J Korean Soc Appl Biol Chem (2015) 58(5):703–714 Online ISSN 2234-344X DOI 10.1007/s13765-015-0094-y Print ISSN 1738-2203

ARTICLE

Purification and characterization of two bacteriocins from Lactobacillus brevis BK11 and Enterococcus faecalis BK61 showing anti-Helicobacter pylori activity

Eun-Seo Lim1

Received: 9 April 2015 / Accepted: 15 June 2015 / Published online: 7 July 2015 Ó The Korean Society for Applied Biological Chemistry 2015

Abstract The aim of this study was to investigate the Keywords Adhesion Á Bacteriocin Á Enterococcus antimicrobial effects of purified bacteriocins isolated from faecalis Á Helicobacter pylori Á Lactobacillus brevis Á Lactobacillus brevis BK11 and Enterococcus faecalis Urease BK61 on Helicobacter pylori. After the final purification step, the molecular weights of the purified bacteriocins from L. brevis BK11 and E. faecalis BK61 were estimated Introduction to be approximately 6.5 and 4.5 kDa, respectively. Sig- nificant decrease in the antimicrobial activity of these The colonization of the mucus layer of the gastric epithelium bacteriocins was observed when they were treated with by Helicobacter pylori is the causative agent of gastric several proteolytic enzymes. However, their antimicrobial disorders such as gastritis, peptic ulcer, nonulcerous dys- activity was stable over long periods of storage and a wide pepsia, and possibly gastric cancer (Malaty and Nyren range of pH and was relatively heat resistant. The inhibi- 2003). The pathogenesis of H. pylori is caused by the tory spectrum of the bacteriocin produced by L. brevis adherence of pathogens against host tissue-specific cell BK11 strain was quite narrow, whereas the bacteriocin receptors, the production of bacterial urease, and cytotoxins produced from E. faecalis BK61 strain exhibited a broad such as VacA, CagA, and endotoxin, and the pathological antimicrobial spectrum against various foodborne patho- changes of gastric epithelial cells (Iwakura et al. 2007). At gens such as Listeria monocytogenes, Escherichia coli least one-third of the world’s population is infected with H. O157, and Salmonella enteritidis. Treatment of H. pylori pylori, so treatment of this pathogen infection is widely with these bacteriocins significantly reduced the number of recommended (Perez–Perez et al. 2004). cells of the pathogen found adhering to the monolayers of H. pylori eradication therapy with proton pump inhibi- cultured human gastric adenocarcinoma epithelial cell line tors and several antibiotics such as ranitidine bismuth (p \ 0.05). Furthermore, the urease activity of adherent H. citrate, amoxicillin, and clarithromycin has been widely pylori after treatment with the bacteriocins was lower than proposed, but these regimens have developed resistance that for the control group, but there appeared to be no strains of H. pylori (Malfertheiner et al. 2012). In addition, significant difference between the competition and dis- antibiotic-associated gastrointestinal side effects are diar- placement groups (p [ 0.05). rhea, nausea, vomiting, bloating, abdominal pain, and esophageal reflux, which were due to an increase in the gastric acidity (De Francesco et al. 2010). Hence, natural food substances, such as apple peel polyphenols and green tea extract, and probiotic foods fermented with lactic acid bacteria (LAB) can be used as an adjuvant for antibiotic- & Eun-Seo Lim antacid treatment to prevent the re-emergence of H. pylori [email protected] infection (Lee et al. 2009; Pastene et al. 2010). 1 Department of Food Science & Nutrition, Tongmyong A probiotic has been defined as a live microbial species, University, Busan 608-735, Republic of Korea which can have a positive effect on intestinal physiological 123 704 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 functions with improved health conditions on the host bovine serum (FBS, Gibco BRL, USA), 0.2 % (w/v) 2,6- when administered in adequate amounts (FAO/WHO di-o-methyl-b-cyclodextrin (CD), and antibiotics (cefsu- 2001). Because probiotics including lactobacilli and bifi- lodine, vancomycin, trimethoprim, and amphotericin B, dobacteria are acid tolerant and able to persist in the Sigma) and the plate was incubated in a gas jar with a stomach longer than other bacteria, some LAB prepara- microaerophilic atmosphere (10 % CO2, Anoxomat sys- tions have been proved to play a crucial role of stabilizing tem, MART Co., Lichtenvoorde, Netherlands) for 48 h at intra-gastric micro-ecological environment (Zheng et al. 37 °C. The strain was maintained as frozen stock held at 2013). The search for new antimicrobial agents showed -80 °C in Brucella broth (Difco.) plus 20 % (v/v) that the implementation of standard anti-H. pylori regimens glycerol. with probiotic LAB could be able to improve mucosal inflammation in animals infected with H. pylori and sup- Purification of bacteriocin press the secretion of interleukin (IL)-8 in gastric cells infected by H. pylori, as they are able to improve the The LAB strains were grown in MRS broth (Difco.) at patient’s compliance by reducing antibiotic-associated 37 °C under aerobic conditions. The cell-free culture adverse events (Armuzzi et al. 2001; Nista et al. 2004). supernatant (CFCS) was derived from fresh overnight LAB LAB intended for antibacterial chemotherapy against cultures adjusted to 1.0 9 109 CFU of the bacteria per mL pathogens may be used as an adjuvant to antibiotic treat- by centrifugation (70009g for 10 min at 4 °C), neutralized ment to prevent the re-emergence of H. pylori infection, to the pH of 6.5 with 1 N NaOH in order to avoid acid side possibly through the inhibition of adherence, as well as by effects, and treated with catalase at a concentration of 200 producing metabolites and antimicrobial molecules (Jack unit/mL. CFCS was transferred in a cold chamber main- et al. 1995). tained at 4 °C and then the crude bacteriocins were In our previous study, the crude bacteriocins of Lacto- obtained by 50 % (w/v) saturated ammonium sulfate pre- bacillus brevis BK11 and Enterococcus faecalis BK61 cipitation overnight with stirring. The precipitated protein strains isolated from Baikkimchi dramatically decreased was separately collected by centrifugation (12,0009g for the viability of H. pylori (Lim 2014). The aim of the pre- 30 min at 4 °C) and suspended in 20 mM sodium phos- sent study was to determine the antimicrobial activity phate buffer (pH 6.5). The pellet containing the crude against H. pylori after purification of the bacteriocins bacteriocins was dialyzed twice against the same buffer for produced by these strains and to investigate the physico- 24 h at 4 °C using a dialysis bag (Spectrum Medical chemical properties, molecular weight, and antimicrobial Industries, Inc., USA) with a molecular weight cut-off of spectrum of the purified bacteriocins. Besides, their abili- 1000 Da for desalting. ties to expel H. pylori from human gastric adenocarcinoma For the bacteriocin purification of L. brevis BK11, epithelial cell line (AGS) and to inhibit the urease activity AKTA purifier-100 system (GE Healthcare, UK) equipped of this pathogen were evaluated. with HiPrep Q HP 16/10 column was used to further purify of the desalted crude bacteriocin. The system was equili- brated with 10 mM citric-phosphate buffer (pH 6.0). The Materials and methods crude bacteriocin solution was eluted by eluent buffer (1 M NaCl in equilibrium buffer) at flow rate of 5 mL/min and Culture conditions of LAB the elution procedure was monitored by UV detector at 280 nm. The eluted fractions were applied to Superdex The bacterial strains used in this study were L. brevis BK11 Peptide 10/300 GL column (GE Healthcare) equilibrated and E. faecalis BK61, which were isolated from with 50 mM trisodium phosphate buffer (pH 5.8). 0.15 M Baikkimchi and these strains showed the antimicrobial NaCl was used for elution at a flow rate of 1 mL/min. The activity in primary experiment. The stock culture collection fractions were automatically collected and pooled accord- of the two strains was maintained at -80 °C in Lactobacilli ing to UV absorbance at 215 nm. The active fractions were MRS broth (Difco., USA) with 20 % (v/v) glycerol, and finally purified by high-performance liquid chromatogra- regenerated twice before being used in the manipulations. phy (LC-8, Shimadzu, Japan) with a reversed-phase (RP) column of C-18 (10 lm, 300 A˚ , Macherey Nagel, France). Culture conditions of H. pylori The mobile phases were solvent A (water:acetonitrile:tri- fluoroacetic acid (TFA) = 95:5:0.1) and solvent B (ace- Helicobacter pylori American type culture collection tonitrile:TFA = 100:0.1). Sample elution program was as (ATCC) 43504 strain used in this study was obtained from follows: 100 % A for 5 min, A linearly decreased to 0 % ATCC. Before experiments, H. pylori was sub-cultured on and B increased to 100 % from 5 to 30 min, and finally B Brucella agar (Difco) plates containing 5 % (v/v) fetal increased to 100 % from 30 to 40 min. Flow rate was 123 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 705

