Isolation, purification, and structural identification of a new bacteriocin made by Lactobacillus plantarum found in conventional kombucha

Journal Pre-proof Isolation, purification, and structural identification of a new bacteriocin made by Lactobacillus plantarum found in conventional kombucha Jinjin Pei, Wengang Jin, A.M.Abd El-Aty, Denis A. Baranenko, Xiaoying Gou, Hongxia Zhang, Jingzhang Geng, Lei Jiang, Dejing Chen, Tianli Yue PII:

S0956-7135(19)30512-2

DOI:

https://doi.org/10.1016/j.foodcont.2019.106923

Reference:

JFCO 106923

To appear in:

Food Control

Received Date: 4 April 2019 Revised Date:

23 September 2019

Accepted Date: 27 September 2019

Please cite this article as: Pei J., Jin W., El-Aty A.M.A., Baranenko D.A., Gou X., Zhang H., Geng J., Jiang L., Chen D. & Yue T., Isolation, purification, and structural identification of a new bacteriocin made by Lactobacillus plantarum found in conventional kombucha, Food Control (2019), doi: https:// doi.org/10.1016/j.foodcont.2019.106923. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Isolation, purification, and structural identification of a new bacteriocin made by

2

Lactobacillus plantarum found in conventional kombucha

3

Jinjin PEI1,2*, Wengang JIN1, A. M. ABD EL-ATY2,3, Denis A. BARANENKO5,

4

Xiaoying GOU1 , Hongxia ZHANG*6, Jingzhang GENG1, Lei JIANG2, Dejing

5

CHEN*1, Tianli YUE*7

6

1

7

Shaanxi University of Technology, Hanzhong, Shaanxi, China

8

2

9

Hai key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau

Shaanxi Key Laboratory of Bioresource, Collage of Bioscience and Bioengineering,

Key Laboratory of Tibetan Medicine Research in Chinese Academy of Sciences, Qing

10

Biology, Chinese Academy of Sciences, Xining, Qinghai, China

11

3

12

12211-Giza, Egypt

13

4

14

25240-Erzurum, Turkey

15

5

16

University, St. Petersburg 191002, Russia

17

6

College of Food Science, Qilu University of Technology, Jinan, Shandong, China

18

7

College of Food Science, Northwest University, Xian, Shaanxi, China

Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University,

Department of Medical Pharmacology, Medical Faculty, Ataturk University,

International Research Centre "Biotechnologies of the Third Millennium", ITMO

19

20

*Corresponding authors:

21

Jinjin Pei: [email protected]

22

Dejing Chen: [email protected]

23

Tianli Yue: [email protected]

24

Hongxia Zhang: [email protected] 1

25

Abstract

26

In recent years, the demand for “natural” products has increased, as customers

27

prefer this type of product over those with added chemical preservatives. The critical

28

issues associated with natural products are how to maintain their safety and quality as

29

well as how to prolong their shelf life. In this study, Lactobacillus plantarum SLG10,

30

isolated from kombucha (a traditional fermented drink in South China), produced a

31

novel bacteriocin, SLG10, which was found to exert antibacterial activity on both

32

Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. An

33

innovative

34

high-performance liquid chromatography (RP-HPLC), was developed for the efficient

35

screening and purification of the bacteriocin found in the cell-free suspension of L.

36

plantarum SLG10. According to matrix assisted laser desorption/ionization-time of

37

flight mass spectrometry (MALDI-TOF-MS), the isolated bacteriocin had a molecular

38

mass

39

Asn-Ile-Val-Trp-Gln-Leu-Ile-Gly-Leu-Pro-Ala-Gln-Al,

40

N-sequencing. Bacteriocin SLG10 showed thermostability and pH tolerant

41

characteristics and was sensitive to most proteases but not trypsin or pepsin. A

42

well-defined linear conformation was suggested by circular dichroism (CD)

43

spectroscopy and 3D structure predictions. The time-kill kinetics curve indicated that

44

bacteriocin SLG10 was bactericidal. The antibacterial mechanism investigation

45

revealed that bacteriocin SLG10 increased cell membrane permeability, causing

46

potassium ion release. We also found that bacteriocin SLG10 can inhibit the formation

of

method,

1422

biochromatography

Da.

The

coupled

amino

2

with

acid as

reversed-phase

sequence

was

determined

by

47

of biofilms. These results suggest that bacteriocin SLG10 has a potential application

48

in the food industry.

49 50

Keywords: Lactobacillus plantarum, bacteriocin, kombucha, purification, mode of

51

action

52 53

1. Introduction

54

Agents that can prevent spoilage or pathogenic bacterial contamination are

55

highly desirable in the food industry (Ayed et al., 2015; Ge et al., 2016). However, as

56

most consumers are concerned about the safety of commonly used food preservatives,

57

there is a high demand for natural and safe alternatives (Ahn et al., 2017, Yue et al.,

58

2013). Bacteriocins are low-molecular-weight peptides with low oral toxicity in

59

humans (Acuna et al., 2012). These compounds show promising applicability in the

60

food industry as bio-preservatives. According to Cotter et al. (2005), bacteriocins are

61

classified into two main categories: Class I, lanthionine-based lantibiotics (molecular

62

weight (Mw) under 5 kDa); and Class II, non-lanthionine bacteriocins (Mw under 10

63

kDa). Class II bacteriocins are divided into 4 subclasses: Class IIa (pediocin-like),

64

Class IIb (two-peptide), Class IIc (cyclic), and Class IId (non-pediocin single linear).

65

The protective effects of bacteriocins/cultures against contamination have been

66

reported for different types of foods, such as fermented dairy food, bakery products

67

and ingredients, alcoholic beverages, meat, fruit, vegetables, and seafood (Gálvez et

68

al., 2008; Viedma et al., 2009). Although a large number of bacteriocins have already

3

69

been discovered, the corresponding mechanisms by which they exert antibacterial

70

action remain unclear, with the exception of the mechanisms of a few Class I and IIa

71

bacteriocins. One such exception is nisin, a Class I lantibiotic, which acts as a

72

pore-forming agent by docking into lipid II molecules as a target, leading to the

73

inhibition of peptidoglycan biosynthesis (Wiedemann et al., 2001). Another

74

bacteriocin with a known mechanism of action is pediocin PA-1/AcH, a class IIa

75

bacteriocin, which initiates the formation of ion-selective pores into the target cell

76

membrane, leading to the loss of intracellular ATP and the loss of proton motive force

77

(Tiwari et al., 2015). Although these pioneering studies have established a solid basis

78

for understanding the mechanisms of action of bacteriocins, additional research is

79

necessitated to further confirm the underlying mechanisms.

