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Received: 10 March 2020    Revised: 15 June 2020    Accepted: 26 June 2020

DOI: 10.1111/jfpp.14779

ORIGINAL ARTICLE

Assessing the impact of the combined application of ultrasound

and ozone on microbial quality and bioactive compounds
with antioxidant attributes of cabbage (Brassica Oleracea
L. Var. Capitata)

Mamadou Bado Traore1,2  | Aidong Sun1,2 | Zhilin Gan1,2 | Wei Yu Long1,2 |

Hamidou Senou1 | Yue Zhu1,2 | Jacques Togo3 | Kankou Hadia Fofana4 |
Aboubacar Modibo Sidibe5

1
College of Biological Sciences and
Technology, Beijing Forestry University, Abstract
Beijing, China Ultrasound (US) and ozone processing allow achieving food technological aims such
2
Beijing Key Laboratory of Forest Food
as the improvement of food safety and preservation. The combination of these tech-
Processing and Safety, Beijing Forestry
University, Beijing, China nologies has been reported to be beneficial in inactivating microorganisms from fruits
3
Department of Biology, University of and vegetables. However, treatment conditions and mechanisms of action of these
Bamako, Bamako, Mali
4
technologies can affect vegetables or fruits structure as well as their phytochemi-
College of Management and Economics,
Beijing Institutes of Technology, Beijing, cal components. This study therefore, aimed to evaluate the antimicrobial capacity
China of ultrasound and ozone alone or combined on artificially inoculated cabbage with
5
Department of Groundwater and
E. coli and Salmonella. The impact of these decontamination processes on cabbage in-
Environment, Jilin University, Changchun,
China tercellular structure, pH, total phenol, total flavonoid and antioxidant activities were
also determined. Results showed that the combination treatment significantly inacti-
Correspondence
Aidong Sun, College of Biological Sciences vated populations of artificially inoculated E. coli and Salmonella compared to individ-
and Technology, Beijing Forestry University,
ual processing. The inactivation effectiveness was greatly enhanced with increased
Beijing 100083, China.
Email: adsun@bjfu.edu.cn ozone concentration (1.5 mg/L) and processing time, with no detectable bacteria in
the washing water after only 8 min of treatment. Furthermore, ultrasound treatment
Funding information
National Key R&D Program, Grant/Award alone for 8 min led to improved cabbage content of phenol, flavonoid and antioxidant
Number: 2016YFD0400302; Chinese
activity without affecting its intercellular structure. However, the combination treat-
Scholarship Council; National Natural
Science Foundation of China, Grant/Award ment exerted a slight decrease of these components though not statistically signifi-
Number: 31871817
cant compared to untreated samples. A significant decrease was observed with the
longest exposure time (20 min). We conclude that ultrasound individual treatment or
its combination with ozone can be used as sanitizer to not only reduce microorgan-
isms from fresh cabbage but also maintain its appearance quality and improve its
bioactive properties and antioxidant activity.
Practical applications
This study was conducted to evaluate the inactivation effectiveness of ultrasound
and ozone on inoculated cabbage leaves. To make an effective disinfection on the
leaves, US and ozone have been combined, which resulted in a reduction of treat-
ment time and maintenance of the bioactive compounds, the antioxidant activity and

