CHAPTER 7 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN PRODUCED BY LACTOBACILLUS PLANTARUM AS1

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1 CHAPTER 7 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN PRODUCED BY LACTOBACILLUS PLANTARUM AS Introduction Bacteriocins produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives (Diep & Nes, 2002). Different bacteriocins have been reported in Lactobacillus plantarum species, which are included in following classes: Plantaricin C and W, included in class I, plantaricin C19 and 423, included in class IIa, plantaricin EF, JK, S, and NC8, included in class IIb, and plantaricin 1.25B included in class IId (Rojo-Bezares et al., 2007). Appam or hoppers are a common type of food in South Indian cuisine especially Tamilnadu and Kerala. Another form of appam is Kallappam which looks like a pan cake. The name originated from Kallu which means toddy (Satish et al., 2010). L. plantarum AS1 was isolated from Kallappam. It showed effective cheese biopreservative property against Salmonella typhi. L. plantarum AS1 showed probiotic properties such as bile salt tolerance, gastric juice tolerance, cholesterol reduction, non-pathogenesis during oral feeding of albino rats for a month period (Satish et al., 2010). L. plantarum AS1 attached efficiently to HT-29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. The attachment to human intestinal cells involved different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surfaces (Satish et al., 2011). In the present work, antimicrobial substance of L. plantarum AS1 was purified to homogeneity and it was characterized by various enzymes and temperature sensitivity to determine the nature of antimicrobial substance. 101

2 7.2. Materials and methods Strains and chemicals Lactobacillus plantarum AS1 (Genbank # GQ468312) was isolated from the Kallappam batter and cultured in MRS broth (Himedia, Mumbai) at 37 C for 16 h before the study. The indicator organisms, viz. Vibrio parahaemolyticus 451, Vibrio vulnificus1145, Vibrio fischeri1738, Vibrio anguillarum, Escherichia coli DH5α, Lactobacillus acidophilus 447, Lactobacillus rhamnosus 1408, Salmonella typhi 734, Listeria monocytogenes 1143 and Proteus vulgaris 426 were procured from the Microbial Type Culture Collection (MTCC) at the Institute of Microbial Technology, Chandigarh, India (Table 11). All strains were maintained as frozen stocks at -80 C. Working cultures were maintained in the agar media and subcultured in liquid media before use. The bacteriological media, analytical grade chemicals, and proteolytic enzymes were obtained from Hi-Media Mumbai, India while molecular weight marker was purchased from Bangalore Genei, Bangalore, India Bacteriocin assay A well diffusion assay procedure was used for antibacterial assay as described previously (5.2.2) Purification of plantaricin AS1 Purification of Enterocin MC13 was carried out as described previously (5.2.4) 102

3 Molecular mass determination Tricine SDS-PAGE: HPLC purified fraction was used to determine the molecular weight of plantaricin AS1 by Tricine SDS-PAGE (10%) (Schägger & Jagow, 1987). A low molecular weight protein marker with sizes ranging from 3.0 kda to 205 kda (Bangalore Genei, Bangalore, India) was used. Gel was stained by silver staining method (Morrissey, 1981). MALDI-TOF Mass Spectrum: The molecular mass of plantarcin AS1 was further confirmed by a mass-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The mass of peptide was carried out by the Proteomic facility of Molecular Biophysics Unit at Indian Institute of Sciences, Bangalore Bacteriocin activity in polyacrylamide gel Tricine SDS-PAGE was run under non-reducing conditions (5.2.6) Effect of heat and enzyme treatment on plantaricin AS1 Dialyzed samples of plantaricin AS1 were used in these experiments. V. parahaemolyticus was used as an indicator organism. About 1 ml aliquots of bacteriocin sample were exposed to temperatures of C for 5 to 30 min and tested for antibacterial activity. Sensitivity to enzymes was determined as described previously (5.2.3) Antibacterial activity spectrum Dialyzed bacteriocin sample (semi-crude preparation) was employed for determination of the antibacterial activity spectrum against indicator strains (Table 11). Bacteriocin assay was performed as mentioned earlier (5.2.2). 103

