Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli

Jun 7, 2017 - *E-mail: [email protected]; [email protected]. ... Glycinin basic peptide (GBP) is an antibacterial ingredient that occurs naturally in the ...
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Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli Guo-Ping Zhao, Ying-Qiu Li, Gui-Jin Sun, and Hai-Zhen Mo J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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Journal of Agricultural and Food Chemistry

Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli

Antibacterial Actions of Glycinin Basic Peptide

Guo-Ping Zhao†, Ying-Qiu Li†,*, Zhao-Sheng Wang‡, Gui-Jin Sun†, Hai-Zhen Mo‡§



School of Food Science & Engineering, Qilu University of Technology, No. 3501 University Road of

Changqing District, 250353, Jinan, Shandong Province, China ‡

School of Food Science and Engineering, Shandong Agricultural University, Taian, 272018, China

§

School of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China

*

Corresponding author: Ying-Qiu Li

E-mail: [email protected]; [email protected] Tel: +86-531-89631195; Fax: +86-531-89631195

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ABSTRACT: Glycinin basic peptide (GBP) is an antibacterial ingredient that occurs

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naturally in the basic parts of soybean glycinin. The antibacterial actions of GBP

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against Escherichia coli ATCC 8739 were investigated in this study. The minimum

4

inhibitory concentration of GBP against E. coli was 200 µg/mL. The exposure of E.

5

coli cells to GBP induced remarkable significant cell damage and inactivated

6

intracellular esterases (stressed and dead cells, 70.9% ± 0.04 for 200 µg/mL of GBP

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and 91.9% ± 0.06 for 400 µg/mL of GBP), as determined through

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staining of in flow cytometry. GBP resulted in the exposure of phosphatidylserine in

9

E. coli cells. The analyses of flow cytometry- manifested GBP treatment led to the

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shrinkage of the cell surface and the complication of cell granularity. The

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observations of in transmission electron microscopy demonstrated that 400 µg/mL of

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GBP severely disrupted the membrane integrity, resulted resulting in ruptures or pores

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of in the membrane, outflows of intracellular contents or aggregation of the cytoplasm.

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Release of alkaline phosphatase, lipopolysaccharide, and reducing sugar further

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verified that the membrane damage was by due to GBP. In addition, GBP treatment

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changed the helicity and base staking of DNA, as determined by

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dichroism spectroscopy. These results showed that GBP had strong antibacterial

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activity against E. coli by the action ofvia membrane damage and DNA perturbation.

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What’s moreAdditionally, GBP had exhibited no cytotoxicity by ondetecting the

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viability of human embryonic kidney cells. Thus, it GBP might may be a promising

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candidate for as a natural antibacterial agents.

through dual

by circular

22

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KEYWORDS: Escherichia coli, antibacterial action, glycinin basic peptide,

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membrane damage, DNA perturbation Formatted: Line spacing: Double

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INTRODUCTION

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Food spoilage is one of the major problems in the food industry. So Thus, there are

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many strategies for preventing pathogenic and spoilage microorganisms, including the

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use of chemical preservatives. However, safety concerns about regarding the

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long-term use of chemical synthetic preservatives has attracted widespread attention,

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due to the potential risk of chemical preservatives to human health.1-2 In fact, the

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antibacterial activities of natural agents have formed the basis of several applications

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in the realms of food preservation.3-5 Thus, natural antibacterial agents will play a

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crucial role in the future of the food industry, although there are many obstacles to

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overcome.6-8

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Glycinin basic peptide (GBP), a basic and hydrophobic subunit (MW=20 kDa) of

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soybean glycinin, is one of thea natural antimicrobial peptides. It has good solubility

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in water, especially in an alkaline environment, to form a uniform dispersion.9 Several

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studies have demonstrated that GBP has antimicrobial activities against bacteria and

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fungi.10-12 The antimicrobial action of GBP may be that due to its GBP disruption ofs

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microbial membranes to that result in damage or , even death of microbial cells.10 Our

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previous findings indicated that GBP might may have additional intracellular

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functional targets.13 However, the underlying antibacterial mechanisms of GBP are

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still not fully understood. Thus, further research about on the antibacterial mechanism

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of GBP is urgently needed before it can be well applied in the food industry as a

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natural preservative.

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The aim of this study was to investigate the antibacterial actions of GBP on

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Escherichia coli ATCC 8739 (E. coli), which included cell damage and morphology

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changes by determined via flow cytometry, membrane damage and structurale

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changes by determined via transmission electron microscopy (TEM), and the release

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of cellular components by determined via quantitative determinations. Moreover, the

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action of GBP on the genomic DNA of E. coli cells was also investigated by using

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circular dichroism (CD) spectroscopy to explore the possible intracellular targeting

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behaviors. In addition, the cytotoxicity of GBP was evaluated in this study. Formatted: Line spacing: Double

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MATERIALS AND METHODS

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Reagents and Chemicals. Peptone, beef extract, and agar for microbial cultivation

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were purchased from Solarbio Life Sciences Ltd. Co. (Beijing, China).

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Glutaraldehyde, 1% osmium tetroxide, 1,2-epoxypropane, epoxy resin, uranylacetate

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and lead citrate for transmission electron microscopy determination were from

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Zhongxingbairui TechonologyTechnology Ltd. Co. (Beijing, China) and were used

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for transmission electron microscopy. Proteinase K, p-nitrophenylphosphate,

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periodicacid, silver nitrate, SDS, β-mercaptoethanol, and bromophenol blue were

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from Sinopharm Chemical Reagent Ltd. Co. (Shanghai, China). Carboxyfluorescein

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diacetate (cFDA), propidium iodide (PI), Annexin V, Dulbecco’s modified Eagle’s

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medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and fetal calf

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serum were also from Solarbio Life Sciences Ltd. Co. (Beijing, China). All other

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reagents and chemicals were of analytical grade.

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Materials. GBP (purity, 99.0%) was isolated from defatted soybean flakes (Scents

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Holding Ltd. Co., Jinan, Shandong) and purified according to our previously reported

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method.11 The purified GBP was dispersed in sterile distilled water to obtain

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dispersions of different concentrations before additioned to the experimental bacterial

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suspensions.

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Bacterial Strain and Cultivation. The bacteria culture of E. coli (ATCC 8739)

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was obtained from the Culture Collection in the, Qilu University of Technology.

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Freeze-dried bacteria were activated according to the ATCC guidelines and

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maintained on a slant of beef extract peptone medium (0.3 g beef extract, 1.0 g

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peptone, 0.5 g NaCl and 2.0 g agar boiled to dissolve in 100 mL of distilled water).