80

Lactobacillus paraplantarum isolated from cheese produces bacteriocin FT259,

81

which has a potential influence on Listeria monocytogenes biofilm formation

82

(Winkelströter et al., 2015). Chopra et al. (2015) investigated a new bacteriocin with

83

the potential to prevent biofilm formation. Because biofilm formation may be

84

regulated by the quorum sensing (QS) system (Algburi et al., 2016; Mhatre et al.,

85

2014), we assumed that certain bacteriocins may also be able to regulate the QS

86

system of sensitive bacteria. Although many bacteriocins have already been isolated

87

and characterized, only two of them (nisin and pediocin PA-1) are commercially

88

available in Europe and the USA (Du et al., 2018).

89

Kombucha has been traditionally used as a functional beverage for thousands of

90

years in South China. Recently, it has gained popularity due to its multiple functional

4

91

properties. It is normally prepared by fermentation of sugared black tea with a

92

symbiotic culture of acetic acid bacteria, yeasts, and other microorganisms known as

93

SCOBY. This beverage can also be brewed using different types of tea and carbon

94

sources. The main acetic acid bacteria isolated from kombucha include: Acetobacter

95

xylinum, Acetobacter xylinoides, Bacterium gluconicum, Acetobacter ketogenum,

96

Acetobacter suboxydans, Gluconobacter liquefaciens, Acetobacter acetiformis, and

97

Acetobacter aceti. Yeasts commonly found in kombucha, include Saccharomyces

98

cerevisiae, Saccharomyces inconspicuous, Lutheran S. ludwigii, Schizosaccharomyces

99

pombe,

Candida

tropicalis,

Candida

krusei,

Debaryomyces

hansenii,

and

100

Zygosaccharomyces bailii et al. On the other hand, Lactobacillus bulagricus,

101

Streptococcus thermophilus, and Lactobacillus plantarum are the main Lactobacillus

102

strains isolated from kombucha. Most of studies focused on fungal flora in

103

kombucha” (Coton et al., 2017; Fu et al., 2014; Yan et al., 2018). In the last few years,

104

researchers have focused on the functional lactic acid bacteria isolated from

105

kombucha, owing to their probiotic benefits. The microbial floras of kombucha made

106

from different sources are different.

107

In this study, a novel bacteriocin named bacteriocin SLG10 was screened and

108

purified from the cell-free supernatant (CFS) of Lactobacillus plantarum SLG10

109

isolated from traditional kombucha in Hanzhong City of China (a city in South China).

110

Along with demonstrating the isolation of this peptide, we also clarified its

111

mechanisms of antibacterial action.

112

5

113

2. Materials and methods

114

2.1 Searching for bacteriocinogenic lactic acid bacteria (LAB)

115

2.1.1 Isolation of LAB

116

Kombucha was purchased from the local market (Hanzhong, Shaanxi, China).

117

One gram of kombucha starter culture was crushed and immersed into 10 mL

118

sterilized distilled water. One loop of the culture was spotted on the De Man, Rogosa

119

and Sharpe agar (MRS, Solarbio, Beijing, China) and cultured at 37

120

medium is consist of peptone (10.0 g), beef paste (10.0g), yeast extract (5.0 g),

121

diammonium hydrogen citrate [(NH4)2HC6H5O7] (2.0 g), glucose (20.0 g), Tween 80

122

(1.0 mL), sodium acetate (CH3COONa·3H2O) (5.0 g), dipotassium hydrogen

123

phosphate (K2HPO4·3H2O) (2.0 g), magnesium sulfate (MgSO4·7H2O) (0.58 g),

124

manganese sulfate (MnSO4·H2O) (0.25 g), agar (20 g/L) and distilled water 1000 mL,

125

pH 6.2~6.6. The Gram staining was manipulated with Gram staining kit (Solarbio,

126

Beijing, China) according to manufacturer’s instruction. The lactic acid bacteria

127

biochemical identification kit (Hopebio, Shanghai, China) was used for catalase- and

128

oxidase- testing. The Gram-positive, catalase-negative, and oxidase-negative colonies

129

were chosen as the possible lactic acid bacteria (LAB) according to Liu et al. (2015).

130

The suspensions of the selected LAB were centrifuged at 8000×g for 20 min at 4

131

remove the cells. Afterward, the supernatants were filtered through 0.22-µm

132

micro-filtering (Millipore Sigma, MA, USA) to obtain the cell free supernatants

133

(CFSs). The antibacterial activity of CFSs towards the Staphylococcus aureus

134

CICC10384 and Escherichia coli CICC 10302 indicator strains was quantified using

6

for 48 h. MRS

to

135

the agar-well-diffusion method (Ayed et al., 2015). Inhibition was recorded as

136

negative if no inhibition zone was observed around the agar well. The standard curve

137

was constructed with the log (antimicrobial activity) and the diameters of the

138

inhibition zone. Antimicrobial activity was expressed as arbitrary units (AU) per mL,

139

and one AU was defined as the reciprocal of the highest dilution showing a clear zone

140

of growth inhibition.

141 142

2.1.2 Identification of strain SLG10

143

The strain was initially identified using a commercially available kit (API 50

144

CHL, BioMerieux, Montalieu Vercie, France) that functions by recognizing the

145

carbohydrate fermentation pattern of a strain according to the kit instructions. The

146

bacterial strain genotype was identified by 16S rRNA gene sequence analysis using

147

7F

148

3’-AGGAGGTGATCCAGCCGCA) (Öz et al., 2017). The DNA of strain SLG10 was

149

extracted with Ezup Column Bacteria Genomic DNA Purification Kit (Sangon,

150

Shanghai, China). The PCR reaction includes: Template DNA (20-50 ng/µL) 0.5 µL,

151

dNTP (2.5 mM) 1 µL, 10 × Buffer (with Mg2+) 2.5 µL, enzyme 0.2 µL, primer 7F 0.5

152

µL, primer 1540R 0.5 µL, and deionized water up to 25 µL. The PCR reaction

153

conditions are as follows: 94

154

and 4

155

purified with SanPrep Column DNA Gel Extraction Kit (Sangon, Shanghai, China).

and

1540R

primers

(5’-CAGAGTTTGATCCTGGCT,

4 min, 94

45s, 55

45s, 72

1min, 72

10 min,

∞ by Sangon Biotech Ltd. Co. (Shanghai, China). The PCR products were

7

156

The similarity search of sequences was performed by conducting a comparison with

157

the NCBI (www.ncbi.nlm.nlh.gov) database.

158 159

2.2 Production of bacteriocins by strain SLG10

160

One loop of stain SLG 10 was added onto 10 mL sterilize MRS broth and

161

cultured at 37℃ for 18 h. After the OD600 value was adjusted to 0.5 and the

162

suspension was inoculated into fresh MRS broth (5%, v/v) and incubated at 37 °C for

163

48h. The optical density at 600 nm (OD600), pH value and the antibacterial acidity of

164

the CFS were monitored every five hours according to Yue et al. (2013). The OD600

165

value was tested by automatic microplate reader (SpectraMax190, Molecular Devices,

166

CA, USA). PH value was tested by pH meter (Rex, Shanghai, China) and the

167

antibacterial activity was tested with the agar-well-diffusion method as described

168

above (Yue et al., 2013). S. aureus CICC 10384 was used as the indicator bacterial

169

strain.