5 mL/min and absorbance was recorded at 280 nm by UV (50 % of the turbidity of the control culture without detector (Zhu et al. 2014). bacteriocin). For the bacteriocin purification of E. faecalis BK61, the desalted solution was subjected to cation exchange chro- Determination of molecular weight matography column [Carboxymethyl (CM) Sepharose (Pharmacia, Sweden)] equilibrated with 0.05 M sodium The molecular weight of the purified bacteriocins was acetate buffer (pH 5.5). Elution was performed at a flow determined using Tricine-sodium dodecyl sulfate–poly- rate of 1 mL/min with a linear gradient of 0–0.6 M NaCl in acrylamide gel electrophoresis described by Scha¨ger and 0.073 M sodium acetate buffer (pH 5.0). The active frac- Von Jagow (1987) with minor modifications. A low- tions were pooled and dialyzed against 20 mM sodium molecular-weight marker (TEFCO, Technical frontier, Co., phosphate buffer (pH 5.8). Ammonium sulfate was then Japan) was used as protein standard. Mini-Protein II elec- added to the pooled fractions to a final concentration of trophoresis system (Bio-Rad Laboratories) was carried out 10 % and subsequently applied to hydrophobic interaction with 4 % stacking gel and 16.5 % separating gel. Elec- chromatography column [Octyl Sepharose CL-4B (Phar- trophoresis was performed at a constant voltage of 110 V for macia)] equilibrated with 20 mM sodium phosphate buffer 90 min. To confirm the purified bacteriocin band, the gel (pH 5.8) containing 10 % (w/v) ammonium sulfate. After which contained only the sample was stained for 30 min washing the column with the same buffer, the activity was with Coomassie brilliant blue R-250 and destained until the eluted with 50 % ethanol in 20 mM sodium phosphate background stain disappeared. The other gel was overlaid buffer (pH 5.8). Then the active fractions were applied to a with Brucella soft agar (0.7 % agar) containing 105 CFU/mL

