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Gallic acid-grafted-Chitosan Inhibits Foodborne Pathogens by a Membrane Damage Mechanism Dae-Sung Lee, and Jae-Young Je J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf401254g • Publication Date (Web): 01 May 2013 Downloaded from http://pubs.acs.org on May 6, 2013

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Journal of Agricultural and Food Chemistry

TOC Graphic Gallic acid-grafted-Chitosan Inhibits Foodborne Pathogens by a Membrane Damage Mechanism (A)

(B)

S. aureus without (A) and with (B) gallic acid-g-chitosan (A)

(B)

E. coli without (A) and with (B) gallic acid-g-chitosan

ACS Paragon Plus Environment


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Gallic acid-grafted-Chitosan Inhibits Foodborne Pathogens by a Membrane

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Damage Mechanism

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DAE-SUNG LEE† AND JAE-YOUNG JE*,‡

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†

POSTECH Ocean Science and Technology Institute, POSTECH, Pohang 790-784, Republic of

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Korea ‡

Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 550-749,

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Republic of Korea

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* Corresponding author.

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Tel.: +82-61-659-7416; fax: +82-61-659-7419. E-mail address: [email protected] (J.-Y. Je).

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ABSTRACT

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In this study, antimicrobial activity of gallic acid-grafted-chitosans (gallic acid-g-chitosans) against

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five Gram-positive and Gram-negative foodborne pathogens was evaluated. The minimum inhibitory

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concentrations (MICs) of gallic acid-g-chitosans were ranged from 16 to 64 µg/mL against Gram-

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positive bacteria and were ranged from 128 to 512 µg/mL against Gram-negative bacteria. These

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activities were higher than those of unmodified chitosan. The bactericidal activity of gallic acid-g-

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chitosan (I), which showed the highest antimicrobial activity, was evaluated by time-killing assay with

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multiples of MIC, and it was recognized to depend on its dose. The integrity of cell membrane, outer

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membrane (OM), inner membrane (IM) permeabilization experiments and transmission electron

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microscopy (TEM) observation were conducted for elucidation of the detailed antimicrobial mode of

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action of gallic acid-g-chitosan. Results showed that treatment of gallic acid-g-chitosan (I) quickly

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increased the release of intracellular components for both Escherichia coli and Staphylococcus aureus.

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In addition, gallic acid-g-chitosan (I) also rapidly increased the 1-N-phenylanphthylamine (NPN)

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uptake and the release of β-galactosidase via increasing the permeability of OM and IM in E. coli.

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TEM observation demonstrated that gallic acid-g-chitosan (I) killed the bacteria via disrupting the cell

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membrane.


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KEYWORDS: Antimicrobial activity; Chitosan; Membrane permeation; Foodborne pathogens

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1. INTRODUCTION

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Consumption of food contaminated with pathogenic bacteria and/or their toxins resulting foodborne

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illness has been of vital concern to public health (1). The microbial contamination problem in food

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industry is of great concern, and controlling pathogens is need to reduce of foodborne outbreaks and

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assure consumers a safe, wholesome, and nutritious food supply. It is well known that the survival of

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microorganisms in food can lead to spoilage and deteriorate the quality of food products or cause

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infection and illness (2, 3). Thus, food preservation is an important in food industry since food

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products require a longer shelf life and greater assurance of freedom from foodborne pathogenic

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organisms (4). Consequently, there is considerable research interest in the development of

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antimicrobial agents in order to prevent the growth of foodborne pathogens or to delay the onset of

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food spoilage (5-7).

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Chitosan is a carbohydrate biopolymer derived from deacetylation of chitin, the main coponent of

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crustacean exoskeletons. Due to the unique biological properties including biocompatible,

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biodegradable, and non-toxic nature, chitosan has been developed for biomaterials since it displays a

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broad spectrum of biological activities such as antioxidant, antitumor, antihypertensive, immuno-

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modulating, and antiinflammatory activity (8-12). In particular, antimicrobial activity of chitosan and

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its derivatives has been well documented. Chemical, physical and biological factors including

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molecular weight, degree of deacetylation, pH, temperature and species of bacteria are affected

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chitosan’s antimicrobial activity (13). However, exact mode of action of chitosan is still not fully

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understood, although several mechanisms have been elucidated for its antimicrobial activity (14).

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Recently, various functionalized chitosans with specific functional groups such as arginine, oleoyl,

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and aminoethyl were developed and evaluated their antimicrobial activity (7, 13, 15). Antimicrobial

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activity of chitosan was improved by conjugating of functional group onto chitosan, indicating the

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conjugation strategy was thought to be good methods for development of novel chitosan derivatives.

