Food safety is an important issue of international

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1 The Asian Journal of Animal Science (December, 21), Vol. 5 Issue 2 : RESEARCH ARTICLE Detection, partial purification and characterization of bacteriocin produced by Lactobacillus pentosus FPTLB13 isolated from freshwater fish S. CHOWDHURY, K.C. DORA AND S.P. BANERJEE Received : Aug., 21; Accepted :Sep., 21 See end of the article for authors affiliations K.C. DORA Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, KOLKATA (W.B) INDIA kc_dora@yahoo.co.in ABSTRACT Lactobacillus pentosus FPTLB13 was isolated from fresh water fish, capable of producing bacteriocin that had broad spectrum of inhibition (64 AU/ml) against Lactobacillus casei, Lactobacillus sakei, Pseudomonus aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. The antimicrobial activity was stable at 8 C for 15 min, and partly active at 121 C for 15 min. It remained active after incubation in ph range of 2-8 for 4 h at 37 C, but only partially inactive in media above ph 1. The bacteriocins produced by the test isolates maintained full stability after storage for 6 days at 2 C; partial stability after storage for 12 days at 4 C; while activity was not detected after storage for 6 to 12 days at 37 C. Its active principle was proteinaceous in nature since the bacteriocin was inactivated by proteolytic enzymes, but not by other non proteolytic enzymes. Mitomycin C and Ultra violet light did not affect the activity of the bacteriocin, while chloroform extraction completely destroyed their activity. The bacteriocin was resistant to treatment with SDS, Tween 2, Tween 8, urea and EDTA. Treatment with Triton X-1 reduced the activity of the bacteriocin and Triton X- 114 had no effect on it. No bacteriocin adsorption was detected at ph 1 to 2, whereas 1% bacteriocin adsorption was found at ph 7.. Based on Tricine SDS-PAGE the estimated molecular mass of bacteriocin was 3. kda. No plasmid was isolated, suggesting that the genes encoding the bacteriocins were located on the genome. Key words : Biopreservation, Lactic acid bacteria, Lactobacillus pentosus, Bacteriocin, Antimicrobial activity Food safety is an important issue of international concern. Prepacked food items available in food market contain variety of chemical preservatives, which may alter chemical constituents, nutritional and organoleptic qualities of food resulting in serious adverse effects on health (Messi et al., 23). Thus, biopreservation of food has emerged as an attractive and safe approach among which, bacteriocins have received increasing attention due to their unique properties of inhibiting food-borne pathogens and spoilage causing microorganisms. Bacteriocins are proteinaceous in nature and are produced by various strains of bacteria, most importantly lactic acid bacteria. They usually exhibit antagonistic activity against microorganisms closely related to the bacteriocin producing microorganisms. Several of these bacteriocins are bactericidal against pathogenic and food spoilage bacteria (Caplice and Fitzgerald, 1999). In the present study, a bacteriocin Chowdhury, S., Dora, K.C. and Banerjee, S.P. (21). Detection, partial purification and characterization of bacteriocin produced by Lactobacillus pentosus FPTLB13 isolated from freshwater fish. Asian J. Animal Sci., 5(2): produced from Lactobacillus pentosus FPTLB13, isolated from fresh water fish, was partially purified and characterized. MATERIALS AND METHODS Lactic acid bacteria screened for bacteriocin production and bacteriocin producing strain Lactobacillus pentosus FPTLB13 were isolated from gut of fresh water fish (Cirrhinus mrigala). Bacterial strains used as indicator organisms and their growth media are shown in Table 1 along with their specific growth medium. Lactic acid bacteria, isolated from gut of Cirrhinus mrigala were cultured in MRS broth (HiMedia, India) and screened for bacteriocins production according to the method described by Van Reenen et al. (1998). The target strains and growth media are listed in Table 1. The antibacterial activity was expressed as arbitrary units per ml (AU/ml). One AU was defined as the reciprocal of the highest serial two fold dilution producing a distinct inhibition of the indicator lawn (Van Reenen et al., 1998). Cell-free supernatants with antimicrobial activity were treated with proteinase K (1mg/ml) to determine if the activity is caused by the presence of bacteriocins.