C2/C18 reverse-phase column (PepRPC HR5/5) by using of H. pylori ATCC 43504, incubated for 48 h at 37 °C, and fast-performance liquid chromatography system. The bac- observed for the formation of inhibition zones. teriocin was eluted from the reverse-phase column with a 55 min linear gradient of 20–40 % (v/v) 2-propanol in Sensitivity to heat, pH, and proteolytic enzymes aqueous 0.1 % (v/v) trifluoroacetic acid at a flow rate of 0.5 mL/min (Cintas et al. 1997). The purified bacteriocins were treated with different During purification, each active fraction was collected, enzymes, heating, storage, and pH to determine the nature of filtered, and tested for the bacteriocin activity against the the compounds responsible for the antimicrobial activity. indicator strains (H. pylori ATCC 43504). In addition, the After physico-chemical treatment, the residual activities protein content for each sample at different steps of the were determined by microtitre plate assay as described purification was estimated using Bradford’s assay (Brad- above. Firstly, the enzymes (Sigma-Aldrich, USA) including ford 1976) reagent as described by the manufacturer (Bio- protease, proteinase K, pepsin, trypsin, lysozyme, catalase, Rad Laboratories, Inc., USA). a-amylase, and lipase were diluted in the most suitable buffer and then the purified bacteriocin was added to the Determination of bacteriocin activity enzyme solutions at a final concentration of 1 mg/mL. After 3 h of incubation at 37 °C, the reaction was stopped by The antimicrobial activity assay of the bacteriocins boiling for 2 min. The control was incubated in only buffer obtained from the purification steps was determined by solution without the enzyme. Meanwhile, the thermal sen- microtitre plate assay (Holo et al. 1991) using H. pylori as sitivity of the purified bacteriocins was evaluated by heating the target organism. Briefly, 48-h-old H. pylori culture was at 100 °C for 10, 20, and 30 min and by autoclaving at centrifuged at 70009g for 10 min at 4 °C. The supernatant 121 °C for 15 min. After cooling, the residual inhibitory was discarded, and the cell pellet was washed twice with activity was measured and compared with the non-treated sterile phosphate buffer saline (PBS, pH 7.0) and diluted to control. The stability of the purified bacteriocins during a approximate 105 CFU/mL in the same buffer. Each well of long-term storage was tested at 25, 4, and -20 °Cover 24-well plate (Falcon, USA) contained 100 lL of Brucella 3 months. The bacteriocin aliquot was taken from storage broth, the bacteriocin fraction (100 lL), and the pathogenic every month and was measured for the antimicrobial culture inoculum (100 lL). After incubation for 24 h at activity. To evaluate the influence of pH, the bacteriocin was 37 °C, the growth inhibition of the indicator strain was adjusted to pH values ranging from 2.0 to 12.0 with 1 M measured spectrophotometrically at 600 nm (Microplate HCl or 1 M NaOH, incubated for 2 h at 37 °C, and then the Reader, BioTek, Inc., Korea). Bacteriocin activity (arbi- pH was re-adjusted to 6.5. After each treatment, the samples trary units, AU) was defined as the reciprocal of the highest were subsequently assayed for antimicrobial activity against dilution inhibiting the 50 % growth of the indicator strain H. pylori ATCC 43504.

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Antimicrobial spectrum of the bacteriocin Meanwhile, H. pylori grown well were cultivated in 10 mL of Brucella broth medium supplemented with 10 % The determination of the inhibitory effects of the bacteri- FBS as previously described. H. pylori strain was diluted to ocins from the tested LAB strains was carried out with agar 108 CFU/mL with sterile PBS (pH 7.0) and then resus- well diffusion assay. The antagonistic activity of the tested pended in RPMI 1640 medium (without serum). The bacteriocins was assessed against the following target inhibition of the H. pylori association with AGS cells by bacteria: Bacillus cereus ATCC 11778, Bacillus subtilis the purified bacteriocin was determined by pre-incubating. ATCC 35421, Lactobacillus acidophilus KCTC (Korean Before the competition assay, the cell suspension of viable Collection for Type Culture) 3168, L. brevis KCTC 3102, H. pylori (100 lL) was co-incubated with the bacteriocin Lactobacillus paracasei ATCC 25302, Lactobacillus solution (100 lL) adjusted to a suitable concentration for plantarum KCTC 1048, E. faecalis KCTC 3206, Listeria 2 h at 37 °C under microaerophilic atmosphere. After AGS monocytogenes KCTC 3569, Staphylococcus aureus cells had grown into a confluent monolayer, the incubation ATCC 6538, Escherichia coli O157 ATCC 43889, Sal- mixture was added to each well of the tissue culture plate monella enteritidis ATCC 13076, and Vibrio para- and plated under 5 % CO2–95 % air at 37 °C for 2 h to haemolyticus KCTC 2471. The bacterial pathogens were allow H. pylori adhesion to cell lines. For the displacement grown in optimal broth [Brain Heart Infusion (BHI, Difco) assay, 100 lL of viable H. pylori at 1 9 108 CFU/mL was or BHI plus 3 % (w/v) NaCl] and all bacterial cultures added to each well and incubated for 2 h to allow H. pylori were incubated at 37 °C. The overnight cultures of these to adhere to the AGS cells. After incubation, the cells in indicator strains were adjusted to 5.0 9 106 CFU/mL with each well were washed three times with sterile PBS (pH PBS (pH 7.0) after centrifugation (70009g, 10 min, 4 °C) 7.0). A 1 mL aliquot of fresh cell culture medium con- and washing and inoculated at 1 % (v/v) in BHI broth, taining 100 lL of the bacteriocin solution adjusted to a containing 0.7 % agar. Once solidified, the dishes were suitable concentration was added to each well and allowed stored for 2 h at 4 °C. Wells (8 mm diameter) were made to incubate for 2 h to remove adherent H. pylori from the into the agar with a sterile glass rod and 50 lL of the AGS cells. Subsequently, the cells in each well were bacteriocin solution was loaded into the wells. The inoc- washed three times with PBS (pH 7.0) to remove non- ulated plates were incubated for 24 h at 37 °C in an aerobic adherent H. pylori. AGS cells were lysed by addition of incubator. The diameter of the inhibition zone surrounding 0.1 % Triton X-100. Appropriate dilutions of the lysate each well was measured, and the zone of inhibition was were plated on antibiotic-selective agar for 48 h at 37 °Cto expressed as a mean diameter for each sample. The inhi- determine the number of viable cell-associated bacteria by bitory effect of target bacteria was ranked as high bacterial colony counts. Bacterial adhesion capacity was ([18 mm), intermediate (14–18 mm), and low (9–13 mm). calculated as percent of adhered bacteria in relation to the total number of bacteria added. Finally, the inhibition (%) Effects of bacteriocin on adhesion of H. pylori of adhesion ability of H. pylori by the bacteriocin treatment against AGS cells was calculated as follows; inhibition (%) = (1 - number of adherent H. pylori after each the bacteriocin treatment/ To test the ability of the bacteriocin to inhibit the adhesion number of adherent H. pylori after PBS treatment) 9 100. of H. pylori against AGS (ATCC CRL 1739), the procedure described by Lim (2014) was used. AGS cells were pur- Effect of bacteriocin on urease activity of H. pylori chased from American Type Culture Collection (USA) and cultured as previously described (Mojtahedi et al. 2007) with The urease activity of H. pylori adhering to AGS cells was minor modification. Briefly, cells were routinely grown in determined by measuring the release of ammonia by a RPMI 1640 medium including L-glutamine, NaHCO3, modified phenol red method (Sgouras et al. 2004). AGS kanamycin (60 lg/mL), and streptomycin (20 lg/mL) cells were grown on microtiter plates to form a confluent (Nikken bio medical, Japan) supplemented with 10 % (v/v) monolayer, and then conditions for incubation of H. pylori inactivated (30 min, 56 °C) FBS at 37 °Cinhumidified against AGS cells with the purified bacteriocin were the atmosphere of 5 % CO2 and 95 % air. Cells were inoculated same as those described previously for the adhesion assay. at a concentration of 1.0 9 105 cells per well to obtain post- The incubated cell cultures were washed five times with confluence and cultured at 37 °C prior to the adhesion assay. PBS (pH 7.0). Urease reaction buffer (200 g/L urea and The culture medium was changed on alternate days. One 0.12 g/L phenol red in PBS, pH adjusted to 6.5) was added milliliter of a suspension of 1.0 9 105/mL AGS cells in to each well. The well multi-dish plates were incubated at fresh tissue culture medium was added to the 24-well multi- 37 °C in order to induce the production of ammonia from H. dish tissue culture plate and then cultured at 37 °Cinthe pylori adhering to AGS cells. After 1 h, the optical density presence of 5 % CO2/95 % air atmosphere. value at 550 nm was measured with a spectrophotometer. 123 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 707

