Journal of Agriculture and Food Research 6 (2021) 100198

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Journal of Agriculture and Food Research

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Use of non-chlorine sanitizer in eliminating bacterial and fungal pathogens

from betel leaves - A field level study
Sharmin Zaman a, Quamrun Nahar b, Arafat al Mamun a, Razu Ahmed b, Md Latiful Bari a, *
a
Center for Advanced Research in Sciences, University of Dhaka, Dhaka, 1000, Bangladesh
b
Plant Quarantine Wing, Department of Agriculture Extension, Ministry of Agriculture, Khamarbari, Farmgate, Dhaka, 1215, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: The presence of foodborne pathogen in betel leaf represents a major threat from the public health perspective
Non chlorine sanitizers and is an important export obstacle for South East Asian countries causing huge economical loss to the betel leaf
Betel leaf value chain farmers. Hence, to comply with national and international food safety requirements, use of non-chlorine sanitizer
Safe fresh betel leaf
and low-cost personal hygiene & awareness tools for producing safe betel leaf were evaluated in this study. The
Low cost approach
Food safety & quality
study results demonstrated that microbial safety measures of the field soil and irrigation water used, and re­
striction in domestic animal entry into the field were able to produce microbiologically safe betel leaf. In
addition, washing the harvested fresh betel leaves with 0.01% non-chlorine sanitizers provided adequate mi­
crobial safety of the fresh betel leaf. Thus, this approach could be applicable in the field for safe fresh betel leaf
production for the consumers.

1. Introduction commodities. Most commercial fresh vegetables producers use nearby

Betel leaf produced largely in South Asia (e.g., Sri-Lanka,
Bangladesh, Nepal and India) and is regularly consumed fresh and
raw, thus can be a common vehicle of transmission of entero-pathogenic
bacteria [1]. As the betel leaves produces in South Asia are exported
throughout the world, thus, the presence of foodborne pathogens in
betel leaf represents major public health concern of importing countries
and is an important export obstacle for Bangladesh [2]. In 2012, the EU
Food regulatory authority detected the presence of Salmonella spp., in
imported betel leaves from Bangladesh and thereafter, betel leaf
exportation to EU was reduced and Bangladesh imposed self-suspension
since 2015 to till date [3–5] to identify the hazard in the supply chain
and looking for low cost methods to eliminate the hazard. Several
studies reported that field soils, irrigation waters, animal and human
solid or liquid waste are the major reservoirs of pathogenic bacteria like
E. coli and Salmonella spp., and frequently occurred on farms through the
entry of wild or domestic animals/birds and the use of contaminated
irrigation water [6–12].). In addition, outbreak incidences data showed
that Salmonella spp. is the number one foodborne pathogen associated
with fresh produce that causes foodborne illness [13,14]. The microbial
quality of irrigation water is an important issue of concern in the
pre-harvest and post-harvest food safety of edible horticulture
pond water or underground water for irrigation purpose, thus potential
microbiological risk associated with fresh produce exists [15]. In addi­
tion, post-harvest contamination of betel leaf can be occurred during
processing & handling, packaging & holding and transportation and
distribution [16–18]. During these practices cross contamination can be
occurred in the betel leaf from other agricultural materials or from the
workers hands [6]. Poor handling practices along the value chain can
contaminate betel leaf, which will offer a favorable environment for
microorganisms to grow. Thus, the use of sanitizers was introduced for
providing adequate safety of fresh produce since its inception. Although
chlorine was the most widely used sanitizer for fresh fruits and vege­
tables, however, as it reacts with organic matter of the fresh produce and
formed trihalomethanes-a precursor of carcinogens; thus, the use of
chlorine in food was banned in several countries including EU since
2005 [19]; Allende et al., 2008; [20,21]. In our previous study, we
showed that non-chlorine sanitizers derived from waste shell powder
were able to eliminate foodborne pathogens, and environmental con­
taminants from the surfaces of betel leaf [22]. In addition, several
research findings showed that waste shell powder (WSP) can be an
effective non-chlorine sanitizer and is safe for use in food and non-food
surfaces [23–27]. WSP was also found to be effective against various
microorganisms, including gram positive, gram negative, and