170 171

2.3 Purification of bacteriocin

172

2.3.1 Biochromatography preparation

173

The biochromatography setup was performed according to a slightly modified

174

version of the method used by Tang et al. (2014). HCl (20%, v/v) was applied for 8 h

175

to activate the silica ((Sangon, Shanghai, China)), followed by washing to neutrality

176

using deionized water and drying at 120 °C. Two grams of Egg-yolk

177

phosphatidylcholine

(EYPC)

(Sangon,

Shanghai,

8

China)

and

1

gram

of

178

1,2-Dimyristoyl-sn-glycero-phosphatidylgly-cerol (Sodium Salt) (DMPG) (Sangon,

179

Shanghai, China) (2:1, w/w) was solubilized in CHCl3/CH3OH (2:1, v/v) (Solarbio,

180

Beijing, China), mixed with 20 g of activated silica (Sangon, Shanghai, China) and

181

shaken at a constant temperature (4 °C) for 120 min. The solvent was removed by

182

rotavapor (RV10, AKI, Staufen, Germany), and a dry residue was purged with N2 and

183

vacuum-dried overnight. The liposomes were formed by soaking the lipid film-coated

184

porous silica gel in phosphate-buffered saline (PBS) for 12 hours followed by

185

centrifugation at 5000×g for 20 min at 4℃. Afterward, silica gel was rinsed 3× with

186

10 mM, pH 7.2 PBS buffer and used as a column-packing material for a 250× 4.6 mm

187

glass column.

188 189

2.3.2 Screening and purification of the bacteriocin

190

The column was washed with PBS (10 mM, pH 7.2, 50 mM NaCl). CFS of strain

191

SLG10 was eluted at 25 °C using PBS as a mobile phase (0.5 mL/min flow rate). The

192

elution was monitored spectrophotometrically (SpectraMax190, Molecular Devices,

193

CA, USA) at 215 nm. The same procedure was repeated twenty times to obtain

194

adequate samples. The amount of proteins was determined by the Bradford method

195

(Bradford et al., 1976). The fractions were collected and freeze-dried and the

196

antibacterial activity was screened against S. aureus CICC 10384. The most potent

197

fraction

198

chromatography (RP-HPLC) (Symmetry C18 column, 250 × 4.6 mm, 5 µm, Waters,

199

Dublin, Ireland) using a gradient elution. The content of the mobile phase was

was

then

subjected

to

reversed-phase

9

high-performance

liquid

200

changed by increasing the content of phase B (100% acetonitrile) from 5 to 100% of

201

the mixture with phase A (0.05% (v/v) trifluoroacetic acid (TFA) over 40 min. A 0.5

202

mL/min flow rate was applied with an injection volume of 1 mL, and the temperature

203

was maintained at a constant level (25 °C).

204 205

2.4 Structural characterization

206

The primary structure of the isolated bacteriocin was determined by an N-amino

207

acid analyzer (Procise491, ABI, Thermofisher Scientific CN, Shanghai China),

208

followed by a homology search in the NCBI database (http://www.ncbi.nlm.nih.gov).

209

The higher-level structure was assessed using circular dichroism (CD) spectroscopy.

210

The CD spectra were acquired on a Jasco J-810 CD spectrometer (Jasco Co., Tokyo,

211

Japan) in a 195–250 nm wavelength range using a 0.5 mm path length cell. The

212

physicochemical descriptors were estimated using ProtParam tools in ExPASy

213

ProtParam (https://web.expasy.org/protparam/). The 3D structure of the bacteriocin

214

was modelled using Hyperchem 7.5 software (Hypercube, Gainesville, Florida, USA).

215 216

2.5 Stability of the bacteriocin

217

The stability of the purified bacteriocin SGL10 was assessed. The effect of pH

218

on the antibacterial activity of the bacteriocin was determined by reconstituting the

219

bacteriocin in distilled water (200 AU/mL). The pH was maintained in between 2 and

220

10 using sterile 1 M HCl and 1 M NaOH for 4 h and readjusted to pH 6.5 (the pH of

221

the unadjusted bacteriocin solution) and tested for the antibacterial peptides as

10

222

described by Yue et al. (2013). The temperature dependence of bacteriocin SGL10

223

activity was determined in the environment for 60 ℃ 30 min, 80 ℃ 30 min, and

224

100ºC for 10 min, respectively (Todorov et al., 2011). To evaluate the sensitivity of

225

bacteriocin SLG10 to proteolytic enzymes, 1.0 mg/mL of trypsin (Sangon, Shanghai,

226

China), proteinase K (Sangon, Shanghai, China), papain (Sangon, Shanghai, China),

227

a-chymotrypsin and pepsin (Sangon, Shanghai, China) were added to the bacteriocin

228

SLG10 solution, and the pH of the sample was set at the optimal value for each

229

enzyme. After incubation at 37 °C for 30 min, the reaction was quenched by 5 min of

230

heating at 100 °C. After adjusting the pH to 6.5, the antibacterial activity of the

231

samples was evaluated according to the method described by Yue et al., (2013). The

232

bacteriocin without any treatment was used as the control.

233 234

2.6 Mode of action

235

2.6.1 Minimal inhibitory concentration (MIC) determination

236

The MIC of bacteriocin against sensitive bacteria was assayed following the

237

Clinical and Laboratory Standards Institute (CLSI) procedures (CLSI: Wayne, PA,

238

2012). Bacteriocin solution of 2048 µg/mL was 2-fold serially diluted in fresh

239

sterilized LB medium. Approximately 106 CFU/mL overnight-culture of different

240

indicator bacteria were added to LB medium with 2-fold serially diluted bacteriocin.

241

The indicator microorganisms used in this study are listed in Table 1. The samples

242

were incubated at 37 °C for 24 h. The MIC is the lowest concentration of bacteriocin

243

that can inhibit the growth of indicator strains. The minimum bactericidal

11

244

concentration (MBC) refers to the minimum bacteriocin concentration required to kill

245

99.9% (down by three orders of magnitude) of the tested microorganisms. The growth

246

of bacteria was monitored by testing the OD600 value. The viability of the bacteria was

247

tested by plate count method. PBS buffer (10 mM, pH 6.5) was used as control

248

instead of bacteriocin solution.

