INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, 2011

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, Patil M.M et al., licensee IPA- Open access - Distributed under Creative Commons Attribution License 2.0 Research article ISSN Patil.M. M., Rajini Kumari, Y., Ramana, K. V. Food Biotechnology Discipline, Defence Food Research Laboratory, Mysore , India. mahantesh_05@yahoo.co.in doi: /ijessi ABSTRACT Lactic acid bacteria are widely known for their inherent ability to produce antimicrobial substances such as bacteriocins, which are ribosomally synthesized low-molecular weight proteins. The bacteriocins have great potential to antagonize food borne pathogens and spoilage microorganisms and they are being used as food biopreservatives. Attempts have been made to isolate bacteriocin producing lactic acid bacteria from rat intestine. Three bacterial strains i.e. DFR8, DFR15 and DFR25 were found to produce substantial amount of bacteriocins. All the 3 bacterial isolates were found to be cocci, homo-fermentative and catalase negative. Growth of these isolates at different temperatures, ph and in the presence of NaCl was studied. Optimum growth was found to be at 37 o C and the strains were able to grow at high ph between 8.6 and 9.2, in the presence of 4% NaCl but not in media containing 6% and 10% of NaCl. Carbohydrate utilization and biochemical tests were performed for their identification. Based on morphological, physiological and biochemical characteristics DFR8 and DFR25 were identified as Enterococcus faecium and DFR15 as Enterococcus faecalis. The bacteriocins produced by these isolates showed broad inhibitory spectrum against various food borne pathogens and some standard lactic acid bacteria. Optimization of medium ingredients to improve the growth and bacteriocin production was accomplished. Partially purified bacteriocins were analyzed by tris-tricine SDS-PAGE indicating the molecular mass of the protein between 3.0 and 3.5 kda. Keywords: Bacteriocins, Enterococcus, Lactic acid bacteria, Intestinal microflora, Biopreservatives. 1 Introduction Lactic acid bacteria (LAB) are widely utilized in the production of various fermented foods and for the production of low molecular weight antimicrobial proteins to be used as biopreservatives. Investigations on LAB in this regard have attracted much attention (Klaenhammer, 1988). The antimicrobial effect of LAB has been shown to extend the shelf life of many foods exerting a strong antagonistic activity against a wide range of microorganisms. LAB are the source of various organic acids, diacetyl, hydrogen peroxide and bacteriocins (Oyetayo and Adetuyi 2003, Sholeva 1998, Zhennai 2000). The bacteriocins from LAB become popular as natural food preservatives to control food-borne pathogens and a variety of spoilage microorganisms, as they possess inhibitory activity towards these microorganisms. Several studies have revealed that different strains of a species or different species in a genus can produce similar type of bacteriocins (Ray and Miller, 2000). In the gastro-intestinal tract of animals, bacteria and yeast maintain a niche producing healthy benefit to the host. Hence an attempt was made to isolate bacteriocin-producing LAB from one of the most popular experimental model such as the rat. Although Received on March, 2011 Published on April

2 a stigma is attributed to the use of bacteriocins extracted from these animals, this type of investigation is more productive to delineate the role or effect of either naturally occurring or other extraneous probiotic bacteria on the well being of the animal and for further translation of the data for application in human being and domestic animals. Furthermore, the bacteriocin genes can be expressed using heterologous expression systems if found suitable for their use as biopreservatives or for clinical/therapeutic applications. Earlier, we have reported isolation and characterization of LAB from rat intestine (Mahantesh et al., ) and the present study deals with isolation and characterization of other bacteriocin producing LAB from rat intestinal flora, optimization of medium for bacteriocin production, purification and preliminary characterization of these bacteriocins. 