LWT - Food Science and Technology 78 (2017) 77e81

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LWT - Food Science and Technology

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

Effectiveness of non-chlorine sanitizers in improving the safety and

quality of fresh betel leaf
Sunzid Ahmed a, Sharmin Zaman a, Razu Ahmed b, Md. Nazim Uddin c,
Antonio Acedo Jr. d, Md. Latiful Bari a, *
a
Center for Advanced Research in Sciences, University of Dhaka, Dhaka, 1000, Bangladesh
b
Department of Agriculture Extension, Khamarbari, Farmgate, Dhaka, 1205, Bangladesh
c
Horticulture Research Center, BARI, Gazipur 7205, Bangladesh
d
AVRDC e The World Vegetable Center, South Asia, Hyderabad, India

a r t i c l e i n f o a b s t r a c t

Article history: Calcined waste shell aggregate (CCa) is a promising non-chlorine sanitizers and 0.01% CCa was found to
Received 10 August 2016 be effective in eliminating pathogens in various fresh produces. This study was designed to evaluate the
Received in revised form efficacy of promising non-chlorine sanitizers including 0.5% (H2O2), 0.01% CCa, and/or 0.5% (CA), in
23 November 2016
enhancing the safety and quality of betel leaf. The presence of Salmonella spp. was observed in the
Accepted 13 December 2016
commercial betel leaf samples. Washing the commercial betel leaf samples with distilled water was able
Available online 14 December 2016
eliminate soil debris, however, was unable to eliminate microorganisms satisfactorily. In contrast,
washing betel leaf sample with 0.01% CCa, 0.5% (H2O2), and 0.5% CA was able to eliminate pathogenic
Keywords:
Food safety & quality
bacteria initially, however, after enrichment the pathogen populations were detected in the commercial
Betel leaf betel leaf samples. When betel leaf samples was artificially contaminated with pathogenic bacteria
Non-chlorine sanitizer [higher (>7.0 log CFU/ml) and lower (>4.0 log CFU/ml) inoculums] and washed with 0.01% CCa, 0.5%
Environmental friendly H2O2, and 0.5% CA solutions, a complete elimination of inoculated bacteria was noticed initially in most of
the cases, but after enrichment, the bacteria was mostly detected in higher inoculums study. This finding
demonstrated that 0.01% CCa, 0.5% H2O2, and 0.5% CA could be useful in improving the safety and quality
of betel leaf, however, use of 0.01% CCa is preferable because of its low cost, availability, biodegradable
and non-hazardous characteristics compared to other sanitizers used. Hence, could be positive alter-
natives of chlorine sanitizers.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction communities and hence an economically important fresh herbs,

The betel leaf commonly called as “paan” in Bangla and Hindi;
belonging to the Piperaceae family is valued for its medicinal
properties and as a mild stimulant. Betel leaf extract has potential
capability to inhibit foodborne pathogens including Escherichia coli,
Staphylococcus aureus and Pseudomonas aeruginosa (Hoque et al.,
2011; Khan & Kumar, 2011). It is mostly consumed in Asia, and
elsewhere in the world by Asian emigrants. The betel has no fruit
and is grown only for the sake of its leaves because, about 60e70%
of Bangladeshi people aged 35e60 years, irrespective of class,
consume betel leaf frequently. Betel leaves is also being exported to
different countries (USA, UK, Middle East, EU etc.) for the ethnic
* Corresponding author.
E-mail addresses: mdlatifulbari@gmail.com, latiful@du.ac.bd (Md.L. Bari).
which export earning averaged USD 8.0 million every year (Karim,
2014), however several foodborne outbreaks have been associated
with the consumption of Betel leaves in recent years. The presence
of foodborne pathogen in betel leaf represent a major threat from
the public health point of view and is an important export obstacle
for Bangladesh. In 2012, the EU regulatory authority detected the
presence of Salmonella spp. in imported betel leaves from
Bangladesh and many outbreaks associated with fresh leaves have
been reported in EU, UK, USA and other countries in recent years. As
a result EU has consequently imposed ban on this fresh herbs that
causes diarrhea and vomiting, which can lead to serious illness to
the consumers (Ahsan, 2012; Chowdhury & Kallol, 2013; Hald &
Baggassen, 2012; Felício et al., 2015).
Sanitization of betel leaf could be one of the best options in
eliminating foodborne pathogens. However, very limited study on
the decontamination of microorganisms in betel leaf was found in