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In our previous report, gallic acid-g-chitosans were developed, and their antioxidant, hepatoprotective

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and enzyme inhibition activity were evaluated (16-18). Biological activities of chitosan were



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agumented by grafting of gallic acid. Thus, as part of our ongoing investigation of biological activity

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of chitosan derivatives, antimicrobial activity of gallic acid-g-chitosans was investigated against

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foodborne pathogens as five Gram-positive and Gram-negative bacteria. In addition, mode of action

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of gallic acid-grafted-chitosan on model Gram-positive bacteria, Staphylococcus aureus and Gram-

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negative bacteria, Escherichia coli, was evaluated using outer membrane (OM) and inner membrane

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(IM) permeability assays, cell membrane integrity, bactericidal activity and transmission electron

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microscopy.

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2. MATERIALS AND METHODS

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Materials. The microorganisms tested for antibacterial activity were obtained from Korean Collection

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of Type Cultures (KCTC, Daejeon, Korea). O-nitrophenyl-β-D-galactoside (ONPG), 1-N-phenyl-

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naphthylamine (NPN) was obtained from Sigma Chemical Co. (St. Loius, MO). All other reagents

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were of the highest grade available commercially.

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Antimicrobial Agents. Four kinds of gallic acid-g-chitosans using different molar ratios of chitosan

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residue and gallic acid for the antimicrobial assay were prepared from our previous method (Scheme 1)

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(19). To confirm successful synthesis, 1H NMR analysis was conducted and the results were compared

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to those of Cho, Kim, Ahn and Je (19). Unmodified chitosan: 1H NMR (400 MHz, D2O) δ: 5.30 (1H,

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H-1), 3.63-4.35 (1H, H-2/6), 2.51 (H-Ac), 4.8 (D2O). Gallic acid-g-chitosan: 1H NMR (400 MHz,

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D2O) δ: 7.63 (phenyl protons of gallic acid), 5.33 (1H, H-1), 3.65-4.36 (1H, H-2/6), 2.51-2.54 (H-Ac),

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4.8 (D2O). The gallic acid contents in the gallic acid-g-chitosans were determined by Folin-Ciocalteau

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method, and the contents were 118.92 mg gallic acid/g gallic acid-g-chitosan for gallic acid-g-

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chitosan (I), 82.91 for gallic acid-g-chitosan (II), 67.62 for gallic acid-g-chitosan (III), and 53.87 for

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gallic acid-g-chitosan (IV).

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Deternimation of MIC Values. Antimicrobial activity of gallic acid-g-chitosans against five Gram-

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positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Enterococcus faecalis, and Listeria


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monocytogenes) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas

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aeruginosa, Salmonella typhimurium and Shigella flexneri) were evaluated using two-fold serial broth

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dilution as follows. Bacteria culture (106-107 CFU/mL) grown in 5 mL Mueller Hinton broth (MHB,

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Difco, MI) which contained 1 mL of antimicrobial agent with various concentrations in 50 mM

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acetate buffer (pH 5.5) was incubated at 37ºC for 18 h. MIC was defined as the lowest concentration

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of antimicrobial agent at which the cell growth was not visible with naked eye.

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Bactericidal Assay. The bactericidal activity of gallic acid-g-chitosan was evaluated with a time-kill

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experiment. Briefly, gallic acid-g-chitosan was added to MHB inoculated with E. coli and S. aureus

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strain, which was adjusted to an estimated cell density of approximately 105 CFU/mL, followed by

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culture at 37°C for 24 h. The final gallic acid-g-chitosan concentrations consisted of the MIC, 2× MIC,

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4× MIC, and 8× MIC. Viable cell counts were estimated at various incubation times by the spread

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plate method. The plates were incubated at 37°C for 24 h, and the colonies were counted.

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Cell Membrane Integrity. Bacterial cell membrane integrity was examined by determination of the

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release of material absorbing at 260 nm (20). Bacterial cultures grown were harvested by

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centrifugation at 11,000 × g for 10 min, washed and re-suspended in 0.5% NaCl solution. The final

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cells suspension was adjusted to an absorbance at 420 nm of 0.7. A 1.5 mL portion of antimicrobial

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agent solution was mixed with 1.5 mL of bacterial cell suspension, and the release over time of

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materials absorbing at 260 nm was monitored with UV spectrometer (SpectraMax® M2e, CA, USA).