J Food Process Preserv. 2020;44:e14779. wileyonlinelibrary.com/journal/jfpp |

© 2020 Wiley Periodicals LLC.     1 of 11
https://doi.org/10.1111/jfpp.14779
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2 of 11       TRAORE et al.
intercellular structure attributes. The results of this research support and suggest
that the combination treatment of US with ozone at 1.5 mg/L for 8 min is the most
suitable processing for sanitizing fresh cabbage without damaging its structure, bio-
active compounds and antioxidant activity.
1 |  I NTRO D U C TI O N
Consumers demand regarding fresh products sanitization with high
nutritional quality such as desirable organoleptic and appropriate shelf
life has been increasing in recent years. Typically, to satisfy this de-
mand many technologies are employed not only for reducing microbial
load but also to minimize the loss of fruits and vegetables’ nutrients
and antioxidant activity. However, some of these technologies such as
thermal processing can cause the loss of some important compound
associated to fresh products. This loss is often due to thermal degra-
dation and leaching into cooking water (Balan, Israel-Roming, Luta, &
Gherghina, 2016). To overcome this problem, many other methods of
decontamination such as ultrasound have emerged (Almeida Duarte
et  al.,  2018). As a non-thermal technique, the use of ultrasound has
been a growing interest in food industry due to its ability to improve
food quality and to prevent the loss of nutrients (Nadeem, Ubaid,
Qureshi, Munir, & Mehmood, 2018). During ultrasound's thermal ef-
fects, the sonication is converted to heat and absorbed by plant tissue.
Therefore, ultrasound's mechanical effects can cause acoustic cavita-
tion thereby creating growing bubbles that result in cell disruption and
improved extraction (Ahn et al., 2003; Altemimi, Choudhary, Watson,
& Lightfoot, 2015; Wu & Wu, 2007). Ultrasound's ability to increase
ascorbic acid content of strawberries and total polyphenol, flavo-
noid, antioxidant activity from spinach has previously been reported
(Altemimi et al., 2015; Cao et al., 2010).
However, significant difference can be observed in the effect of
ultrasound on the chemical composition and physical properties of
fresh vegetables and fruits depending on their type and processing
conditions. Regarding to food pathogenic inactivation, many studies
reported that treatment using ultrasound alone is not effective enough
to reduce pathogenic from fresh products (Piyasena, Mohareb, &
McKellar, 2003; Sango, Abela, McElhatton, & Valdramidis, 2014).
Ultrasound can be combined with other technologies such as
ozone to enhance the efficiency at the industrial level, thereby re-
ducing processing time. Ozone has been recognized as a “Generally
Safe Substance” (GRAS) (Khadre, Yousef, & Kim, 2001; KIM, Yousef,
& Chism, 1999) due to its capacity of effectively inactivating bac-
teria, viruses, fungi and mycotoxins without harmful residues (Coll
Cardenas, Andres, Giannuzzi, & Zaritzky, 2011; Papachristodoulou,
Koukounaras, Siomos, Liakou, & Gerasopoulos, 2018). Ozone pro-
cessing has been found to be effective in extending the shelf-life of
oranges, raspberries, grapes, pears and apples (Skog & Chu, 2001).
Furthermore, ozone has been shown to induce oxidative stress in
fresh fruit, which promotes various physiological responses, includ-
ing synthesis of antioxidants, phenolic compounds, polyamines, eth-
ylene, and other secondary metabolites (Forney, 2003).
However, there is currently very limited data on cabbage's bioac-
tive compounds and antioxidant activity changes during ultrasound
and ozone treatment.
Cabbage (Brassica oleracea L. var. capitata) is one of the most im-
portant vegetables grown worldwide and leading leafy green veg-
etables for commercial production (Burkness & Hutchison, 2009;
Rokayya, Li, Zhao, Li, & Sun, 2013). Wennberg et al reported that ap-
proximately 6.3 kg of this variety are consumed per person annually
(Wennberg, Ekvall, Olsson, & Nyman, 2006). Due to the properties
related to its bioactive compound with antioxidant and anti-inflam-
matory activities, cabbage is widely used in traditional medicine in
alleviation of symptoms associated with gastrointestinal disorders
such as duodenal ulcers, irritable bowel syndrome, gastritis and
