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Purification and Characterization of Two Bacteriocins from Lactobacillus

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Purification and Characterization of Two Bacteriocins from Lactobacillus 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).
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