Statistical analysis 1575.4 AU/mg. After final purification step, the bacteriocin from L. brevis BK11 strain was purified 3137.3-fold with a All experiments were performed at least in triplicate. recovery of 6 %. Results are expressed as the mean and standard deviations. In E. faecalis BK61 strain, the precipitated protein was Statistical analyses were performed using one-way analysis further purified using CM Sepharose cation exchange of variance in the SPSS 11.0 software (SPSS Inc, USA). chromatography column. It was revealed that the specific Differences between the means of the test and control antimicrobial activity increased from 74.7 to 900.2 AU/ groups were evaluated by Student’s t test, and p \ 0.05 mg. And then, the specific activity of the bacteriocin was considered statistically significant. purified by hydrophobic interaction chromatography was 4096 AU/mg of protein, which corresponds to a 1780.9- fold increase. The final specific activity of the purified Results and discussion bacteriocin from this strain was increased approximately 12,367-fold compared to that in the culture supernatant and Purification and molecular weight of bacteriocin the recovery was 2 %. Meanwhile, the molecular weights for the purified bac- The purification steps and the recovery values of the bac- teriocins of L. brevis BK11 and E. faecalis BK61 were teriocins produced from the tested two strains are shown in estimated to be about 6.5 and 4.5 kDa, respectively, Table 1. The bacteriocins of L. brevis BK11 and E. faecalis (Fig. 1). These bacteriocins displayed the antimicrobial BK61 were recovered following the 50 % saturation of the activity on a lawn of H. pylori ATCC 43054 which cor- culture broths with ammonium sulfate with an increase to responded to those bands. specific activity of 29.1 and 74.7 AU/mg protein, respec- Bacteriocin is a form of protein that is produced by tively. In L. brevis BK11 strain, the second step in the certain bacteria and kills or inhibits the growth of closely purification protocol was to concentrate the activity from related species or other strains within the same species the precipitated protein by cation exchange chromatogra- (Cleveland et al. 2001). Bacteriocins, antimicrobial sub- phy equipped with HiPrep Q HP 16/10 column and resulted stances obtained from LAB that need complex nutritional in an approximately 496-fold concentration and 32 % exigencies to produce these compounds are ribosomally recovery. In the subsequent purification step on a Superdex synthesized peptides having low molecular weight (rarely peptide 10/300 GL column, the specific activity was over 10 kDa) and often produced only in small amounts

Table 1 Purification of the bacteriocins obtained from the tested strains Strain Purification stage Vol. Total protein Bacteriocin Total Specific Purification Recovery (mL) (mg/mL) activity activity activity (fold) (%) (AU/mL) (AU) (AU/mg)

Lactobacillus brevis BK11 Cell culture 1000 26.7 32 32,000 1.2 1 100 supernatant Ammonium sulfate 120 17.6 512 61,440 29.1 24.3 66 precipitation Cation exchange 10 8.6 5120 51,200 595.3 496.1 32 chromatography Gel chromatography 2 6.5 10,240 20,480 1575.4 1312.8 24 RP-HPLC 1 1.7 6400 6400 3764.7 3137.3 6 Enterococcus faecalis BK61 Cell culture 1000 56.8 128 128,000 2.3 1 100 supernatant Ammonium sulfate 150 13.7 1024 153,600 74.7 32.5 24 precipitation Cation exchange 20 9.1 8192 163,840 900.2 391.4 16 chromatography Hydrophobic 5 2.5 10,240 51,200 4096.0 1780.9 4 interaction chromatography RP-FPLC 1 0.9 25,600 25,600 28,444.4 12,367.1 2

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kDa M (1) (2) (3) (4) hydrophobic nature. Both enterocin C1 and enterocin C2 205 produced from this strain had been displayed similar molecular weight (ca. 2.5 kDa). 116 97.4 Effects of enzyme, heating, storage, and pH 69 on bacteriocin activity 55 Effects of enzyme, heating, storage, and pH on the bacte- 36.5 riocin activity of L. brevis BK11 and E. faecalis BK61 29 strains are shown in Table 2. Significant decrease of 20.1 antimicrobial activity of the bacteriocin obtained from L.