http://dx.doi.org/10.1016/j.lwt.2016.12.025
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
78 S. Ahmed et al. / LWT - Food Science and Technology 78 (2017) 77e81
the literature. Vinegar was effective in eliminating foodborne
pathogens in betel leaf (Khan & Kumar, 2011). Research showed
that, washing the betel leaf with 2% acetic acid was able to reduce
individual pathogens population by 4.0 log CFU/g and mixed
pathogens by 2.0 log CFU/g (Singla, Ganguli, Ghosh, & Sohal, 2009).
Chlorine is the most widely used sanitizer for fresh vegetables and
fruits, however, it reacts with organic matter in the produce to form
highly carcinogenic trihalomethanes; consequently, use of chlorine
is being phased out in several countries including EU. Thus, there is
a need for alternative non-chlorine sanitizers for washing fresh
produces. Therefore, the objective of this study was to assess the
effectiveness of calcinated calcium (0.01%), food grade hydrogen
peroxide (0.5%) and citric acid (0.5%) in eliminating food borne
pathogens and improving the safety and quality of fresh betel leaf.
2. Materials and methods
2.1. Sample collection
Commercial betel leaves were either purchased from the ordi-
nary market of Dhaka city, or supplied by betel leaf businessmen
and brought to the laboratory by maintaining temperature using a
cool box. Injured or dirty betel leaf samples were discarded and
fresh samples were used for experiment on the same day at room
temperature. Twenty five (25) grams of betel leaf samples were
weighted by an electric balance (Shimadzu Corporation, Japan) and
used for each experiment. Each trial was conducted at least three
times.
2.2. Preparation of sanitizer solutions
Sanitizer solutions were prepared immediately prior to appli-
cation and used within 1.0 h of preparation. The calcinated calcium
(CCa) solution containing 0.01% concentration, with a pH of
11.4 ± 0.2, was prepared using tap water; H2O2 (0.5%) solution was
prepared by adding deionized water (DW) to commercial 30% H2O2.
Citric acid (CA) 0.5% solution was prepared using DW and DW water
wash served as control. The pH was determined using a pH meter
(HORIBA Ltd, Tokyo, Japan).
2.3. Challenge test with high and low inoculums
Three foodborne pathogens (E. coli O157:H7, S. aureus and
S. typhi) and two spoilage bacteria (P. aeruginosa, V. metschnikovii)
isolated from various fresh produces were chosen for the challenge
test. Nalidixic acid resistant strains were prepared by adopting the
above mentioned bacteria individually in tryptic soya broth, as
described by Inatsu, Bari, and Kawamoto (2007). Briefly, each in-
dividual bacterium was grown in TSB media supplemented with
50 mg/ml of nalidixic acid, after three successive inoculations,
bacterial cells were collected by centrifugation (3000 rpm; 10 min)
and were washed three times with sterile saline water and finally
re-suspended in sterile water and initial bacterial count was
determined. Similarly all the inoculums were prepared and pooled
together to get a cocktail of 5 bacterial strains and hold for 30 min.
Both the high (5.0e6.0 log CFU/ml individual marker bacteria) and
low (4.0e5.0 log CFU/ml individual marker bacteria) inoculum
cocktail solution were prepared and used for the experiment. One
hundred (100) grams of betel leaf samples were dipped into both
inoculums separately, stir slowly for 5 min and then transferred to a
sterile perforated tray and dried in bio-safety cabinet for 4 h at
22 ± 2
C. The dried high and low inoculum betel leaf samples were
then stored at 4