2 S. CHOWDHURY, K.C. DORA AND S.P. BANERJEE Bacteriocin producing strains were identified on the basis of Gram staining, catalase reaction and carbohydrate fermentation pattern using API 5CHL test strips and API web (Biomerieux, France). To determine the molecular mass of the bacteriocin, the isolated strain was grown in MRSm broth for 18 h at 3 C. Cells were harvested (8 X g, 1 min, 4 C) and the bacteriocin precipitated from the cell-free supernatant with 4% saturated ammonium sulphate. The precipitate was resuspended in one tenth volume of 25 mm ammonium acetate buffer (ph 6.5), desalted by using a 1 Da cut off dialysis membrane (Himedia, India). The molecular mass of bacteriocin produced by Lb. pentosus FPTLB13 was estimated by Tricine SDS-PAGE system as described by Schagger and Von Jagow (1987). Electrophoresis was performed in vertical gels (8 mm X 7 mm X.75 mm) in a Mini-protean II cell (Himedia) at 2 ma for 3 h. After electrophoresis, the gel was cut in two vertical parts. One part was fixed and stained with Coomassie brilliant blue R-25. The other part was assayed for antimicrobial activity according to Bhunia et al. (1987) with modifications. Briefly, the gel was fixed for 3 min (25% isopropanol, 1% acetic acid), rinsed with distilled water (1 h initial rinsed followed by two washes of 5 min), and overlaid with 25 ml of soft (1%) Nutrient agar seeded with 1 5 CFU/ml of indicator strain. After incubation at 37 C for 24 h the gel was examined for the presence of an inhibitory zone. Molecular Mass Markers for Peptides (HiMedia) were used for mass standards. In order to assess the bacteriocin production by the isolated strain at different times of their growth MRS broth was inoculated with an overnight culture (1% v/v) of strain FPTLB13. Incubation was done at 3 C, without agitation. Sample was taken at 1 h intervals to determine the optical density at 6 nm (OD 6 ), ph of the culture and the antimicrobial activity (AU/ml) of the bacteriocin produced by the strain, as described previously (Todorov and Dicks, 25). The effect of ph on adsorption of bacteriocin onto producer cells was evaluated as reported by Yang et al. (1992). After 18 h of growth at 3 C, the culture broth was adjusted to ph 6. and centrifuged (2 X g) at 4 C for 15 min. It is then washed with sterile.1 M phosphate buffer (ph 6.5). The cells were re-suspended in 1 ml of 1 mm NaCl (ph 2.), agitated for 1 h at 4 to allow delaminating bacteriocin from the cells and then harvested (2 X g) at 4 C for 15 min. The cell free supernatant was neutralized to ph 7. with sterile 1 M NaOH and tested for activity. To study the cell lysis ability, 2 ml cell free supernatant (64 AU/ml, ph 6.) was added to a 1 ml-culture of Lactobacillus sakei in early exponential (OD 6 =.6). The optical density of the culture was determined every hour for 9 h (Todorov et al., 1999). For isolation of plasmid the procedure of Ivanova et al. (1998) was used. The cells of a 2 ml 18 h culture were collected and washed in 1 mm Tris-HCl, ph 8.2 and resuspended in 5 ml 2 mm Tris-HCl, ph 8.2. Then 3 mg lysozyme was added, followed by the addition of 24% polyethylene glycol (PEG 2 ) and 5 ml diethylpyrocarbonate (DEP). After 6 min incubation at 37 C and centrifugation, the pellet was resuspended in 9 ml 1 mm Tris-HCl, 1 mm Na 2 EDTA, ph 8.5. Cell lysis was achieved by the addition of 1 ml 1% SDS and incubation and incubation at 37 ºC for 15 min. The lysate was adjusted to ph 12.2 with 5 M NaOH and stirred for 15 min. at room temperature, followed by the addition of 2 ml of 2 M Tris-HCl, ph 7 and 1.2 ml of 5 M NaCl. The deproteinization was carried out once with phenol, saturated with 3% NaCl and once with chloroform. The plasmid DNA was precipitated with two volumes of cold ethanol and dissolved in 2 ìl of 1 mm Tris-HCl; 1 mm Na 2 EDTA, ph 8. and treated with RNAase (1 ìg/ml) (All from HiMedia, India). The CSF of Lb. pentosus FPTLB13 was characterized with respect to thermal and ph stability, susceptibility to denaturation by enzymes, stability during storage and UV light induction. For preparation of CSF, the isolated strains were grown in 2 ml of MRSm broth, under anaerobic conditions in order to avoid H 2 O 2 formation, up to stationary (48 h at 3 C). Cultures were