249 250

2.6.2 Time-kill kinetics

251

One loop of stain S. aureus CICC 10384 was added onto 10 mL sterilize LB

252

broth and cultured at 37℃ for 18 h. After the OD600 value was adjusted to 0.5 and the

253

suspension was inoculated into the fresh LB broth (5%, v/v) and incubated at 37 °C

254

for 18 h. The cells were washed two times by centrifugation (8000×g) at 4 ℃ for 20

255

min. Then the cells were dissolved into 10 mM pH 6.5 PBS containing 1× or 2× the

256

MIC of bacteriocin with concentration of approximate 108 CFU/mL. The samples

257

were cultured at 37℃. Cell viability was determined by the plate count method every

258

hour. The S. aureus CICC 10384 suspension without bacteriocin treatment was used

259

as a control.

260 261

2.6.3 Leakage of K+

262

The Log-phase S. aureus CICC 10384 cells were collected by centrifugation and

263

dissolved in PBS as described above. After the OD600 of samples were adjusted to 0.5,

264

bacteriocin of 1× or 2× the MIC were added in, respectively. The samples were

265

cultured at 37 ℃for 0, 5, 10, 20, 30, 60, and 90 min. PBS-only treated cells were used

12

266

as controls. Supernatants were obtained after centrifugation at 8000 ×g for 20 min

267

(4 °C) and passed through a 0.22-µm filter (MilliporeSigma, MA, USA). Then the

268

potassium ions of the supernatants were quantified using an Optima 8000 ICP-OES

269

(PerkinElmer Inc., Waltham, MA, USA) (Han et al., 2017).

270

2.6.4 Inhibition of biofilm formation

271

Bacterial biofilms of S. aureus CICC 10384 were cultured using the microporous

272

plate method (Cui et al., 2012). The effect of bacteriocin on the biofilm formation of

273

S. aureus was quantitatively analyzed by staining with crystal violet (Sangon,

274

Shanghai, China). The activated S. aureus was diluted to approximately 108 CFU/mL,

275

and 200 µL cultures were added to each pore of the sterilized 96-well plate.

276

Bacteriocin, with final concentrations of 0.5×, 0.7×, and 0.9× the MIC (to prevent the

277

possibility that the inhibition of biofilm synthesis was due to the reduction in living

278

cells caused by bacteriocin, under-MIC concentrations were used), was added

279

(bacteriocin was not added to the control group). The S. aureus biofilm was cultured

280

at 37 °C for 5 days. The supernatant was removed and the biofilm was washed 3 times

281

with distilled water. Then, the biofilm was fixed with 250 µL of formalin for 5 min,

282

dyed with 250 µL of 0.3% crystal violet for 30 min, rinsed with distilled water 3 times

283

and left to dry. Then, 250 µL of 95% ethanol was added to dissolve the crystal violet

284

bound to the biofilm. A total of 200 µL was absorbed from each pore and placed in

285

another clean 96-well plate to test the absorbance at 595 nm. The habitation rate of the

286

biofilm was calculated as follows: I%=(1-A/A0) × 100%, where A is the absorbance

287

of the sample treated with bacteriocin and A0 is the absorbance of the control.

13

288 289 290

2.7. Statistical analyses Data analysis was performed using the SPSS 18.0 program (IBM SPSS Statistics,

291

Amund, NY, USA) (Tang et al., 2014). All data are the average of three replicates and

292

are represented as the mean ± SD. T-test was used to calculate whether there are

293

significant differences in statistics for the group of data. t>t0 (P<0.05) was considered

294

that the differences are significant. The accurate molecular mass of bacteriocin was

295

determined using the Mass Lynx4.1 software (Waters, Milford, MA, USA).

296 297

3 Results and discussion

298

3.1 Isolation and identification of SLG10

299

Seven strains isolated from traditional kombucha in South China were potentially

300

active strains of LAB. Among them, strain SLG10 was the only one that was able to

301

act on both Gram-positive and Gram-negative bacteria (S. aureus CICC10384 and E.

302

coli CICC10302). After eliminating the possibility that the inhibitory potency of strain

303

SLG10 was due to the organic acid content, strain SLG10 was selected as the

304

bacteriocin-producing strain. From the metabolic profiles of the carbohydrates

305

obtained by the API 50 CHL system, strain SLG10 was identified as Lactobacillus

306

plantarum. Phylogenetic tree based on the 16S rDNA sequences of the strain SLG10

307

was shown on Fig. 1. According to Fig.1, L. plantarum is the closest species to strain

308

SLG10. It is suggested that strain SLG10 is the L. plantarum; the results which are in

309

line with the metabolic identification kit. Thence, we named strain SLG10 as

310

Lactobacillus plantarum SLG10. 14

311

For thousands of years, kombucha has been believed to improve gastric health in

312

Chinese people and to contain a varied microbial community (Coton et al., 2014).

313

Acetic acid bacteria, yeast, and lactic acid bacteria are the main microorganisms in

314

kombucha. In the past years, researchers pay much attention on fungal compositions

315

of kombucha (Fu et al., 2014.). However, kombucha may also be considered as a

316

potentially rich source of functional lactic acid bacteria (Yan et al., 2018). The

317

application of L. plantarum in food is well documented; the majority of studies

318

address its safety profiles (Domingos-Lopes et al., 2017; Seddik et al., 2017).

319

Nowadays, L. plantarum, probiotic strain, is generally regarded as safe. SLG10 as a L.

320

plantarum, may not only be used as a bacteriocin-producing strain but also has

321

potential as a starter culture for fermented foods.

322 323

3.2 Production of bacteriocin SLG10

324

Bacteriocin SLG10 production started at 20 hours and the maximal bacteriocin

325

SLG10 production was recorded after 30 hours of growth in MRS broth (Fig. 2).

326

Similar results were also observed for other LAB bacteriocins, such as plantaricin

327

GZ1-27, plantaricin JLA-9, and plantaricin K25 (Du et al., 2018; Wen et al., 2016;

328

Zhao et al., 2016). This observation leads to the idea that bacteriocin production is a

329

secondary metabolic production that is dependent upon the cell number. However,

330

other study has already reported that some lactic acid bacteria, such as Bacillus

331

amyloliquefaciens An6, could produce bacteriocins as primary metabolites (Ayed et

332

al., 2015). It seems there is no specific rule to infer whether a bacteriocin produced

15

333

from lactic acid bacteria is a primary or a secondary metabolite.

334 335

3.3 Purification of bacteriocin

336

The biochromatography elution is shown in Fig. 3A. Only one elution was

337

obtained, and the antibacterial activity test suggested that this elution was the

338

bacteriocin fraction. The results indicated that biochromatography has good

339

specificity for identifying the bacteriocin. The elution was pooled and further

340

separated using RP-HPLC with the main peak shown in Fig. 3B. The retain time is

341

21.3 min.