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The bacteria were inoculated from the beef extract peptone medium into a flask

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containing 100 mL of sterile beef extract peptone broth. Then, they were cultivated

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with shaking (130 rpm) at 37 °C for 9 h to yield a final cell concentration of 107-108

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CFU/mL that was used for further study.

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Determination of the Minimum Inhibitory Concentration. The mMinimum

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inhibitory concentration was visually identified as the lowest concentration of GBP

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which that inhibited the visible growth. The minimum inhibitory concentration of

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GBP against E. coli cells was measured according to the report of Li et al.14 A series

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of quantityamounts of GBP were added to the bacterial suspensions (approximate

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106-107 CFU/mL) to get obtain final concentrations of 0, 50, 100, 150, 200, 250, 300,

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350 and 400 µg/mL. The bacteria treated with GBP were incubated at 37 °C for 24 h.

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Then, the OD600 values of all of the treated samples were measured. All tests were

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performed in triplicate.

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Flow Cytometric Measurement by Double Staining of Carboxyfluorescein

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Diacetate and Propidium Iodide. Flow cytometric assessments were performed by

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dual staining of each sample to differentiate viable, stressed and dead cells. Before

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staining, three copies of the E. coli cell suspensions that were treated with 0, 200 and

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400 µg/mL GBP for 4 h were centrifuged at 4,500 g to remove significant traces of

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interfering media components, respectively. Then, the cell pellets were suspended in

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sterile saline buffer (0.85%) to reach approximately 106 CFU/mL. An aliquot (1 mL)

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of the resulting suspension was firstly stained with 30 µL of carboxyfluorescein

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diacetate (cFDA, 50 µg/mL) at 37 °C for 10 min and , then stained with 40 µL of

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propidium iodide (PI, 50 µg/mL) for penetration at 25 °C for 15 min. Both of the

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staining procedures were carried outconducted in dark conditions. Furthermore, cells

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treated by with heat (boiled for 30 min) were prepared as the positive control.

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The treated samples were performed analyzed usingon an imaging flow cytometer

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(ImageStreamX Mark II, Merck Millipore Inc., Germany), equipped with an 200-mW,

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488-nm excitation laser. The condition was set at the slowest flow rate with the

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biggest magnification. For each sample, a total of 10,000 events were collected. The

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gGreen fluorescence of cells stained with carboxyfluorescein was collected in the FL2

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channel (emission wavelength of 533 ± 35 nm) and the red fluorescence of cells

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stained with PI was collected in the FL4 channel (emission wavelength of 610 ± 30

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nm).15-16

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Determination of the Morphologies of E. coli. The morphologies of E. coli were

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also measured by using an the imaging flow cytometer (ImageStreamX Mark II,

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Merck Millipore Inc., Germany).17 The cells of E. coli cells were treated with 0, 200

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and 400 µg/mL of GBP at 37 °C and shaken constantly (130 rpm) for 4 h. Then, the

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treated cells were washed and resuspended in phosphate buffer solution (PBS, 10 mM,

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pH 7.4). The resuspended cells were illuminated by with 488- nm of excitation light,

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and the size (area) and granularity (intensity) of the cells were measured.

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Determination of Phosphatidylserine Exposure by Staining of Annexin V-FITC.

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The exposure of phosphatidylserine was detected by using the flow cytometer

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(ImageStreamX Mark II, Merck Millipore Inc., Germany) using Annexin V-FITC

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stianingstaining, following the kit instructions with some modifications (Solarbio Life

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Sciences Ltd. Co., China). After treatmented with GBP (0, 200 and 400 µg/mL) at

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37 °C for 4 h, the cells of E. coli cells were washed and resuspended in PBS (10 mM,

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pH 7.4) by via a gentle vortex. The resuspended cells were bound with binding buffer

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and , then stained with Annexin V-FITC for 10 min in the dark at room temperature

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(25 °C). Finally, the stained cells were analyzed by via flow cytometry at in the FL2

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channel (emission wavelength of 530 ± 30 nm).

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TEM Determination of E. coli Cell Ultrastructure. The cells of E. coli cells were

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treated with GBP of 0, 200 and 400 µg/mL concentrations for 4 h at 37 °C. Then, the

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treated cells were harvested by via centrifugation at 4,500 g for 15 min and

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transferred into eppendorfEppendorf tubes, where the cells were washed twice with

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PBS. The resulting cell pellets with had a thickness of no more than 2 mm and were

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fixed with glutaraldehyde (2.5% in 0.1 M PBS, pH 7.4) for at least 2 h at 4 °C. The

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fixed pellets in the eppendorfEppendorf tubes was were oxidated by adding to 200 µL

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of 1% osmium tetroxide for 3 h at 4 °C. After oxidation, the bacterial pellets were

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rinsed twice for 10 min with PBS and dehydrated sequentially using 30%, 50%, 70%

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and 90% acetone for 15 min each. Subsequently, 30 min of’s dehydration with 100%

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acetone was carried outperformed three times. Finally, the dehydrated pellets were

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transferred into an embedding plate, and were then permeated twice with 1,

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2-1,2-epoxypropane for 10 min and with epoxy resin (DER: DMAE: ERL: NSA =

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1.80: 0.06: 2.00: 5.00) for 45 min at 4 °C before drying (70 °C, 18 h) to form

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specimen blocks.

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The specimen blocks were hand-trimmed with a razor blade and sectioned with

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using an ultra microtome (Leica EM UC7, Germany). Thin sections (approximately

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60 nm) were placed on copper grids of that were 300 mesh. The sections were stained

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in uranylacetate for 30 min and incubated in lead citrate for 10 min. The images of the

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prepared samples were observed by using TEM (JEOL-JEM-1200 EX, Japan).18

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Determination of Alkaline Phosphatase Leakage. The leakage of alkaline

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phosphatase (ALP) between the outer membrane and inner membrane of the E. coli

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cells was measured using the modified method reported by Chung and Chen.19 ALP

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was exposed to the medium when the cell membrane was damaged. It could catalyze

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the hydrolyzationhydroxylation of the ester (p-nitrophenylphosphate) to produce a

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phosphate and yellow p-nitrophenol. The activity of ALP was evaluated by measuring

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the produced p-nitrophenol, which had a maximal absorbtionabsorption at 405 nm.