2. Materials and Methods 2.1. Chemicals and cultures Analytical grade chemicals were obtained from SRL, India and S.D Fine Chemicals, India, while the molecular weight markers and bacteriological media were obtained from Sigma, USA and HiMedia, India, respectively. Indicator organisms comprising Staphylococcus aureus, Yersinia enterocolitica, Aeromonas hydrophila, Clostridium perfringens, Micrococcus luteus, Listeria monocytogenes, Lactococcus cremoris, Lactobacillus planatarum, Lactobacillus rhamnosus, Leucanostoc mesenteroides, Lactococcus diacetylactis and Lactococcus lactis were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. The bacterial strains Escherichia coli DFR-262 and Pseudomonas DFR-219 were isolated at Defence Food Research Laboratory, Mysore, India Isolation and screening of LAB for bacteriocin production LAB were isolated from Wistar albino rat (6 months old and approximately 200g weight). The animal was sacrificed under the effect of anesthesia and intestinal contents were collected by dissecting the animal. One-gram of intestinal contents were suspended in 9 ml of sterile saline and centrifuged at 5000 rpm for 15min. The supernatant was serially diluted and spread plated on MRS (demann, Rogosa and Sharpe) agar and incubated anaerobically at 37 o C for 48 h. The isolated colonies were transferred to MRS broth and streaked on MRS agar to check the purity of the isolates and stored in MRS soft agar (0.5%) overlaid with 50% glycerol at -20 o C. A total of 26 isolates were screened for antimicrobial activity. LAB colonies on MRS agar plates were overlaid with nutrient agar (0.7%) containing indicator organisms viz. Staphylococcus aureus, Listeria monocytogenes and Lactobacillus cremoris. Overlaid plates were incubated at 37 o C for 24 h and examined for the presence of inhibition zone. Three isolates exhibiting substantial zone of inhibition were selected for further study Morphological, physiological and biochemical tests All the 3 isolates were examined microscopically for their cellular morphology and Gram s stain phenotype. Growth of these isolates was studied at different temperatures (15, 37 and 45 o C), ph (3.9, 4.5, 8.6 and 9.2) and in presence of 4%, 6% and 10% (w/v) NaCl. Catalase activity of the isolates was tested by spotting colonies with 3% hydrogen peroxide. Production of acid and CO 2 from glucose was checked in MRS broth (without citrate) 1403

3 containing Durham s tubes (Schillinger and Luke, 1987). Production of ammonia was ascertained in MRS broth (without glucose and meat extract) containing arginine (0.3%) and sodium citrate (0.2%), using Nessler s reagent. Homo and Hetero-fermentative differentiation (HHD) test was carried out according to the method reported by Zuniga et al., (1993). Their ability to ferment various carbohydrates was checked by using HiCarbohydrate TM kit HiMedia, India Bacteriocin extraction and antibacterial assay Bacteriocin extraction was done using cell adsorption-desorption method (Yang et al., 1992) with suitable modifications. Two liters of 48 h culture broth was heated at 70 0 C for 20 min (to inactivate proteases), adjusted the ph to 6.5 with 10N NaOH and kept for stirring for 3 h at low speed for adsorption of bacteriocin over the cells. The cells were pelleted (6000 rpm, 20 min), washed with 5mM sodium phosphate buffer (ph 6.5) and desorbed with 0.1N NaCl (ph 2.0) by stirring overnight at 5 o C. Cells were removed by centrifugation at 6000 rpm for 20 min at 4 o C. The supernatant was concentrated, adjusted to ph and the antimicrobial activity was determined by agar well diffusion assay (Tagg and Mc Given, 1971) Partial purification of bacteriocins A column (110 X 1.5 cm) was packed with