peptic as well as in treatment of minor cuts, wounds and mastitis
(Rokayya et al., 2013). Therefore, in most of the cases, this cabbage
is consumed either raw or with minimal process, and is often not
subjected to proper decontamination during post-harvest washing.
The present study was designed to evaluate the effect of ultra-
sound and ozone treatment on the cabbage's bioactive compounds,
antioxidant activity and surface structure. Furthermore, the effect
of these treatments on the inactivation efficacy of artificially inoc-
ulated E. coli and Salmonella were investigated. Finally, the levels of
both bacteria in water were also determined in order to avoid cross
contamination.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Materials
Cabbage (Brassica oleracea var. capitata) has been purchased in the
local supermarket (Wumart) and stored in darkness at 4°C no more
than 2 days before each treatment. Salmonella enterica subsp. enterica
21,594 serotype Typhi and Shiga toxin negative NCTC 12,900 strain of
E. coli O157:H7 were used in this study. Tryptic soy agar (TSA), tryptic
soy broth (TSB), sterile peptone water (SPW), phosphate buffered sa-
line (PBS); sodium thiosulfate (Na2S2O3), sodium chloride (NaCl), Folin–
Ciocalteu reagent, methanol, sodium carbonate (Na2CO3), sodium
nitrite (NaNO2), gallic acid (GA), aluminium chloride (AlCl3), formic acid;
quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) were utilized.
2.2 | Preparation of inoculum
Stock cultures of both strains were maintained in tryptic soy broth
and stored at  −°C. To obtain a working culture, both strains were
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individually cultivated in TSA (Difco Co. containing 0.85% of NaCl).
The plates were thereafter incubated overnight at 37°C and single
colonies of each strain were transferred into 100 ml of TSB contain-
ing 0.85% of NaCl and cultured in a shaking incubator (150 rpm/min)
for 18–24 hr at 37°C. Twenty five milliliter of each culture was har-
vested by centrifugation (6000 g for 15 min) at 4°C. The resulting cell
pellet was used as inoculum with 0.1% of SPW to obtain a population
of 7.8 log CFU/mL in the suspension.
2.3 | Preparation and inoculation of cabbage
Debris and other particles of cabbage leaves were removed and
discarded, while the rest of the leaves were washed for 30–60  s
using deionized water followed with 70% alcohol wash to eliminate
natural microbiota load. Samples were dried with absorbing paper,
chopped into squares with a sterile knife, placed inside a biological
safety cabinet (BSC) and exposed to UV for 15  min. Spot inocula-
tion was performed and slices of leaves were placed in Petri dishes
(150  mm  × 15  mm). 500 microliter of the well-mixed bacteria sus-
pension was dropped onto each leaf surface at different locations
by using a sterile serological micropipette. Samples were then main-
tained at room temperature in a BSC for 90 min for inoculum to dry.
The dried leaves were then turned over and the same inoculation
procedure was repeated on the other side.
saline (PBS) and mixed for 20 s (Froehling & Schlueter, 2015). Three
replicates of each treatment were analyzed. The control treatment
was designated with sterilize water for 4 min.
2.5 | Microbiological analysis
Treated samples were aseptically transferred into sterile stom-
acher bag containing 0.1% of SPW and homogenized for 70  s, of
which 15  ml were transferred into a sterile test tube and allowed
to stand for 2  min. After vortexing for 2  min with a vortex mixer
(Fisher Scientific, Waltham, Massachusetts), the supernatant of each
homogenate was serially diluted with 0.1% SPW. Surviving bacte-
rial populations were determined in duplicate onto tryptic soy agar
(TSA) containing 0.85% NaCl. The plates were dried in the BSC until
no visible diluent was noted, and incubated for 48 hr at 37°C before
enumerating surviving population. After each treatment, the treated
water was also analyzed briefly by transferring 10 ml of water sam-
ples into 15 ml sterile centrifuge tubes, diluted in 0.1% SPW and ana-
lyzed for each target microorganism. Results are reported as log10
colony forming per mL (log CFU/mL). Each experiment was carried
out in three replicates.
2.6 | Analysis of physicochemical properties