C for overnight and subjected to wash with
different sanitizers.
2.4. Washing protocol
In each experimental condition, 25.0 g of betel leaf was washed
separately with 1.0 L of the washing solution in a sterile glass
beaker (2 L). Washing was carried out for 90 s at room temperature
by hand rubbing. After washing, the solution was decanted; a
second wash was done with 1.0 L of sterile distilled water to remove
the residual washing solution. After washing experiment betel leaf
were placed on a sterile perforated tray in a laminar flow biosafety
cabinet for 4 h at 22 ± 2
C to facilitate drying. Microbial analysis
was done before and after washing with these sanitizers at
different intervals and the surviving bacterial population was
determined on both of selective and nonselective culture media.
Physical parameters including surface browning, color change,
cumulative weight loss and rotting percent of betel leaf were also
observed.
2.5. Microbiological analysis
Before and after wash with each sanitizer, 25 g of betel leaf
samples were placed in a stomacher bag with 225 mL of sterile
saline water. The mixture was pummeled for 90 s and serial decimal
dilutions were prepared with sterile saline water. The diluted and
undiluted samples (0.1 mL) were then surface plated on both se-
lective and nonselective agar media. Tryptic Soy Agar (TSA; Oxoid,
England) medium was used for the total bacterial count and se-
lective medium such as Sorbitol MacConkey (SMAC; Oxoid, En-
gland) agar for E. coli, Chromocult Agar (Merck, Germany) for
coliform, Bismuth Sulfite Agar (BSA; Oxoid, England) for Salmonella
spp., Sabouraud's Dextrose Agar (Active Fine Chemicals Ltd.,
Bangladesh) for yeast & mold were used. Identification of E. coli and
Salmonella spp. was confirmed through set of biochemical tests
with API 20E system.
In addition, Tryptic soy agar (TSA; Oxoid) and Sorbitol Mac-
Conkey (SMAC) agar supplemented with 50 mg/ml Nalidixic acid
was used as non-selective and selective medium, respectively, for
the recovery of E. coli O157:H7. On the other hand, Cetrimide Agar
(Oxoid, England), Thiosulphate Citrate Bile-Salt Sucrose Agar (Nis-
sui, Japan), Mannitol Salt Agar (Merck, Germany), and Bismuth
Sulfite Agar (BSA; Oxoid, England) was supplemented with 50 mg/
ml nalidixic acid for the recovery of Pseudomonas aeruginosa, Vibrio
metschnikovii, Staphylococcus aureus, and Salmonella typhi, respec-
tively from the inoculated betel leaf samples.
Enterobacter Enrichment Broth- Mossel (Oxoid, England), and
Rappaport Vassiliadis Broth (Eiken Chemical, Japan) was always
used for the enrichment procedure of E. coli, and Salmonella spp.
respectively. Antibiotic was supplemented in the enrichment broth
when it was necessary for challenge test.
2.6. Statistical analysis
All trials were replicated three times. Reported plate counts
determined on all agar media were converted to Log CFU/gm and
numbers represent the mean values obtained from three individual
trials, with each of these values being obtained from duplicated
samples. Data were subjected to analysis of variance using the
Microsoft Excel program (Redmond, Washington DC, USA.). Sig-
nificant differences in plate count data were established by the
least-significant difference at the 5% level of significance.
3. Results
The microbial quality of commercial betel leaf samples was
determined and the results were presented in Table 1. It was
observed that on an average 4.0e5.0 log CFU/g of total aerobic
 S. Ahmed et al. / LWT - Food Science and Technology 78 (2017) 77e81 79

