Accepted Manuscript

Insight into the antibacterial property of chitosan nanoparticles against Escherichia

coli and Salmonella Typhimurium and their application as vegetable wash disinfectant

Pathompong Paomephan, Apinya Assavanig, Soraya Chaturongakul, Nathaniel C.

Cady, Magnus Bergkvist, Nuttawee Niamsiri

PII: S0956-7135(17)30455-3
DOI: 10.1016/j.foodcont.2017.09.021
Reference: JFCO 5793

To appear in: Food Control

Received Date: 14 June 2017

Revised Date: 8 September 2017
Accepted Date: 17 September 2017

Please cite this article as: Paomephan P., Assavanig A., Chaturongakul S., Cady N.C., Bergkvist M. &
Niamsiri N., Insight into the antibacterial property of chitosan nanoparticles against Escherichia coli and
Salmonella Typhimurium and their application as vegetable wash disinfectant, Food Control (2017), doi:
10.1016/j.foodcont.2017.09.021.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

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1 Insight into the antibacterial property of chitosan nanoparticles against
2 Escherichia coli and Salmonella Typhimurium and their application as
3 vegetable wash disinfectant
4
5 Pathompong Paomephan1, Apinya Assavanig1, Soraya Chaturongakul2,
6 Nathaniel C. Cady3, Magnus Bergkvist3, and Nuttawee Niamsiri1*

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8 Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road,

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9 Ratchathewi, Bangkok 10400, Thailand
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10 Department of Microbiology, Faculty of Science, Mahidol University, 272 Rama VI Road,

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11 Ratchathewi, Bangkok 10400, Thailand
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12 College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller

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13 Road, Albany, NY 12203, USA
*Corresponding author; Tel: +66-22015301; Fax: +66-23547160; E-mail:
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15 nuttawee.nia@mahidol.ac.th
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17 ABSTRACT
18 This study investigated the influence of molecular weight (Mw) and particle size
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19 characteristics on the antibacterial property of chitosan nanoparticles (CNs) for application as

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20 a vegetable wash disinfectant. Formulations prepared from two different Mw of chitosan

21 resulted in three different size ranges of CNs; 300-400 nm (i.e. LS and HS), 500-600 nm (i.e.
22 LL) and 700-800 nm (i.e. HL). A time-dependent antibacterial assay against Escherichia coli
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23 was used as a model and showed that CNs with smaller size (i.e. LS and HS) produced from
24 either low or high Mw of chitosan were effective antibacterial agents, leading to an
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25 approximate 2 log reduction in the number of bacteria within 12 h. Once demonstrated to

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26 have good antibacterial activity, all CNs were formulated as vegetable wash disinfectants in
27 citric acid and evaluated using an in vitro inactivation assay with E. coli and a pathogenic
28 bacterium (Salmonella Typhimurium), known to be possible contaminates on fresh
29 vegetables. The results showed that the smallest CNs could significantly reduce the number
30 of E. coli at 3.38 log CFU/mL within 15 min. On the other hand, the number of S.
31 Typhimurium was significantly reduced 2.83 log CFU/mL within 15 min using the largest
32 CNs. Finally, the formulations with the highest antibacterial activity were selected to evaluate
33 their ability to reduce the number of inoculated bacteria under simulated vegetable washing

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34 condition on fresh lettuce. The wash solution containing CNs was found to be the most
35 effective in killing more than a 1 log reduction of both inoculated E. coli and S. Typhimurium
36 populations, suggesting their potential use as effective disinfectant in washing fresh
37 vegetables.
38 Keywords: chitosan nanoparticles, ionotropic gelation, particle size, antibacterial agent,
39 lettuce, vegetable wash disinfectant

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41 1. Introduction

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42 Prevention of bacterial contamination is a constant concern for producers in order to
43 supply safe food for consumers. Fresh fruits and vegetables are an important part of a healthy