2.6.1 | Extraction procedures
2.4 | Equipment characterization and
decontamination procedure Cabbage was divided into two different sections namely inner and
outer leaves and denoted as C1 and C2 respectively. 10 g of each
A bench-top ultrasonic cleaner (model KQ3200DE with 650W and section after each treatment were ground with 25 ml 80% methanol
40 kHz, Kunshan CO LTD, China) and Feili ozone generator (FL- and 0.35 µl formic acid by using Chopper Grinder (Multi-Function
802A) which allows oxygen and ozone by producing 2 g/h of ozone Food Fruit Processor 3 in 1 Electric Blender Mixer) for 1 min. For
were used to perform ultrasound and ozone processing. Treatment
conditions and time were selected based on our preliminary studies
(Traore et al., 2019) with slight modification. Ultrasound treatment
was conducted by transferring 25 g of inoculated leaves into steri-
lized beaker containing five hundred milliliter of sterilized distilled
water. Samples were then sonicated at 40 kHz and 100 W at 50°C
for various time intervals (4, 8, 12, 16 and 20 min). Ozone treatment
was performed by interconnecting a container containing sterile
deionized water (25 liter), forming a closed circuit ring apparatus.
Appropriate ozone concentrations (0.7 and 1.5 mg/L) were obtained
by continuously incorporating bubble into water for 90 and 150 min,
respectively. A kit was used to determine its concentration (25180-
50 Ozone AccuVac color disc kit). For individual treatments, 10 g of
inoculated cabbage were used in aqueous ozone at 0.7 and 1.5 mg/L.
For combined treatment, the same inoculated weight was placed in
sterilized beaker and filled with 500 ml volumes of above mentioned
concentrations of ozone, and sonication was carried out in the same
conditions as ultrasound individual processing. After each period of
treatment, ozone reaction was stopped by adding 30 ml of 0.6 M so-
dium thiosulfate (Na2S2O3) containing 50 mM of phosphate buffered
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4 of 11       TRAORE et al.
extraction, the treated samples (C1 and C2) were filtered under
vacuum to remove the mashed leaves. The filtrates were thereafter
passed through 0.45 µm filters, and immediately subjected to total
phenolic, flavonoid and DPPH radical scavenging activity analysis.
The untreated cabbage was designed as control and analyzed fol-
lowing the same procedures.
2.6.2 | Total phenolic content
Total phenol content (TPC) was performed by using the Folin–
Ciocalteu reagent, following the methodology described by
Singleton et al with a slight modifications (Singleton, Orthofer, &
Lamuela-Raventós, 1999). The reaction mixture was prepared into a
tube by combining 450 µl methanolic sample extract with 2,250 µl of
Folin—Ciocalteu solution (previously diluted 1:10 vol/vol, with dis-
tilled water). After 3 min of incubation at room temperature, 1,800 µl
of an aqueous Na2CO3 solution (7.5% wt/vol) were added, and the
reaction mixture was incubated for 1 hr under the same conditions.
The absorbance was measured at 765 nm using a spectrophotome-
ter (UV-6100 spectrophotometer METASH). Values were expressed
as (GAE) mg/g (gallic acid (GA)) and prepared under the same condi-
tions as the samples. Linearity curve of the calibration was obtained
from 0.3 to 3 g/ml concentration for gallic acid (R 2 = 0.9998).
2.6.3 | Total flavonoid content
The total flavonoid content (TFC) was determined by following a
methodology described by Zhishen et al. with slight modifications
(Zhishen, Mengcheng, & Jianming,  1999). Briefly, 450  µl of metha-
nolic extract was added to 1,935  µl of an aqueous NaNO2 solu-
tion 0.35% (wt/vol), and incubated for 5 min at room temperature.
135  µl of AlCl3 solution (10% wt/vol) were added and the mixture
was incubated for 1 min. 1,980 µl of NaOH 0.454 M was also added.
4,500 µl of the mixture sample were pipetted into a micro tube, and
the absorbance was read at 496 nm using a spectrophotometer. TFC
was calculated from a calibration curve prepared with quercetin as
a standard, and the results are expressed as mg of quercetin equiva-
lents (QE)/100 g and linearity of the calibration curve was obtained
from 0.001 to 0.021 mg/ml amount for quercetin (R 2 = 0.9468).
2.6.4 | Determination of in vitro antioxidant activity
Antioxidant activity was evaluated by using a model of scavenging
with stable DPPH before and after treatment as previously reported
(Brand-Williams, Cuvelier, & Berset, 1995). DPPH solution was pre-
pared by dissolving the DPPH radical in pure methanol, and adjust-
ing the absorbance of the solution (515 nm). 300 µl methanol sample
extract before and after treatment were pipetted into a tube and
4200 µl of the DPPH radical were then added. The mixture was in-
cubated in the dark for 30 min. The absorbance was read at 515 nm
using a spectrophotometer. The percentage of scavenging activity
was determined and compared with that of L-ascorbic acid (100 μg/
ml), which was used as the standard. The inhibition of the DPPH rad-
ical scavenging effect was calculated using the following equation:
%In = [(Abso − Abss)∕𝐴𝑏𝑠𝑜] × 100%
where Abso was the absorbance of the blank (containing only
DPPH) and Abss, the absorbance of DPPH in the sample solution.
The measures were performed in triplicate for each sample and the
values were averaged. The results were expressed as mg AAE/g.
2.7 | Leaves histological analysis
Samples structural changes following treatment were performed
according to our previous study (Traore et al., 2019). Briefly, the
unprocessed and processed samples were immersed in FAA fixa-
tive containing 3.7% v/v formaldehyde, 50% ethanol, 5% acetic
acid. Samples were placed in xylene I, xylene II for 20 min followed
with anhydrous ethanol and 75% alcohol for 5 min. The fixed sam-
ples were then immersed into the saffron staining for 1-2 hr and
washed with tap water for dye excess removing. For discoloration,
the samples were placed into 50%, 70% and 80% gradient alcohols
sequentially for 3–8 s each. Slices were then placed for 30-60 s in
solid green dyeing solution and dehydrated in anhydrous ethanol
(Dehydrator Wuhan Junjie Electronics Co., Ltd. JJ-12J). These were
finally cut into thin sections and placed onto microscope (Upright
Optical Microscope Japan Nikon, NIKON ECLIPSE E100) for further
observation. Images were taken using Nikon NIKON DS-U3 camera.
2.8 | The changes of pH
Cabbage pH was determined according to a previously described
method (Lee et al., 2014). 10 grams of unprocessed (leaves washed
with DW) and processed samples were drained from treated liquid to
remove surface water by using paper towel. Leaves were then ground
by using a grinder (Magic Bullet, CA) in 50 ml of deionized water for
1 min. The pH of the homogenate was determined using a pH meter
with glass electrode (Orion Star A111, Thermo Scientific) at 23°C.
2.9 | Statistical analysis
The experiments were performed in replication for each processing
time. Data were graphed using Pad Prism software 6.01 (Graph pad
Software, Inc., La Jolla, California). Analysis of variance (ANOVA)
was used to determine statistic significance and Tukey's multi-
ple comparisons test was used for comparison of means between
groups. Unpaired t-test was also used for means comparison be-
tween 2 groups. All data points are expressed as mean ± standard
error of the mean (SEM), and p value of ≤.05 is considered significant.
TRAORE et al. |
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TA B L E 1   Surviving populations of E. coli and Salmonella, on cabbage leaves, after treatment with ultrasound for 20 min