Table 1
Microbiological quality of commercial betel leaf samples of Dhaka City (n ¼ 6) and efficacy of sanitizers in reducing bacterial populations.

Microorganisms Bacterial population (Log CFU/g)

Non- wash Distilled water 0.01% CCA wash 0.5% H2O2 wash (pH 0.5% CA wash (pH
wash (pH 7.142) (pH 11.564) 5.920) 2.514)

BEc AEd BE AE BE AE BE AE BE AE

Total bacterial count 4.88a NDb 3.21a ND 1.96a ND 1.23a ND 1.14a ND

Total coliform count 2.00a ND 1.10b ND <1.0 <1.0 <1.0 <1.0 <1.0 <1.0
Escherichia coli <1.0a <1.0 <1.0 A <1.0 A <1.0 A <1.0 A
Salmonella spp. <1.0 P <1.0 P <1.0 A <1.0 A <1.0 A
Total fungal count 4.93a ND 3.64c ND 2.01c ND 1.54b ND 1.69b ND

The mean values of different letters are significantly different (a  0.003; b  0.003; c  0.005). While mean values with the same letter are not significantly different.
A ¼ Absent.
P ¼ Present.
a
<1.0: less than detection limit; the detection limit was 1.0 log CFU/g.
b
ND ¼ Not Done.
c
BE ¼ Before enrichment.
d
AE ¼ After enrichment.

bacteria population; 2.0 log CFU/g of coliform bacteria; and 4.5e5.0
log CFU/g of yeast & mold count was recorded in commercial betel
leaf samples. Furthermore, presence of Salmonella was observed in
the betel leaf samples analyzed. This finding suggested that com-
mercial betel leaves were of poor quality. Washing the betel leaf
samples with DW removed some soil or other debris, and were able
to reduce around 1.5 log CFU/g resident bacterial load (P > 0.05). On
the other hand, washing betel leaf with 0.01% CCa or 0.5% H2O2, or
0.5% CA (Citric Acid) followed by a distilled water wash was able to
reduce 2.0e3.5 log CFU/g of viable bacteria, coliform bacteria and
yeast & mold count (Table 1). In addition, these sanitizer washes
was able to completely eliminate Salmonella spp. population from
commercial betel leaf samples. Absence of Salmonella spp. were
noticed in some samples initially on selective agar plates, however,
after enrichment all the sample were found positive for Salmonella
spp., this finding suggested that enrichment steps were necessary
for the detection of Salmonella spp. in betel leaf. Challenge test was
performed by inoculating 5 microorganisms (3 pathogens and 2
spoilage bacteria) individually to see the effectiveness of each
sanitizer in reducing each pathogen and spoilage bacteria. Higher
and lower inoculum was analyzed to mimic the real practice
(Tables 2 & 3).
In higher inoculum study, on an average 6.18 log CFU/g of total
marker bacterial count; 4.0 log cfu/g of E. coli, and 4.0e4.5 log CFU/g
of P. aeruginosa; V. metschnikovii, S. aureus and S. typhi was recorded
in before wash betel leaf samples (Table 2). Washing with distilled
water was able to reduce approximately 1.5e2.0 log CFU/g of the
inoculated bacteria, and washing with 0.01% CCa reduce 3.0 log
CFU/g of total marker bacterial count (TMBC), S. aureus and E. coli,
however, complete reduction of P. aeruginosa, V. metschnikovii and
S. typhi was noticed initially, but after enrichment presence of all
the inoculated bacteria was detected. Similar experimental results
were observed while washing betel leaf with 0.5% H2O2 or 0.5% CA
(Table 2). In case of low inoculum study, on an average 3.5 log CFU/g
of TMBC, S. aureus, and S. typhi; and approximately, 3.0 log CFU/g of
E. coli O157:H7, and P. aeruginosa; V. metschnikovii was recorded in
non-wash samples (Table 3). Washing these low inoculum betel
leaf samples with distilled water was able to reduce approximately
1.5e2.0 log CFU/g of the inoculated bacteria, and washing with
0.01% CCa reduced 3.0 log CFU/g of TMBC, and S. aureus. On the
other hand, E. coli, P. aeruginosa V. metschnikovii and S. typhi was
reduced to below detectable level and after enrichment none of
these pathogens were detected. Similar experimental results were
recorded while washing betel leaf with 0.5% H2O2. In contrast,
lower effectiveness of pathogen reduction was observed with 0.5%
CA. This finding suggested that 0.01% CCa or 0.5% H2O2 could be
able to reduce microorganisms better compared to 0.5% CA. How-
ever, use of 0.01% CCa is preferable because CCa is an organic
compound, biodegradable and cause minimum harm to environ-
ment compared to other two sanitizers used in this study.