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The E. coli cells that were centrifuged at 4,500 g for 15 min were washed twice and

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re-suspended with in sterile saline (0.85%) and to be treatedthen treated with 0, 200

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and 400 µg/mL GBP, respectively. The samples were incubated at 37 °C with shaking

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(130 rpm). Every one hour, 1 mL of GBP-treated bacterial suspension was centrifuged

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at 4,500 g for 10 min, and the supernatant was mixed with 5 mL of

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p-nitrophenylphosphate (0.1% in 0.5 M of Tris-HCl buffer, pH 8.0). Then, the ALP

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leakage of E. coli was determined by measuring the OD405 of the resulting samples

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that were incubated at 28 °C for 30 min , using a spectrophotometer (V-1100,

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Meipuda Instrument Ltd. Co., Shanghai, China).

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Determination of Lipopolysaccharide Release. The lLipopolysaccharide (LPS)

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release from the cell outer membrane of the E. coli cells was determined by via the

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method of electrophoresis.20 The E. coli cultures were centrifuged at 4,500 g for 15

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min, washed three times and resuspended in Tris-HCl buffer (10 mM, pH 7.2). The

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GBP solution was added to the E. coli suspension to get obtain the final

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concentrations of 0, 200 and 400 µg/mL. These suspensions were incubated at 37 °C

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for 4 h, followed by centrifugation at 6,500 g for 30 min. Aliquots (10 µL) of the

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cell-free supernatants were dissolved in 10 µL of sample buffer (0.1 M of Tris-HCl,

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20% sucrose, 2% SDS, 0.1% β-mercaptoethanol, and 1% bromophenol blue, pH 6.8)

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and boiled for 10 min. After each sample was cooled to room temperature (25 °C), 10

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µL of proteinase K solution (2.5 mg/mL) was added. Then, the mixture including LPS

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was electrophoresed through a 5% stacking gel at 10 mA for 30 min and through a

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12% resolving gel at 25 mA for 2 h.

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The gel was oxidized with 0.7% periodic acid in 30% ethanol-10% acetic acid at

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22 °C for 20 min and , washed three times with distilled water for 5 min. Then, the gel

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was stained with freshly prepared silver nitrate solution (1 mg/mL) at 30 °C for 30

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min and , washed quickly with distilled water for 10 s. The color was developed with

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a 3% pre-cooling NaCO3 solution (4 °C) containing 0.02% fresh formaldehyde. After

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the color reaction was stopped with 10% acetic acid, the LPS gel was photographed

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immediately.

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Determination of the Reducing Sugar Leakage. The leakage of cellular reducing

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sugar from the E. coli cells was determined with via a reducing sugar analyzer

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(SGD-IV, Precision & Scientific Instrument Ltd. Co., Shanghai, China).21 After

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centrifugation at 4,500 g for 15 min, the precipitated E. coli cells were washed triply

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and re-suspended with PBS (10 mM, pH 7.0). An aAppropriate GBP solution was

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mixed with the bacterial suspension to get obtain final concentrations of 0, 200 and

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400 µg/mL, respectively. The mixtures were incubated at 37 ºC with shaking (130

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rpm), and the leakage of reducing sugar was measured at 0, 1, 2, 3, 4, 5, 6, 7 and 8 h,

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respectively.

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Determination of Genomic DNA in E. coli. The gGenomic DNA of the E. coli

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was investigated by via CD measurements.22 E. coli cells at that were in the

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mid-stationary phase were treated with GBP of 0, 200 and 400 µg/mL of GBP for 4 h

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at 37 ºC. Then, the intracellular DNA was extracted using a bacterial genomic DNA

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extraction kit (BioTeke

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(approximatapproximatelye 100 µL) was dissolved in 200 µL of Tris-HCl buffer (10

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mM, pH 8.0). The CD spectra of DNA in the absence and presence of GBP at

200

different concentrations was measured using theon CD spectrometer (Chirascan,

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Applied Photophysics Ltd. Co., England) using via a 1.0- mm quartz cell under

Ltd.

Co., Beijing, China). The extracted DNA

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nitrogen atmosphere. All observed CD spectra were the average of three scans

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recorded at a bandwidth of 1.0 nm and corrected for the buffer signal.

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Cytotoxicity Assay. The cytotoxicity of GBP was evaluated against the human

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embryonic kidney cell line HEK293, which was

obtained from the Shanghai Ccell

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Bbank of the Chinese Aacademy of Ssciences. The viability of cell viabilitiess were

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detected by converting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

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to formazan.23

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The kidney cells were cultured at 37 ºC in Dulbecco’s modified Eagle’s medium

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(supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution)

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with 5% CO2. The cultured cells were harvested and resuspended in growth medium

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to a final concentration of 105 cells/mL. Then, they were treated with various

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concentrations of GBP (0, 100, 200, 300 and 400 µg/mL) in a 96-well culture

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microplate (200 µL per well) for 48 h at 37 ºC and centrifuged at 300 g for 5 min. The

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obtained kidney cells were treated with 200 µL of growth medium containing 0.5

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mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for 4 h at 37

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ºC. Then, the growth medium was replaced with 100 µL of 2-propanol in order to

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dissolvesolve the converted purple dye. The absorbances of the samples s waswere

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measured using a microplate reader (Multiskan Ascent, Thermo Scientific Ltd. Co.,

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America) at 570 nm with 690 nm of as the background subtraction. The IC50 value

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represented the concentration of GBP that inhibited 50% of cell growth.

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Statistical analysis. All experiments were performed in triplicate,s and the results

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were expressed by the mean plus the standard error. The fFlow cytometric data were

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analysedanalyzed with using the software IDEAS Analysis 5.0 software (Merck

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Millipore Inc., Germany). Quantitative assessment of each bacterial subpopulation

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was performed by counting the number of events that were included inside the

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corresponding regions. The Oother data were statistically analyzed using ANOVA

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variance analysis by in Eexcel 2007. Least significant differences were used to

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separate the means at p < 0.05. Formatted: Line spacing: Double

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RESULTS AND DISCUSSION

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Minimum Inhibitory Concentration of GBP against E. coli Cells. The inhibitory

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effect of GBP against E. coli was is depicted in Figure 1. The OD600 values decreased

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with the an increase of in GBP concentration. For GBP, 200 µg/mL GBP madecaused

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the OD600 value of E. coli to decrease sharply. Thus, the minimum inhibitory

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concentration of GBP against E. coli was is 200 µg/mL.