14.3 brevis BK11 was observed after treated by protease, pro- 6.5 kDa teinase K, trypsin, and lysozyme. However, the activity of 6.5 4.5 kDa the bacteriocin was completely retained after treatment with pepsin. The bacteriocin activity of E. faecalis BK61 3.5 was completely inactivated by protease, and relative loss of activity was observed after treatment with proteinase K, pepsin, and trypsin. Unlike L. brevis BK11, treatment with Fig. 1 SDS-PAGE of the purified bacteriocins from L. brevis BK11 lysozyme did not affect significantly on the bacteriocin (1) and E. faecalis BK61 (3) and the gel overlaid with a lawn of H. activity of E. faecalis BK61. Catalase treatment did not pylori ATCC 43504 cells to determine the band corresponds to the destroy the antimicrobial activity of the bacteriocins from antimicrobial activity of the purified bacteriocin from L. brevis BK11 the two strains, which proves that the antimicrobial activity (2) and E. faecalis BK61 (4). (M) molecular weight marker made by TEFCO is not due to the hydrogen peroxide production. The antimicrobial activity of two bacteriocins was found to be insensitive to a-amylase and lipase. This result indicated during incubation process. Bacteriocins are in general that these substances might not contain a lipid or carbo- cationic, amphipathic molecules as they contain an excess hydrate component, which, at least, can be affected by a- of lysyl and arginyl residues (Zacharof and Lovitt 2012). amylase and lipase used in this study. The sensitivity of the Purification methods that have been employed in the bacteriocin obtained from L. brevis BK11 to heat treatment purification of bacteriocins from LAB are varied showed that the inhibitory activity was not altered by (Carolissen-Mackay et al. 1997). Typically, the major heating for 10, 20, and 30 min at 100 °C. However, the methods of the purification of bacteriocins (curvacin A, activity was reduced by up to 25 % after heating for sakacin P, lactosin S, and bavaricin A) by LAB to homo- 15 min at 121 °C. The bacteriocin from E. faecalis BK61 geneity can be done by ammonium sulfate precipitation, also showed good heat stability even treated for 10 and ion exchange, hydrophobic interaction, gel filtration, and 20 min at 100 °C, but its activity was partly reduced when RP-HPLC (De Vuyst and Leroy 2007). heated for 30 min at 100 °C and for 15 min at 121 °C. In accordance with this report, each step during the These results suggested that the antimicrobial substances purification procedures of L. brevis OG1 resulted in a produced by the tested strains were relatively heat resistant. considerable loss of protein concentration, while specific Meanwhile, the antimicrobial activity of these two activity increases. The optimal bacteriocin recovery was bacteriocins remains stable during the storage period of achieved by including ammonium sulfate precipitation and 3 months at 25 and 4 °C. These bacteriocin activities were Trichloroacetic acid precipitation. Ultrafiltration experi- completely stable for 60 days of incubation at -20 °C ments showed that the bacteriocins were unable to pass (data not shown), but there was approximate 50 % loss of through 1000 and 10,000 Da molecular weight cut-off residual activity of the bacteriocin after 90 days of storage membrane (Ogunbanwo et al. 2003). Our results disagreed at the same temperature. The purified bacteriocin from L. with a previous study by Gautam and Sharma (2009); they brevis BK11 was found to be stable over a wide range of reported that the relative molecular mass of the purified pH from 2 to 12. The bacteriocin produced by E. faecalis bacteriocin from L. brevis MTCC 7539 was 93.74 kDa, BK61 had a good stability over a pH range from 4.0 to while specific activity and recovery were 35.52-folds and 10.0. However, significant difference was found at acidic 17.13 %, respectively. Maldonado-Barragan et al. (2009) condition, there was reduction in the bacteriocin activity of demonstrated that the antimicrobial compound produced about 75 and 50 % at pH 2.0 and pH 3.0, respectively. by E. faecalis C901 precipitated in the presence of Our results were similar to the previously reported ammonium sulfate and showed a cationic and highly studies; the inhibitory activity of oral bacteria directed 123 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 709