Table 2
Effectiveness of non-chlorine sanitizers in reducing bacterial population in high inoculum Betel leaf samples.

Microorganisms inoculated Bacterial population (Log CFU/g)

Initial bacterial inoculums (cocktail) load Non-wash DW wash 0.01% CCA wash 0.5% H2O2 0.5% CA wash
(pH 7.130) (pH 11.325) wash (pH 2.931)
(pH 5.821)

BEc AEd BE AE BE AE BE AE

Total bacterial count 8.16a 6.18b 4.09a NDb 3.12 ± 0.31 ND 3.01b ND 3.01a ND
Escherichia coli 6.31c 4.13b 2.19b ND 1.00a P <1.0a A <1.0 A
Pseudomonas aeruginosa 5.84a 4.42a 3.16c ND <1.0 P 2.00b A <1.0 A
Vibrio metschnikovii 5.69b 4.30c 3.30c ND <1.0 P <1.0 P <1.0 P
Staphylococcus. aureus 5.57a 4.31c 4.07c ND 1.12b P <1.0 P 1.00c P
Salmonella typhi 5.27b 4.34c 2.69c ND <1.0 P <1.0 P <1.0 A

The mean values of different letters are significantly different (a  0.002; b  0.002; c  0.005). While mean values with the same letter are not significantly different.
A ¼ Absent.
P ¼ Present.
a
<1.0: less than detection limit; the detection limit was 1.0 log CFU/g.
b
ND ¼ Not Done.
c
BE ¼ Before enrichment.
d
AE ¼ After enrichment.
80 S. Ahmed et al. / LWT - Food Science and Technology 78 (2017) 77e81

Table 3
Effectiveness of non-chlorine sanitizers in reducing bacterial population in low inoculum Betel leaf samples.

Microorganisms inoculated Bacterial population (Log CFU/g)

Initial bacterial inoculums load Non- wash DW wash 0.01% CCA 0.5% H2O2 0.5% CA wash
(7.121) wash wash (pH 2.7)
(pH 11.162) (pH 6.637)

BEc AEd BE AE BE AE BE AE

Total bacterial count 5.43b 3.85a 2.42b NDb 1.54a ND 1.15b ND 1.15b ND
Escherichia coli 5.60c 2.92a 1.30c ND <1.0a A <1.0 A <1.0 P
Pseudomonas aeruginosa 4.00a 2.98a 1.93b ND <1.0 A <1.0 P <1.0 P
Vibrio metschnikovii 4.23b 2.72c 1.93a ND <1.0 A <1.0 A <1.0 A
Staphylococcus aureus 4.00a 3.75a 2.15b ND 1.30a P <1.0 A 1.54a P
Salmonella typhi 4.91b 3.76c 1.69c ND <1.0 A <1.0 A <1.0 A

The mean values of different letters are significantly different (a  0.004; b  0.004; c  0.005). While mean values with the same letter are not significantly different.
A ¼ Absent.
P ¼ Present.
a
<1.0: less than detection limit; The detection limit was 1.0 log CFU/g.
b
ND ¼ Not Done.
c
BE ¼ Before enrichment.
d
AE ¼ After enrichment.