* Corresponding author. Food Analysis and Research Laboratory, Center for Advanced Research in Sciences, University of Dhaka, Dhaka, 1000, Bangladesh.
E-mail address: latiful@du.ac.bd (M.L. Bari).

https://doi.org/10.1016/j.jafr.2021.100198
Received 4 July 2021; Received in revised form 18 August 2021; Accepted 19 August 2021
Available online 24 August 2021
2666-1543/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Zaman et al. Journal of Agriculture and Food Research 6 (2021) 100198

multi-drug resistant bacteria; mycobacteria; enveloped and
non-enveloped, large and small viruses; and fungi [28–34]. Further­
more, WSP sanitization is cost-effective based on cost-benefit analysis; it
is also environment-friendly and enhances food safety [35].
Therefore, in this study, we evaluated the microbial safety of irri­
gation water, and field soil, monitoring of wild and domestic animals’
entry into the field during production and harvest. In addition, intro­
duction of non-chlorine sanitizer for postharvest washing as additional
safety measures for safe betel leaf production.
2. Materials and methods
2.1. Study area & betel leaf farm selection (may 9–11, 2017)
The betel leaf study area was selected by considering the accessibility
from Dhaka to reach the study area and come back to Dhaka in a day.
There are many betel leaf farms in the proposed study districts. For farm
selection, willingness of the farmers to improve, & willingness and
helping attitude of local agriculture extension officials was also taken
into account before selecting three study area in three districts, they are:
1) Kushtia district, Veramara, thana, Kulchera village; 2) Jhenaidah
district, Shailkupa, Thana, Uttar Putia Village; 3) Barisal district, Ujirpur
Thana, Gutia village.
2.2. Farmer selection
Forty (40) farmers (relatively new) from each location/village who
were willing to produce safe betel leaf for getting premium price were
selected. Thus, total 120 farmers were selected to produce safe betel leaf
as per our guidance.
2.3. Field study
The expert team members consist of a soil scientists, microbiologists,
horticulturists and agriculture extension officials were unanimously
decided to visit Jhinaidah & Kushtia on May 19–21, 2017 & Barishal on
June 6–7, 2017 to accomplish 1) conduct survey in project area to check
the baseline information of the project area; 2) identify the sources of
contamination and collection of samples to evaluate the existing quality
of the field soil, irrigation water and betel leaf samples.
2.4. Sample collection
Betel leaf samples were collected from the study areas (Jhenaidah,
Kushtia, Barisal district) and commercial betel leaf samples were pur­
chased from the market and analyzed. These farms were visited seven
times during the period of April 2017 to June 2018 to collect various
samples. Field soil, irrigation water and field betel leaf samples were
collected and taken to the laboratory using sample collection bags by
maintaining temperature (4 ◦ C) in a cool box. Injured or dirty betel leaf
samples were discarded and visually looked fresh betel leaf samples
were used for experiment on the following day of collection at room
temperature (25 ◦ C). Tween five (25 g) grams of betel leaf samples were
weighted by an electric balance (ABS 220-4 N, Shimadzu Corporation,
Japan) and used for each lab experiment according to ISO 6576
methods. Each experiment was repeated at least three times and the
average results were presented in Table 1.
2.5. Preparation of waste shell powder (WSP)
The aggregate (scallop/crab/oyster/egg) waste shell was collected
and cleaned thoroughly with warm water (50 ◦ C) several times. Then it
was dried overnight in an oven at 80 ◦ C and crushed to powder using
grinder and after that grinded powder were sieved (150 μm) and sub­
jected to pyrolysis in a furnace at 1000 ◦ C in air atmosphere. The
calcined waste shell powder was further sieved with 15 μm and stored in
plastic container until use.
2.6. Preparation of sanitizer solutions
The best non chlorine sanitizers were selected on the basis of pre­
vious study [22] and used for these experiments. Sanitizer solutions
were prepared immediately prior to application and used within 1.0 h of
preparation. The waste shell powder (WSP) solution containing 0.01%
and 1.0% concentration, with a pH of 11.4 ± 0.2, was prepared using tap
water; H2O2 (0.5%) solution was prepared by adding distilled water
(DW) to commercial 30% H2O2., Chlorine solution 200 ppm was pre­
pared by adding commercial hypochlorite (5.25%) into distilled water,
and DW water served as control. The pH was determined using a pH
meter (HORIBA Ltd, Tokyo, Japan).
2.7. Washing protocol
In each experimental condition, 25.0 g betel leaf was dipped sepa­
rately in 1.0 L of each sanitizer solution in previously sterile plastic
beaker (2 L). Washing was carried out for 1 min at room temperature
with gentle agitation by hand rubbing. After washing, the solution was
decanted & the sample was rinsed with sterile tap water to remove the
residual washing solution. After that the betel leaves were placed
distinctly on specifically labelled sterile perforated tray to drain off the
excessive water & placed in laminar flow biosafety cabinet for 4 h
drying. Microbiological analysis was done before and after washing with
these sanitizers at different intervals and survival bacterial population
was determined by different selective and non-selective culture media,
as shown in Fig. 1.
2.8. Microbiological analysis of soil, irrigation water and betel leaf
2.8.1. Total aerobic bacterial, total coliform count and presence of
Escherichia coli, & Salmonella spp.
Ten (10 g) grams of betel leaf and 25 g of soil sample was placed in a
stomacher bag separately with 90 ml or 225 ml of sterile saline water.