Table 2 Effects of enzymatic Treatment Bacteriocin activity (AU/mL) and physico-chemical treatment on the activity of the purified Lactobacillus brevis BK11 Enterococcus faecalis BK61 bacteriocin from L. brevis BK11 and E. faecalis BK61 toward the Control 6400 25,600 indicator strains H. pylori Enzyme Protease 320 ND ATCC 43504 Proteinase K 640 12,800 Pepsin 6400 10,240 Trypsin 1280 5120 Lysozyme 3200 25,600 Catalase 6400 25,600 a-Amylase 6400 25,600 Lipase 6400 25,600 Heating 100 °C, 10 min 6400 25,600 100 °C, 20 min 6400 25,600 100 °C, 30 min 6400 10,240 121 °C, 15 min 1600 6400 Storage 25 °C, 3 months 6400 25,600 4 °C, 3 months 6400 25,600 -20 °C, 3 months 3200 12,800 pH 2.0 6400 6400 3.0 6400 12,800 4.0–12.0 6400 25,600 ND not detected against H. pylori strains was affected by heating and pro- molecular-weight bacteriocin may be due to the formation tease treatment, except for those of Fusobacterium of small globular structures and stable disulfide intra- nucleatum ATCC 25586 (Ishihara et al. 1997). The molecular bonds and the possession of a high glycine antimicrobial compounds isolated by Collado et al. (2005) content and hydrophobicity (De Vuyst and Vandamme were able to inhibit the growth of both antibiotic-sensitive 1994). The heat-stable bacteriocin can be a very useful for and -resistant H. pylori strains and related to heat-stable application as a food preservative in food processing pro- proteinaceous compounds; resistant to heating at 100 °C cedures involving a heating step such as pasteurized for 10 min, but sensitive to protease. Proteolytic enzymes products or canned foods and as a biopreservative in could destroy part of the active site in the structure of the combination with thermal processing to preserve the food bacteriocins of BK11 and BK61 strains, indicating that the products (De Vuyst and Vandamme 1994). antimicrobial substance which inhibited the growth of H. This study showed that the antimicrobial activity of the pylori strain was proteinaceous nature. And these bacteri- two bacteriocins was stable over long periods of storage. ocins may be readily inactivated by the action of digestive Similar results were observed in many previous reports. proteases in the gastrointestinal tract. Whereas, the Simova et al. (2009) reported that the bacteriocin preserved antimicrobial activity of the bacteriocins was not sensitive the maximum activity during 90 days of storage at -20 °C to a-amylase or lysozyme, indicating that the substance and an insignificant reduction in the bacteriocin activity may be an attractive candidate for oral applications, espe- was observed during storage at 4 °C. The activity of the cially for prevention and/or treatment of oral diseases. partially purified bacteriocin from E. faecalis KT2W2G Heat stability of the bacteriocins of L. brevis BK11 and isolated from the mangrove forest in southern Thailand was E. faecalis BK61 is in agreement with that of the bacteri- maintained in full stability after storage -20, 4, and 37 °C ocin by LAB isolated from traditional Bulgarian dairy for 2 months. Optimum temperature and time of storage products. The antimicrobial substance of Lactobacillus depend on the kind of bacteriocin (H-Kittikun et al. 2015). bulgaricus BB18 strain showed inhibition action against H. This study demonstrated that the bacteriocins could be pylori strains revealed high thermostability, surviving active in a broad pH range from 2.0 to 12.0. Simova et al. treatments for 60 min at 100 °C, but the activity of this (2009) also reported the similar findings; the antimicrobial strain slowly decreased for 15 min at 121 °C (Simova et al. substances of stain L. bulgaricus BB18 remained maximally 2009). The heat stability, which is a major feature of low- active within a wide pH range from 3.0 to 11.0. The acidic

123 710 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 range pH tolerance indicates that such bacteriocin would be any activity against B. cereus, B. subtilis, L. plantarum, beneficial for its use as a preservative of acidic foods due to and V. parahaemolyticus. Among the indicator strains their stability under high acidity affecting the viability of the used, highest growth inhibition of the bacteriocins pro- bacteriocin-producing strains (Banerjee et al. 2013). duced from L. brevis BK11 and E. faecalis BK61 was recorded against H. pylori ATCC 43504. Antimicrobial spectrum Kim et al. (2003) noted that lacticins A164 and BH5 produced by Lactococcus lactis subsp. lactis showed the The antimicrobial spectrum of the purified bacteriocins strongest antibacterial activity against H. pylori strains and obtained from L. brevis BK11 and E. faecium BK61 was therefore would have potential application in treating evaluated against various closely related species, harmful stomach ulcers. Besides, pediocin PO2 was less active than and pathogenic strains by using the agar well diffusion the lacticins against four strains of H. pylori, and leucocin method. It is shown in Table 3 that the inhibitory spectrum K was the least active peptide, with no inhibition toward H. of the bacteriocin produced by L. brevis BK11 strain was pylori ATCC 43504. According to previous reports, the quite narrow. This strain displayed the inhibitory activity release of bacteriocins active against H. pylori has been against L. acidophilus KCTC 3168, L. brevis KCTC 3102, studied chiefly in Lactobacillus, but probiotic bifidobacte- L. plantarum KCTC 1048, H. pylori ATCC 43504, and S. ria may also produce them. The supernatant of a culture of enteritidis ATCC 13076; however, no antibacterial activity Lactobacillus johnsonii NCC533 was shown to inhibit both was detected against B. cereus ATCC 11778, B. subtilis the urease activity and growth of H. pylori free or adherent ATCC 35421, L. paracasei ATCC 25302, E. faecalis to epithelial cells (Mladenova-Hristova 2013). A bacteri- KCTC 3206, L. monocytogenes KCTC 3569, S. aureus ocin-like inhibitory substance with anti-H. pylori activity ATCC 6538, E. coli O157 ATCC 43889, and V. para- was confirmed in probiotic L. johnsonii strain LA1 and L. haemolyticus KCTC 2471. Meanwhile, the spectrum of acidophilus strain LB (Giri and Singh 2013). Other pro- inhibition by E. faecalis BK61 was significantly wider than biotic bacteria, such as Weissella confuse, Lactobacillus L. brevis BK11. Namely, the bacteriocin produced from lactis, and B. subtilis were shown to secrete bacteriocins BK61 strain exhibited a broad antimicrobial spectrum inhibited H. pylori growth in vitro (Lesbros-Pantoflickova against most of the tested strains, Gram-positive bacteria et al. 2007). Tsai et al. (2004) noted that Enterococcus (L. acidophilus, L. brevis, L. paracasei, E. faecalis, L. faecium TM39 strain may secrete a heat-stable and pro- monocytogenes, and S. aureus) and Gram-negative bacteria tease-resistant antimicrobial substance(s) active against (E. coli O157, H. pylori, and S. enteritidis). It did not show Helicobacter infection in vitro.