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44 diet, however, there are some concerns due to foodborne illness outbreaks related to the
45 consumption of fresh products (Allende, Mcevoy, Tao, & Luo, 2009; Harris et al., 2003;
46 Sivapalasingam, Friedman, Cohen, & Tauxe, 2004). Recently, there were major cases of
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foodborne illness from bacteria caused by eating fresh vegetables such as lettuce, sprouts, and
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48 spinach, as noted by the Centers for Disease Control and Prevention (CDC) (CDC, 2015).
49 Lettuce is one of the most consumed fresh vegetables worldwide. More than a third of the
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50 population in Europe and the United States (US) eat lettuce (USDA-NASS, 2016). Between
51 1998 and 2011, over 170 lettuce-associated outbreaks were reported to CDC (Kim et al.,
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52 2013; Sivapalasingam et al., 2004). In the US, foodborne disease outbreaks associated with
53 fresh lettuce is mainly due to Gram-negative bacteria including Escherichia coli O157:H7,
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54 and Salmonella spp. (CDC, 2015). E. coli O157:H7 contamination can be problematic with a
55 low infective dose and severe consequences. Symptoms can manifest in several ways. For
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56 example, symptoms of hemorrhagic colitis begin with a sudden onset of severe cramps and
57 abdominal pain followed within 24 h by watery diarrhea (Doyle, 1991). S. Typhimurium is
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58 also a common serotype associated with foodborne illness with symptoms including diarrhea,
59 abdominal pain, mild fever, and chills (Baird-Parker, 1990).
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60 One major factor contributing to foodborne illness from fresh vegetables is bacterial
61 surface contamination (Doyle & Erickson, 2008). The best approach to eliminate surface
62 pathogens is a straightforward washing process (López-gálvez, Allende, Selma, & Gil, 2009).
63 Disinfectants used in washing procedures in the food industry include acidified sodium
64 chlorite, citric acid (Allende et al., 2009), chlorine, lactic acid (López-gálvez et al., 2009),
65 sumac, oregano (Gündüz, Gönül, & Karapinar, 2010), and hydrogen peroxide (Back, Ha, &
66 Kang, 2014). Whether washing can reduce pathogen levels to lower than infective dose
67 depended on the efficacy of the washing solution (López-gálvez et al., 2009). Some chemical
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68 agents used in food wash solutions are controversial as they can leave residual material on the
69 products that are over the regulation limit of some countries (Seymour & Appleton, 2001).
70 Thus, there is still a great interest in finding biocompatible disinfectants from natural sources
71 that can be formulated into new vegetable wash solutions.
72 Chitosan is a naturally derived polycationic biopolymers produced from chitin, which
73 is found in exoskeletons of crustaceans, arthropods, and cell wall of fungi. It is a linear

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74 polysaccharide (poly-(b-1/4)-2-amino-2-deoxy-D-glucose) obtained from deacetylation of
75 chitin (Rinaudo, 2006). Chitosan and many of its derivatives are biocompatible,

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76 biodegradable, and lack toxicity to human (Kumar, 2000). It can be processed and used in
77 many forms such as gel, capsule, micro- and nanoparticles, as well as applied as an additive

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78 in pharmaceutical, food, agricultural, textile, and cosmetic industries (Rinaudo, 2006). In
79 addition, chitosan has been demonstrated to have antimicrobial properties toward a broad-
80 spectrum of Gram-positive and Gram-negative bacteria (Helander, Nurmiaho-lassila,
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Ahvenainen, Rhoades, & Roller, 2001; Kumar, 2000; Sarwar, Katas, & Mohamad Zin, 2014).
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82 Chitosan in the form of nano-scale particles have gained attraction as a novel
83 antibacterial agent, which is attributed in part to their high surface area and charge density
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84 (Hajipour et al., 2012; Whitesides, 2005). Previous studies have shown that the antibacterial
85 property of chitosan nanoparticles (CNs) in terms of its minimum inhibitory concentration
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86 (MIC) against Gram-negative bacteria varies from less than 1 to 120 µg/mL (Du, Xu, Xu, &
87 Fan, 2008; Katas, Mohamad, & Zin, 2011; Qi, Xu, Jiang, Hu, & Zou, 2004; Sarwar et al.,
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88 2014). For Gram-positive bacteria, the MIC varies from 0.1 to 15 µg/mL (Chen, Kung, Chen,
89 & Lin, 2010; Katas et al., 2011; Sarwar et al., 2014). The MIC value of CNs depends on
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90 several factors such as a particle size and zeta potential, bacterial strain tested, and pH of
91 testing environment (Sarwar et al., 2014). CNs can be prepared through a variety of methods
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92 including ionotropic gelation, complex coacervation, microemulsion, emulsion-droplet