Table 1
Distribution of aerobic bacteria, coliform bacteria and presence of pathogens in field soil, irrigation water and betel leaf samples produced in three study districts of
Bangladesh (n = 6).
Microorganisms of interest Betel leaf samples produced in the Field soil samples where betel leaf was Irrigation water samples used for betel leaf
Boraj produced production

Jhenaidah Kushtia Barishal Jhenaidah Kushtia Barishal Jhenaidah Kushtia Barishal

Bacterial population log CFU/g

Total aerobic bacterial count (TABC) 5.4 ± 0.1 5.3 ± 0.1 6.2 ± 0.3 6.9 ± 0.5 6.8 ± 0.2 7.1 ± 0.4 5.0 ± 0.06 4.9 ± 0.05 4.0 ± 0.06
Total coliform count (TCC) 3.9 ± 0.3 3.9 ± 0.1 3.9 ± 0.2 5.2 ± 0.3 5.3 ± 0.2 5.8 ± 0.1 2.2 ± 0.04 2.3 ± 0.04 2.1 ± 0.02
Presence of E. coli 1 (11)a 0 (11) 0 (5) 5 (11) 4 (11) 2 (5) 0 (11) 0 (11) 0 (5)
Presence of Salmonella spp. 0 (11) 0 (11) 1 (5) 4 (11) 2 (11) 3 (5) 0 (11) 0 (11) 1 (5)

Mean ± SD value for three replicate experiments.

a
Parenthesis indicates number of betel leaf field positive for pathogen (total number of betel leaf field sample tested).