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OM Permeabilization Assay. OM permeabilization activity of water-soluble chitosans was

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determined by the NPN assay described by Ibrahim, et al. (21). Bacterial cultures grown were

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harvested by centrifugation at 11,000 × g for 10 min, washed and re-suspended in 0.5% NaCl solution.

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The final cells suspension was adjusted to an absorbance at 420 nm of 1.0. A 20 µL of 1 mM NPN

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was added into 1 mL of bacteria in a quartz cuvette, and background fluorescence was record using a

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SpectraMax® M2e, with 1 cm path length cuvettes, at excitation wavelength of 350 nm and emission



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wavelength of 429 nm. Then, various concentrations of antimicrobial agent were added. Increase in

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fluorescence due to partitioning of NPN into the OM was recorded as a function of time until no

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further increase in intensity. Control tests were performed to verify that the enhanced fluorescence

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was due to NPN uptake by bacteria.

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IM Permeabilization Assay. IM permeabilization was determined by measuring the release of

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cytoplasmic β-galactosidase activity from E. coli into the culture medium using ONPG as the

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substrate (21). Logarithmic-phase bacteria grown in nutrient broth containing 2% lactose were

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harvested, washed, and resuspended in 0.5% NaCl solution in order to adjust an absorbance at 420 nm

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of 1.2. A 200 µL of bacterial suspension was pipetted into the wells of a standard microtiter plate

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followed by 10 µL of ONPG (30 mM) added to each well. The production of o-nitrophenol over time

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was determined by monitoring the increase in absorbance at 420 nm using a spectrophotometer.

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Transmission Electron Microscopy (TEM). E. coli and S. aureus were inoculated into MHB in the

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absence or presence of the gallic acid-g-chitosans and then further incubated at 37°C for 18 h. After

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the incubation, 1 mL aliquots were centrifuged at 11,000 rpm, washed twice with PBS (pH 7.2), and

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fixed with 2.5% glutaraldehyde in 0.1 M PBS. Samples were post-fixed with 1% (w/v) OsO4 in 0.1 M

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PBS for 2 h at room temperature, washed once with the same buffer, dehydrated in a graded series of

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ethanol solutions, and embedded in Spurr low-viscosity embedding medium. Thin sections of the

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specimens were cut with a diamond knife on an Ultracut ultramicrotome and double-stained with

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uranyl acetate and lead citrate.

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3. RESULTS AND DISCUSSION

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Antimicrobial Activities of Gallic acid-g-Chitosans. The antimicrobial activity of chitosan has been

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well documented and its activity was influenced by a number of factors including the type of chitosan,

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molecular weight and some of its other physicochemical properties (22). Recently, chitosan

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derivatives with specific moiety were developed to improve its antimicrobial activity (13, 23, 24), and


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the results showed that chitosan derivatives exhibited better antimicrobial activity than that of

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unmodified chitosan. These results indicate that chemical modification is a good strategy for

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improving antimicrobial activity of chitosan.

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Gallic acid is a naturally occuring phenolic compound, which is noteworthy for their antioxidant

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activity (25). Currently, antimicrobial activity of gallic acid was reported (26-28). In this study,

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therefore, we conjugated gallic acid onto chitosan backbone in order to improve antimicrobial activity

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of chitosan. For that, different molar ratios were used to conjugate gallic acid onto chitosan, thereby

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we produced four kinds of gallic acid-g-chitosans bearing different amounts of gallic acid, and it

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could be expected to show enhanced antimicrobial activities. Ten bacteria strains, including five

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Gram-positive bacteria and Gram-negative bacteria, were used to determine the antimicrobial

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spectrum of gallic acid-g-chitosans by two-fold serial dilution method as describe above. The activity

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appeared to vary among the tested bacteria. The unmodified chitosan showed the MICs of 64-128

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µg/mL against five Gram-positive bacteria and the MICs of 512-1024 µg/mL against five Gram-

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negative bacteria (Table 1). However, gallic acid-g-chitosans showed higher antimicrobial activities

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than that of unmodified chitosan, indicating the conjugation of gallic acid improved the antimicrobial

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activity of chitosan. The MICs of four gallic acid-g-chitosans were ranged from 16 to 64 µg/mL

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against Gram-positive bacteria and were ranged from 128 to 512 µg/mL against Gram-negative

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bacteria. In addition, gallic acid-g-chitosan (I) possessed the highest antimicrobial activities than those

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of other gallic acid-g-chitosans. As described above, gallic acid content in gallic acid-g-chitosans was