93 coalescence, emulsion-solvent extraction, and self-assembling of hydrophobically modified
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94 chitosan (Peniche & Peniche, 2011). One particularly attractive method for preparation of
95 CNs is ionotropic gelation as it is non-toxic, controllable, convenient, and organic solvent
96 free (Agnihotri, Mallikarjuna, & Aminabhavi, 2004; Peniche & Peniche, 2011). This
97 technique is based on the ionic crosslinking between positive charges residing on the primary
98 amino groups in chitosan and negatively charged residues of a polyanion. Frequently sodium
99 tripolyphosphate (TPP), which is a non-toxic multivalent anion, is used as a crosslinker (Shu
100 & Zhu, 2002). Several factors can influence the formation of CNs such as the molecular
101 weight of chitosan, the absolute and relative chitosan and TPP concentrations, temperature,
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102 and pH (Aydin & Pulat, 2012; Fan, Yan, Xu, & Ni, 2012; Gan, Wang, Cochrane, &
103 McCarron, 2005; Katas et al., 2011). For example, increasing molecular weight and
104 concentration of chitosan can produce larger particles (Aydin & Pulat, 2012; Gan et al., 2005;
105 Katas et al., 2011). CNs formulated with higher mass ratio of chitosan to TPP (3:1 to 7:1)
106 showed a linear increase of particle size and zeta potential (Gan et al., 2005).
107 In this work, we investigated the antibacterial properties of four different CNs

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108 formulations, with respect to polymer molecular weight and CNs size, against Gram negative
109 bacteria models. The selected CNs preparations were then formulated using citric acid into

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110 vegetable wash disinfectants and evaluated for their antibacterial activity against E. coli and
111 S. Typhimurium. Finally, CNs formulations with the highest antibacterial activity against

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112 both bacteria were examined under a simulated washing condition with lettuce and compared
113 with commercial food-grade detergents. Due to their excellent antibacterial activity and
114 relative ease of preparation, the CN solutions have great potential as a disinfectant wash for
115 fresh vegetables.
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117 2. Materials and methods
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118 2.1 Preparation and characterization of chitosan nanoparticles (CNs)

119 CNs were prepared by a slightly modified ionic gelation method (Fan et al., 2012).
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120 Briefly, two different molecular weights (Mw) of chitosan polymers (30 and 2,100 kDa) with
121 94% of degree of deacetylation (Marine Bio Resources, Thailand) were separately dissolved
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122 in 2% (v/v) acetic acid at 2.5 mg/mL by stirring for 4 h after which 1 mg/mL solutions were
123 prepared by diluting with sterile distilled water. Subsequently, both chitosan solutions were
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124 adjusted to pH 4.7 using 5 M sodium hydroxide. Sodium tripolyphosphate (TPP) (Sigma-
125 Aldrich, Germany) was dissolved in sterile distilled water to produce 2.5 mg/mL and 1
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126 mg/mL stock solutions, and stored at 4 °C. To prepare CNs formulations, three volumes of a
127 specific chitosan solution was prepared in a 60 °C water bath for 10 min and immediately
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128 placed on a stirrer set at 700 rpm. Subsequently, 6.7 mL volume of cold TPP stock solution
129 (at the same concentration as the chitosan) was quickly added and stirred for 10 min to
130 produce CNs with a 3:1 mass ratio of chitosan to TPP. The CNs suspensions were left to
131 stabilize overnight at room temperature. The CNs were pelleted using centrifugation at
132 35,000 x g for 30 min and re-suspended in 0.1% acetic acid (Sarwar et al., 2014). The
133 suspended final stock solutions were stored at 4 °C. Each CNs solution was centrifuged,
134 washed three times in sterile distilled water, and then the defined volume (i.e. 100 µL) of

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135 CNs was freeze-dried for 24 h to gravimetrically determine the final CNs concentration in
136 each solution. This information was used to prepare working solutions for antibacterial
137 testing. The characteristics of CNs with regards to mean size, size distribution (i.e.
138 polydispersity), and surface charge (zeta potential) were measured as in Fan et al., (2012)
139 using a Zetasizer Nano-ZS90 (Malvern Instruments, UK). Three batches of each CNs
140 formulations were prepared and measured in triplicates for statistical analysis. Four types of

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141 CNs were obtained regardless size of CNs including 300-400 nm (i.e. LS and HS), 500-600
142 nm (i.e. LL) and 700-800 nm (i.e. HL). Here, the first letter; L and H, indicate CNs prepared

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143 from low and high molecular weight, respectively, while the second letter; S and L indicate
144 small and large size of CNs, respectively.