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Cell Damage by GBP. cFDA is a non-fluorescent precursor which that can easily

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permeate intact cell membranes. Inside the cell, diacetate groups were hydrolyzed by

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unspecific esterases and transformed into a membrane-impermeant fluorescent

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compound, called

240

488 nm and an emission wavelength of 525 nm. Thus, retention of carboxyfluorescein

241

in the cell could indicate activities of cytoplasmic enzymes so asto to assess bacterial

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viability.24-25 The nucleic acid dye, PI, could bind to the DNA of destroyed cells to

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form a red fluorescent DNA-complex, which has a maximum emission wavelength of

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620 nm. PI-stained cells correspond to the cells having that have compromised or

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destroyed membranes and , which are considered as stressed or dead.15 Whereas

carboxyfluorescein, with a maximum excitation wavelength of

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However, double stained cells correspond to cells having that have both enzymatic

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activity and compromised membranes, which are considered to be as stressed cells.16

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The region designation and the a possible explanation of for the involved cellular

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mechanisms were has been previously reported according byto Ananta et al.24 The

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membrane damage of due to GBP to in E. coli was is displayed as dual-parameter

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fluorescence density plots (Figure 2). After double staining with cFDA and PI, three

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subpopulations were identified, including cFDA positive (R2), cFDA and PI positive

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(R3), and PI positive (R4), which represented viable, stressed and dead cells,

254

respectively. It was clearly showed that Clearly, the distribution patterns of

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GBP-treated E. coli cells were significantly differed from the negative control. As

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seen shown in Figure 2 (a-d) and Table 1, the subpopulation rates of R2 (viable cells)

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treated with 0, 200 and 400 µg/mL of GBP was were 94.4% ± 0.07, 20.4% ± 0.10 and

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6.91% ± 0.09, respectively, which indicated that the esterases viabilities of

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GBP-treated E. coli cells were much weaker than in untreated cells. At the same

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timeMeanwhile, the subpopulation rates of R4 (dead cells) treated with 0, 200 and

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400 µg/mL of GBP was were 0.40% ± 0.02, 8.40% ± 0.08, and 47.8% ± 0.05,

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respectively., The subpopulation rate (62.5% ± 0.08) in R3 (stressed cells) of in the

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200- µg/mL GBP -treated grouptreatment group was higher than that in the 400-

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µg/mL GBP (44.1 ± 0.12) and the control (0.12% ± 0.01) groups. These results

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showed that GBP treatment could can induce stressed and dead cells. Moreover, the

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with a higher GBP concentration was, there were the more dead cells there were.

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It is well known that bacterial cell membranes (outer and inner membranes) can

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protect cells from the surroundings and are responsible for transportation of

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nutritinutrients that are on ingredients necessary to for cell growth and metabolism.26

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Once the cell membranes were damaged, bacterial growth and metabolism were

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inhibited. Our results indicated that GBP- damaged cell membranes and enzymes of

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in E. coli, which made cause the an unbalance of in cell growth and metabolism,

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eventually leading lead to the death of E. coli. Similarly, the membrane damage of

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several bacteria by nisin and other bacteriocins was investigated using flow cytometry,

275

which

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concentrationsintensity.27 A novel lactoferricin B- like peptide from adult centipedes

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was found to cause microbial membrane permeabilization by via PI staining on in

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flow cytometry.28

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showed

increasing

damaginge

effects

Effects of GBP on the E. coli Morphologies.

with

increasing

treatment

of E. coli. The morphological

280

changes of in E. coli after GBP treatment were are presented by plotting the cell

281

intensity and area. Area (x-axis) is an indicator of size, and intensity (y-axis) is an

282

indicator of granularity.28 In our study, the area/intensity characteristics in for different

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concentrations of GBP-treated cells were changed (Figure 3). In the untreated group,

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E. coli cells accounted for 91.66% in the uniform R region. However, the R region

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cells treated with 200 and 400 µg/mL GBP were 40.96% and 36.10%, respectively.

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The area (plumpness of cells) of GBP-treated cells decreased, while whereas the

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intensity (complexity of the cell structure) took on increased phenomenon. These

288

results demonstrate thatd GBP caused the cell structure to shrink and the cell

289

granularity to become complicated.

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A similar phenomenon of microbial cell shrinkage was observed after addition of

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perillaldehyde.17 It was has also been reported that a lactoferricin B- like peptide,

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derived from centipede Scolopendra subspinipes mutilans, decreased the cell size and

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caused shrinkage of Candida albicans.28

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Effects of GBP on Phosphatidylserine Exposure of to E. coli. In normal cells,

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phosphatidylserine is located inside the cell membrane. At During the stages of cell

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death, phosphatidylserine can be eversed from the inside to the surface of the cell

297

membrane and exposed to the extracellular environment. Annexin V is a

298

calcium-dependent protein to that specifically binds phosphatidylserine with a high

299

affinity. Annexin V was labeled with the fluorescein, FITC, as a fluorescent probe.29

300

Figure 4 showedFigure 4 shows the effects of GBP on phosphatidylserine exposure

301

of to E. coli. The exposure amount of phosphatidylserine in E. coli cells treated with 0,

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200 and 400 µg/mL of GBP was 16.7%, 47.8% and 60.8%, respectively. In

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comparisonCompared to with the control, there was a significant difference (p < 0.05)

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with between 200 and 400 µg/mL of GBP. This showed that GBP could effectively

305

induce damage to the bacterial cell membrane to damage, leading to the exposure of

306

phosphatidylserine exposure, which was is coincident with the result of cFDA/PI

307

double staining (Figure 2).

308

Effect of GBP on the Cell Ultrastructure of in E. coli. To investigate the

309

membrane damage and cellular structure changes of E. coli in response to GBP, the

310

GBP-treated bacteria were observed using TEM (Figure 5). The An image of the

311

control manifested shows intact cells with a uniform cytoplasmic appearance and

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well- defined membranes (Figure 5a). Compared to with the control, the bacterial

313

surface of E. coli treated with 200 µg/mL GBP (Figure 5b) was slightly damaged and

314

began to shrivel, with an outflow of some portion of the cytoplasm from the cells.

315

BesidesAdditionally, the nucleic acid substances of in the 200- µg/mL GBP-treated E.

316

coli were not clear, and some of the cytoplasm in the cells exhibited a phenomenon of

317

aggregation. Moreover, as shown in Figure 5c, bacterial cells treated with 400 -µg/mL

318

of GBP were more severely damaged than that those of the of 200- µg/mL GBP group,

319

and cell membranes in most of the E. coli were no longer complete with notable

320

ruptures or pores on the surface. The bacteria were vacuolated and transparent with

321

the release of intracellular contents or aggregation of the cytoplasm. These results

322

demonstrate thatd GBP damaged and perforated membrane of E. coli membranes,

323

leading which led to the leakage of the protoplasm to the exterior of the cells.