centrifuged at 8 X g for 1 min, the supernatants were collected, adjusted to ph 6.5, sterilized by microfiltration (.22 ìm pore size filter paper, Himedia, Inidia). The solution thus obtained was stored at -2 C until further use. CSF of Lb. pentosus FPTLB13 (4µl) was exposed to various heat treatments: 4, 6, 8, 1 and 121 C. Aliquot volumes of each fraction were then removed after, 3, 6 or 9 min and assayed for bacteriocin activity (Rattanachaikunsopon and Phumkhachorn, 26). CSF of Lb. pentosus FPTLB13 (4 µ l) were adjusted to ph 2, 4, 6, 8, 1, and 12 with hydrochloric acid (HCl) and sodium hydroxide (NaOH), incubated for 4 h at room tempera ture and simila rly assayed (Rattanachaikunsopon and Phumkhachorn, 26). CSF of Lb. pentosus FPTLB13 was stored at 2, 4 and 37 C. At different time intervals, samples were taken from the stored material to determine bacteriocin activity (Corsetti et al., 24). [Asian. J. Animal Sci. (Dec., 21) Vol. 5 (2) ] 175

3 PARTIAL CHARACTERIZATION OF BACTERIOCIN FROM Lb pentosus FPTLB13 Sensitivity of the CSF to enzymes was tested by treatment with proteinase K, protease B, chymotrypsin, tripsine, papain, pepsin, pronase E, lysozyme, lipase, catalase and á-amylase at a final concentration of 1 mg/ ml in phosphate buffer (ph 6.5). The supernatants were incubated with these enzymes at 37 C for 2 h after which remaining activity was determined (Corsetti et al., 24). Mitomycin C was added at a final concentration of 1. µ g/ml to CSF of Lb. pentosus FPTLB13 and incubation was carried out at 3 C. Samples were removed at 6, 12, 24 and 36 min. and analysed by the well diffusion method (Wanda and Bonita, 1991). A 1 ml aliquot of CSF from Lb. pentosus FPTLB13 was placed in a sterile Petri dish and exposed to short wave uv light from a 15 W germicidal bulb at a distance of 3 cm. Times of exposure ranged from to 5 min. (Wanda and Bonita, 1991). After each time interval, bacteriocin activity was analysed. Various organic solvents including iso amylalcohol, chloroform, n propanol, hexane, Di ethyl ether, petroleum ether were added to CSF of Lb. pentosus FPTLB13 in 1:1 ratio. After thorough mixing, separation was achieved by centrifugation (1 min at 5 rpm). When propanol was used for the extraction, 5 g/l NaCl was added to the mixture in order to obtain separation. The organic and the aqueous were collected and solvent removed by evaporation at 45 C. The residue from the organic was resuspended in an amount of saline (8.5 g/l NaCl) equal to the starting volume of the original supernatant fluid. Bacteriocin activity of both preparations was then determined (Corsetti et al., 24). The effect of surfactants on the bacteriocin was tested by adding sodium dodecyl sulphate (SDS), Tween 2, Tween 8, urea, Triton X-114 and Triton X-1 (1%, v/v, final concentration) to the bacteriocin. EDTA was added to the bacteriocin to final concentrations of.1 mm, 2. mm and 5. mm, respectively. Untreated bacteriocin and the detergents at these respective concentrations were used as controls. All samples were incubated at 37 C for 5 h and then tested for antimicrobial activity (Todorov and Dicks, 24). All the experiments were carried out in duplicate and the results were shown as mean ± standard deviation (SD). The significance of differences amongst treatments after each day of storage during storage stability study was determined by analysis of variance using Statistical Package for Social Sciences (SPSS) program for Windows, version All other calculations were performed using Microsoft Excel, version 5, statistical functions (Microsoft Corp., Redmond, WA, USA). Differences were considered significant at the P<.5 level. RESULTS AND DISCUSSION Lactic acid bacteria isolated from mrigala were initially screened for bacteriocin production against the test strains as listed in Table 1. The isolated strain inhibited various bacteria with a spectrum of activity seen toward both gram positive and gram negative bacteria. To confirm that the inhibition was due to bacteriocin, neutralized cell free supernatant was treated with proteinase K and assessed for antimicrobial activity which was found to be absent suggesting the proteinaceous nature of the bacteriocin. Similar results were reported by Todorov and Dicks (24 and 