Sephadex G-50 (Sigma, USA) and equilibrated with 0.05 mm ammonium acetate buffer, ph 4.5. The same was used to elute the sample. Void volume was determined by passing blue dextran (2000 kda) through the column. Two ml of partially purified bacteriocin preparations obtained from cell adsorption and desorption technique was loaded to the column. Fractions were collected at a flow rate of 0.2 ml/min at 6 min intervals. Absorbance was measured at 280 nm using a Shimadzu spectrophotometer. Various fractions around peaks, shoulders, and valleys were pooled separately and assayed for antimicrobial activity against Staphylococcus aureus SDS-PAGE analysis Tris-tricine SDS-PAGE (Schagger and Von Jagow, 1987) was employed to determine the molecular weight of partially purified bacteriocin samples. A vertical slab gel electrophoretic apparatus with 16% separating gel, 10% spacer gel and 6% stacking gel was used. The molecular weight was calculated from the relative mobility of the standard molecular weight markers run simultaneously Protein assay Protein quantification for cell adsorbed-desorbed samples and column passed samples was carried out by Biuret method (Gornall et al., 1949) using bovine serum albumin as standard Optimization of media for bacteriocin production Production of bacteriocins from the 3 isolates was evaluated using different media viz, MRS, double strength MRS (2X-MRS), 1 % TGE (Tryptone, Glucose and Yeast extract), double strength TGE (2% TGE), and M17. All media were inoculated with 1% (v/v) of 16-h cultures and incubated at 37 o C for 48 h. After 48 h of incubation, viable counts of isolates were determined by pour plate technique and bacteriocin extraction from all the culture broths was 1404

4 carried out by cell adsorption-desorption method. Antibacterial activity of the bacteriocin samples was determined by agar well diffusion assay as mentioned earlier Inhibitory spectrum of bacteriocins Antibacterial activity of GPC purified bacteriocins was determined against various indicator organisms (Table 4) by agar well diffusion assay. 3. Results 3.1. Morphological, Physiological and Biochemical tests All the 3 isolates were found to be Gram-positive cocci. Optimum temperature for their growth was found to be 37 o C. All the isolates grew luxuriously at ph values 8.6 as well as 9.2 but could not grow at ph 4.4. Weak growth was reported for all isolates in presence of 6% NaCl and none could tolerate 10% NaCl concentration (Table 1). The isolates were catalase negative and did not produce CO 2 from glucose. All the 3 isolates produced ammonia from arginine and were found to be homo-fermentative (Table 1). Table 1: Morphological, physiological and biochemical characteristics of Enterococcus isolates. Enterococcus Isolates Tests E. faecium DFR8 E. faecalis DFR15 E. faecium DFR25 Morphology and cell arrangement Growth at temperature ( o C) Growth at ph Growth in NaCl (%) Cocci, Pairs & chains Cocci, Pairs Cocci, Pairs & chains W W W CO 2 from Glucose Acid from Glucose NH 3 from Arginine HHD Medium Homo Homo Homo Catalase Legend: Growth (+), Luxurious growth (++), Weak growth (W), No growth (-), Homofermentative (Ho). Fructose, galactose, maltose, mannitol, mannose, salicin and sorbitol were utilized by these isolates however they could not ferment adonitol, arabinose, dulcitol, malonate and raffinose (Table 2). Based on their morphological, physiological and biochemical characteristics the isolates were assigned to the genus Enterococcus. The ability to ferment melizitose and inability to ferment mellibiose is a special character of E. faecium, while E. faecalis could 1405