324

A similar report showed that bacterial cells treated with glycinin basic subunit were

325

evidentlyobviously deformed, following by cell wall and cell membrane

326

disintegration and separation, as well as the loss of regular cellular shapes, in

327

comparison to with the control, which had an

328

(Listeria monocytogenes and Salmonella enteritidis).10 The sSynergistic antibacterial

329

agents, of ε-polylysine and nisin, damaged the morphology and cell structure of

330

Bacillus subtilis cells, which led to hollow cells with leaking cell contents that , and

331

eventually resulted in cell death, as determined via through observation of TEM.2

with intact and smooth morphology

332

Effect of GBP on the Leakage of ALP in E. coli. ALP of in Gram-negative

333

bacteria forms a strong barrier in the periplasmic space, which cannot be determined

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334

due to the protection of outer membranes in intact cells. WhereasHowever, ALP can

335

be determined immediately when the outer membranes of bacterial cells are destroyed.

336

Therefore, the leakage of ALP is used to evaluate the amount of damage there is to

337

degree of bacterial outer membranes.30 The leakage of ALP from E. coli cells treated

338

with GBP was is shown in Figure 6. The OD405 of GBP-treated samples increased

339

significantly from 0.021 to 0.710 with the an increase of in GBP treatment time from

340

0 to 8 h. However, the OD405 values of in the control almost didn’t changedid not

341

significantly change. The OD405 values of both samples treated with 200 and 400

342

µg/mL of GBP increased quite rapidly from 0.02 to 0.50 and to 0.67 during from 0-5

343

h, respectively. After 5 h, the values increased slowly from 0.50 to 0.53 (200 µg/mL

344

GBP) and from 0.67 to 0.71 (400 µg/mL GBP). These results demonstrated that the

345

for longer GBP treatment time swas, there is more ALP leakage.

346

BesidesAdditionally, GBP could destroy quickly destroy the outer membrane of the E.

347

coli cells in during the initial 5 h andto caused ALP the leakage of ALP.

of ALP was.

348

This present study was is in agreement with the finding that the antibacterial

349

peptide, pyrrhocoricin, could can cause the ALP leakage of ALP, and inhibit the

350

activity of ALP in E. coli cells.31 The ALP of E. coli cells treated with an antibacterial

351

agent could can be released quickly into the medium and reach a steady state by 2 h.19

352

Similarly, low-molecular-weight phlorotannins from Sargassum thunbergii could can

353

inhibit the growth of thalli during theat logarithmic phase and lead to the leakage of

354

ALP by damaging the outer membrane of Vibrio parahaemolyticus.32

355

Effect of GBP on the LPS of the E. coli Outer Membrane. LPS is a major

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constituent of the outer membrane of Gram-negative bacteria. LPS can be released

357

from the bacteria to the medium due to damage and decomposition of the membrane

358

structure.33 The profile of LPS released from E. coli cells that were treated with GBP

359

was is shown in Figure 7. Lanes 1, 2, and 3 were show samples treated withwith the

360

control, 200 and 400 µg/mL of GBP, respectively. As seen shown in the LPS -gel,

361

there were are more kinds types and amounts of bands on in lanes 2 and 3 than in lane

362

1. Moreover, there moare many more low-molecular-weight bands of for LPS in the

363

400- µg/mL GBP-treated sample (lane 3) were much more than those in the of 200-

364

µg/mL GBP-treated group (line 2). These results demonstrated that GBP could release

365

and decompose LPS macromolecules of LPS into small molecules, , therefore, leading

366

to damage of to the bacterial outer membrane. Moreover, the with a higher GBP

367

higher concentration of GBP was, there was more serious the damage of to the

368

membrane was.

369

Chensinin-1b, a native peptide, could can bind with LPS by via electrostatic

370

interactions and induce disassociation of LPS.34 The release of LPS was also observed

371

when E. coli cells were treated with antibacterial agent essential oil components as

372

well as chitosan.20,35

373

Effects of GBP on the Leakage of Reducing Sugar in E. coli Cells. The leakage

374

of reducing sugar, a intracellular substance in E. coli cells, is regarded as another

375

additional evidence of the membrane damage in membrane ofin bacterial cells.36 As

376

shown in Figure 8, the reducing sugar content of in the control was constant (0.010%)

377

during the period of the 8- h treatment. However, obvious upward trends appeared in

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the GBP-treated samples during the same period. Among them, the reducing sugar

379

content of the samples treated with 200 and 400 µg/mL of GBP increased quickly

380

from 0.010% to 0.051% and to 0.053% during the initial 6 h, respectively. After 6 h,

381

the reducing sugar contentthose of the GBP-treated samples hardlybarely changed.

382

These results indicated that GBP could disrupt quickly disrupt the bacterial cell

383

membrane structure in 6 h, to resulting in the leakage of reducing sugar from the E.

384

coli cells.

385

Reducing sugar leakage could be initiated by adding the an antibacterial agent.37

386

Similarly, hypocrellin A could was found to change the membrane permeability and

387

induce the leakage of reducing sugar from Staphylococcus aureus cells.21 Moreover,

388

Kagohashi et al. reported that the polyene antibiotic, amphotericin B, facilitated

389

glucose across bilayer lipid membranes, and the transportation of glucose was

390

observed immediately after treatment of with amphotericin B.38

391

Interaction between GBP and the Genomic DNA of E. coli Cells. Besides In

392

addition to affecting the cell membrane, GBP might may affectperform the

393

intracellular biomacro-molecules, such as DNA. CD spectroscopy is an effective way

394

to study the properties of the corresponding DNA conformation.39 As shown in Figure

395

9, the CD spectra of E. coli DNA exhibited a negative band at 248 nm due to the

396

right-handed helicity and a positive band at 277 nm due to base stacking, which were

397

are characteristic of a typical CD spectra of the B-DNA structure.22,39 In the presence

398

of GBP (200 and 400 µg/mL of concentrations), the negative band of DNA shifted

399

slightly toward shorter wavelengths, with an obvious decrease in intensity. At For the

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positive band, an inconspicuous shiftexcursion was observed toward shorter

401

wavelengths. However, the intensity of the base stacking band had remarkable

402

attenuationwas significantly decreased in the GBP-treated groups. These results

403

manifested thatThus, GBP treatment changed the helicity and base staking of DNA.