26). The Gram-positive, catalase negative FPTLB13 was identified as Lactobacillus pentosus FPTLB13 by API 5 CHL test kit. The neutralized cell fr ee supernatant produced by Lactobacillus pentosus FPTLB13 showed an antimicrobial activity of 64 AU/ml against indicator organisms. The partially purified bacteriocin was analyzed by the tricine SDS-PAGE for determination of the molecular weight. One part of the gel was assayed for antimicrobial activity using agar overlaying technique with nutrient soft agar containing indicator organisms. A zone of inhibition was observed at the position equivalent to a protein band, having a molecular mass of about 3 kda, formed in the other part of the stained SDS-PAGE gel. On the basis of these, it may be concluded that the antibacterial bacteriocin produced by Lb. pentosus FPTLB13 was a peptide with a molecular mass of 3 kda. Similar molecular mass (3 kda) for Lb. pentosus ST151BR was reported by Todorov and Dicks (24). Detectable level of bacteriocin was recorded after 3 h of growth in MRS broth, indicating that the peptides are primary metabolites. Similar results were reported for plantaricin Y (Chin et al., 21) and bacteriocin produced by Lactobacillus pentosus ST151BR (Todorov and Dicks, 24). Production of bacteriocin FPTLB13 increased throughout logarithmic growth (i.e. the first 17h) and stabilized at 64 AU/ml during the following 12 h of slow growth (Fig. 1). As observed for sakacin K and a bacteriocin produced by Enterococcus faecium RZS C5, extended growth does not necessarily lead to increased production of activity levels (Leroy and De Vuyst, 22). In the present study the maximum level of the bacteriocin is produced towards the end of logarithmic growth. The activity level of bacteriocin remained at 64 AU/ml for the duration of incubation (Fig. 1), suggesting that they [Asian. J. Animal Sci. (Dec., 21) Vol. 5 (2) ] 176

4 S. CHOWDHURY, K.C. DORA AND S.P. BANERJEE Table 1: Growth medium and incubation temperature of indicator strains and spectrum of antimicrobial activity recorded for bacteriocin produced by Lactobacillus pentosus FPTLBL13 Indicator organisms Strain no. Specific medium Incubation temperature Zone diameter (mm) Lactobacillus casei MTCC 1423 MRSm 3 C 7±.3 Lactobacillus sakei ATCC MRSm 3 C 8±.3 Staphylococcus aureus ATCC Baird Parker 37 C 5±.2 Pseudomonus aeruginosa MTCC 3163 BHI 37 C 7±.3 Escherichia coli MTCC 1563 Tergitol 7 37 C 8±.3 Enterococcus faecalis MTCC 2729 MRS 37 C 1±.5 are not affected by changes in culture conditions (e.g. lowering of ph from 6.5 to 3.5). OD (6 nm) Bacteriocin production by FPTLBL time (h) ph AU/ml Fig. 1: Bacteriocin production by Lb. pentosus FPTLBL. ( ) showing the increasing OD 6 value of the culture, ( ) showing the decreasing ph value and ( ) showing the bacteriocin activity (AU/ml) AcH, 6.5 for nisin, 5.5 and above for sakacin A, and 5.5 for leuconocin Lem 1, while no bacteriocin adsorption was detected at ph below 1.5 for pediocin AcH, below 3 for nisin, below 2 for sakacin A, below 3 and above 8 for leuconocin Lem 1. Addition of the bacteriocin (64 AU/ml), produced by FPTLB13, to early logarithmic- cells of Lactobacillus sakei (3 h-old; aprox OD 6 =.6) resulted in a significant (P<.1) growth inhibition after 1 h, followed by complete growth inhibition for the remaining 6 h (Fig. 3). Addition of same activity of CSF to stationary cells of Lactobacillus sakei, resulted in no inhibition suggesting that the older cells of L. sakei became resistant to the bacteriocin or that the bacteriocin was destroyed by extracellular proteolytic enzymes. Similar observation was reported by Todorov and Dicks (24). Adsorption of Lb. pentosus FPTLB13 bacteriocin onto producing cells was strongly influenced by the ph of the suspending environment. The bacteriocin adsorption increased from 5 to 1% as the ph increased from 4 to 7. and decreased thereafter at higher ph. No bacteriocin adsorption was detected at ph 1 to 2 (Fig. 2), suggesting that the bacteriocins did not adhere to the surface of the producer cells. Yang et al. (1992) reported that the highest percentage of bacteriocin adsorption onto producing cells occurred at ph 6 and 6.5 for pediocin Bacteriocin adssorption OD (6 nm) Fig. 3: Time (H) control Effect of bacteriocin