Table 3 Antimicrobial spectrum of the bacteriocin obtained from L. brevis BK11 and E. faecalis BK61 against Gram-positive and Gram- negative bacteria Strain Source Antimicrobial activity Lactobacillus brevis BK11 Enterococcus faecalis BK61

Gram-positive Bacillus cereus ATCC 11778 – – Bacillus subtilis ATCC 35421 – – Lactobacillus acidophilus KCTC 3168 ??? Lactobacillus brevis KCTC 3102 ?? ? Lactobacillus paracasei ATCC 25302 – ? Lactobacillus plantarum KCTC 1048 ?? – Enterococcus faecalis KCTC 3206 – ?? Listeria monocytogenes KCTC 3569 – ?? Staphylococcus aureus ATCC 6538 – ? Gram-negative Escherichia coli O157 ATCC 43889 – ? Helicobacter pylori ATCC 43504 ??? ??? Salmonella enteritidis ATCC 13076 ?? Vibrio parahaemolyticus KCTC 2471 _ – – no inhibition; ? 9–13 nm; ?? Diameter of the inhibition zone 14–18 mm; ??? Diameter of the inhibition zone is larger than 18 mm

123 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 711

The antimicrobial activity of bacteriocins against target Adhesion of H. pylori to AGS cells in the presence microorganisms usually result from blockage of cell wall of bacteriocin synthesis, change in the membrane permeability, depletion of the transmembrane potential (4W) leading the leakage Table 4 shows the inhibition of the adhesion of H. pylori of cellular materials, and/or formation of pores in the ATCC 43504 by the bacteriocins tested. Treatment of H. membrane causing the death of the target cells (Cleveland pylori with the bacteriocins obtained from L. brevis BK11 et al. 2001). Generally, the cell wall of Gram-positive and E. faecalis BK61 significantly reduced the number of bacteria allows passage of bacteriocins, so these antimi- the pathogen adhering to the monolayers of cultured human crobial substances inhibit only closely related species or gastric epithelial AGS cells (p \ 0.05). These antibacterial other Gram-positive spoilage microorganism and food- substances inhibited H. pylori in a concentration-dependent borne pathogens. However, some LAB bacteriocins are manner. Considering that the adherence rate of H. pylori unable to reach their targets/receptors in the cellular without the bacteriocin treatment was 100 %, this rate membrane of Gram-negative strains. The inherent resis- dropped to approximately 30.2–62.1 % after co-treatment tance of Gram-negative bacteria to many microbicidal with the bacteriocin from BK11 strain and H. pylori. The agents is due to the lipopolysaccharide layer of the outer levels of H. pylori competition varied between 15.2 and membrane, which is a constituent of the cell envelope in all 49.8 % depending on the concentration of the bacteriocin Gram-negative bacteria, and acts as an efficient perme- produced by E. faecalis BK61 strain. Meanwhile, the ability barrier against macromolecules and hydrophobic bacteriocin (5120 AU/mL) of L. brevis BK11 was the most substances (Jack et al. 1995; Helander et al. 1997). Cintas effective on displacement of H. pylori. The competition et al. (1997) reported that enterocin P from E. faecium P13 rate of the tested bacteriocins toward this pathogen was did not show inhibition zones against Gram-negative bac- also slightly higher than their displacement rate under the teria such as Salmonella typhimurium, E. coli, Pseu- same concentration. This finding implies that the pro- domonas fluorescens, Yersinia enterocolitica, Enterobacter teinaceous antimicrobial substances of LAB play a key role aerogenes, and Aeromonas hydrophila. in inhibiting the adhesion of H. pylori to AGS cells. On the other hand, the Gram-negative bacteria (Pseu- Similar results were noted in other studies; certain lac- domonas aeruginosa and E. coli) as well as the pathogenic tobacilli such as L. johnsonii La1 or L. acidophilus LB can Gram-positive bacteria (B. subtilis, Bacillus pumilus, and exert their anti-adhesion activity by secreting antimicrobial Listeria sp.) were inhibited by the enterocin CD1 of E. substances (Lesbros-Pantoflickova et al. 2007). Lin et al. faecalis CD1 isolated from commercial curd (Sarika et al. (2009) reported that the spent culture supernatants from 2011). The inhibition spectrum of the bacteriocin from L. LAB (LAB-SCS) inhibit H. pylori infection and adhesion brevis BK11 was different from that of the bacteriocin to AGS cells. In addition, treatment by the bacteriocin or produced from L. brevis NM 24, which was able to inhibit cells of E. faecium TM39 significantly reduced the binding the growth of B. subtilis, E. coli, E. faecalis, L. monocy- of H. pylori to monolayers of AGS cell line (Tsai et al. togenes, L. innocua, Lactobacillus fermentum, Lacto- 2004). The ability to adhere to mucosal surfaces is bacillus reuteri, Lactobacillus delbrueckii, S. typhimurium, important for bacterial maintenance in the human gas- and S. aureus, while Candida albicans, Streptococcus trointestinal tract (Chen et al. 2012). pyogenes, and P. aeruginosa were resistant to their action (Mojgani et al. 2008). The bacteriocin produced by L. Bacteriocin inhibits H. pylori urease activity brevis FPTLB3 inhibited the growth of E. coli MTCC 1563, E. faecalis MTCC 2729, Lactobacillus casei MTCC After treatment with the bacteriocin, the urease activity of 1423, Lactobacillus sakei ATCC 15521, and S. aureus H. pylori ATCC 43504 adhered to AGS cells was exam- ATCC 25923 (Banerjee et al. 2013). In the LAB having ined. As shown in Table 5, the urease activity of H. pylori antagonistic effect against Gram-negative bacteria, the was significantly (p \ 0.05) inhibited by the presence of outer membrane of target microorganisms allows the pen- the bacteriocin (2560 and 5120 AU/mL) obtained from L. etration of relatively small hydrophilic compounds through brevis BK11 strain. The urease activity of adherent H. porin proteins, which form water-filled pores traversing the pylori after treatment with the bacteriocin was lower than membrane. In other words, the low-molecular-mass bac- that of the control group, but there appeared to be no sig- teriocins, provided that they are hydrophilic enough, may nificant difference between competition and displacement thus have access to the deeper parts of the Gram-negative groups (p [ 0.05). When adhered to AGS cells after co- bacterium without any alteration to the permeability of the incubation of H. pylori with the bacteriocin (5120 AU/mL) outer membrane (Helander et al. 1997). Compared with the of E. faecalis BK61, we found that the urease activity of other results, the antimicrobial spectrum of bacteriocins this pathogen was significantly reduced (p \ 0.05). obtained from LAB was found to be species dependent. Therefore, the bacteriocins obtained from the LAB strains 123 712 J Korean Soc Appl Biol Chem (2015) 58(5):703–714