2
S. Zaman et al.
Fig. 1. Post harvest washing, packaging and transportation of betel leaf.
The mixture was then gently hand rubbed for 90 s for betel leaf samples
and soil samples and serial decimal dilution up to 10− 6 was prepared
with sterile normal saline for each sample and both the diluted and
undiluted samples (0.1 ml) were then spread plated onto Tryptic soy
agar (Oxoid Ltd., Hampshire, England) for total aerobic bacterial count,
Chromocult coliform agar (CHR) medium for coliform and E. coli count,
Bismuth sulfite agar (BSA, Oxoid, England) medium for Salmonella count
and incubated at 37 ◦ C for 24 h. On the following day colonies were
counted and target bacteria was isolated from petridishes and enrich­
ment broth. Next day the pure target colonies were picked from the SAP
(selective agar plate) and streak onto NSAP (Non selective agar plate).
Then identification of E. coli and or Salmonella was confirmed through
set of biochemical tests (TSI Test, Indole Test, MR Test, VP Test, Citrate
Test, and EMB Test) and further confirmation was also done using API
20 E immunoassay kits.
2.9. Isolation, purification and identification of fungi associated with
fresh betel leaf
Fungi associated with the fresh betel leaf were isolated following
tissue planting method [36]. Isolation steps are as follows:
(a) Preparation of inoculums
(b) Surface sterilization
(c) Plating
Total ​ number ​ of ​ inocula ​ from ​ which ​ a ​ fungal ​ isolate ​ was ​ observed
% ⋅ frequency =
Total number of inocula
3
Journal of Agriculture and Food Research 6 (2021) 100198
(d) Inoculation
For both the methods of isolation, surface sterilized inocula were
used. One hundred pieces inocula, each measuring 2 mm are cut with a
sterilized scissors from the diseased betel leaf parts and kept in a sterile
petri plate. The inocula were washed with sterile water and then surface
sterilized by dipping in 10% chlorine solution, WSP and H2O2 for 2.0
min separately followed by rinse with sterile water for 3 min. Finally, the
inocula were placed inside a sterile folded blotting paper to remove the
excessive surface water. The surface sterilized inocula thus prepared
were used for the isolation of fungi.
2.9.1. Tissue planting method
The associated fungi were isolated from the infected surface steril­
ized inocula following the above mentioned process. Briefly, a total of 60
inocula were placed in 20 sterilized petri-plates containing solidified
potato dextrose agar (PDA) medium. Each petri-plate contained 15 ml of
PDA medium with an addition of 1 drop (ca 0.03 ml) of lactic acid which
was used to check the bacterial growth incubated in an incubator (25 ±
2ᵒ C) for 7 days. In both the methods fungi growing out of the inocula
were identified in situ whenever possible and transferred to PDA slants.
The isolates were purified following dilution plate method, maintained
on PDA slants and stored at 10 ± 0.5ᵒC in an incubator for future studies.
Cultures were maintained by sub-culturing after 4 weeks of intervals.
Percentage frequency of occurrence of the fungal isolates was calculated
by adopting the following formula of Spur and Welty [37]:
S. Zaman et al. Journal of Agriculture and Food Research 6 (2021) 100198