5 ferment mellibiose and not melizitose. This character helped in distinguishing between E. faecalis from E. faecium (Mundt, 1986). Table 2: Carbohydrate utilization pattern of Enterococcus isolates. Name of the sugar E. faecium DFR8 E. faecalis DFR15 E. faecium DFR25 Adonitol Arabinose Cellobiose Citrate Dextrose Dulcitol Esculin Fructose Galactose Malonate Maltose Mannitol Mannose Melezitose Melibiose ONPG Raffinose Ribose Rhamnose Salicin Sorbitol Sorbose Sucrose Xylitol Xylose Legend: Positive (+), Negative (-), Weak positive (W) Extraction, partial purification and SDS-PAGE analysis of bacteriocins The cell adsorption-desorption method for extraction of bacteriocins resulted in a partially pure bacteriocins. Further purification was achieved by gel permeation chromatography (GPC) technique using Sephadex G-50 column, and was revealed by the presence of a single diffused band on Tris-tricine SDS-PAGE analysis. E. faecium DFR8 and DFR 25 bacteriocins were found to have a molecular weight of ~3.5 kda, while it was ~3.0 kda for E. faecalis. DFR Effect of media on growth and bacteriocin production Among the various media tried, maximum growth for all the 3 isolates was reported in double strength MRS (2XMRS) medium. Bacteriocin production from the isolates was found to increase substantially in double strength MRS medium (Table 3) as revealed by the antimicrobial activity in the form of zone of inhibition against Staphylococcus aureus, Listeria monocytogenes, Lactobacillus rhamnosus (Table 3). 1406

6 Table 3: Effect of media on growth and bacteriocin production. Isolates E. faecium DFR8 E. faecalis DFR15 E. faecium DFR25 Zone of inhibition Zone of inhibition Zone of Growth Growth Growth Media (cm) (cm) inhibition (cm) (cfu/ml) (cfu/ml) (cfu/ml) SA LM LR SA LM LR SA LM LR TGE %TGE 1.9X MRS 6.7X XMRS 2.1X M X Legend: SA (Staphylococcus aureus), LM (Listeria monocytogenes), LR (Lactobacillus rhamnosus) Antimicrobial spectrum The GPC purified bacteriocin preparations showed inhibitory spectrum against a wide range of food borne pathogens comprising of both Gram-positive and Gram-negative bacteria, as well as some selected lactic acid bacteria as depicted in Table 4. Indicator organism Table 4: Antimicrobial spectrum of bacteriocins. E. faecium DFR8 Zone of inhibition (cm) E. faecalis DFR15 E. faecium DFR25 Staphylococcus aureus Pseudomonas Yersinia enterocolitica Micrococcus luteus Clostridium perfringens Escherichia coli Aeromonas hydrophilla Bacillus subtilis Listeria monocytogenes Lactococcus cremoris Lactobacillus rhamnosus Nil Nil Nil Lactobacillus brevis Nil Nil Nil Leucanostoc mesenteroides Lactococcus diacetylactis Discussion The bacteriocins produced by LAB have a narrow inhibitory spectrum (Barefoot and Klaenhammer, 1983 and Holo et al., 1991) however the bacteriocins of Enterococcus sp. isolates exhibited a broad inhibitory spectrum including Gram-negative bacteria. Bacteriocins acting against Gram-negative bacteria have also been reported by Skytta et al., (1993) and Jamuna et al., (2004). 1407

7 These bacteriocins were found to inhibit food borne pathogens notably, Aeromonas hydrophila, Yersinia enterocolitica, Escherichia coli and Staphylococcus aureus. It is of interest to the food industry that these strains exhibited such a wide range inhibitory effect on various food borne pathogens, as well as spoilage organisms. In addition a strong inhibitory effect is an interesting and unusual finding since inhibitory affects of bacteriocins from LAB is less prevalent on Gram-negative bacteria such as Aeromonas hydrophila, Yersinia enterocolitica, and Escherichia coli (Arihara et al., 1996 and Messi, 2001). The inhibitory activity exerted by these strains appears to be the result of a low molecular weight protein as GPC purified fraction showed substantial antibacterial activity against indicator organisms, and firmly suggests to be a bacteriocin. LAB produces bacteriocins either spontaneously or by induction. The genetic determinants for most bacteriocins are located on plasmids, with a few exceptions of chromosomally encoded ones. Bacteriocins from Gram- positive bacteria, do not bind to specific receptors for adsorption, are generally of lower molecular weight and demonstrate a broader spectrum of antimicrobial activity. The bacteriocins that are