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146 2.2 Bacterial strains
147 Escherichia coli ATCC 25922 was used as a bacterial model strain of Gram-negative
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bacteria to examine antibacterial activity. Salmonella Typhimurium strain LT2 was chosen as
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149 a representative pathogenic bacterium for surface contamination of lettuce to test the efficacy
150 of CNs formulations as vegetable wash disinfectant. Working cultures were streaked on
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151 tryptic soy agar (Bacto, France) and incubated at 37 °C for 24 h. Subsequently, a
152 representative bacterial colony was inoculated in tryptic soy broth (Bacto, France) and
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153 incubated at 37 °C for 20 h. The optical density (OD) of the cultures were measured at 600
154 nm to determine the viable cell count (CFU/mL) using a previously established relation
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155 between OD600 and CFU/mL. The bacterial cultures were centrifuged at 4000 x g for 10 min.
156 The cell pellets were washed three times with phosphate buffer saline (PBS) pH 7.4, re-
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157 suspended, and diluted in PBS to desirable concentration for use in antibacterial tests.
158 Mueller Hinton agar (MHA) (Scharlau, Spain) was used to assess a viable bacterial count
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159 after antibacterial testing.

160
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161 2.3 Antibacterial testing with time-dependent killing efficacy by viable count technique
162 The CNs (LS, HS, LL and HL formulations) were diluted in PBS pH 5.0 to 100, 400,
163 and 1,600 µg/mL concentrations for antibacterial testing. The concentrations were based on
164 the minimum bactericidal concentration value of CNs against Gram-negative bacteria
165 reported by Sarwar et al., (2014). The procedure was modified from Pfaller et al., (2004). In
166 brief, inoculum of E. coli ATCC 25922 was prepared by adjusting an overnight culture to
167 1×106 CFU/mL in PBS pH 5.0. One mL of inoculum was mixed with 1 mL of CNs at each
168 concentration. Thus, the final CNs concentrations were half of the starting concentration (i.e.
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169 50, 200, and 800 µg/mL) with a bacterial load of 5×105 CFU/mL. The tubes were incubated
170 at 37 °C for 12 h under shaking condition at 150 rpm. Samples were collected at 0, 2, 4, 8,
171 and 12 h and the reduction of bacterial viability was determined by standard plate count
172 technique. The tubes with non-treated and treated with 50 mg/mL ampicillin were used as
173 negative and positive control samples, respectively. All plates were incubated aerobically at
174 37 °C for 24 h, after which the colonies of E. coli and S. Typhimurium were enumerated.

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176 2.4 In vitro inactivation assay of formulated vegetable wash disinfectant

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177 E. coli and S. Typhimurium were used in this experiment. The CNs stock preparations
178 (LS, HS, LL and HL) were diluted in citric acid to 100 and 1600 µg/mL for antibacterial
testing. Both bacteria were diluted to 1×106 CFU/mL with PBS pH 7.4. One mL of each

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180 bacterial suspension was transferred to a tube and mixed with 1 mL of CNs at each
181 concentration. The tubes were incubated at 37 °C under shaking condition at 150 rpm.
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Samples were collected at 0, 15, and 30 min and the reduction of bacterial viability was
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183 measured by standard plate count techniques using MHA. Solutions with PBS and 1% citric
184 acid were used as controls.
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186 2.5 Preparation of artificially inoculated lettuce and washing procedure
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187 Thai lettuce (Lactuca sativa) was purchased from a local market in Bangkok,
188 Thailand. The outer leaves and core were removed from the lettuce head and discarded. The
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189 remaining leaves were then washed with tap water for 1 min. Leaves were cut into pieces
190 with size 3×3 cm, approximately 0.2 g each for inoculation experiments. These lettuce pieces
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191 were treated by UV light (30 W, 50 cm irradiation distance) in a class II biosafety cabinet
192 (International Scientific Supply, Huaykwang, Thailand) for 30 min (15 min for each side) to
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193 reduce the native microflora. The UV treated lettuce pieces were inoculated with tested
194 bacteria using a modified sprinkle inoculation method (Singh, Singh, Bhunia, & Stroshine,
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195 2002). The bacterial cells (i.e. E. coli and S. Typhimurium) were prepared as described above
196 in section 2.2 and adjusted to 1×106 CFU/mL. Lettuce leaf pieces (3 pieces) were placed in a
197 plastic bag and 500 µL of bacterial cocktail was added and then shaken manually for 1 min.
198 After that, pieces of lettuce were air-dried on a tray in class II biosafety cabinet for 1 h. After
199 air-drying, inoculated leaves were placed in zip-lock bag and then stored in a refrigerator at 4
200 °C. The CNs formulated in 1% citric acid was used for simulated vegetable washing
201 experiments. Individual inoculated lettuce piece was immersed in a flask containing 50 mL of
202 washing solution and incubated in a shaker at 150 rpm for 15 min. After that, the lettuce
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203 leave was dipped immediately in sterile distilled water to remove residual material and then
204 homogenized in PBS pH 7.4 for 2 min with a stomacher (Seward, UK). The reduction of
205 bacteria on lettuce was measured by standard plate count technique using MHA as mentioned
206 in Section 2.3.
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209 2.6 Statistical analysis
210 One-way analysis of variance (ANOVA) was performed between and within groups
211 of all data using SPSS 18, and differences were considered to be significant at a level of p ≤