Morphological studies of the fungal isolates were made in order to betel leaf samples with 0.01% WSP for 60 s followed by rinse with tap
determine their identity. For microscopic observation, fungal structure water was able to reduce resident aerobic bacteria, total coliform bac­
like mycelia, spore bearing structures and spores were mounted in lac­ teria significantly and reduced pathogenic bacterial population to below
tophenol. In case of hyaline structures, a little amount of aniline blue detection limit and was not evident after enrichment. This finding sug­
(cotton blue) was added to the mounted fluid. A clean cover slip was gested that 0.01% WSP solution were able to eliminate pathogens from
placed over the material and excess fluid was removed by blotting paper the betel leaf surfaces and thus could be useful in improving the safety
and examined under microscope (Motic BA200 Trinocular Compound and quality of betel leaf.
Microscope, with resolution 40–1000X). The microscopic structural While doing the field study, we noticed some diseased betel leaf plant
view of the fungi was taken by a digital camera. The diagram of (fungi infected) in Barishal districts and the fungus was identified as
microscopic structures was drawn with the aid of camera lucida and Phytophthora palmivora, which was not found in the uninfected green
identities of the isolates were determined following standard literature parts of the betel leaf plant. Therefore, decided to evaluate the types and
[38–43]. percentages of fungi present in fresh betel leaves of the study districts.
Thus, the field grown betel leaf samples were collected from the study
area and predominant non-pathogenic and pathogenic fungi were iso­
2.10. Statistical analysis
lated, identified and their percent frequencies were presented in Fig. 2.
The results demonstrated that irrespective of the study area, non-
All trials were replicated three times. Reported plate counts observed
pathogenic Aspergillus flavus was the predominant fungus in fresh betel
in all agar media were converted into CFU/g and the numbers represent
leaf samples. However, the prevalence of Aspergillus flavus (40%),
the mean values obtained from three individual trials, with each of these
Nigrospora spp. (42%). Pestaloptiosis guepinii (8.5%) and sterile mycellia
values obtained from duplicated samples. Data were subjected to anal­
(39%) was observed in fresh Betel leaf samples of Jhenaidah districts
ysis of variance using Microsoft Excel program (Redmond, Washington,
and Aspergillus flavus (55%), Colletotrichum gloeosporioides (17%) and
DC, USA). Significant differences of plate count data were established by
sterile mycelia (29%) were evident in Kushtia districts and similarly,
the least significant difference at the 5% level of significance.
Aspergillus flavus (60%), Cladosporium spp. (18%) and sterile mycelia
(28%) was seen in Barishal districts (Fig. 2). This observation suggested
3. Results and discussion
that, the highest percentage of pathogenic fungi Colletotrichum gloeo­
sporioides was found in Jhenaidah > Barishal > Kushtia, respectively,
Irrespective of study area selected, the farm produced betel leaf
and the other pathogenic fungi (Curvularia lunata) was found in Kushtia
possess moderate level of total aerobic bacteria (TABC) ranging from 5.3
> Jhenaidah > Barishal respectively. In addition, the frequency ratio of
to 6.2 log CFU/g and 3.9 log CFU/g of total coliform bacteria. Both
pathogenic fungi (Pestalotiopsis guepinii) was observed in Jhenaidah >
Salmonella spp. and E. coli was evident in one field betel leaf of Barishal
Barishal and Kushtia, respectively. Now, to eliminate the pathogenic
and Jhenaidah district, respectively (Table 1). On the other hand, only
fungi from fresh betel leaf, the same non-chlorine sanitizers (0.01%
one irrigation water sample (3.7%) of 27 was found contaminated with
WSP) along with 0.5% H2O2, & 200 ppm chlorine water, was used to
Salmonella spp. in Barishal districts. Furthermore, the total aerobic
compare the effectiveness of the sanitizers in eliminating pathogenic
bacterial count in field soil was recorded as 6.8–7.1 log CFU/g and
fungi from the betel leaf.
coliform bacteria as 5.2–5.8 log CFU/g, however, these values were less
The results showed that washing the betel leaf with 0.5% hydrogen
than the standard agriculture soil. The pH of the soil sample was
peroxide and 200 ppm chlorine water was able to reduce the fungi to an
recorded as 6.3–7.5, which also falls within the standard pH level for
undetectable level but after enrichment presence of fungi were evident
agriculture soil. On the other hand, E. coli -an indicator bacterium of
(Table 3). However, when washing the betel leaf samples with WSP
fecal contamination was observed in 12 field soil samples (44% field) of
(0.01%), the fungus was found eliminated effectively compared to
27 field analyzed, and Salmonella spp. was found in 10 fields soil samples
hydrogen peroxide and chlorine water (Table 3 and Fig. 3).
(37% field). Of them, 7 field soil samples (26% field) were found
Furthermore, a field monitoring study for the domestic or wild ani­
contaminated with both Escherichia coli and Salmonella spp. (Table 1).
mal entry into the field were also conducted and ten volunteers (4 fe­
This observation suggested that the field soil was grossly contaminated
male and 6 male) were contacted through Unnyan Dhara Bangladesh a
with fecal materials. Surprisingly, the field grown betel leaf samples
local NGO, for doing this experiment and trained them how to monitor
were found less contaminated, despite the irrigation water & field soil
the animal entry into the betel leaf field. The volunteers were grouped in
were found grossly contaminated.
2 volunteers for daytime and 3 volunteers for night-time and female
The field contaminated betel leaf samples were brought to the lab­
volunteers were assigned during daytime and male volunteers were
oratory for post-harvest washing with freshly prepared 0.01% WSP so­
assigned during night time. The volunteers were asked to monitor any
lution followed by rinse with tap water and the results were presented in
domestic or wild animal entry into the field and noted in the monitoring
Table 2. The results of this study suggested that, washing betel leaf with
assessment sheet. The observation results showed that small domestic
distilled water was able to clean the surfaces of betel leaf but were un­
animals such as hens and stray dogs or cats sometimes entered into the
able to reduce the bacterial population. On the other hand, washing