released are species specific. The majority of bacteriocins produced by LAB have been characterized according to their activity as a proteinaceous inhibition, on the estimation of their molecular mass, and on the determination of their spectrum of inhibition. The co-occurrence of LAB in animals thus has a role to play in the form of safe guarding the health system from invading pathogens. The requirement for enhanced concentration of nutrients (2XMRS medium) and bacteriocin activity against some Gram-negative pathogens indicates LAB isolated from animals are somewhat different than the LAB of that appears in fermented foods. Acknowledgement Authors are thankful to Dr. A.S. Bawa, Director, Defence Food Research Laboratory, Mysore for providing the necessary facilities, guidance and constant encouragement. 5. References 1. Arihara, K., Ogihara, S., Mukai, T., Itoh, M., Kondo, Y. (1996). Salivicin 140, a novel bacteriocin from Lactobacillus salivarius sub sp. salicinius T140 active against pathogenic bacteria, Letters in Applied Microbiology, 22, pp Barefoot, S. F., Klaenhammer, T. K. (1983). Detection and activity of lacticin B, a bacteriocin produced by Lactobacillus acidophilus, Applied and Environmental Microbiology, 45, pp Gornall, A. G., Bardwill, G. J., David, M. M. (1949). Determination of serum proteins by means of Biuret reaction, Journal of Biological Chemistry, 117, pp Holo, H., Nilssen, O., Nes, I. F. (1991). A new bacteriocin from Lactococcus lactis sub sp. cremoris: isolation and characterization of the protein and its gene, Journal of Bacteriology, 173, pp Jamuna, M., Jeevaratnam, K. (2004). Isolation and partial characterization of bacteriocins from Pediococcus species, Applied Microbiology and Biotechnology, 65, pp

8 6. T. R. Klaenhammer. (1988). Bacteriocins from lactic acid bacteria, Biochimie, l 70, pp Mahantesh, P., Ajay, P., Vijai, P., Rajini Kumari, Y. ( ). Isolation of bacteriocinogenic lactic acid bacteria from rat intestine, Journal of Culture Collections, 5, pp Messi, P., Bondi, M., Sabia, C., Battini, R., Manicardi, G. (2001). Detection and preliminary characterization of a bacteriocin (Plantaricin 35d) produced by a Lactobacillus plantarum strain, International Journal of Food Microbiology, 64, pp O. J. Mundt. (1986). Enterococci In: Bergey s manual of systematic bacteriology, Baltimore: The Williams & Wilkins Co., 2, pp Oyetayo, V. O., Adetuyi Akinyo soye, F. (2003). Safety and protective effect of Lactobacillus acidophilus and Lactobacillus casei used as probiotic agent in vivo, African Journal of Biotechnology, 2, pp Ray, B., Miller, K. W. (2000). Pediocin, In a Text book of Natural food antimicrobial systems, Boca Raton, CRC press, pp Schagger, H., Von Jagow, G. (1987). Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from kda, Annals of Biochemistry, 166, pp Schillinger, U. R., Lucke, F. K. (1987). Identification of lactobacilli from meat and meat products, Food Microbiology, 4, pp Sholeva, Z., Stefanova, S., Chipeva, V. (1998). Screening of antimicrobial activities among Bulgaricin Lactobacilli strains, Journal of Culture Collections, 2, pp Skytta, E., Haikara, A., Mattila-Sandholm, T. (1993). Production and characterization of antibacterial compounds produced by Pediococcus damnosus, Journal of Applied Bacteriology, 74, pp Tagg, J. R., Mc Given, A. R. (1971). Assay systems for bacteriocins, Applied Microbiology, 21, pp Yang, R., Johnson, M. C., Ray, B. (1992). Novel method to extract large amounts of bacteriocins from lactic acid bacteria, Applied and Environmental Microbiology, 58, pp Y. Zhennai. (2000). Antimicrobial compounds and extracelluar polysaccharides produced by LAB: structure and properties, Academic dissertation. Department of Food Technology, University of Helsinki, pp Zuniga, M., Pardo, I., Ferrer, S. (1993). An improved medium for distinguishing between homofermentative and heterofermentative lactic acid bacteria, International Journal of Food Microbiology, 18, pp