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212 0.05, means were separated using the Duncan’s multiple range tests. All microbial
experiments were performed with triplicate samples and averages of duplicate plate counts.

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214
215 3. Results and discussion

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216 3.1 Characteristics of chitosan nanoparticles (CNs)
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217 CNs were prepared by ionic gelation method using polyanionic TPP. The size and
218 surface charge characteristics of CNs were found to be influenced by the formulation
219 parameters (chitosan molecular weight, concentration of each component, and buffer
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220 conditions). The size of CNs prepared from low molecular weight chitosan (30 kDa, LMw) in
221 PBS increased from 328.6 ± 10.6 to 526.3 ± 15.6 nm when the concentration was increased
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222 from 1.0 to 2.5 mg/mL (Table 1, p ≤ 0.05). A similar trend was found for high molecular
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223 weight chitosan (2,100 kDa, HMw) where particle size significantly increased from 335.0 ±
224 5.7 to 717.9 ± 25.1 nm (Table 1, p ≤ 0.05). Thus, the size of CNs can be modulated by the
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225 formation conditions, where lower Mw and concentration resulted in smaller CNs. One
226 possible explanation for this is because of reduced viscosity of lower concentration of
227 chitosan in acetic acid (Fan et al., 2012; Gan et al., 2005; Ing, Zin, Sarwar, & Katas, 2012). In
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228 general, LMw chitosan produced smaller size particles with narrower size distribution
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229 compared to particles produced from HMw chitosan. The zeta potential of CNs prepared
230 from LMw or HMw chitosan at equal mass ratio (i.e. chitosan per TPP) at either
231 concentration was not significantly different (Table 1). As expected, different zeta potential
232 resulted when the particles were dissolved in solutions with different pHs. When CNs were
233 dissolved in PBS pH 5.0, the surface charge was around 19 mV, while the surface charge of
234 CNs when dissolved in 1% citric acid (pH 2.0) increased around 3 times as shown in Table 1.

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235 3.2 Time-dependent killing assay against Escherichia coli
236 The antibacterial property of CNs formulations in phosphate buffer saline (PBS) (pH
237 5.0) having different sizes, including 300-400 nm (i.e. LS and HS), 500-600 nm (i.e. LL), and
238 700-800 nm (i.e. HL) were tested using a time-dependent killing assay with E. coli ATCC
239 25922 as a bacterial model. For the LS CNs formulation, E. coli was reduced from its initial
240 load by 1 log CFU/mL at 4 h for all CNs doses and gradually reduced by 2 log CFU/mL

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241 within 12 h. The reduction of bacteria for the highest vs. lowest dosage was significant (p ≤
242 0.05) as shown in Figure 1 (as in supplement data, Table S1). The results showed that the

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243 smaller size CNs (i.e. LS and HS) reduced E. coli around 2 log CFU/mL from its initial load,
244 where the larger size CNs (LL and HL) had a reduction of 1.73 log CFU/mL and 1.50 log