Table 2
Effectiveness of non-chlorine sanitizer (WSP 0.01%) in eliminating E. coli and Salmonella population in field contaminated betel leaf samples.
Sample ID Treatment Bacterial population log CFU/g

Total aerobic bacterial count (TABC) Total coliform count (TCC) Presence of E. coli Presence of Salmonella spp

BE AE BE AE BE AE

Jhenaidah (J9) Unwashed 4.23 ± 0.3 4.07 ± 0.2 ND <1.0 P <1.0 P

0.01% 2.08 ± 0.1 1.36 ± 0.1 ND <1.0 A <1.0 A
WSP wash
Barishal(B4) Unwashed 4.13 ± 0.3 4.23 ± 0.2 ND <1.0 P <1.0 P
0.01% 2.03 ± 0.1 1.87 ± 0.1 ND <1.0 A <1.0 A
WSP wash

TABC = Total aerobic bacterial count; TCC = Total coliform count; BE = before enrichment; AE = afterenrichment; ND= Not done; P= Present; A = Absent; WSP =
waste shell powder.

4
S. Zaman et al. Journal of Agriculture and Food Research 6 (2021) 100198

Fig. 2. Mean percent frequency of fungi associated with fresh betel leaf in Barishal, Jhenaidah, and Kushtia study area.

Table 3
Comparison of different sanitizers’ efficacy in eradicating fungi isolated from fresh betel leaf.
Sampling Area Non wash Distilled water wash Different sanitizers

0.01% WSP 0.5% H2O2 200 ppm Cl2

BW AW BW AW BW AW BW AW

Barishal P P P P A p P P p
Jhenaidah P P P P A P P P P
Kushtia p P P p A P P p P

BW = Before wash, AW = After wash, WSP = waste shell powder, H2O2 = hydrogen peroxide, P = Presence of fungi, A = absence of fungi.

betel leaf fields, which might contribute to contamination (Table 4). In
addition, birds or group of birds were seen flying during the sunset over
the field in Jhenaidah and Kushtia area, which might excrete feces and
consequently contaminate the soil. It was also observed that the betel
leaf field was located nearby the farmer’s house in Jhenaidah and
Kushtia area, but in Barishal area, the betel leaf field was little far from
the locality, which might be the reason of frequent entry of domestic
animals into the betel leaf field in Jhenaidah and Kushtia area compared
to Barishal area. This finding suggested that the betel leaf field should be
established little far from the locality to avoid animal entry.
In Bangladesh, the existing traditional and cultural production
practices of betel leaf, irrigation water uses, betel leaf holding con­
tainers, transport vehicles, and personal hygiene of farmers, vendor and
other stakeholder throughout the value chain may contribute to
contaminate betel leaves. In addition, besides production practices,
post-harvest washing is the last step to ensure safety of the betel leaf,
thus have importance in reducing fresh produce borne illness, because
washing step can either reduce or may contribute to contamination of
fresh produce depending on the quality of wash water. Furthermore,
many researchers identified washing is the critical point of contamina­
tion by bacteria including coliform, Escherichia coli, Salmonella spp., to
the produce. Thus, washing fresh produce with supply water/clean
water may eliminate soil and other debris, but is not sufficient to
eliminate the microorganisms from the surfaces of fresh produce
[44–46]. Therefore, monitoring soil and water quality on a regular basis
and maintaining improved personal hygiene practices along the value
chain could produce microbiologically safe betel leaf; however, for
adequate protection in ensuring food safety of the betel leaf, intro­
duction/use of non-chlorine sanitizer during post-harvest washing is
preferable [47,50].
Since, washing betel leaf samples with 0.01% WSP solution was
found suitable in eliminating both bacterial and fungal pathogens from
the betel leaves and thus contributed in improving the safety and quality
of betel leaf compared to distilled water, 0.5% H2O2, and 200 ppm
chlorine [51]. Hands on training in post-harvest washing practices,
availability of non-chlorine sanitizers and improved personal hygiene