404

The change of in the DNA space structure possibly interfered with cell metabolic

405

functions, including gene expression or protein synthesis, which was verified in our

406

previous study.13

407

The

changes

in

helicity

and

base

stacking of

genomic

DNA

were

408

confirmedpresented by analysis of CD spectra when of Staphylococcus aureus cells

409

that were treated with the antibacterial agent, carvacrol.22 A peptide analog derived

410

from a cell-penetrating peptide strongly altered the CD spectra of Salmonella

411

typhimurium and Streptococcus pyogenes genomic DNA, indicating that this peptide

412

interacted with bacterial DNA and changed the DNA conformation.40

413

Cytotoxicity of GBP. The cytotoxicity of GBP against human embryonic kidney

414

cells was is outlined in Figure 10. The viabilities of kidney cells treated with 0, 100,

415

200, 300 and 400 µg/mL of GBP were 100%, 97.966%, 96.580%, 94.725% and

416

94.504%, respectively. There was no significant difference (p > 0.05) between the

417

GBP-treated groups and the control. This indicated that GBP had no apparent

418

cytotoxic effect on human embryonic kidney cells.

419

In summary, this work showed that GBP exerted well good antibacterial

420

characteristics against E. coli. GBP treatment could severely destroy bacterial cell

421

membrane integrity, including shrinkage, rupture and perforation of the membrane.

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And Additionally, there were alsowe observed

423

complications, the disappearance of viability, and aggregation of the cytoplasm in

424

GBP-treated E. coli cells. Meanwhile, GBP gave rise to a lose loss of ALP, LPS, and

425

reducing sugar, as well as inactivation of intracellular esterases. BesidesMoreover, the

426

change of in the DNA space structure of E. coli treated by with GBP possibly

427

perturbed the cell metabolism including gene expression or protein synthesis.

428

Moreover, GBP had no cytotoxicity on human embryonic kidney cells. Thus, GBP

429

may be as a safe and valuable biopreservative to bethat can be applied in the

430

agricultural and food industriesy.

431

AUTHOR INFORMATION

432

Corresponding Author

433

*Ying-Qiu Li, Telephone: +86-531-89631195; Fax: +86-531-89631195; E-mail:

434

phenomena of granularity

[email protected]; [email protected]

435

Notes

436

The authors declare no competing financial interest.

437

ACKNOWLEDGEMENTS

438

The authors would like to express their gratitude to the National Natural Science

439

Foundation of China (31371839), Major Plan of Studying and Developing

440

(2015GSF120006), Science and Technology Development Project of Shandong

441

Province (2014GSF121039), as well as the Program for Science and Technology

442

Innovation Team in Universities of Henan Province (16IRTSTHN007).

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REFERENCES

444

(1) Meyer, A. S.; Suhr, K. I.; Nielsen, P.; Holm, F. Natural food preservatives. Innov.

445

Food. Sci. Emerg. 2002, 6, 130-131.

446

(2) Liu, H. X.; Pei, H. B.; Han, Z. N.; Feng, G. L.; Li, D. P. The antimicrobial

447

effects and synergistic antibacterial mechanism of the combination of ε-Polylysine

448

and nisin against Bacillus subtilis. Food Control 2015, 47, 444-450.

449

(3) Küçükgülmez, A.; Kadak, A. E.; Gökçin, M. Antioxidative and antimicrobial

450

activities of shrimp chitosan on gilthead sea bream (Sparus aurata) during

451

refrigerated storage. Int. J. Food Sci. Technol. 2013, 48, 51-57.

452

(4) Bonilla, J.; Vargas, M.; Atarés, L.; Chiralt, A. Effect of chitosan essential oil

453

films on the storage-keeping quality of pork meat products. Food Bioprocess Technol.

454

2014, 7, 2443-2450.

455

(5) Chen, Z. F.; He, B.; Zhou, J.; He, D. H.; Deng, J. D.; Zeng, R. H. Chemical

456

compositions and antibacterial activities of essential oils extracted from Alpinia

457

guilinensis against selected foodborne pathogens. Ind. Crop. Prod. 2016, 83, 607-613.

458

(6) Shih, I. L.; Shen, M. H.; Van, Y. T. Microbial synthesis of poly(ε-lysine) and its

459

various applications. Bioresource Technol. 2006, 97, 1148-1159.

460

(7) Arauza, L. J.; Jozalaa, A. F.; Mazzolab, P. G.; Penna, T. C. V. Nisin

461

biotechnological production and application: a review. Trends Food Sci. Technol.

462

2009, 20, 146-154.

463

(8) Zhao, G. P.; Li, Y. Q.; Sun, G. J.; Mo, H. Z. Effects of glycinin basic peptide on

464

physicochemical characteristics and microbial inactivation of pasteurized milk. J.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

465

Dairy Sci. 2016, 99, 5064-5073.

466

(9) Dias, K.; Myers, D. J.; Bian, Y.; Lihono, M. A.; Wu, S.; Murphy, P. A.

467

Functional properties of the acidic and basic subunits of the glycinin (11S) soy protein

468

fraction. J. Am. Oil Chem. Soc. 2003, 80, 551-555.

469

(10) Sitohy, M. Z.; Mahgoub, S. A.; Osman, A. O. In vitro and in situ antimicrobial

470

action and mechanism of glycinin and its basic subunit. Int. J. Food Microbiol. 2012,

471

154, 19-29.

472

(11) Li, Y. Q.; Hao, M.; Yang, J.; Mo, H. Z. Effects of glycinin basic polypeptides

473

on sensory and physicochemical properties of chilled pork. Food Sci. Biotechnol.

474

2016, 25, 803-809.

475

(12) Yang, J.; Sun, G. J.; Li, Y. Q.; Cui, K. Y.; Mo, H. Z. Antibacterial

476

characteristics of glycinin basic polypeptide against Staphylococcus aureus. Food Sci.

477

Biotechnol. 2016, 25, 1477-1483.

478

(13) Li, Y. Q.; Sun, X. X.; Feng, J. L.; Mo, H. Z. Antibacterial activities and

479

membrane permeability actions of glycinin basic peptide against Escherichia coli.

480

Innov. Food Sci. Emerg. 2015, 31, 170-176.

481

(14) Li, Y. Q.; Han, Q.; Feng, J. L.; Tian, W. L.; Mo, H. Z. Antibacterial

482

characteristics and mechanisms of ε-poly-lysine against Escherichia coli and

483

Staphylococcus aureus. Food Control 2014, 43, 22-27.

484

(15) Chang, S. S.; Paparella, A.; Taccogna, L.; Aguzzi, I.; Chaves-Lo´pez, C.; Serio,

485

A.; Marsilio, F.; Suzzi, G. Flow cytometric assessment of the antimicrobial activity of

486

essential oils against Listeria monocytogenes. Food Control 2008, 19, 1174-1182.