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245 CFU/mL as shown in Figure 1. These findings suggest that the antibacterial property of CNs
246 is governed mainly by their particle size, in which the smaller particles being more effective
247 in killing bacteria, and not influenced by molecular weight of original chitosan polymers,.
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In addition, the bacterial growth inhibition of different CNs sizes at similar zeta
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249 potential were observed. All four CNs (LS, HS, LL and HL) at 50 and 200 µg/mL exhibited
250 similar killing efficiency leveling off at 8-12 h, while the small CNs (LS and HS) at 800
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251 µg/mL exhibited a continued reduction up to 12 h. Normalization of total particle surface area
252 with the number of viable cells after treatment indicated that the antibacterial activity of CNs
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253 against E. coli was surface area dependent and was not due to the size (Data not shown). The
254 generally suggested mode of action for CNs against bacteria is destabilization and disruption
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255 of bacterial membranes, leading to leakage of cellular components (Sarwar et al., 2014).
256 Considering that smaller size CNs would have a higher surface area compared to larger size
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257 CNs (at the same mass concentration), the smaller particles would be more effective at killing
258 bacteria (O’Callaghan & Kerry, 2016). This agrees with our findings that LS and HS CNs
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259 produced from different Mw’s of chitosan but with similar size and zeta potential have the
260 same antibacterial activity against E. coli (Figure 1), thus the nanoparticle size, not Mw of
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261 the chitosan is a critical factor for antibacterial activity

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262 3.3 In vitro inactivation assay of CNs formulated as vegetable wash disinfectant
263 While CNs in PBS show antibacterial properties, it requires several hours to be fully
264 effective. Citric acid is a common additive in food products as it has an ability to enhance the
265 antimicrobial power of a wide range of disinfectants and antibiotics (OCC, 2015). Previous
266 work has shown that citric acid can kill Gram-negative bacteria with a 1-2 log reduction
267 within 20 min (Sagong et al., 2011), and has been studied as an antibacterial agent against E.

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268 coli and S. Typhimurium (Seo et al., 2013). The mode of action of citric acid is the diffusion
269 of fully protonated form of acid into bacterial cells that can cause cell death (Bjornsdottir,

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270 Breidt, & Mcfeeters, 2006). Thus we wanted to study if chitosan combined with citric acid
271 would show improved antibacterial efficacy.

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272 The four CNs preparations (LS, HS, LL, and HL) were used to formulate vegetable
273 wash solutions in 1% citric acid and tested to their antibacterial property. Citric acid is a

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274 common additive in food products as it has an ability to enhance the antimicrobial power of a
275 wide range of disinfectants and antibiotics (OCC, 2015). As a result, citric acid alone at 1%
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276 was used as a control. As shown in Figure 2 the results with E. coli showed that the smaller
277 size CNs (LS and HS) led to about 3.30 log CFU/mL reduction from the initial load with
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278 significant difference (p ≤ 0.05). In contrast, the larger size CNs (LL and HL) reduced the
279 initial bacterial load by 1.81 log CFU/mL at both concentrations of 50 and 800 µg/mL at 30
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280 min. Results of the S. Typhimurium study showed that the largest CNs (HL) reduced the
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281 initial bacterial load around 1.16 log CFU/mL within 15 min and 2.83 log CFU/mL within 30
282 min with a significant difference when compared to the smaller CNs (p ≤ 0.05) (Table 1 and
283 Figure 3). Here, the citric acid solution alone showed less efficacy at about 1 log reduction
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284 (at 15 and 30 min). This finding agrees with previous studies showing that citric acid can
285 reduce the number of certain bacteria around 1 to 2 log at 1% of citric acid (Francis & Beirne,
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286 2002; Seo et al., 2013). The increased antibacterial activity for CNs in citric acid is expected
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287 as a higher surface charge (Table 1) could allow stronger electrostatic interactions between
288 the CNs and the bacterial cell wall, leading to destabilization and disruption of the cell. Based
289 on their killing efficiency, both LS and HL were selected for a further antibacterial study to
290 simulate vegetable washing conditions against E. coli and S. Typhimurium, respectively.
291 The solution containing CNs was switched to citric acid to better replicate a standard
292 vegetable wash disinfectant, therefore the antibacterial activity of CNs was increased, which
293 was exhibited by a short killing time (within 15-30 min). Previous study revealed that citric
294 acid has the ability to kill Gram-negative bacteria with a 1-2 log reduction within 20 min

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295 (Sagong et al., 2011). Further, increased antibacterial activity of CNs in citric acid is expected
296 since higher surface charge of CNs was also found (Table 1). This would create stronger
297 electrostatic interactions between the CNs and the bacterial cell wall, leading to cell
298 disruption.
299 S. Typhimurium was selected for additional antibacterial testing as it is a pathogenic
300 Gram-negative bacteria often found in contaminated vegetables (Back et al., 2014; Gündüz et