5
S. Zaman et al. Journal of Agriculture and Food Research 6 (2021) 100198

Fig. 3. Evaluation of effectiveness of different sanitizers in eradicating fungi isolated from fresh betel leaf (A) WSP 0.01% treated betel leaves; (B) H2O2 (0.5%)
treated betel leaves; (C) Cl2 (200 ppm) treated betel leaves.

Table 4
Monitoring of domestic or wild animal entry into the production field results.
Sailokupa, Jhenaidah Veramara, Kushtia UjirpurBarishal

Animal types Morning to Noon to Sunset to Morning to noon Noon to Sunset to Morning to Noon to Sunset to
noon sunset sunrise 6:00AM-12:00 sunset sunrise noon sunset sunrise
6:00AM- 12:00–20; 00 20:00–6:00 a. 12:00–20; 00 20:00–6:00 a. 6:00AM- 12:00–20; 00 20:00–6:00 a.
12:00 m. m. 12:00 m.

Hens/goose/ Hen with 13 Hens 3 Birds 2 Hens 3 Hens A group of - Birds flying -
birds kids together 6:00 11:00 2:30PM birds 6:20 p.m.
4:00PM 4:23 p.m. 6:30PM
Cats/dogs – – Cats 2 cats run over - - - - -
6:15PM 8:00AM
1 Dog at 9:10AM
Goats/cows - - - - - - - - -

practices along with monitoring soil and water quality on a regular
basis, might be the major contributory factors in eliminating pathogens
in betel leaf.
4. Conclusion
The microbial safety of the irrigation water used, field soil and re­
striction in domestic animal entry into the field were able to produce
microbiologically safe betel leaf. In addition, introduction of non-
chlorine sanitizers in post-harvest washing showed additional protec­
tion in producing safe fresh betel leaf. Furthermore, Good agricultural
practices along with improving personnel hygiene practices during betel
leaf production could further improve the situation. Thus, this approach
could be applicable in the field for safe fresh betel leaf production for the
consumers.
Declaration of competing interest
The authors declare that there is no conflict of interest in the content
of this manuscript and all the authors agreed for its submission in any
Elsevier journal.
Acknowledgement
This study was financially supported by NATP-2-PIU-BARC, CRG-
project no-452, using the research fund of World Bank, IFAD and GoB
through Ministry of Agriculture. We would like to thanks to the World
Bank for arranging the grand fund and supervising the CRGs by BARC. It
is worthwhile to mention the cooperation and quick responses of PIU-
BARC, NATP-2, in respect of field implementation of the sub-project
in multiple sites. The authors would like to thanks Mr. Talib Bashar
Nayon, Unnayan Dhara Bangladesh for their enormous support in
finding and selecting betel leaf farmers at Jhenaidah and Kushtia dis­
tricts, and Ms. Kazi Munni in finding and selecting betel leaf farmers at
Barishal district.
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