342

The traditional methods for the isolation of bacteriocin from complex mixtures

343

frequently involve complicated chromatographic separations (Heredia-Castro et al.,

344

2015; Yi et al., 2010). Such methods are effective but too complex (Hwanhlem et al.,

345

2017; Panagiota et al., 2016). It is believed that the initial step of the antibacterial

346

action of any bacteriocin demands an exchange with the cell membrane of the

347

bacterial target (Paiva et al., 2012; Snyder et al., 2014). Based on that, several

348

bio-chromatography techniques were developed to isolate antimicrobial peptides. For

349

instance, Tang et al. (2014) developed an efficient immobilized bacterial membrane

350

liposome chromatography for successful isolation of potential antimicrobial peptides

351

from boiled-dried anchovies. On the other hand, Ge et al. (2016) constructed

352

bio-chromatography and successfully purified novel bacteriocin synthesized by

353

Lactobacillus paracasei HD1-7 isolated from Chinese Sauerkraut juice. In this study,

16

354

biochromatography was able to quickly screen and isolate bacteriocin from the cell

355

free supernatants (CFS) of strain SLG10.

356 357

3.4 The structural depiction of bacteriocin SLG10

358

MS analyses revealed a characteristic peak associated with a species having a

359

mass of 1422 Da (Fig. 4A). The primary structure of this compound was identified as

360

a decapeptide with the sequence Asn- Ile- Val- Trp- Gln- Leu- Ile- Gly- Leu- Pro- Ala-

361

Gln- Ala (NIVWQLIGLPAQA). The sequence was different from those of any of the

362

known bacteriocins in the NCBI database, as shown by the BLAST analysis, so the

363

isolated peptide is a novel bacteriocin named SLG10. Bacteriocin SLG10 falls into

364

the category of Class IId bacteriocins because it does not contain lanthionine or

365

YGNGVXC (characteristics of Class IIa bacteriocins).

366

The CD spectrum suggested that bacteriocin SLG10 exhibits an irregular coil

367

formation (Fig. 4B), and the predicted three divisions of the structure of bacteriocin

368

SLG10 showed that it exhibits an irregular linear formation (Fig. 4C). The theoretical

369

mass of bacteriocin SLG 10 is 1422.26 Da, which is consistent with the findings of

370

the MS testing (Fig. 4C).

371

Although bacteriocins are generally over 2 kDa in Mw, lower-weight bacteriocins

372

such as bifidocin A (1198.68 Da), plantaricin JLA-9 (1044 Da), plantaricin GZ1-27

373

(975 Da), and plantaricin K25 (1772 Da) have also been reported (Du et al., 2018; Liu

374

et al., 2015; Wen et al., 2016; Zhao et al., 2016). Very recently, the mode of action of

375

small size bacteriocins attracts researchers’ attention. Because of the small size,

17

376

bacteriocins might not be able to cause “pore formation” on sensitive bacteria. They

377

might inhibit cell wall/membrane synthesis through binding with precursors of cell

378

wall/membrane synthesis, destroying integrity of wall/membrane, or interacting with

379

enzyme system, or DNA in cells (Miao et al., 2014; Zhao et al., 2016). Structurally,

380

bacteriocin SLG10 resembles other small and hydrophobic bacteriocins with a

381

random coil but well-defined conformation such as bacteriocin F1, Plantaricin JLA-9

382

and so on. These structural features might increase the stability of bacteriocins in

383

complex environments (Zhao et al., 2016).

384 385

3.5 Stability of bacteriocin SLG10

386

The bacteriocin retained its antibacterial activity after heating treatments in this

387

study and still retained its inhibitory activity after storage at 37 °C for 14 days or even

388

after 2 months at 4 °C (Fig.5.A). Bacteriocin SLG10 was stable in a pH range of

389

2.0–7.0, but the activity decreased at pH 8.0 and above (Fig.5). This finding is in

390

accordance with previous reports showing that the alkaline medium easily inactivates

391

most bacteriocins, such as bacteriocin RC20975, plantaricin JLA-9, and plantaricin

392

GZ1-27 (Due et al., 2018; Yue et al., 2013; Zhao et al., 2016). These results suggested

393

that bacteriocin SLG10 would remain notably stable during food processing. It is

394

interesting to note that bacteriocin SLG10 is insensitive to trypsin and pepsin

395

(Fig.5.C). Bacteriocins, such as bifidocin A, paracin C, and bacteriocin RC20975, are

396

always sensitive to proteases (Liu et al., 2015; Yue et al., 2013). However, several

397

bacteriocins are insensitive to several proteases. For example, plantaricin JLA-9 and

18

398

plantaricin K25 are also insensitive to pepsin and trypsin (Wen et al., 2016; Zhao et al.,

399

2016). The lack of sensitivity might be attributed to the small sizes of peptides, such

400

as plantaricin JLA-9 (1004 Da, Wen et al., 2016), plantaricin K25 (1772 Da, Wen et

401

al., 2016), and bacteriocin SLG10 (1422 Da, in this study). Additionally, the

402

sequences of these peptides are lacking the Phe, Trp, Try, Lys, and Arg residues,

403

which are the endonuclease sites of trypsin (Lys and Arg) and pepsin (Phe, Trp, Try).

404

It might also refer to the advanced conformation of these peptides. The exact reasons

405

remain unknown and this necessitated further research works.

406 407

3.6 Mode of action of bacteriocin SLG10

408

3.6.1 Antimicrobial spectrum and MICs

409

The antimicrobial activity of bacteriocin SLG10 is shown in Table 1. Bacteriocin

410

SLG10 inhibited both Gram-positive (Bacillus subtilis, Bacillus cereus, Bacillus

411

megaterium, Micrococcus luteus, Brochothrix thermosphacta, Clostridium butyricum,

412

S. aureus, Listeria innocua, and L. monocytogenes) and Gram-negative bacteria (E.

413

coli). Importantly, bacteriocin SLG10 is also active against methicillin-resistant S.

414

aureus. This activity may be due to the fact that the bacteriocin has a different mode

415

of action than antibiotics. It is also worth noting that bacteriocin SLG10 is also active

416

against the Gram-negative bacteria E. coli, because most bacteriocins were reported to

417

be active only against Gram-positive bacteria (Du et al., 2018；Liu et al., 2015; Yue et

418

al., 2013). Most of the new bacteriocins (reported over the last 10 years) belong to the

419

Class I and Class II (Ahn et al., 2017; Todorov et al., 2011). The antibacterial

19

420

spectrum was narrow with activity only against Gram-positive bacteria. Nisin, a Class

421

I lantibiotic and pediocin PA-1/AcH, a Class IIa bacteriocin, are working through

422

interaction with Gram-positive bacterial cell membrane. Firstly, bacteriocins are

423

linked with bacterial outer membrane receptor and then introduced into the cell

424

forming pores and leakage of intracellular materials (Tiwari et al., 2015; Wiedemann

425

et al., 2001). On the other hand, multiple studies have suggested that some

426

bacteriocins have the ability to interact with intracellular enzyme system and nucleic

427

acid, besides damaging the integrity of the bacterial cell membrane. These

428

bacteriocins may be active against Gram-negative bacteria, even though some fungi

429

(Acuna et al., 2012; Du et al., 2018; Hwanhlem et al., 2017). The MICs of bacteriocin

430

SLG10 ranged from 16-32 µg/mL according to the sensitive bacteria (Table 1).