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301 al., 2010; Olasupo, Fitzgerald, Gasson, & Narbad, 2003). After preliminary tests, we found
302 that this pathogenic strain was more susceptible to CNs compared to E. coli. Therefore, the

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303 inoculum of S. Typhimurium was increased 3 log of the initial load of E. coli. The higher
304 susceptibility of S. Typhimurium to CNs might be attributed to the more negative cell

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305 membrane composition. Additional negative charges on the outer membrane of S.
306 Typhimurium can facilitate the interaction with positively charged CNs resulting in a higher
307 degree of cell death over the same time interval (Chung et al., 2004). In previous studies, S.
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Typhimurium was also found to be more susceptible to various antimicrobial compounds
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309 than E. coli. For instance, organic acids (such as citric acid) (Sagong et al., 2011), natural
310 organic compound (i.e. thymol) (Olasupo et al., 2003), polymyxin (Peterson, Fesik, &
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311 McGroarty, 1987), and quinolones (Hirai, Aoyama, Irikura, Iyobe, & Mitsuhashi, 1986) had
312 higher killing efficacy on S. Typhimurium than E. coli.
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313 Interestingly, according to our results, the most effective antibacterial activity of CNs
314 against S. Typhimurium was exhibited in the largest size of CNs. Further investigation into
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315 the mechanism of CNs are required to better understand this phenomena. Nevertheless, many
316 previous studies have shown that the composition of the cell envelope of S. Typhimurium and
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317 E. coli are quite different and that might lead to different sensitivity towards different CNs
318 (Chung et al., 2004; Hirai et al., 1986; Peterson et al., 1987).
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320 3.4 Antibacterial testing of CNs under simulated vegetable washing conditions
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321 In this study, the antibacterial activities of selected CNs (i.e. LS for E. coli and HL for
322 S. Typhimurium) under simulated vegetable washing condition were evaluated and compared
323 to commercial food-grade detergents. Pre-cut lettuce leaves artificially inoculated with E. coli
324 or S. Typhimurium was used as a vegetable testing model, with a slightly modified protocol
325 from previous work (Singh et al., 2002). After washing, the viable bacteria on the lettuce
326 leaves were enumerated by standard plate counting method. The results showed that washing
327 with citric acid alone reduced the numbers of bacteria by 1.22 and 0.43 log CFU/g E. coli and
328 S. Typhimurium, respectively. For CNs solutions, the numbers of viable bacteria were
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329 reduced by 1.63 and 1.16 log CFU/g for E. coli and S. Typhimurium, respectively, with a
330 significant reduction over the citric acid alone at p ≤ 0.05, as shown in Figure 4. Washing
331 with the commercial food-grade detergents at their recommended concentration, including
332 Brand#A (active ingredients: sodium lauryl ether sulfate 4.3%), Brand#B (active ingredients:
333 citrus oil, sodium citrate and grapefruit seed extract), and water resulted in less reduction of
334 bacteria than the other treatments. In comparison, Back et al. (2014) treated E. coli and S.

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335 Typhimurium on lettuce leaves using 1% (i.e. 10 mg/mL) hydrogen peroxide vapor (HPV)
336 where the levels of E. coli and S. Typhimurium were reduced by 1.62 log CFU/g and 1.48 log

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337 CFU/g, respectively, after 10 min treatment (Back et al., 2014). A variety of commercial
338 sanitizers have also been used for washing lettuce. E. coli populations were reduced by about

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339 2 log CFU/g after washing with chlorine and Tsunami 100, where Citrox and Purac reduced
340 E. coli counts by approximately 1.50 log CFU/g (López-gálvez et al., 2009). According to all
341 previous data, our CNs caused a decrease in bacteria populations on lettuce after washing
342

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with the level as effective vegetable wash disinfectants used worldwide. These findings thus
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343 suggest that CNs in citric acid could be used to formulate an effective washing solution for
344 vegetable disinfection against relevant pathogens.
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346 4. Conclusion
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347 Chitosan nanoparticles (CNs) were demonstrated as a potential vegetable wash