Surviving population (log CFU/g) at the following time (min)

Microorganisms 0 4 8 12 16 20
A B C C C
E. coli 7.3 ± 0.04 5.8 ± 0.32 5.2 ± 0.21 5.2 ± 0.09 5.1 ± 0.05 4.5 ± 0.00 D
Salmonella 7.27 ± 0.02 A 5.4 ± 0.04B 5.2 ± 0.14B 5.1 ± 0.10 B 5.1 ± 0.01B 4.4 ± 0.07C

Note: Values are means of triplicate measurements ± standard deviations. Means that do not share letters are significantly different.

F I G U R E 1   Logarithmic reduction of E.
coli O157:H7 and salmonella during ozone
treatment. (a) E. coli (two way ANOVA,
F(4,20) = 21.99, p < .0001); (b) salmonella
(two way ANOVA F(4,20) = 25.86,
p < .0001). Logarithmic reduction of E. coli
O157:H7 and salmonella after US+ ozone
treatment. (c) E. coli (two way ANOVA,
F(4,20) = 84.21, p < .0001); (d) salmonella
(two way ANOVA, F(4,20) = 145.2,
p < .0001). Each data point represents an
average of three replicates. ns indicates
not significance, **p < .01; ***p < .001;
****p < .0001. Values are presented as
mean ± standard Error. CTR, control

3 | R E S U LT S
3.1 | Decontamination of E. coli and Salmonella on
fresh-cut cabbages surface
The efficiency of ultrasound against E. coli and Salmonella at vari-
ous time intervals were investigated and results are summarized in
Table 1. The mean initial population of E. coli and Salmonella upon
inoculation was 7.29 ± 0.04 and 7.27 ± 0.02 log CFU/g, respectively.
There is significant effect of ultrasound treatment on bacterial inac-
tivation (two way ANOVA, F(5,12) = 472.0, p < .0001; F(5,12) = 103.8,
p < .0001 for E coli and Salmonella respectively). Furthermore 4 min
of treatment resulted in a reduction of 1.45 ± 0.36 and 1.83 ± 0.01
log CFU/ g of E. coli and Salmonella respectively. This reduction was
positively correlated to the time of treatment with longer time re-
sulting in higher reduction (20 min led to the highest reduction in
both strains with 2.83 ± 0.03 and 2.85 ± 0.05 log CFU/ g for E. coli
and Salmonella respectively). However, there was no significant dif-
ference between 8, 12 and 16 min of ultrasound treatment (one way
ANOVA, F(2,6) = 0.8494, p = .4733) for E. coli and (one way ANOVA,
F(2,6) = 1.364, p = .3250) for Salmonella.
The reduction of E. coli and Salmonella population under different
ozone concentrations and its combination with ultrasound is shown
in Figure 1a,b. Upon inoculation the initial bacterial population was
evaluated at 4.38 ± 0.09 log CFU/g and 4.32 ± 0.06 log CFU/g for
E. coli and Salmonella, respectively. Microbial reduction loads were
found not significantly different after 4 and 8 min with increasing
the ozone concentration from 0.7 to 1.5  mg/L (two way ANOVA,
F(1,8) = 3.349, p = .1046 and F(1, 8) = 1.345, p = .2796) for E. coli and
Salmonella respectively. However, after 12 min and above, bacte-
rial population was significantly reduced for both concentrations
of ozone. The highest reduction rate was observed at 1.5 mg/L of
ozone. Furthermore, synergistic reduction effects against the strain
were observed for 0.7–1.5 mg/L ozone and ultrasound (combination
treatment) as indicated in Figure 1c,d. The combination treatment
with 0.7 mg/L of ozone exerted a significant reduction of bacterial
population (3.34 ± 0.09 and 3.28 ± 0.06 for E. coli and Salmonella)
after 16 min. Therefore, synergistic reduction values of both strains
were obtained with ozone concentration of 1.5  mg/L after only
8 min of treatment. There was no significant difference afterward
compared to longer treatment time (12–20 min).
3.2 | The changes of microbial quality in processing
wash water
To evaluate the risk of potential cross-contamination, E. coli and
Salmonella populations in processing water are summarized in
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6 of 11       TRAORE et al.