431 432

3.6.2 Kill kinetics curve of bacteriocin SLG 10 against S. aureus

433

After treatment with 2× the MIC of bacteriocin SLG10, the number of S. aureus

434

cells decreased quickly within 30 min, while the log10 CFU decreased below 1 after

435

60 min (Fig. 6). Furthermore, 1× the MIC of bacteriocin SLG10 destroyed all S.

436

aureus cells within 120 min (Fig. 6). These results indicated the fast and sustained

437

action of bacteriocin SLG10 against S. aureus. The effect of bacteriocin SLG10 on

438

the sensitive bacterial strains was bactericidal. Almost all bacteriocins, such as

439

bifidocin A, enterocin FH99, plantaricin GZ1-27, and bacteriocin BacC1 (Du et al.,

440

2018; Goh et al., 2015; Kaur et al., 2013; Liu et al., 2015), have bactericidal effect

441

(Hwanhlem et al., 2017; Todorov et al., 2011). Although the mechanisms of action of

20

442

different types of bacteriocins are not the same, we might suggest that they may have

443

a common way of action.

444 445

3.6.3 Membrane permeability detection

446

The permeability of drugs through the bacterial cell membrane can be studied by

447

quantifying the amount of K+ ions released into the surroundings upon treatment. Ten

448

minutes after the treatment of cells with bacteriocin SLG10, a severe loss of

449

intracellular potassium ions was observed, while the control cells were mostly intact

450

(Fig. 7). The treatment of cells with 1× the MIC of SLG10 elevated the extracellular

451

potassium ion level up to 0.63 mg/mL during the first hour, and the amount remained

452

stable in the next hour.

453

These results confirmed the ability of bacteriocin SLG 10 to make S. aureus cell

454

membranes more permeable, leading to potassium ion efflux. A similar mode of

455

action is found for bifidocin A, Bacteriocin RC20975, bificin C6165, paracin C,

456

Enterocin FH99 and bacteriocin BacC1 (Goh et al., 2015; Kaur et al., 2013; Liu et al.,

457

2015; Yue et al., 2013). It seems like that damaging the integrity of cell membranes of

458

the sensitive bacteria and cause the leakages of intracellular contents and eventually

459

death of cells is one of the common mode of action of bacteriocins from lactic acid

460

bacteria, no matter which classes the bacteriocins belonged to (Ahn et al., 2017; Du et

461

al., 2018).

462 463

3.6.4 Inhibition of S. aureus biofilm formation by bacteriocin SLG10

21

464

As shown in Fig.8, the rates of bacteriocin SLG10 inhibition of S. aureus biofilm

465

formation reached 16.8%, 45.6%, and 56.1% at 0.5× the MIC, 0.7×the MIC and 0.9×

466

the MIC, respectively (Fig. 8). It is postulated that 99% of all bacterial cells exist as

467

biofilms and that only 1% live in a planktonic state (Winkelströter et al., 2015). The

468

biofilm state allows the bacteria that are integrated into the biofilm to be protected

469

from fluctuations in environmental conditions such as humidity, temperature, pH and,

470

in the case of infections, antibacterial preparations applied to the host organism,

471

lengthening the infection and providing concentrated nutrients and waste disposal

472

mechanisms (Chopra et al., 2015). Food spoilage or pathogens can form biofilms on

473

the surface of containers or packages, causing contamination. Bacteriocin SLG10 may

474

provide a good way to inhibit the biofilm formation of food spoilage bacteria or

475

pathogens in the food industry. Researches on mode of action of bacteriocins from

476

lactic acid bacteria mostly focused on the effect of bacteriocins on sensitive bacterial

477

cells. Very recently, some studies have shown that bacteriocins from LAB might also

478

inhibit the formation of biofilm of sensitive bacteria. In this context, Chopra et al.

479

(2015) suggested a new bacteriocin, which is able to inhibit the formation of E. coli

480

biofilm. On the other hand, Winkelströter et al. (2015) found that bacteriocin from

481

Lactobacillus

482

monocytogenes biofilm formation

paraplantarum,

FT259

was

483 484

4. Conclusions

22

able

to

influence

on

Listeria

485

In conclusion, the SLG10 strain, identified as Lactobacillus plantarum, was

486

isolated from traditional kombucha in South China. Biochromatography coupled with

487

RP-HPLC was used for the purification of bacteriocin SLG10 from the CFS of the

488

SLG10 strain. This novel Class IId bacteriocin was active against both G+ and G–

489

bacteria. Bacteriocin SLG10 acts by increasing the permeability of the cell membrane,

490

which eventually leads to bacterial cell death. Bacteriocin SLG10 can also inhibit S.

491

aureus biofilm formation. The novel bacteriocin discovered herein is a promising

492

antibacterial agent with potential to be used in the food preservation or

493

pharmaceutical industries.

494 495

Acknowledgements

496

This study was funded by the National Natural Science Foundation of China

497

(31801563), the Research Foundation of Science and Technology Bureau of Shaanxi,

498

China (2015SZS-15-05), and scientific funding from the Collaborative Innovation

499

Center of Biological Resources Comprehensive Development (QBXT-17-3).

500 501

References

502

Acuna, L., Picariello, G., Sesma, F., Morero, R.D., Bellomio, A. (2012). A new

503

hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against

504

pathogenic Gram-positive and Gram-negative bacteria. FEBS Open Biology, 2,

505

12-19.

23

506

Ahn, H., Kim, J., Kim, W.J. (2017). Isolation and characterization of

507

bacteriocin-producing Pediococcus acidilactici HW01 from malt and its

508

potential to control beer spoilage lactic acid bacteria. Food Control, 80, 59-66.

509

Algburi, A., Zehm, S., Netrebov, V. (2016). Subtilosin Prevents Biofilm Formation

510

by Inhibiting Bacterial Quorum Sensing. Probiotics & Antimicrobial Proteins,

511

2016, 1-10.

512

Ayed, H. B., Maalej, H., Hmidet, N., Nasri, M. (2015). Isolation and biochemical

513

characterisation of a bacteriocin-like substance produced by Bacillus

514

amyloliquefaciens An6. Journal of Global Antimicrobial Resistance, 3(4),

515

255-261.

516

Bradford, M.M.1976. A rapid and sensitive method for the quantification of

517

microgram quantities of protein utilizing the principle of protein-dye binding.

518

Analytical Biochemistry, 72, 248-254.

519

Chopra, L., Singh, G., Kumar, J. K. (2015). Sonorensin: A new bacteriocin with

520

potential of an anti-biofilm agent and a food biopreservative. Scientific Reports,

521

5, 13412.