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in order of gallic acid-g-chitosan (I)> gallic acid-g-chitosan (II)> gallic acid-g-chitosan (III)> gallic

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acid-g-chitosan (IV). These results indicated that gallic acid content in gallic acid-g-chitosans by the

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conjugation of gallic acid was crucial role for improvement of antimicrobial activity of chitosan. The

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antimicrobial activity of gallic acid-g-chitosan (I) was 4-times stronger than that of unmodified

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chitosan. We also tested the antimicrobial activity of gallic acid alone as the same concentration of

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gallic acid calculated from gallic acid-g-chitosan (I), which is containing 118.92 mg gallic acid/g

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gallic acid-g-chitosan (I), compared to the MICs of gallic acid-g-chitosan (I). However, gallic acid

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does not showed antimicrobial activity against the tested bacteria (data not shown). Taguri, Tanaka



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and Kouno (28) reported the MIC values of gallic acid against Bacillus cereus (1067 µg/mL), Bacillus

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subtilis (1600 µg/mL), Listeria monocytogenes (1600 µg/mL), Staphylococcus aureus (533 µg/mL),

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Escherichia coli (600 µg/mL), Klebsiella pneumoniae (400 µg/mL), Shigella flexneri (267 µg/mL),

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and Pseudomonas aeruginosa (533 µg/mL), respectively. In this study, the MIC values of gallic acid-

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g-chitosan (I) were ranged of 16 of 32 µg/mL against Gram-positive bacteria and were ranged of 128

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of 256 µg/mL against Gram-negative bacteria. Considering gallic acid content in these concentrations,

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gallic acid content in gallic acid-g-chitosan (I) was very lower than that of the literature MIC values.

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Thus, the gallic acid in gallic acid-g-chitosans was not solely responsible for the growth inhibition of

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the tested bacteria, and the synergy effect maybe existed by the conjugation of gallic acid onto

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chitosan.

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Several researches revealed that polyphenols with gallate such as catechin gallate, epicatechin gallate

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and epigallocatechin gallate possess higher affinity to cell membranes than those without gallate (29,

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30). In addition, epigallocatechin gallate showed higher antimicrobial activity via damaging the lipid

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bilayer of liposomes than that of epicatechin, but epicatechin does not damaged the lipid bilayer (31).

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These results indicate that gallic acid would also have high affinity to the cell membrane. This might

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be the reason for why the antimicrobial activity of unmodified chitosan was improved by the

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conjugation of gallic acid. In other words, the affinity of chitosan to the bacterial cell membranes was

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improved by the conjugation of gallic acid.

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In comparison with the MIC values of food antiseptics, gallic acid-g-chitosans showed higher

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antimicrobial activities that those of sorbic acid (6055 µg/mL for E. coli and 9262 µg/mL for S.

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aureus), benzoic acid (10171 µg/mL for E. coli and 16788 µg/mL for S. aureus), and propionic acid

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(15000 µg/mL for E. coli and S. aureus) (32, 33). This comparison indicates that gallic acid-g-

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chitosans may be useful for food preservative.

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Bactericidal Assay. Considering the MIC values of gallic acid-g-chitosans, the bactericidal effects of

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gallic acid-g-chitosan (I) against E. coli and S. aureus were confirmed by a time-kill curve experiment.

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As shown in Figure 1, E. coli (5.8 × 105 CFU/mL) suspension without gallic acid-g-chitosan (I) was

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significantly increased to 1.4 × 109 CFU/mL after 24 h. However, the incubation with gallic acid-g-


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chitosan (I) at MIC suppressed bacterial growth for 24 h, indicating a bacteriostatic effect. Over the

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MIC, gallic acid-g-chitosan (I) killed the bacteria, and 2.8-log reduction (2 × MIC), 5.0-log reduction

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(4 × MIC) and no viable cells were observed after being exposed to 8 × MIC for 24 h (Figure 1A). In

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the case of S. aureus, viable cells without gallic acid-g-chitosan (I) were increased from 2.2 × 105

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CFU/mL to 5.6 × 108 CFU/mL after 24 h. However, gallic acid-g-chitosan (I) treatment decreased

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viable cells, and 1.2-log reduction (MIC) and 3.4-log reduction (2 × MIC) were observed after 24 h.

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No viable cells were observed after being exposed to 4 × MIC for 18 h and 8 × MIC for 12 h (Figure

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1B).