TA B L E 2   E. coli and Salmonella (log

Ozone concentration (mg/L) US+ Ozone (mg/L)
Exposure CFU/ml) in processing wash water after
Microorganisms (min) 0.7 1.5 0.7 1.5 washing fresh-cut cabbages
ns
E. coli 4 2.83 ± 0.04 2.61 ± 0.24 1.09 ± 0.22 ND
ns
8 2.56 ± 0.29 2.36 ± 0.31 ND ND
12 1.93 ± 0.45 1.79 ± 0.40 ns ND ND
ns
16 1.81 ± 0.36 1.54 ± 0.32 ND ND
20 1.39 ± 0.21 1.13 ± 0.13ns ND ND
ns
Salmonella 4 2.58 ± 0.36 2.15 ± 0.05 1.02 ± 0.08 ND
8 2.33 ± 0.20 1.94 ± 0.37ns ND ND
ns
12 1.75 ± 0.40 1.67 ± 0.50 ND ND
16 1.67 ± 0.19 1.34 ± 0.20 ns ND ND
ns
20 1.06 ± 0.34 1.07 ± 0.20 ND ND

Note: ND not detected (below the limit of detection of 1.0 CFU/ml).

ns indicates not significance,(ANOVA, GLM). Means were compared according to the concentration
of ozone (row) rather than processing time (column).

F I G U R E 2   pH of fresh-cut cabbages
after treatment (one way ANOVA F(6,7) =
0.8889, p = .5491) for C1 Figure 2 a and
(one way ANOVA, F(6,7) = 5.259, p = .0233)
for C2 Figure 2b. Duplicate of each data
point represents as mean ± standard
Error. CTR, control

Table 2. No statistical difference was observed regarding to both

pathogens even when ozone concentration was increased from
0.7 to 1.5  mg/L, (two way ANOVA, F(1, 20) = 3.397 p  = .0802 and
F(1, 20) = 4.388 p = .0501, respectively for E. coli and Salmonella). The
combination treatment exerted the maximum reduction of bacterial
population in water with no detection (ND) of pathogens. Therefore,
combination processing with 1.5  mg/L of ozone for 4  min is suffi-
cient to avoid cross contamination.
3.3 | Cabbages pH after treatment
The changes of pH values in the cabbage during individual and com-
bination processing for 8 and 20 min are shown in Figure 2. The
pH values were ranged from 6.25  ±  0.007 to 6.17  ± 0.03 for C1
Figure  2a and 6.4  ±  0.06 to 6.3  ± 0.02 for C2 Figure 2b. No sta-
tistically significant difference (one way ANOVA, F(6,7) = 0.8889,
p = .5491) was observed in the pH level between CTR and processed
samples for 8 and 20 min in C1. In C2 we noticed a slight decrease
but not significant (one way ANOVA, F(6,7) = 5.259, p = .0233) of pH
value after treatment by comparing control to treated samples.
3.4 | Histological observation and physicochemical
properties after treatment
The histological cross sections revealed no damage on the structural
anatomy of the leaves after individual treatment at 8 min compared
to raw cabbage (Figure 3). Similar results were also observed with
combination treatment with both concentrations of ozone during
the same processing time Figure 4.
However, ultrasound alone and its combination with ozone (0.7
and 1.5  mg/L) for 20  min revealed some leaves cells disruption as
indicated in Figures 3 and 5. Cabbage total flavonoid, phenol content
with antioxidant activity before and after treatments were deter-
mined and results are summarized in Table 3.
Compared with control samples cabbage, the ultrasound
(40 kHz, 100 W) alone for 8 and 20 min of processing significantly
increased (p < .05) total phenolic content resulting in 4.3 ± 0.00 and
4.1 ± 0.00 in C1; 3.31 ±  0.01 and 3.06  ±  0.05 in C2 respectively.
Similar results were observed with combination treatment at 8 min
for both C1 and C2 with 0.7 mg/L as ozone concentration. However,
for the same treatment time, 1.5  mg/L of ozone with ultrasound
TRAORE et al.
F I G U R E 3   Light microscopic images of untreated (C1, C2), US treated (C1, C2 8 min) and US treated (C1, C2 20 min) cabbage samples
F I G U R E 4   Light microscopic images of
combination treatment of US with Ozone
at 8 min
resulted in significant decrease of phenol content. Ultrasound alone
for 8 min has been found to significantly increase total flavonoid
content, resulting in 1.5 ± 0.1 and 0.85 ± 0.1 in C1 and C2 respec-
tively. However, there is no significant difference between treated
and untreated samples after the combined treatment with both con-
centrations of ozone after 8 min. In contrast a slight decrease in TF
was observed at 20 min in combination treatment.
Cabbage antioxidant activity was evaluated before and after
treatment by the DPPH assays. In the DPPH test, ultrasound in-
dividual process had significantly increased the antioxidant activ-
ity of cabbage (C1) at 8 min, with a value of 49.1 ± 0.9 for C1.
However, no difference was found in C2 compared with unpro-
cessed samples. The individual processing of ultrasound at 20 min
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and its combination with ozone at both processing times (8 and
20 min) exerted a significant decrease of antioxidant activity as
shown in Table 3. The lowest antioxidant activity was obtained
when ultrasound was combined with 1.5 mg/L as ozone concen-
tration for 20 min.
4 | D I S CU S S I O N
The purpose of the present study was to provide a comprehensive
and structured examination of the effect of individual and combi-
nation treatment of ultrasound and ozone on cabbage structure
and bioactive properties. In addition, we also evaluated the ability
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8 of 11       TRAORE et al.