Table 4 Exclusion and Bacteriocin-producing strain Concentration of bacteriocin (AU/mL) Inhibition (%) displacement effects of the bacteriocin of L. brevis BK11 Competition Displacement and E. faecalis BK61 against the adhesion of H. pylori ATCC Control 100.0 ± 3.4 100.0 ± 4.9 43504 to AGS cells L. brevis BK11 1280 30.2 ± 4.3a 25.9 ± 2.4a 2560 47.4 ± 6.2a 41.1 ± 6.0a 5120 62.1 ± 3.3a 60.8 ± 5.6a E. faecalis BK61 1280 15.2 ± 5.1a 11.1 ± 1.9a 2560 31.6 ± 7.0a 24.6 ± 3.2a 5120 49.8 ± 4.7a 42.5 ± 2.8a Data were presented as mean ± SD from three independent experiments a Statistical analysis preformed with s Student t test between control and treated H. pylori with the bacteriocin showed a highly significant difference (p \ 0.05)

Table 5 Inhibition of the Bacteriocin-producing strain Concentration of bacteriocin (AU/mL) Urease activity (OD550 nm) bacteriocin against the urease activity of H. pylori ATCC Competition Displacement 43504 Control 0.32 ± 0.03 0.33 ± 0.01 L. brevis BK11 1280 0.28 ± 0.03 0.29 ± 0.02 2560 0.24 ± 0.02a 0.25 ± 0.01a 5120 0.19 ± 0.03a 0.21 ± 0.02a E. faecalis BK61 1280 0.30 ± 0.01 0.32 ± 0.02 2560 0.27 ± 0.03 0.30 ± 0.03 5120 0.23 ± 0.02a 0.28 ± 0.04 Data were presented as mean ± SD from three independent experiments a Statistical analysis preformed with s Student t test between control and treated H. pylori with the bacteriocin showed a highly significant difference (p \ 0.05) having anti-H. pylori activity inhibited the urease activity one-tenth of its original level. Thus, the inhibitory activity of this pathogen in a concentration-dependent manner. of the antibacterial compound(s) secreted from strain Urease is an important product produced by H. pylori, TM39 could result in the inhibition on H. pylori urease which is a surface protein component of H. pylori and is activity. able to survive from the acid in the stomach by neutralizing The detection rate of drug-resistant H. pylori strains has the acidic environment. Urease converts urea into bicar- recently been increasing, and the side effects caused by bonate and ammonia, leading to increased pH in the triple therapy are serious in some patients (Khani et al. stomach which promotes colonization of this pathogen 2012). In a previous study, adjuvant therapy with bacteri- (Adamsson et al. 2000). A similar inhibition pattern was ocin-producing probiotic LAB can reduce adverse effects found in the other studies. Yoon and Won (2002) suggested induced by H. pylori and may improve eradication rates, that both Lactobacillus helveticus CU631 and CFCS thus have beneficial effects in H. pylori-infected individuals obtained from this strain had strong inhibitory activities in (Egan et al. 2007). The release of heat-stable, proteinaceous urease of H. pylori NCTC 11637 and CJH12. Lin et al. bacteriocins with anti-H. pylori activity has been identified (2009) found that there was an inverse relationship in probiotic strains of Lactobacillus, E. faecium, B. subtilis, between the exclusion rate and urease activity; that is the and Bifidobacterium (Gotteland et al. 2006). point when the exclusion rate of LAB-SCS against H. In this study, the two LAB strains isolated from pylori infection was highest (41.1 %), the urease activity of Baikkimchi, namely L. brevis BK11 and E. faecalis BK61, H. pylori was lowest. Therefore, LAB-SCS is able to showed significant anti-H. pylori activity, and these inhi- inhibit H. pylori infection in AGS cells. Tsai et al. (2004) bitory activities may be contributed by the proteinaceous demonstrated that after 2 h contact of the H. pylori cells antimicrobial substance (bacteriocin) in vitro. These bac- with the bacteriocin from E. faecium TM39, the urease teriocins could also inhibit the adhesion ability to AGS activity of H. pylori adhered to AGS cells decreased to cells and the urease activity of H. pylori. The anti-H. pylori

123 J Korean Soc Appl Biol Chem (2015) 58(5):703–714 713 activities of the antimicrobial substances secreted by L. resistance: a systematic review. J Gastrointest Liver Dis brevis and E. faecalis are unusual among the known LAB 19:409–414 De Vuyst L, Leroy F (2007) Bacteriocins from lactic acid bacteria: bacteriocins. These bacteriocins may be useful adjuvant production, purification, and food applications. J Mol Microbiol agent for prevention or alleviation of chronic gastritis and Biotechnol 13:194–199 peptic ulcer disease caused by H. pylori. Besides, the De Vuyst L, Vandamme EJ (1994) Nisin, a lantibiotic produced by antagonistic effect of the bacteriocins against the food- Lactococcus lactis subsp. lactis: properties, biosynthesis, fer- mentation and applications. In: De Vuyst L, Vandamme EJ (eds) borne pathogens indicated its usefulness in the preservation Bacteriocins of lactic acid bacteria. Blackie Academic & of different food products enhancing their shelf life. 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