348 disinfectant with good antibacterial properties against both Escherichia coli and Salmonella
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349 Typhimurium, two common species of foodborne pathogens often found contaminating fresh
350 vegetables. The formulation of CNs as an effective vegetable disinfectant was the
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351 combination of CNs in citric acid. The results showed that the smallest CNs (i.e. LS) was the
352 most effective in killing 3 log reduction of E. coli within 15 min, while the largest CNs (i.e.
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353 HL) was the most effective against S. Typhimurium. Thus far, the ability of CNs as
354 antibacterial agent appears to be particle size-dependent to the targeting bacteria species
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355 rather than surface charge or Mw of chitosan polymers. Nevertheless, the clear mechanism
356 underlying the efficacy of different size CNs against different species of bacteria needs to be
357 investigated further. Future studies should include Gram-positive bacteria to confirm broad-
358 spectrum antibacterial activity of CNs, and stability studies for CNs formulations with respect
359 to their shelf-life. In summary, a vegetable washing solution formulated with CNs in citric
360 acid was the most effective and capable of reducing both E. coli and S. Typhimurium
361 populations more than 1 log reduction of their initial number, suggesting its potential
362 application as an effective vegetable wash disinfectant.
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363
364 5. Acknowledgement
365 This research was supported by Faculty of Science, Mahidol University, National
366 Science and Technology Development Agency (NSTDA) through Coordinating Center for
367 Thai Government Science and Technology Scholarship Students (CSTS): A New Researcher
368 Scholarship of CSTS-MOST, and Thailand Research Fund (IRG5980001).

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369
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Figure captions:

Figure 1. Survival ability of E. coli ATCC 25922 treated with CNs in PBS pH 5.0 with
different doses including 50 µg/mL (A), 200 µg/mL (B), and 800 µg/mL (C) at 37 °C for 12
h with the initial load at 6.12 log CFU/mL. The results are means of triplicate experiments
and error bars indicate standard errors. Different letters between treatments within the same

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time indicate significant difference (p ≤ 0.05).

Figure 2. Survival ability of E. coli ATCC 25922 treated with CNs formulated at two

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concentrations (i.e. 50 and 800 µg/mL) as vegetable wash disinfectant at 37 °C for 30 min
with the initial load at 6.79 log CFU/mL. The results are means of triplicate experiments and

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error bars indicate standard errors. Different letters between treatments within the same time
point indicate significant difference (p ≤ 0.05).

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Figure 3. Survival ability of S. Typhimurium LT2 treated with CNs formulated at two
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concentrations (i.e. 50 and 800 µg/mL) as vegetable wash disinfectant at 37 °C for 30 min
with the initial load at 9.18 log CFU/mL. The results are means of triplicate experiments and
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error bars indicate standard errors. Different letters between treatments within the same time
point indicate significant difference (p ≤ 0.05).
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Figure 4. Reduction of E. coli ATCC 25922 (A) and S. Typhimurium LT2 (B) on lettuce
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pieces treated with CNs formulated as vegetable wash disinfectant at 25 °C for 15 min. The
results are means of triplicate experiments and error bars indicate standard errors. Different
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letters between treatments indicate significant difference (p ≤ 0.05).

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Table 1. CNs particle size and zeta potential of LS, HS, LL, and HL (n = 3).

Dissolved in Dissolved in
phosphate buffered saline pH 5.0 1% citric acid pH 2.0
Conc.
CNs Zeta Zeta
(mg/mL) Particle size Particle size
potential potential
(nm) (nm)
(mV) (mV)

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LS (LMw) 1 328.6 ± 10.6a 18.7 ± 0.4a 352.7 ± 2.8a 55.4 ± 4.3a
HS (HMw) 1 335.0 ± 5.7a 19.6 ± 0.8a 454.6 ± 4.1b 58.2 ± 3.3a

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LL (LMw) 2.5 526.3 ± 15.6b* 18.9 ± 0.6a 525.0 ± 10.7c* 56.7 ± 3.6a
HL (HMw) 2.5 717.9 ± 25.1c 19.5 ± 0.5a 865.9 ± 15.3d 57.1 ± 3.8a

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Data represent means ± standard deviations.
Means with the different superscript lowercase letters in the same column are significantly

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*Within each characteristic of CNs (i.e. particle size and zeta potential), means with an
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asterisk in the same row are not significantly different.
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Highlights:
• Chitosan nanoparticles (CNs) was formulated as a vegetable wash disinfectant.
• Smaller size CNs was more effective at killing Escherichia coli.
• Bigger size CNs was more effective at killing Salmonella Typhimurium.
• Vegetable wash formulated with CNs could effectively reduce bacteria population.

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