Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli

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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

ACS Paragon Plus Environment


1

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

2

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

3

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

7

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

8

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

10

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

11

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

12

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

13

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

14

Release of alkaline phosphatase, lipopolysaccharide, and reducing sugar further

15

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

16

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

17

dichroism spectroscopy. These results showed that GBP had strong antibacterial

18

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

19

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

20

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

21

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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26

INTRODUCTION

27

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

28

many strategies for preventing pathogenic and spoilage microorganisms, including the

29

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

30

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

31

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

32

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

33

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

34

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

35

overcome.6-8

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

37

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

38

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

39

studies have demonstrated that GBP has antimicrobial activities against bacteria and

40

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

41

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

42

previous findings indicated that GBP might may have additional intracellular

43

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

44

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

45

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

46

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

50

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

51

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

52

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

53

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

54

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

57

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

58

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

59

and lead citrate for transmission electron microscopy determination were from

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

reagents and chemicals were of analytical grade.

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

69

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



70

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

71

dispersions of different concentrations before additioned to the experimental bacterial

72

suspensions.

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

CFU/mL that was used for further study.

82

Determination of the Minimum Inhibitory Concentration. The mMinimum

83

inhibitory concentration was visually identified as the lowest concentration of GBP

84

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

85

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

86

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

87

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

88

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

89

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

90

performed in triplicate.

91

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

The treated samples were performed analyzed usingon an imaging flow cytometer

104

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

105

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

106

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

107

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

108

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

109

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

110

nm).15-16

111

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

112

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

113

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



114

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

115

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

116

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

117

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

118

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

channel (emission wavelength of 530 ± 30 nm).

128

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

129

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

130

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

131

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

132

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

133

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

135

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%

137

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

139

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 =

141

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

142

specimen blocks.

143

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

144

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

145

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

146

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

147

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

148

Determination of Alkaline Phosphatase Leakage. The leakage of alkaline

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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



158

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

159

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

160

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

161

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

162

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

163

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

164

Determination of Lipopolysaccharide Release. The lLipopolysaccharide (LPS)

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

12% resolving gel at 25 mA for 2 h.

177

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

178

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

179

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

181

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

182

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

183

immediately.

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

respectively.

193

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

194

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

195

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

196

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

197

extraction kit (BioTeke

198

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

199

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,

201

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

Ltd.

Co., Beijing, China). The extracted DNA



202

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

203

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

206

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

207

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

208

to formazan.23

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

225

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

226

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

227

corresponding regions. The Oother data were statistically analyzed using ANOVA

228

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

229

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

230

RESULTS AND DISCUSSION

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

unspecific esterases and transformed into a membrane-impermeant fluorescent

239

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

242

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

243

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

244

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

245

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

carboxyfluorescein, with a maximum excitation wavelength of


Formatted: Font: Not Italic


246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

269

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

270

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

271

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

272

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

273

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

274

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

275

which

276

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

277

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

278

flow cytometry.28

279

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

283

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

284

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

285

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

286

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

287

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.



290

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

291

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

292

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

293

caused shrinkage of Candida albicans.28

294

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

295

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

296

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,

302

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

303

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

304

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



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


Page 18 of 42

Page 19 of 42


356

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



378

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


Page 20 of 42

Page 21 of 42


400

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.



422

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).


Page 22 of 42

Page 23 of 42


443

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

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

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2014, 7, 2443-2450.

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

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compositions and antibacterial activities of essential oils extracted from Alpinia

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

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

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

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

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

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2009, 20, 146-154.

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physicochemical characteristics and microbial inactivation of pasteurized milk. J.



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

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

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

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154, 19-29.

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

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

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

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

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Biotechnol. 2016, 25, 1477-1483.

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

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membrane permeability actions of glycinin basic peptide against Escherichia coli.

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

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

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

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

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

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

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


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

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

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

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35-45.

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

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

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

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Food Chem. 2016, 64, 7404-7413.

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

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

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

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

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

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

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

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3590-3595.

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

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

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28, 3151-3157.

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(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



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

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2016, 64, 6355-6363.

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

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

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2005, 53, 170-175.

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

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

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567-577.

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

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

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

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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.

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

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

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

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

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of lactoferricin B like peptide derived from centipede Scolopendra subspinipes

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

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

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


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

532

Food Chem. 2016, 64, 7404-7413.

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

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

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1897-1905.

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

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antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents

538

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

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

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membrane permeability and cell death of Vibrio parahaemolyticus caused by

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

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

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

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selectivity and LPS-neutralizing activity of short antimicrobial peptides designed

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

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

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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,

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

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

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



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

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(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

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(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.

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(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

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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

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

566

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

567


Page 29 of 42


568

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


Formatted: Font: Not Bold


590

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


Page 30 of 42

Page 31 of 42

613


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



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


Page 33 of 42

619


Figure 2

620

621 622



623

Figure 3

624

625 626


Page 34 of 42

Page 35 of 42

627


Figure 4

628

629



630

Figure 5

631 632

a

b

c

633

b


Page 36 of 42

Page 37 of 42

634


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



637

Figure 7

638 639 640


Page 38 of 42

Page 39 of 42

641


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



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


Page 40 of 42

Page 41 of 42

649


Figure 10

650

651



652

Page 42 of 42

Formatted: Font: 12 pt

Table of Contents Graphic

Formatted: Justified, Line spacing: Double Formatted: Font: 12 pt Formatted: Line spacing: Double

653


Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli

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