F I G U R E 5   Light microscopic images of

combination treatment of US with Ozone
at 20 min

TA B L E 3   Contents of total flavonoids and total phenolic, and DPPH scavenging activity of cabbages (Brassica Oleracea L. Var. Capitata)
treated with ultrasound

Total phenol mM (TEAC) Total flavonoid mg Qe/g Total antioxidant (mg/g)

C1 C2 C1 C2 C1 C2
D C B AB B
Untreated 3.2 ± 0.02 2.8 ± 0.02 1.2 ± 0.01 0.79 ± 0.01 44.9 ± 0.1 41.5 ± 0.7A
US (8 min) 4.3 ± 0.00A 3.31 ± 0.01A 1.5 ± 0.1A 0.85 ± 0.1A 49.1 ± 0.9A 40.9 ± 0.8A
C C B AB D
US+ 0.7 mg/L O3 (8 min) 3.9 ± 0.00 2.8 ± 0.00 1.16 ± 0.02 0.77 ± 0.01 23.9 ± 0.9 22.5 ± 0.3C
US+ 1.5 mg/L O3 (8 min) 2.8 ± 0.00 E 2.6 ± 0.00 D 1.17 ± 0.00 B 0.69 ± 0.08AB 21.1 ± 0.2E 18.1 ± 1.0 D
B B C AB C
US (20 min) 4.1 ± 0.00 3.06 ± 0.05 0.84 ± 0.01 0.74 ± 0.06 30.8 ± 0.3 29.4 ± 0.3B
US+ 0.7 mg/L O3 (20 min) 2.9 ± 0.01E 2.5 ± 0.05D 0.81 ± 0.02C 0.63 ± 0.08B 17.9 ± 1.2F 17.3 ± 0.8D
F E C C G
US+ 1.5 mg/L O3 (20 min) 2.7 ± 0.00 2.3 ± 0.00 0.79 ± 0.02 0.40 ± 0.03 15.1 ± 0.4 11.4 ± 1.6E

Note: US: Ultrasound.

Means that do not share letters are statistically significant. (ANOVA, GLM).

