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.

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 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

1
6 Shaanxi Key Laboratory of Bioresource, Collage of Bioscience and Bioengineering,

7 Shaanxi University of Technology, Hanzhong, Shaanxi, China

2
8 Key Laboratory of Tibetan Medicine Research in Chinese Academy of Sciences, Qing

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

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

3
11 Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University,

12 12211-Giza, Egypt
4
13 Department of Medical Pharmacology, Medical Faculty, Ataturk University,

14 25240-Erzurum, Turkey
5
15 International Research Centre "Biotechnologies of the Third Millennium", ITMO

16 University, St. Petersburg 191002, Russia

6
17 College of Food Science, Qilu University of Technology, Jinan, Shandong, China
7
18 College of Food Science, Northwest University, Xian, Shaanxi, China

19

20 *Corresponding authors:

21 Jinjin Pei: jinjinpeislg@163.com

22 Dejing Chen: 851268574@qq.com

23 Tianli Yue: yuetl305@nwsuaf.edu.cn

24 Hongxia Zhang: zhanghongxia326@hotmail.com

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 method, biochromatography coupled with reversed-phase

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 of 1422 Da. The amino acid sequence was

39 Asn-Ile-Val-Trp-Gln-Leu-Ile-Gly-Leu-Pro-Ala-Gln-Al, as determined by

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

2
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 for 48 h. MRS

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 to

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
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 and 1540R primers (5’-CAGAGTTTGATCCTGGCT,

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 4 min, 94 45s, 55 45s, 72 1min, 72 10 min,

154 and 4 ∞ by Sangon Biotech Ltd. Co. (Shanghai, China). The PCR products were

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

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, China) and 1 gram of

8
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 was then subjected to reversed-phase high-performance liquid

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

9
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 2.7. Statistical analyses

290 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 paraplantarum, FT259 was able to influence on Listeria

482 monocytogenes biofilm formation

483

484 4. Conclusions

22
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

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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

Gram-negative bacteria

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 - -

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