ACS Paragon Plus Environment

Page 24 of 42

Page 25 of 42

Journal of Agricultural and Food Chemistry

487

(16) Majeed, H.; Antoniou, J.; Shoemaker, C. F.; Zhong, F. Action mechanism of

488

small and large molecule surfactant-based clove oil nanoemulsions against food-borne

489

pathogens and real-time detection of their subpopulations. Arch. Microbiol. 2015, 197,

490

35-45.

491

(17) Tian, J.; Wang, Y. Z.; Lu, Z. Q.; Sun, C. H.; Zhang, M.; Zhu, A. H.; Peng, X.

492

Perillaldehyde, a promising antifungal agent used in food preservation, triggers

493

apoptosis through a metacaspase-dependent pathway in Aspergillus flavus. J. Agrc.

494

Food Chem. 2016, 64, 7404-7413.

495

(18) Jung, W. K.; Koo, H. C.; Kim, K. W.; Shin, S.; Kim, S. H.; Park, Y. H.

496

Antibacterial activity and mechanism of action of the silver ion in Staphylococcus

497

aureus and Escherichia coli. Appl. Environ. Microbiol. 2008, 74, 2171-2178.

498 499

(19) Chung, Y. C.; Chen, C. Y. Antibacterial characteristics and activity of acid-soluble chitosan. Bioresource Technol. 2008, 99, 2806-2814.

500

(20) Helander, I. M.; Alakomi, H. L.; Latva-Kala, K.; Mattila-Sandholm, T.; Pol, I.;

501

Smid, E. J.; Gorris, L. G. M.; Von, W. A. Characterization of the action of selected

502

essential oil components on Gram-negative bacteria. J. Agrc. Food Chem. 1998, 46,

503

3590-3595.

504

(21) Du, W.; Sun, C. L.; Liang, Z. Q.; Han, Y. F.; Yu, J. P. Antibacterial activity of

505

hypocrellin A against Staphylococcus aureus. World J. Microbiol. Biotechnol. 2012,

506

28, 3151-3157.

507 508

(22) Wang, L. H.; Wang, M. S.; Zeng, X. A.; Zhang, Z. H.; Gong, D. M.; Huang, Y. B. Membrane destruction and DNA binding of Staphylococcus aureus cells induced

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

509

by carvacrol and its combined effect with a pulsed electric field. J. Agrc. Food Chem.

510

2016, 64, 6355-6363.

511

(23) Liu, B. H.; Wu, T. S.; Su, M. C.; Chung, C. P.; Yu, F. Y. Evaluation of citrinin

512

occurrence and cytotoxicity in Monascus fermentation products. J. Agrc. Food Chem.

513

2005, 53, 170-175.

514

(24) Ananta, E.; Heinz, V.; Knorr, D. Assessment of high pressure induced damage

515

on Lactobacillus rhamnosus GG by flow cytometry. Food Microbiol. 2004, 21,

516

567-577.

517

(25) Hayouni, E. A.; Bouix, M.; Abedrabba, M.; Leveau, J.; Hamdi, M. Mechanism

518

of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil on six LAB

519

strains as assessed by multiparametric flow cytometry and automated microtiter-based

520

assay. Food Chem. 2008, 111, 707-718.

521 522

(26) Teixeira, V.; Feio, M. J.; Bastos, M. Role of lipids in the interaction of antimicrobial peptides with membranes. Prog. Lipid Res. 2012, 51, 149-177.

523

(27) Budde, B. B.; Rasch, M. A comparative study on the use of flow cytometry

524

and colony forming units for assessment of the antibacterial effect of bacteriocins. Int.

525

J. Food Microbiol. 2001, 63, 65-72.

526

(28) Choi, H.; Hwang, J. S.; Lee, D. G. Antifungal effect and pore-forming action

527

of lactoferricin B like peptide derived from centipede Scolopendra subspinipes

528

mutilans. BBA-Biomembranes 2013, 1828, 2745-2750.

529

(29) Tian, J.; Wang, Y. Z.; Lu, Z. Q.; Sun, C. H.; Zhang, M.; Zhu, A. H.; Peng, X.

530

Perillaldehyde, a promising antifungal agent used in food preservation, triggers

ACS Paragon Plus Environment

Page 26 of 42

Page 27 of 42

Journal of Agricultural and Food Chemistry

531

apoptosis through a metacaspase-dependent pathway in Aspergillus flavus. J. Agrc.

532

Food Chem. 2016, 64, 7404-7413.

533

(30) Zheng, C. C.; Zhou, L. X. Antibacterial potency of housefly larvae extract

534

from sewage sludge through bioconversion. J. Environ. Sci-China. 2013, 25,

535

1897-1905.

536

(31) Kragol, G.; Lovas, S.; Varadi, G.; Condie, B. A.; Hoffmann, R.; Otvos, L. The

537

antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents

538

chaperone-assisted protein folding. Biochemistry-US 2001, 40, 3016-3026.

539

(32) Wei, Y. X.; Liu, Q.; Xu, C. J.; Yu, J.; Zhao, L.; Guo, Q. Damage to the

540

membrane permeability and cell death of Vibrio parahaemolyticus caused by

541

Phlorotannins with low molecular weight from Sargassum thunbergii. J. Aquat. Food

542

Prod. Technol. 2016, 25, 323-333.

543

(33) Nan, Y. H.; Bang, J. K.; Jacob, B.; Park, I. S.; Shin, S. Y. Prokaryotic

544

selectivity and LPS-neutralizing activity of short antimicrobial peptides designed

545

from the human antimicrobial peptide LL-37. Peptides 2012, 35, 239-247.

546

(34) Sun, Y.; Dong, W. B.; Sun, L.; Ma, L. J.; Shang, D. J. Insights into the

547

membrane interaction mechanism and antibacterial properties of chensinin-1b.

548

Biomaterials 2015, 37, 299-311.

549

(35) Helander, I. M.; Nurmiaho-Lassila, E. L.; Ahvenainen, R.; Rhoades, J.; Roller,

550

S. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative

551

bacteria. Int. J. Food Microbiol. 2001, 71, 235-244.

552

(36) Tasi, G. J.; Su, W. H. Antibacterial activity of shrimp chitosan against

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

553

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Escherichia coli. J. Food Protect. 1999, 62, 239-243.

554

(37) Diao, W. R.; Hua, Q. P.; Zhang, H.; Xu, J. G. Chemical composition,

555

antibacterial activity and mechanism of action of essential oil from seeds of fennel

556

(Foeniculum vulgare Mill.) Food Control 2014, 35, 109-116.

