pressure processing. Journal of Food Measurement
and Characterization,12(3), 1962–1971. https://doi.org/10.1007/
s11694-018-9811-7
Tiwari, B. K., Muthukumarappan, K., O'Donnell, C. P., & Cullen, P. J.
(2008). Kinetics of freshly squeezed orange juice quality changes dur-
ing ozone processing. Journal of Agricultural and Food Chemistry,56
(15), 6416–6422. https://doi.org/10.1021/jf800515e
Tiwari, B. K., O'Donnell, C. P., Brunton, N. P., & Cullen, P. J. (2009). Degra-
dation kinetics of tomato juice quality parameters by ozonation. Inter-
national Journal of Food Science and Technology,44(6), 1199–1205.
https://doi.org/10.1111/j.1365-2621.2009.01946.x
Tiwari, B. K., O'Donnell, C. P., Muthukumarappan, K., & Cullen, P. J.
(2009). Anthocyanin and colour degradation in ozone treated black-
berry juice. Innovative Food Science & Emerging Technologies,10(1),
70–75. https://doi.org/10.1016/j.ifset.2008.08.002
Tiwari, B. K., O'Donnell, C. P., Patras, A., Brunton, N., & Cullen, P. J.
(2009a). Anthocyanins and color degradation in ozonated grape juice.
Food and Chemical Toxicology,47(11), 2824–2829. https://doi.org/10.
1016/j.fct.2009.09.001
Tiwari, B. K., O'Donnell, C. P., Patras, A., Brunton, N., & Cullen, P. J.
(2009b). Effect of ozone processing on anthocyanins and ascorbic acid
degradation of strawberry juice. Food Chemistry,113(4), 1119–1126.
https://doi.org/10.1016/j.foodchem.2008.08.085
Torlak, E. (2014). Efficacy of ozone against Alicyclobacillus acidoterrestris
spores in apple juice. International Journal of Food Microbiology,172,
1–4. https://doi.org/10.1016/j.ijfoodmicro.2013.11.035
Torres, B., Tiwari, B. K., Patras, A., Wijngaard, H. H., Brunton, N.,
Cullen, P. J., & O'Donnell, C. P. (2011). Effect of ozone processing on
the colour, rheological properties and phenolic content of apple juice.
Food Chemistry,124(3), 721–726. https://doi.org/10.1016/j.
foodchem.2010.06.050
Tzortzakis, N. (2016). Ozone: A powerful tool for the fresh produce preser-
vation. In Postharvest management approaches for maintaining quality of
fresh produce (pp. 175–207). Cham: Springer. https://doi.org/10.1007/
978-3-319-23582-0_11
Williams, R. C., Sumner, S. S., & Golden, D. A. (2004). Survival of
Escherichia coli O157:H7 and salmonella in apple cider and orange
PANIGRAHI ET AL.13 of 14

juice as affected by ozone and treatment temperature. Journal of Food
Protection,67(11), 2381–2386. https://doi.org/10.4315/0362-028x-
67.11.2381/10.4315/0362-028x-67.11.2381
Yeoh, W. K., Ali, A., & Forney, C. F. (2014). Effects of ozone on major anti-
oxidants and microbial populations of fresh-cut papaya. Postharvest
Biology and Technology,89,56–58. https://doi.org/10.1016/j.
postharvbio.2013.11.006
Zia, S., Khan, M. R., Zeng, X. A., Shabbir, M. A., & Aadil, R. M. (2019). Com-
bined effect of microwave and ultrasonication treatments on the qual-
ity and stability of sugarcane juice during cold storage. International
Journal of Food Science and Technology,54(8), 2563–2569. https://doi.
org/10.1111/ijfs.14167
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Panigrahi C, Mishra HN, De S. Effect
of ozonation parameters on nutritional and microbiological
quality of sugarcane juice. J Food Process Eng. 2020;e13542.
https://doi.org/10.1111/jfpe.13542
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