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Integrity of Bacterial Cell Membranes. The cytoplasmic cell membrane is a structural component,

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which may become damaged and functionally invalid during exposure to antimicrobial agents. Thus,

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release of intracellular components is a good indicator of membrane integrity. If bacterial membrane

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became compromised by interact with antimicrobial agents, firstly, small ions such as potassium and

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phosphate tend to leach out, and followed by large molecules such as DNA, RNA and other materials.

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These intracellular components are easily detected by UV at 260 nm as an indication of membrane

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damage (20).

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As shown in Figure 2, total nucleotide leakage from E. coli and S. aureus as a function of incubation

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time with gallic acid-g-chitosan (I) was plotted. Upon addition of gallic acid-g-chitosan (I) to E. coli,

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there was dramatically increased in OD260 within 30 min and thereafter OD260 was slightly increased

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up to 90 min. The extents of nucleotide leakage by treatment of gallic acid-g-chitosan (I) were dose-

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dependent manner, which is agreeable with the findings for bactericidal activity. In case of S. aureus,

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the extents of nucleotide leakage by treatment of gallic acid-g-chitosan (I) was higher than that of E.

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coli. This was probably due to the differences in cell wall structure and composition, and S. aureus

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does not have the OM to prevent the influx of foreign molecules (20). These results indicate that gallic

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acid-g-chitosan (I) can bind to membrane components and cause membrane permeabilization to

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varying extents.

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OM permeabilization assay. Gram-negative bacteria such as E. coli have two cell envelope

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membranes. Thus, we examined the ability of gallic acid-g-chitosan (I) to interact with both OM and



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IM. To determine OM permeabilization, hydrophobic NPN probe is used. NPN is normally excluded

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from OM. However, when OM was damaged and functionally invalid by interaction with

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antimicrobial agents, NPN could partitioned into perturbed OM exhibiting increased fluorescence.

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Therefore, the increase of NPN fluorescence intensity can be used as an indicator for increased cell

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membrane permeability.

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Based on this principle, the change of NPN fluorescence upon the addition of gallic acid-g-chitosan (I)

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is shown in Figure 3. The fluorescence increase by the addition of gallic acid-g-chitosan (I) to E. coli

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suspensions was dose- and time-dependent manner, it is indicated that E. coli cell membranes were

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damaged or functionally invalid by gallic acid-g-chitosan (I).

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OM contains polyanionic lipopolysaccharide (LPS) stabilized by divalent cations, thus polycationic

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antimicrobial agents could be bind to the negatively charged O-specific oligosaccharide units of E.

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coli LPS thus disrupting the integrity of OM resulting in loss of the barrier function or blocking the

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nutrient flow with concomitant bacterial death due to depletion of the nutrients. Chitosan is a

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polycationic biopolymer due to the primary amines at C-2 position; this is a major factor affecting

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antimicrobial activity of chitosan. Moreover, we conjugated gallic acid, which has high affinity to

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lipid bilayer in the cell membrane, onto chitosan. Thus gallic acid-g-chitosan could be interacted with

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LPS and lipid bilayer in the cell membrane; thereby the antimicrobial activity of gallic acid-g-chitosan

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was appeared by disruption of OM.

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IM permeabilization assay. Destabilization of OM is necessary to gain access to IM. Gallic acid-g-

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chitosan (I) could interact with IM because OM permeation was demonstrated. Thus, we further

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examined IM permeabilization of E. coli as a function of cytoplasmic β-galactosidase release, with

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bacteria grown in lactose-containing medium in order to operate lac operon in E. coli. As shown in

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Figure 4, the control suspensions, there was a lag of about 40 min before cytoplasmic β-galactosidase

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was released very slowly. Whereas, the suspensions with gallic acid-g-chitosan (I) showed immediate

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release of cytoplasmic β-galactosidase followed by a progressive release up to 70 min to reach a

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steady state. The release of cytoplasmic β-galactosidase in E. coli was dose-dependent manner, which

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was agreeable with the assay of integrity of cell membranes and OM pemeabilization.


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Transmission Electron Microscopy. To further elucidate the nature of the killing mechanisms of

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gallic acid-g-chitosan (I), E. coli and S. aureus were treated with gallic acid-g-chitosan (I) for 18 h,

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and the bacteria were analyzed by TEM. Compared to the control, gallic acid-g-chitosan (I) treatment

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resulted in clear morphological changes (Figure 5 and 6). Untreated cells displayed a smooth and

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compact surface, without release of intracellular components and notable ruptures or pores on the cell

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surface (Figure 5A and 6A). However, the cells shape changed compared to those of untreated cells. S.