522

Clinical and Laboratory Standards Institute. (2012). Methods for dilution

523

antimicrobial susceptibility tests for bacteria that grow aerobically, approved

524

standards, 9th ed.; Document M07-A9; CLSI: Wayne, PA.

525

Coton, M., Pawtowski, A., Taminiau, B., Burgaud, G., & Coton, E. (2017).

526

Unraveling microbial ecology of industrial-scale kombucha fermentations by

527

metabarcoding and culture-based methods. Fems Microbiology Ecology, 93(5).

24

528 529

Cotter, P. D., Hill, C., Ross, R.P. (2005). Bacteriocins: developing innate immunity for food. Nature Review in Microbiology, 3, 777-788.

530

Cui, Y. Zhao, Y., Tian, Y., Zhang, W., Li, X., Jiang, X. (2012). The molecular

531

mechanism of action of bactericidal gold nanoparticles on Escherichia coli.

532

Biomaterials, 33(7), 2327-2333.

533

Domingos-Lopes, M.F.P., Stanton, C., Ross, P.R., Dapkevicius, M.L.E., Silva, C.C.G.

534

(2017).Genetic diversity, safety and technological characterization of lactic acid

535

bacteria isolated from artisanal Pico cheese. Food Microbiology, 63, 178-190.

536

Du, H., Yang, J., Lu, X., Lu, Z., Bie, X., Zhao, H., Zhang, C., Lu F. (2018).

537

Purification, characterization, and mode of action of action of Plantaricin

538

GZ1-27, a novel bactriocin against Bacillus cereus. Journal of Agricultural and

539

Food Chemistry, 66, 4716-4724

540

Fu, C., Yan, Fen, Cao, Zeli, Xie, Fanying, & Lin, Juan. (2014). Antioxidant activities

541

of kombucha prepared from three different substrates and changes in content of

542

probiotics during storage. Food Science & Technology, 34(1), 123-126.

543

Gálvez, A., López, R., Abriouel, H., Valdivia, E., Omar, N. B. (2008). Application of

544

bacteriocins in the control of foodborne pathogenic and spoilage bacteria.

545

Critical Review in Biotechnology, 28, 125-152.

546

Ge, J., Sun, Y., Xin, X., Wang, Y., Ping, W. (2016). Purification and Partial

547

Characterization of a Novel Bacteriocin Synthesized by Lactobacillus paracasei

548

HD1-7 Isolated from Chinese Sauerkraut Juice. Scientific Report. DOI:

549

10.1038/srep19366.

25

550

Goh, H.F., Philip, K. (2015). Isolation and mode of action of bacteriocin BacC1

551

produced by nonpathogenic Enterococcus faecium C1. Journal of Dairy Science,

552

98(8), 5080- 5090.

553

Han, J., Zhao, S., Ma, Z., Gao, L., Liu, H., Muhammad, U., Lu, Z., Lv, F., Bie, X.

554

(2017). The antibacterial activity and modes of LI-F type antimicrobial peptides

555

against Bacillus cereus in vitro. Journal of Applied Microbiology, 123, 602-614.

556

Heredia-Castro, P.Y., Méndez-Romero, J.I., Hernández-Mendoza, A., Acedo-Félix,

557

E., González-Córdova, A.F., Vallejo-Cordoba, B. (2015). Antimicrobial activity

558

and partial characterization of bacteriocin-like inhibitory substances produced by

559

Lactobacillus spp. isolated from artisanal Mexican cheese, Journal of Dairy

560

Science, 98(12), 8285-8293.

561

Hwanhlem, N., Ivanova, T., Biscola,V.,Choiset, Y., Haertlé, T. (2017). Bacteriocin

562

producing Enterococcus faecalis isolated from chicken gastrointestinal tract

563

originating from Phitsanulok, Thailand: Isolation, screening, safety evaluation

564

and probiotic properties. Food Control, 78, 187-195.

565

Kaur, G., Singh, T. P., Malik, R. K. (2013). Antibacterial efficacy of Nisin, Pediocin

566

34 and Enterocin FH99 against Listeria monocytogenes and cross resistance of

567

its bacteriocin resistant variants to common food preservatives. Brazil Journal of

568

Microbiology, 14(44), 63-71.

569

Liu, G., Ren, L., Song, Z., Wang, C., Sun, B. (2015). Purification and characteristics

570

of bifidocin A, a novel bacteriocin produced by Bifidobacterium animals BB04

571

from centenarians’ intestine. Food control, 50, 889-895.

26

572

Mhatre, E., Monterrosa, R. G., Kovács, Á. T. (2014). From environmental signals to

573

regulators: Modulation of biofilm development in Gram positive bacteria.

574

Journal of Basic Microbiology, 54(7), 616-632.

575

Miao, J., Guo, H., Ou, Y., Liu, G., Fang, X., Liao, Z., Ke, C., Chen, Y., Zhao, L., Cao,

576

Y. (2014). Purification and characterization of bacteriocin F1, a novel

577

bacteriocin produced by Lactobacillus paracasei subsp. tolerans FX-6 from

578

Tibetan kefir, a traditional fermented milk from Tibet, China. Food Control, 42,

579

48-53.

580 581

Öz, E., Kaban, G., Barış, Ö., Kaya, M. (2017). Isolation and identification of lactic acid bacteria from pastırma. Food Control, 77, 158-162.

582

Paiva, A. D., Irving, N., Breukink, E., Mantovani, H. C. (2012). Interaction with lipid

583

II induces conformational changes in bovicin HC5 Structure. Antimicrobial

584

Agents and Chemotherapy, 56(9), 4586-4593.

585

Panagiota, K., Kyriakou, E. B., Kristiansen, P. E., Kaznessis, Y. N. (2016).

586

Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated

587

through molecular dynamics simulations. Biochimica et Biophysica Acta (BBA) –

588

Biomembranes, 1858(4), 824-835.

589

Seddik, H. A., Bendali, F., Gancel, F., Fliss, I., Spano, G., Drider, D. (2017).

590

Lactobacillus plantarum and its probiotic and food potentialities. Probiotics

591

Antimicrobial Protein, 9, 111-122.

27

592

Snyder, A. B., Worobo, R. W. (2014). Chemical and genetic characterization of

593

bacteriocins: antimicrobial peptides for food safety. Journal of Science of Food

594

and Agriculture, 94(1), 28-44.

595

Tang, W. T., Zhang, H., Wang, L. (2014). New cationic antibacterial peptide screened

596

from boiled-dried anchovies by immobilized bacterial membrane liposome

597

chromatography. Journal of Agricultural and Food Chemistry, 62(7), 1564-1571.

598

Tiwari, S. K., Noll, S.K., Cavera, V. L., Chikindas, M. L. (2015). Improved

599

antimicrobial activities of synthetic-hybrid bacteriocins designed from enterocin

600

E50-52 and pediocin PA-1. Applied Environmental Microbiology, 81,

601

1661-1667.