produced by Lb. pentosus FPTLBL on indicator organism bac+ % of bacteriocin adsorbed ph Fig. 2: Influence of ph of bacteriocin absorption In the present, study no plasmid was isolated from the test isolate. In most cases the genes encoding bacteriocins are harboured on plasmids (Klaenhammer, 1988; Huot et al., 1996) although for a number of bacteriocins the genes have been located on the genome (Diop et al., 1994; Todorov et al., 1999). Since no plasmid was isolated from the strain Lb. pentosus FPTLB13, it may be concluded that the genes encoding the bacteriocin was located on its genome. During characterization of CSF of Lb. pentosus [Asian. J. Animal Sci. (Dec., 21) Vol. 5 (2) ] 177

5 PARTIAL CHARACTERIZATION OF BACTERIOCIN FROM Lb pentosus FPTLB13 FPTLB13, antimicrobial activity was determined using E. faecalis as indicator organism. The inhibitory compound (bacteriocin) produced by the isolated strain was considered to be heat stable. The activity of the bacteriocin produced by Lactobacillus pentosus FPTLB13 remained constant (64 AU/ml) after heating at 8 C for 15 min and partly active at 121 C for 15 min (Fig. 4). Ogunbanwo et al. (23) and Corsetti et al. (24) also reported loss of activity of antibacterial substances produced by Lactobacillus spp. after heat treatment at 121 C for different time duration. Activity (AU/ml It was observed that bacteriocin produced by FPTLB13 strain was stable at ph 2 to 8 for 4 h at 37 C, but only partially inactivated above ph 1 (Table 2). Todorov and Dicks (26), Corsetti et al. (24), Rattanachaikunsopon and Phumkhachorn (26) also reported the similar findings. Table 2: Effect of ph treatment on bacteriocin activity (AU/ml) produced by the test isolate ph AU/ml ph AU/ml 2 64±. 8 64±. 4 64±. 1 32±. 6 64±. 12 4±. Zone diam eter (m Fig. 4: Fig. 5: 4 6 Temperature 8 ( o C) 1 12 Da ys min 3 min 6 min 9 min Effect of time and temperature on antimicrobial activity In the present study, effect of time and temperature of storage on bacteriocin activity was also carried out. Bacteriocin produced by FPTLB13 maintained fully stability after storage for 6 days at -2 C; partial stability " -2 C 4 C 37 C Effect of storage time and temperature on antimicrobial activity after storage for 12 days at 4 C, while no activity was detected after storage for 6 to 12 days at 37 C (Fig. 5), indicating that cold temperature may be the most appropriate preservation technique. Ogunbanwo et al. (23) reported the similar findings while working with L. brevis OG1 and L. plantarum F1. Corsetti et al. (24) also reported the similar results while working with Sourdough lactic acid bacteria. Results from enzyme inactiva tion studies demonstrated that antimicrobial activity was lost or unstable after treatment with all the proteolytic enzymes, whereas treatment with lipase, catalase, lysozyme, á- amylase, mitomycin C and uv light did not affect the activity of bacteriocin produced by the test isolates (Table 3), confirming the protein status of bacteriocins. Antimicrobial activity of the bacteriocins produced by the organisms in this study was not due to hydrogen peroxide or acidity, as activity was not lost after treatment with catalase or peroxidase or adjustment of ph to 7.. Similar results were reported by Todorov and Dicks (26), Corsetti et al. (24), Rattanachaikunsopon and Phumkhachorn (26) and many other researchers. Table 3: Effect of enzymes, mitomycins and uv light treatments on activity (AU/ml) of bacteriocin produced by the test isolate Treatments AU/ml Treatments AU/ml Control 64±. α - Amylase 64±. Lipase 64±. Papain ND Chymotrypsin ND Pepsin ND Protease B ND Pronase E ND Catalase 64±. Lysozyme 64±. Trypsin ND Mitomycin C 64±. Proteinase K ND UV light 64±. ND = non-detectable Various organic solvents were tested for the extraction of bacteriocin produced by the test isolates. It was observed that extraction with polar solvents such as hexane, di-ethyl ether, and petroleum ether did not result in the removal of bacteriocin produced at the aqueous to the organic, while chloroform extraction completely destroyed the bacteriocins activity. However, when different alcohols such as n-propanol and Iso-amyl alcohol were used in the extraction procedure, bacteriocin was removed from the aqueous and recovered from the organic (Table 4). Extraction of bacteriocin in this study using organic solvents indicated that bacteriocin was removed from the aqueous and could be recovered from the organic. This suggests that at least part of the bacteriocin molecule has a [Asian. J. Animal Sci. (Dec., 21) Vol. 5 (2) ] 178