4. Discussion
Pathogenic and spoilage microorganisms are critical postharvest
hurdle that have been causing human sufferings, food losses and
economic erosion. Cleaning and sanitation have thus become
standard postharvest handling operation to ensure quality and
safety of the fresh produce. Cleaning must be done to remove
adhering soil and other debris and sanitation must be done to
remove spoilage and pathogenic microorganisms from the surfaces
of the produce. Unfortunately, in Bangladesh, fresh produces are
hardly clean, and not sanitized at all before entering into the
marketing channel, and thus contribute to unsafe, poor quality
fresh produce. In this study, presence of Salmonella spp. was
evident in almost all the commercial betel leaf samples, this results
were consistent with the results of literature, which reported the
presence of 17.34% E. coli, 25.51%, Salmonella spp., 19.39%, Vibrio
spp., and 19.39%, Staphylococcus spp., in commercial betel leaf of
Bangladesh (Hoque et al., 2011). Fresh leaves can become
contaminated with pathogens from a number of sources, including:
1) contaminated water used for irrigation or to apply pesticides,
fungicides or plant feed; 2) human or animal sewage used as fer-
tilizer; 3) contaminated water used to wash the crop after har-
vesting; 4) domestic or wild animal contact with the crop; 5)
contaminated equipment used to wash, process or package the
crop; 6) contaminated vehicles, crates or storage areas used in
distribution; 7) cross-contamination during preparation in other
food businesses or in the home; and 8) infected food handlers.
On the other hand, presence of undesirable pathogens, envi-
ronmental contaminants, and pesticide residues on the surfaces of
fresh produces is a common concern worldwide. The consumptions
of fresh herbs or leaves, due to many health benefits, have increased
many folds and that simultaneously increases the food safety risk
worldwide. Chlorine has been widely used sanitizers for fresh fruits
and vegetables worldwide; however, recent report revealed that
the chloramines compound formed, while in contact with fresh
herbs, is a precursor for cancer. Many organic and inorganic sani-
tizers were studied to wash fresh produce include, acidified NaOCl,
Ca(OCl)2, acidified Ca(OCl)2(Annous, Sapers, Mattrazzo, & Riordan
2001; Inatsu et al., 2011; Yasmin, Bari, Inatsu, & Kawamoto, 2008;
Elano et al., 2010), Na3PO4, Tsunami™, Vortex™, H2O2 (Bari et al.,
2005), gaseous acetic acid (Bari et al., 2005; Ukuku, Bari,
Kawamoto, & Isshiki, 2005), organic acid ozone (Inatsu et al.,
2011), ozonated water & ozone nano bubble (Kim, Yaptenco, &
Lim, 2006), electrolyzed water (Bari, Sabina, Isobe, Uemura, &
Isshiki, 2003; Inatsu et al., 2007; Koseki, Yoshida, Isobe, & Itoh,
2001), acidified electrolyzed water (Haq, Sugiyama, & Isobe,
2005; Laíd, Cuartas, Mercado, Díaz, & Carrascal, 2005; Rahman,
Ding, & Oh, 2010), peroxy acetic acid (Beuchat, 1998; Beuchat,
Alder, & Lang, 2004), Combination of EDTA and organic acid, and
competitive inhibition (Bari et al., 2005) etc. However, most of
these sanitizers, were either expensive or non-user friendly or in-
volves some risk. Furthermore, ozone, electrolyzed water, mild heat
treatment, organic acid, and other natural sanitizers alone or in
combinations have been applied to various fresh-cut vegetables
and are acceptable for non-organic fresh produce. In Bangladesh,
the primary barrier for producing safe and high quality fresh pro-
duce for premium domestic & export market is the use of sanitizers
during post-harvest handling. The cleaning and sanitization prac-
tices usually improve the safety, quality, and shelf life of the fresh
produce. Furthermore, the introduction of novel sanitizer will
eradicate the use formalin solution -an illegal chemical and highly
carcinogens.
5. Conclusion
The results of this study suggested that, washing betel leaf
samples with 0.01% CCa, 0.5% H202, and 0.5% CA was able to reduce
resident aerobic bacteria, total coliform bacteria and yeast & mold
significantly and reduced pathogenic bacterial population to below
detectable level. This finding suggested that all the non-chlorine
sanitizers used in this experiment could be useful in improving
the safety and quality of betel leaf. However, use of 0.01% CCa is
preferable because CCa is an organic material, biodegradable and
cause minimum environmental damage compared to other two
sanitizers used in this study. Thus, the results of this study
demonstrate the great potential of CCa as a substitute of chlorine
for sanitizing fresh betel leaf.
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