of those treatments in inactivating E. coli and Salmonella. Individual
processing of ultrasound was found to significantly inactivate E. coli
and Salmonella viable cells based on plate counting assays when the
treatment time was increased. The trend observed in this study is
similar to the results reported by Kang, Seymour and coworkers
after washing fresh product for 10 min as processing time (Huang,
Wrenn, Tikekar, & Nitin, 2018; Seymour, Burfoot, Smith, Cox, &
Lockwood, 2002). It has also been reported that ultrasound treat-
ment for 10 min can achieve maximal removal of attached bacteria
from fruits and vegetable surfaces without significant damage to the
fresh product quality (Bilek & Turantas, 2013). In addition Scherba
and coworkers reported that the intensity of acoustic energy and
frequency, effectiveness of ultrasound treatment is dependent on
the treatment time with extended treatments being generally more
effective (Scherba, Weigel, & O'Brien, 1991). In accordance of those
literatures, 20 min of treatment in the present study led to a signifi-
cant removal of bacteria from inoculated cabbage. However, there
was no significant difference between 8, 12 and 16  min of treat-
ment. Both strains represented a similar trend during ultrasound
treatment, indicating that ultrasound has same mode of action on
inoculated E. coli and Salmonella.
The individual treatment of ozone in the present report has
been found to significantly reduce bacterial from leaves surface
with increasing concentrations (0.7 and 1.5  mg/L) and exposure
time. Taking into account its strong oxidant activity, ozone has been
reported to elicit physiological, chemical and microbial changes in
fresh product (Yeoh, Ali, & Forney, 2014). Furthermore, according to
Karaca and Velioglu, ozone destroys microorganisms by the progres-
sive oxidation of vital cellular components (Karaca & Velioglu, 2007).
As a result, accumulation of oxidation effect with increasing treat-
ment time may contribute to higher log reduction in product treated
with ozonate water (Xu & Wu, 2014).
Based on these findings, we suggest that the application of
both treatments separately is not effective to inactivate bacte-
ria from cabbage surface. In order to increase the efficacy of the
process, both treatments should be simultaneously combined. A
synergistic effect of bacterial inactivation was observed when
the concentration of ozone tested was 1.5  mg/L after only 8  min
TRAORE et al.
of processing. Additionally, in the current study, the presence of
E. coli and Salmonella in the ozone water was assessed and results
have shown that although the levels of reduction on the leaves
are pronounced, a high amount of bacteria is transferred to the
ozone water. Kristen et al. stated that ozone >0.3 ppm can prevent
cross-contamination through inactivation of microorganisms in the
water once the microbes are no longer attached to the product in
the batch wash ozone sanitation system (BWOSS) (Gibson, Almeida,
Jones, Wright, & Lee, 2019). However, combination treatment with
1.5 mg/L of ozone for 4 min can prevent cross-contamination by in-
activating bacterial population in washing water below the limited
of detection of 1.0 log CFU/ml. Similar observations were reported
in numerous studies which have investigated the ability of combina-
tion treatment of ultrasound to inactivate microorganisms on fresh
product (Alves do Rosario et  al.,  2017; Gomez-Lopez, Gil, Allende,
Vanhee, & Selma, 2015; Huang et al., 2018; Millan-Sango, Garroni,
Farrugia, Van Impe, & Valdramidis, 2016).
The consumers’ interest in functional food with basic nutrient func-
tions and properties have been increasing, because that can promote
health and prevent diseases. Vegetables and Fruits have these proper-
ties. The total phenolic, total flavonoid and antioxidant activity in both
parts of cabbages before and after treatments were determined. Total
phenolic and flavonoid content in ultrasound (40 kHz for 8 and 20 min)
as well as ultrasound + ozone at 0.7 mg/L for 8 min increased as com-
pared to the control (C1 and C2). The maximum content was observed
for only ultrasound treatment, which support Khandpur, Dadi and
coworkers's observation where they determined TPC and TFC from
Moringa stenopetala and also from different fruits and vegetable juices
by using ultrasound at different time and temperature (Dadi, Emire,
Hagos, & Eun, 2019; Khandpur & Gogate, 2015; Nadeem et al., 2018).
Other technologies such as non-isothermal auto hydrolysis were also
found to improve the phytochemical component in natural broccoli with
increasing operation temperature up to 150°C (Jesus Gonzalez-Munoz,
Conde, Dominguez, & Dolores Torres, 2019). However, steaming pro-
cessing has been reported to decrease the total phenol and flavonoid
content with increasing the processing time (Li, Hao, & Zhu,  2019).
Therefore, the possible destruction of the extracted TPC and TFC
may also occur with longer exposure time and higher temperature of
ultrasound. Antioxidant activity for ultrasound individual processing
(8 min) was also higher as compared to the combination and individual
processing of ultrasound for (20 min). According to Khandpur and co-
workers the initial increase in the antioxidant activity can be attributed
to the fact that ultrasound improves the release of compounds from
the cell matrices (Khandpur & Gogate, 2015). Muhammad Nadeem and
coworkers reported that, the physical effects of sonication can dam-
age fruit tissues and cell walls, resulting in the diffusion of water into
fruit cells (Nadeem et al., 2018). However, according to Artur Wiktor
and coworkers in such cases, visual inspection of microscopic images
can be very helpful (Wiktor et al., 2018). Image of ultrasound at 20 min
treated leaves tissue show destroyed cell walls and more inordinate
inner structure. Such impact of US is even more evident when sonica-
tion was combined with ozone at both concentrations for 20 min. 8 min
of processing has been found to have a positive impact on bioactive
|
      9 of 11
compounds, antioxidant activity of cabbage as compared to the un-
treated or combination and individual ultrasound (20 min).
Furthermore, the pH which is an important parameter for qual-
ity and shelf stability of vegetables and fruits was determined. We
found that the pH was not significantly affected during the whole
process of our treatment, which is also in line with the fact that pro-
cessing should not affect the pH of the cabbage solution.
In summary, we evaluated the role of ultrasound with and
without its combination with ozone as sanitizers and the impact
of these treatments on antioxidant and bioactive compounds of
fresh cabbage. The reduction in bacterial concentration from inoc-
ulated leaves samples increased with ultrasound treatment time.
When compared to individual treatments, the combination of ultra-
sound with ozone significantly increased the removal of E. coli and
Salmonella from the cabbage. Combination treatment with 1.5 mg/L
for 8 min was found to be the most suitable processing for washing
cabbage without damaging its structure. In these optimal process
conditions, most of the artificially inoculated E. coli and Salmonella
were reduced below the detection level. Furthermore, ultrasound
alone and its combination with ozone for 8 min as processing time
were found to be the optimum treatments to maintain and improve
cabbage bioactive compounds such as phenol and flavonoid.
AC K N OW L E D G E M E N T S
The authors greatly appreciate the support for the study by the
project of the National Key R&D Program (No. 2016YFD0400302),
Chinese Scholarship Council and National Natural Science
Foundation of China (No. 31871817).
C O N FL I C T O F I N T E R E S T
The authors have declared no conflicts of interest for this article.
ORCID
Mamadou Bado Traore  https://orcid.org/0000-0002-3491-6530
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