557

(38) Kagohashi, H.; Shirai, O.; Kubota, S.; Kitazumi, Y.; Kano, K. Facilitated

558

transport of ions and glucose by amphotericin B across lipid bilayers in the presence

559

or absence of cholesterol. Electroanal. 2014, 26, 625-631.

560

(39) Liu, Z. G.; Xiang, Q. S.; Du, L. H.; Song, G.; Wang, Y. T.; Liu, X. B. The

561

interaction of sesamol with DNA and cytotoxicity, apoptosis, and localization in

562

HepG2 cells. Food Chem. 2013, 141, 289-296.

563

(40) Li, L.; Shi, Y.; Cheserek, M. J.; Su, G.; Le, G. Antibacterial activity and dual

564

mechanisms of peptide analog derived from cellpenetrating peptide against

565

Salmonella typhimurium and Streptococcus pyogenes. Appl. Microbiol. Biotechnol.

566

2013, 97, 1711-1723. Formatted: Indent: First line: 1 ch

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Figure legends

569

Figure 1. Effect of different GBP concentrations on the OD600 of E.coli. OD600

570

decreased gradually decreased with the an increase of in the GBP concentration with ,

571

which athe minimum inhibitory concentration was of 200 µg/mL.

572

Figure 2. Various distribution patterns (%) of the subpopulation cells obtained by

573

cFDA/PI double staining of E. coli after 4 h of GBP treatment. (a) negative control; (b)

574

200 µg/mL GBP; (c) 400 µg/mL GBP; and (d) positive control (treated by heat).

575

There were much fewer subpopulations of in the GBP-treated group in the R2 (viable

576

cells) region were much less than in the negative control. Whereas However, the

577

subpopulations in the R3 (stressed cells) and R4 (dead cells) regions showed exhibited

578

the adverse opposite phenomena.

579

Figure 3. Effect of GBP on the morphologies of E. coli morphologies. (a) control;

580

(b) 200 µg/mL GBP; and (c) 400 µg/mL GBP. The area of cells decreased, and the

581

intensity enhanced increased with the an increase of in GBP concentration.

582

Figure 4. Effect of GBP on phosphatidylserine exposure of to E. coli. (a) control;

583

(b) 200 µg/mL GBP; and (c) 400 µg/mL GBP. The phosphatidylserine exposure in the

584

exposed quantity of GBP-treated groups was higher than in the control.

585

Figure 5. TEM images observation of the GBP-treated E. coli at a magnification of

586

40,000 times. (a) control; (b) 200 µg/mL GBP; and (c) 400 µg/mL GBP. The image of

587

the control exhibited regular cells with a uniform cytoplasm and intact membrane (a).

588

While However, the cell membrane treated with 200 µg/mL GBP was partially broken.

589

There occurred was aggregation of some portion of the cytoplasm, with the

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vacuolization of the cells (b). These phenomena of in cells treated with 400 µg/mL of

591

GBP (c) were much more obvious than in the cells treated with 200 µg/mL GBP.

592

Figure 6. Release of alkaline phosphatase in E. coli cells treated with different

593

concentrations of GBP. (◆) control; (■) 200 µg/mL GBP; and (▲) 400 µg/mL GBP.

594

The OD405 of GBP-treated samples increased significantly increased, whereas .

595

Whereas that of the control almost didn’t did not change.

596

Figure 7. Silver-stained LPS electrophoresis of E. coli cells treated with different

597

concentrations of GBP. Lane 1 (control); Lane 2 (200 µg/mL GBP); and Lane 3 (400

598

µg/mL GBP). There were more kinds and amounts of bands on in lanes 2 and 3 than

599

in lane 1, especially the low-molecular-weight bands of LPS.

600

Figure 8. The leakage of reducing sugar of from E. coli cells treated with different

601

concentrations of GBP. (◆) control; (■) 200 µg/mL GBP; and (▲) 400 µg/mL GBP.

602

The content of reducing sugar of in the control was constant (0.010%) during the

603

period of treatment period. However, obvious upward trends (0.051% and 0.053%)

604

appeared inwere observed in the GBP-treated samples during the same period.

605

Figure 9. CD spectra of intracellular DNA in E. coli treated with different

606

concentrations of GBP. The base linebaseline was balanced and was not presented.

607

Both of the negative band and positive band of DNA in the GBP-treated samples

608

shifted toward shorter wavelengths, with obvious decreasing intensitiesy.

609

Figure 10. Cytotoxic effect of different concentrations of GBP on human

610

embryonic kidney cells. There was no apparent cytotoxicity between the GBP-treated

611

groups and the control.

612

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613

Journal of Agricultural and Food Chemistry

Figure 1

614 0.9 0.8

OD (600 nm)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

50

100

150

200

250

300

350

400

GBP (µg/mL)

615 616

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617

Table 1. Subpopulation rate of E. coli cells after treatment with different

618

concentrations of GBP for 4 h.

Page 32 of 42

Formatted: Line spacing: Double

Microorganism

Program

sample

Stained cells (%)

Unstained (R1)

Viable (R2)

Stressed (R3)

Dead (R4)

Control

2.50 ± 0.02

94.4 ± 0.07

0.12 ± 0.01

0.40 ± 0.02

200 µg/mL GBP

6.73 ± 0.06

20.4 ± 0.10

62.5 ± 0.08

8.40 ± 0.08

400 µg/mL GBP

0.28 ± 0.07

6.91 ± 0.09

44.1 ± 0.12

47.8 ± 0.05

Heat- treated

0.30 ± 0.01

0.52 ± 0.03

2.45 ± 0.07

94.7 ± 0.13

Formatted: Line spacing: Double

E. coli

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619

Journal of Agricultural and Food Chemistry

Figure 2

620

621 622

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623

Figure 3

624

625 626

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Journal of Agricultural and Food Chemistry

Figure 4

628

629

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Figure 5

631 632

a

b

c

633

b

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634

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Figure 6

635

0.8 0.7

OD (405 nm)

0.6 0.5 0.4 0.3 0.2 0.1 0 0

1

2

3

4

5

6

7

8

9

Time (h)

636

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637

Figure 7

638 639 640

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641

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Figure 8

642

Reducing sugar content (%)

0.06 0.05 0.04 0.03 0.02 0.01 0 0

1

2

3

4

5

6

7

8

Time (h)

643

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Journal of Agricultural and Food Chemistry

644

Figure 9

645

646 1 control

200 µg/mL GBP

400 µg/mL GBP

CD (mdeg)

0.5

0

-0.5

-1

-1.5 220

240

260

280

300

320

Wavelength (nm)

647 648

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Figure 10

650

651

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