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aureus cells treated with gallic acid-g-chitosan (I) appeared to undergo cell membrane damage,

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resulting in the release of their cellular components into surrounding environment, and finally became

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empty (Figure 5B). In case of E. coli, the cell membrane was shrunk and irregular by treatment of

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gallic acid-g-chitosan (I), and the cellular components were release. These results correlate with

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integrity of cell membrane, OM and IM permeabilization.

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The present study suggests that gallic acid-g-chitosans had good antimicrobial activity against

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foodborne pathogens. The integrity cell membrane, OM and IM permeabilization experiments

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indicated that gallic acid-g-chitosans influenced the structure of membrane and speculated to interact

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with lipids on the cell membrane. TEM observations demonstrated the disruption of the cell

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membrane by gallic acid-g-chitosans. Chitosan and gallic acid are regarded as relatively safe, thus

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these conjugation products, gallic acid-g-chitosans, could be used for food preservatives.

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ACKNOWLEDGMENT

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This research was supported by the Basic Science Research Program through the National Research

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Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-

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0009244).

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345

FIGURE CAPTION

346

Figure 1. Bactericidal activity of gallic acid-g-chitosan (I) against E. coli. (A) and S. aureus (B).

347

MIC, ●; MIC x 2, ○; MIC x 4, ▼; MIC x 8, △; Control, ■. The values represent means ± S.D.

348

(n=3).

349 350 351

Figure 2. The release of intracellular components of E. coli (A) and S. aureus (B). The values represent means ± S.D. (n=3).

352 353 354

Figure 3. The uptake of NPN by E. coli suspensions treated with gallic acid-g-chitosan (I). The values represent means ± S.D. (n=3).

355 356 357

Figure 4. The release of cytoplasmic β-galactosidase activity of E. coli cells treated with gallic acid-g-chitosan (I). The values represent means ± S.D. (n=3).

358 359 360

Figure 5. TEM of S. aureus treated with 128 µg /mL of gallic acid-g-chitosan (I) for 18 h. (A) Untreated S. aureus; (B) Gallic-g-chitosan treated S. aureus. Scale bar: 100 nm

361 362 363

Figure 6. TEM of E. coli treated with 1024 µg /mL of gallic acid-g-chitosan (I) for 18 h. (A) Untreated E. coli; (B) Gallic-g-chitosan treated E. coli. Scale bar: 100 nm

364 365



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Table. Minimum inhibitory concentrations (MICs) of gallic acid-g-chitosans and unmodified chitosan MIC (µg /mL) Strain

gallic acid-g-chitosan gallic acid-g-chitosan gallic acid-g-chitosan gallic acid-g-chitosan

Unmodified

(I)

(II)

(III)

(IV)

chitosan

Staphylococcus aureus (KCTC 1927)

32

32

32

32

128

Bacillus subtilis (KCTC 1028)

16

32

32

32

64

Bacillus cereus (KCTC 3624)

32

32

64

64

128

Enterococcus faecalis (KCTC 2011)

16

16

32

32

64

Listeria monocytogenes (KCTC 3569)

16

32

64

64

128

Escherichia coli (KCTC 1682)

256

512

512

512

1024

Klebsiella pneumoniae (KCTC 2242)

128

256

256

256

512

Pseudomonas aeruginosa (KCTC 1637)

256

256

512

512

512

Salmonella typhimurium (KCTC 1925)

128

256

256

512

512

Shigella flexneri (KCTC 2998)

256

256

512

512

1024


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A

10

Log CFU (cells/mL)

8

6

4

2

0

B

10

Log CFU (cells/mL)

8

6

4

2

0 0

5

10

15

20

25

Time (h)

Figure 1.



A

0.035 MIC MIC x 2 MIC x 4

Absorbance (260 nm)

0.030 0.025 0.020 0.015 0.010 0.005 0.000

B

0.07 MIC MIC x 2 MIC x 4

Absorbance (260 nm)

0.06 0.05 0.04 0.03 0.02 0.01 0.00 0

20

40

60

80

100

Time (min)

Figure 2.


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40

Relative fluorescence

MIC MIC x 2 MIC x 4

30

20

10

0 0

2

4

6

8

10

Time (min)

Figure 3.



Absorbance (420 nm)

0.08 Control MIC MIC x 2 MIC x 4

0.06

0.04

0.02

0.00 0

20

40

60

80

100

Time (min)

Figure 4.


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Figure 5.



A

B

Figure 6.


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Gallic Acid-Grafted-Chitosan ... - ACS Publications

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