6 S. CHOWDHURY, K.C. DORA AND S.P. BANERJEE Table 4: Influence of organic solvents in the extraction of bacteriocin produced by the test isolates solvent Aqueous solvent Aqueous Control - 1±.5 Di I amylalcohol hydrophobic character, and shares this property with most other bacteriocins (Klaenhammer, 1993). The bacteriocin FPTLB13 was resistant to treatment with 1% (m/v, final conc.) SDS, Tween 2, Tween 8 and urea, and.1 mm, 2. mm or 5. mm EDTA. Treatment with Triton X-1 reduced the activity of the bacteriocin. However, Triton X-114 had no effect on bacteriocin produced by FPTLB13 (Table 5). Similar results were recorded for pediocin ST18 (Todorov and Dicks, 25), plantaricin ST31 (Todorov et al., 1999) and bozacin B14 (Ivanova et al., 2). Table 5: Factors affecting the activity of bacteriocin produced by the test isolate Treatments Bacteriocin activity Treatments Bacteriocin activity Surfactants (1%, final Protease inhibitor: EDTA concentration) SDS +.1 mm + Tween mm + Tween mm + Urea + Triton X Triton X = active; - = no activity ethylether - 7±.3 8±.3 8±.3 Hexane - 9±.5 Choloform - - Petroleum N 8±.3 5±.3 ether - 6±.3 propanol Zone of inhibition (mm) Control = bacteriocin without addition of organic solvent The present investigation indicates that Lactobacillus pentosus FPTLB13 isolated from fresh water fish, Cirrhinus mrigala produced bacteriocin like substances that has spectrum of antimicrobial activity against Lactobacillus casei, Lactobacillus sakei, Pseudomonus aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. Partially purified bacteriocin is characterized by high heat stability, wide ph tolerance and sensitivity to various enzymes. Molecular weight of the antimicrobial substance is about 3 kda. The strong antagonistic effect of CSF against the food borne pathogens indicated its usefulness in the preservation of different food items enhancing their shelf-life and thus providing future scope for its application as food preservative. Acknowledgement: The support from CFTRI (Mysore, India) and IMTECH (Chandigarh, India) is gratefully acknowledged for enriching this study with literatures support and providing microbial cultures, respectively. Authors affiliations: S. CHOWDHURY AND S.P. BANERJEE, Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, KOLKATA (W.B.) INDIA LITERATURE CITED Bhunia, A.K., Johnson, M.C. and Ray, B. (1987). Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulphate-polyacrilamide gel electrophoresis. J. Indian Microbiol., 2: Caplice, E. and Fitzgerald, G.E. (1999). Food fermentations: role of microorganisms in food production and preservation. Internat. J. Food Microbiol., 5: Chin, H.S., Chin, J.S., Kim, J.M., Yang, R. and Yoon, S.S. (21). Detection and antibacterial activity of a bacteriocin produced by Lactobacillus plantarum. Food Sci. Biotechnol., 1: Corsetti, A., Settanni, L. and Van Sinderen, D. (24). Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity. J. Appl. Microbiol., 96: Diop, D.B., Havarstein, L.S., Nisen-Meyer, J. and Nes, I.F. (1994). The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an Arg-like regulatory system. Appl. Environ. Microbiol., 6: Huot, E., Maghrous, J. and Barena-Gonzalez, C. (1996). Bacteriocin J46, a new bacteriocin produced by Lactococcus lactis subsp. cremoris J46: Isolation and characterization of the protein and its gene. Anaerobe., 2: Ivanova, I., Miteva, V., Stefanova, T. S., Pantev, A., Budakov, I., Danova, S., Moncheva, P., Nikolova, I., Dousset, X. and Boyaval, P. (1998). Characterization of a bacteriocin produced by Streptococcus thermophilus 81. Internat. J. Food Microbiol., 42: Ivanova, I., Kabadjova, P., Pantev, A., Danova, S. and Dousset, X. (2). Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza-bulgarian traditional cereal beverage. Biocatal.-Vestnik Moskov. Univ., Kimia., 41: [Asian. J. Animal Sci. (Dec., 21) Vol. 5 (2) ] 179

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