602

Todorov, S. D., Prévost, H., Lebois, M.., Dousset, X., LeBlanc, J. G., Franco, B. D.

603

G. M. (2011). Bacteriocinogenic Lactobacillus rhamnosus ST16Pa isolated from

604

papaya (Carica papaya) - From isolation to application: Characterization of a

605

bacteriocin.

606

Food Research International, 44, 1351-1363.

Viedma, P. M., Abriouel, H., Omar, N. B., Lopez, R. L., Galvez, A. (2009).

607

Antistaphylococcal

608

of

609

antimicrobials. Journal of Food Science, 74, 384-389.

vegetable

effect

origin,

of enterocin alone

and

AS-48 in

in

bakery

combination

with

ingredients selected

610

Wen, L. S., Philip, K., Ajam, N. (2016). Purification, characterization and mode of

611

action of plantaricin K25 produced by Lactobacillus plantarum. Food Control,

612

60, 430-439.

28

613

Wiedemann, I., Breukink, E., van Kraaij, C., Kuipers, O. P., Bierbaum, G., de Kruijff,

614

B., Sahl, H. G. (2001). Specific binding of nisin to the peptidoglycan precursor

615

lipid II combines pore formation and inhibition of cell wall biosynthesis for

616

potent antibiotic activity. Journal of Biology Chemistry, 276, 1772-1779.

617

Winkelströter, L.K., Tulini, F.L., De Martinis, E.C.P. (2015). Identification of the

618

bacteriocin produced by cheese isolate Lactobacillus paraplantarum, FT259 and

619

its potential influence on Listeria monocytogenes biofilm formation. LWT-Food

620

Science and Technology, 64(2),586-592.

621

Yan, H., Xi, G., Yun, R. , Yixiao, C., & Meiqin, F. (2018). Isolation and biological

622

characterization of lactic acid bacteria from kombucha. Journal of Dairy Science

623

and Technology.

624

Yi, H. X., Zhang, W. L., Tuo, Y. F., Han, X., Du, M. (2010). A novel method for

625

rapid detection of class IIa bacteriocin-producing lactic acid bacteria．Food

626

Control, 21, 426-430.

627

Yue, T. L., Pei, J. J., Yuan, Y. H. (2013). Purification and Characterization of

628

Anti-Alicyclobacillus Bacteriocin Produced by Lactobacillus rhamnosus.

629

Journal of Food Protection, 76, 1575-1581.

630

Zhao, S. G., Han, J. Z., Bie, X. M., Lu, Z.X., Zhang, C., Lv, F. X. (2016). Purification

631

and Characterization of Bacteriocin JLA-9: A Novel Bacteriocin against Bacillus

632

spp. Produced by Lactobacillus rhamnosus JLA-9 from Suan-Tsai, a Traditional

633

Chinese Fermented Cabbage. Journal of Agricultural and Food Chemistry, 64,

634

2754-2764.

635 29

636

Figure captions

637

Fig. 1 Phylogenetic tree based on the 16S rDNA sequences of the isolate

638

Fig. 2 Production of bacteriocin SLG10

639

Fig. 3 Purification of bacteriocin SLG10. A: spectrum of biochromatography; B:

640

spectrum of RP-HPLC.

641

Fig. 4 Structure of bacteriocin SLG10. A: Mass spectrometry of bacteriocin SLG10

642

by MALDI-TOF MS; B: CD spectrum of bacteriocin SLG10; C: The predicted

643

three-dimensional structure and information of bacteriocin SLG10.

644

Fig 5 Effect of enzymes, temperature, and pH on bacteriocin SLG10.A: T1- Lipase;

645

T2-a-amylase; T3-proteinase K; T4-papain; T5- a-chymotrypsin; T6-trypsin;

646

T7-pepsin; T8-Catalase; B: T1-60 ℃；T2-80℃；T3-100℃；T4-37℃for 14 d; T5-2

647

month at 4 ℃; C: T1-pH2; T2-pH3; T3-pH4; T4-pH5; T5-pH6; T6-pH7; T7-pH8;

648

T8-pH9; T9-pH10. Three replicates were same so there is no Standard Deviation.

649

Fig. 6 The viability of S. aureus CICC 10384 cells after treatment with 1× the MIC

650

(▲), 2× the MIC (●) and without (▇) bacteriocin SLG10.

651

Fig. 7 Leakage of K+ ions from S. aureus CICC 10384 cells treated with 1× the MIC

652

(▲), 2× the MIC (●) and without (▇) bacteriocin SLG10.

653

Fig. 8 inhibition rate of biofilm formation for 0.5× the MIC, 0.7× the MIC and 0.7×

654

the MIC bacteriocin SLG10.

655

30

656

Table 1 Antimicrobial activity of bacteriocin SLG10 Microorganisms

MICs

MBCs

Gram-positive bacteria

µg/mL

µg/mL

Bacillus subtilis CICC 10034

16

32

B. cereus CICC 2155

16

32

Micrococcus luteus CICC 10209

16

32

Brochothrix thermosphacta CICC 10509

16

32

Clostridium butyricum CICC 10350

32

64

Staphylococcus aureus CICC 10384

16

32

S. aureus CICC 10201

16

32

Methicillin-resistant S. aureus*

16

32

Listeria innocua CICC 10416

8

16

L. monocytogenes CICC 21529

8

16

Escherichia coli CICC 10302

32

64

E. coli CGMCC 3373

32

64

E. coli CICC 10300

32

64

Pseudomonas aeruginosa CICC 21636

-

-

Enterobacter cloacae CICC 21539

-

-

Salmonella paratyphi β CICC 10437

-

-

-

-

Gram-negative bacteria

Funal Aspergillus niger CICC 2124

31

Candida albicans CICC 1965

-

-

Saccharomyces cerevisiae CICC 1002

-

-

657

CICC: China Center of Industrial Culture Collection

658

* Methicillin-resistant S. aureus was provided by the local hospital in Xi’an, Shaanxi,

659

China

660

MIC: is the lowest concentration of bacteriocin that can inhibit the growth of indicator

661

strains.

662

MBC: the minimum bacteriocin concentration required to kill 99.9% (down by three

663

orders of magnitude) of the tested microorganisms.

664

32

665

Fig. 1

666 667

33

668

Fig. 2

669 670

34

671

Fig. 3. A

672 673

B

674 675

35

676

Fig. 4. A

677 678

B.

679 680

C.

681 682

36

683

Fig. 5.A

684 685

B.

686 687

C.

688

37

689

Fig. 6

690 691

38

692

Fig. 7

693 694

39

695

Fig. 8

696 697 698 699

40

1. A bacteriocin- producer strain was isolated from kombucha 2. Bacteriocin SLG10 was purified using biochromatography and RP-HPLC 3. Bacteriocin SLG10 was active against both G+ and G- bacteria 4. Bacteriocin wasable to inhibit the formation of biofilms of S. aureus