4 7.3. Results Purification of Enterocin MC13 The active fraction of Sephadex G-25 purified sample was lyophilized and dissolved in minimum volume of Solvent A (0.1% (w/v) trifluoro-acetic acid (TFA) in 5% (v/v) acetonitrile in water) for HPLC analysis. On injection of SephadexG-25 purified sample to reverse phase HPLC, a distinct peak was eluted at 35% of acetonitrile, corresponding to retention time of min (Fig. 8). The peak was shown to be active against V. parahaemolyticus Molecular mass determination Tricine SDS-PAGE of the active fraction collected from reverse phase-hplc analysis yielded a peptide band of approximately 3.3 kda (Fig. 9). This result was confirmed by MALDI-TOF mass spectrum and a sharp peak corresponding to kda was obtained (Fig. 10). So, this revealed the molecular mass of plantaricin AS1 to be kda Bacteriocin activity in polyacrylamide gel A clear zone in native PAGE was observed, which was corresponding to plantaricin AS1 band (Fig. 9). This result further validated molecular mass of plantaricin AS1 as kda. 104

5 Fig. 8. HPLC chromatogram of L. plantarum AS1 bacteriocin A distinct peak of purified bacteriocin was obtained at min. 105

6 Fig. 9. Tricine SDS-PAGE of L. plantarum AS1 bacteriocin A - Bacteriocin activity in native PAGE against Vibrio parahaemolyticus (An activity zone corresponding approximately to 3.3 kda bacteriocin band) B - Purified bacteriocin band of approximately 3.3 kda C Molecular weight marker ( kda) 106

7 Fig. 10. MALDI-TOF Mass Spectrometry of L. plantarum AS1 Bacteriocin A peak of 3.35 kda molecular mass was obtained when a HPLC purified bacteriocin sample was subjected to MALDI-TOF mass spectrometry. 107

8 Effect of heat and enzymes on plantaricin AS1 Plantaricin AS1 was found to be heat resistant until the temperature of 100 C for 30 min (Table 12). Plantaricin AS1 was sensitive to chymotrypsin, trypsin and protease but insensitive to catalase confirming that inhibition was due to proteinaceous molecule, i.e. bacteriocin and not due to hydrogen peroxide or diacetyl. Its activity was not reduced by lipase indicating that there was no structural modification by lipid moiety (Table 12) Antibacterial spectrum of plantaricin AS1 Inhibition was found against a wide spectrum of bacterial species which includes both grampositive as well as gram-negative bacteria (Table 11). Dialyzed bacteriocin sample (semi-crude preparation) of L. plantarum AS1 was found to be inhibitory against important pathogens of food and livestock (fish and shrimp): L. monocytogenes, V. parahaemolyticus, V. harveyi, V. vulnificus, V. anguillarum, V. fisheri, Salmonella typhi, and Proteus vulgaricus. 108

9 Table 11. Antibacterial spectrum of Plantaricin AS1 Indicator bacteria Growth medium Antibacterial activity Vibrio parahaemolyticus TSA Vibrio vulnificus TSA Vibrio fischeri TSA Vibrio aungullarum TSA E.coli DH5 α TSA Lactobacillus acidophilus MRS Lactobacillus rhamnosus MRS Salmonella typhi TSA Listeria monocytogenes BHI Proteus vulgaris TSA, inhibition zone less than10mm in diameter;, inhibition zone 10mm - 20mm in diameter; inhibition zone larger than 20mm in diameter; -, no inhibition zone recorded. TSA Tryptone soy agar; BHI Brain heart infusion; MRS de Man, Rogosa and Sharpe agar 109

10 Table 12. Effect of heat and enzymes on plantaricin AS1 activity Treatment Bacteriocin activity* Heat 50 C for 30 min 60 C for 30 min 70 C for 30 min 80 C for 30 min 90 C for 30 min 100 C for 30 min 121 C for 30 min Enzymes Trypsin Catalase Protease Lipase Chymotrypsin - - -, inhibition zone;, no inhibition zone * Bacteriocin activity was compared against that of V. parahaemolyticus 110

11 7.4. Discussion For the first time, we isolated Lactobacillus plantarum from South Indian fermented food Kallappam. L. plantarum strains were isolated earlier from various sources such as fermented milk Amasi (Todorov et al., 2007), kefir (Powell et al., 2007), Papaya (Todorov et al., 2011), Mexican corn Tortilla (Hata et al., 2010), Sorghum beer (Verellen et al., 1998), Koumiss (Xie et al., 2011), Bulgarian salami (Todorov & Velho, 2008), fermented cucumbers (Atrih et al., 2001), fermented Italian sausages (Messi et al., 2001), sourdough (Todorov et al., 1999), fermented cream Jiaoke (Gong et al., 2010), fermented carrot (Andersson et al., 1988), Pineapple (Kato et al., 1994), Beloura and Chaurico (Todorov et al., 2010), fermented green olives (Diaz et al., 1993), Munster Cheese (Ennahar et al., 1996), Grass silage (Maldonado et al., 2003), Algerian fermented olives (Mourad et al., 2005). L. plantarum AS1 is a potential probiotic strain. Its safety evaluation was performed by feeding 10 8 CFU/ml/day of viable cells to Wistar rats. Also, it was tolerant to bile salt, artificial gastric juice, and assimilated cholesterol provided in the growth medium (Satish et al., 2010). L. plantarum AS1 effectively colonized HT-29 cell line and also prevented adhesion of enteropathogen V. parahaemolyticus to HT-29 cells (Satish et al., 2011). Dialyzed sample (semicrude preparation) of L. plantarum AS1 had shown broad spectrum antibacterial activity. It was inhibitory towards pathogens such as L. monocytogenes, Vibrio parahaemolyticus, V. harveyi, V. vulnificus, V. anguillarum, V. fischeri, Salmonella typhi, and Proteus vulgaris. Similarly, Suma et al., (1998), reported a broad spectrum bacteriocin Plantaricin LP84 that showed inhibitory activity towards Bacillus cereus, E. coli, Staphylococcus aureus, B. licheniformis, B. subtilis and Pseudomonas aeruginosa. Lash et al., (2005), described a bacteriocin produced by L. plantarum (ATCC 8014) that showed antibacterial activity towards S. aureus, E. coli, L. 111

12 innocua, P. aeruginosa. Xie et al.,(2011) reported a bacteriocin active against strains of Listeria, Lactobacillus, Streptococcus, Pediococcus, and Escherichia. Plantaricin 35d produced a L. plantarum strain showed a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and Aeromonas hydrophila (Messi et al., 2001). Plantaricin MG produced by L. plantarum KLDSI 0391 isolated from Jiaoke showed a broad inhibitory activity against Grampositive and Gram-negative bacteria including L. monocytogenes, S. aureus, S. typhimurium, and E. coli (Gong et al., 2010). Valenzuela et al., (2008), described a bacteriocin that inhibited food poisoning and pathogenic bacteria B. cereus, E. coli, Salmonella enterica. Plantaricin 423 showed inhibitory activity towards B. cereus, Clostridium sporogenes, Listeria spp. and Staphylococcus spp.(reenen et al., 1998). Planataricin AS1 was thermostable as it was active up to 100 C for 30 min. Also, it was sensitive to proteolytic enzymes, trypsin, chymotrypsin, protease but not to lipase, catalase confirming the proteinaceous nature of antibacterial substance. These properties are reported to be similar in all the bacteriocins of L. plantarum. Plantaricin AS1 was purified to homogeneity by multiple step purification. MALDI-TOF mass spectrometry analysis confirmed the purity of the sample and determined the molecular mass to be 3.3 kda. Previous reports on molecular mass of bacteriocins of L. plantarum strains did not exactly corresponded to plantaricin AS1 molecular mass. The molecular mass varied among L. plantarum bacteriocins e.g. Bacteriocin AMA-K was reported to be 2.9 kda (Todorov et al., 2007), BacST8KF was 3.5 kda in size (Powell et al., 2007), Bacteriocin ST16Pa from L. plantarum ST16Pa reported to be 6.5 kda (Todorov et al., 2011). Bacteriocin ATCC 8014 s molecular mass assessed by SDS-PAGE and reported to be 122 kda which is highest among L. plantarum bacteriocins (Lash et al., 2005). Similarly, plantaricin ASM1 was reported to be 5.05 kda (Hata et al., 2010). Some of the 112

13 bacteriocins were very close in molecular mass to plantaricin AS1 such as plantaricin ST8SH of 3 kda (Todorov & Velho, 2008), plantaricin C19 of 3.8 kda size (Atrih et al., 2001), bacst202ch of 3.5 kda mass (Todorov et al., 2010), and plantaricin C and 423 of 3.5 kda each (Gonzalez et al., 1994; Reenen et al., 1998). The combination of antibacterial and probiotic properties of L. plantarum AS1 suggests it to be a putative human probiotic strain. Nevertheless, further work on identification of bacteriocin genes and animal trials are mandatory to confirm L. plantarum AS1 to be a human probiont. 113

CHAPTER 8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA STRAINS FOR PROBIOTIC CHARACTERISTICS

CHAPTER 8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA STRAINS FOR PROBIOTIC CHARACTERISTICS 8.1. Introduction Probiotic bacteria must overcome physical and chemical barriers such as acid and bile