ABSTRACT The bacteriocinsproduced by Pediococcus sp DFR6, Pediococcus sp DFR8 and Lactobacillus fermentum DFR13 were investigated for their efficacy to control some commonly occurring food contaminating bacteria. The orange juice was spiked with an initial count of 5.27 log 10 CFU/ml of Staphylococcus aureus, 4.38 log 10 CFU/ml of Bacillus cereus and 5.76log 10 CFU/ml of Salmonella typhimureum along with the addition of bacteriocins at various concentrations, 41.5/83.0/124/ and 168 AU/ml of Pediococcus sp. and 38.17/76.34/114.5/ and 152.6 AU/ml from Lactobacillus fermentum. The final bacterial count was enumerated after 0.6, 12.0, 24.0, 36.0, 48.0, 60.0 and 72.0 hr revealed substantial reduction (Staphylococcus aureus, Bacillus cereus and Salmonella typhimureum) in the number of bacteria with direct proportional to time. Further more in this study activated charcoal was used for the removal of dark brown colour imparting substances from bacteriocin preparations. The dark brown constituents of bacteriocins were found to reduce the quality of orange juice and paneer by way of changing their natural colour and taste. Activated charcoal was found to be advantageous in entrapping these coloured impurities produced as a result of carbohydrate and protein interaction at high temperatures. It was found that 5% of charcoal could effectively remove the colour from cell free supernatant without affecting the bacteriocin activity. 121
INTRODUCTION Food safety remains a major challenge to food producers and legislators endeavoring to provide adequate consumer protection. In spite of our knowledge of microbiology and the implementation of safety procedures, such as hazard analysis and critical control point (HACCP), the worldwide incidence of food poisoning cases is increasing (Hugas et al., 2002). One important and possible way to limit the growth of undesirable microorganisms in minimally processed food products is the use of protective cultures, especially, the bacteriocin producing strains of lactic acid bacteria (LAB), or use of antimicrobial metabolites extracted from these organisms. With the aim of achieving biological preservation without changing sensory characteristics of the food products, improve food safety and naturalness of bacteriocins are being investigated worldwide. In their search for a food preservative system, investigations on certain antibacterial proteins (bacteriocins) from LAB have been very popular. Growth of undesirable bacteria in fruit juices during storage process and manufacture of beverages may cause significant losses to the food and beverage industry. One of the most common forms of spoilage is the formation of slime, which gives the juice a highly undesirable ropy appearance. Among the preferred methods that have been suggested to avoid spoilage of food and beverages is the application of bacteriocins. (Cleveland et al., 2001; Devlieghere et al., 2004; Stiles, 1996) in the form of biopreservatives. The contamination of food with various pathogenic bacteria has, in several instances been seen to occur in post process stages, often during slicing or packaging. It is important at this point to avoid contamination and the use of bacteriocins produced by LAB has been suggested to minimize the risk of out growth of various pathogenic 122
bacteria. Bacteriocins, especially those with a broad antibacterial spectrum are bactericidal, or they can be made bactericidal to many spoilage and pathogenic bacteria with certain measures are important in this direction. Decolorization simply means colour removal. The chemical nature of activated charcoal combined with its high surface area and porosity, makes it an ideal medium for the adsorption. Brown colour imparting completed mainly that result from protein and carbohydrate reaction in the process of media preparation. MATERIALS AND METHODS Bacteriological media: Nutrient agar, Potato dextrose agar, Brain heart infusion broth (BHI) and Tryptic soya agar (TSA) were obtained from Himedia Laboratories pvt ltd, India, for enumeration of bacteria in orange juice and paneer products. Bacterial strains: The indicator microorganisms, viz., Staphylococcus aureus MTCC 737 and Bacillus cereus MTCC 1272 were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India, where as Salmonella typhimureum was isolated for this study at Defence Food Research Laboratory, Mysore, India, and was maintained in the laboratory as frozen stock culture in MRS agar (0.7%) containing 50% glycerol at 40 0 C. Bacteriocin preparation: Extraction of bacteriocin from the three LAB isolates was done by cell adsorptiondesorption method (Yang et al.,1992) followed by gel permeation chromatography (GPC) using sephadex G-50 and G-25 columns. Bacteriocin concentrates were sterilized by passing through a 0.22μM membrane filter (Millipore). Samples were 123
serially diluted and tested for antibacterial activity against the indicator organisms viz; Staphylococcus aureus, Bacillus cereus and Salmonella typhimureum, by agar well diffusion assay (Tagg & McGiven, 1971). One arbitary unit was defined as the highest dilution producing a visible zone of inhibition (9mm). Preparation of inoculum: The Staphylococcus aureus, Salmonella typhimureum and Bacillus cereus were grown in BHI broth for 12 hr at 37 0 C and were used to spike the orange juice and paneer in specified numbers. Treatment of orange juice with bacteriocin samples: Fresh orange juice was prepared using oranges, procured from the local market. The juice was obtained by macerating the inner portion of the fruit with the help of laboratory blender. The juice was further centrifuged at 7000rpm for 20min to remove fibrous residues, and it was sterilized by passing through a 0.22μM porosity membrane filter (Millipore). The juice was adjusted to ph 4.5 with sterile 0.1M NaOH solution. Liquid cultures of Salmonella typhimureum, Staphylococcus aureus and Bacilluscereus were inoculated in duplicate in to orange juice at a final concentration of 5.29, 4.39 and 5.76 log 10 CFU/ml of Staphylococcus aureus, Bacillus cereus and Salmonella typhimureum respectively. The samples were further supplemented with bacteriocins produced by Pediococcus sp. DFR6, Pediococcus sp. DFR8 and Lactobacillus fermentum DFR13 at the desired final concentrations i.e., 41.5, 83.0, 124.5, 166.0 AU/ml for Pediococcus sp. DFR6, Pediococcus sp. DFR8 and 38.17, 76.34, 114.5, 152.6 AU/ml for Lactobacillus fermentum DFR13. The orange juice was incubated at 30 0 C for 72 hr. At different 124
intervals of incubation, controls and bacteriocin treated samples were serially diluted in sterile saline solution with vortexing, 100 μl was plated in triplicates in to nutrient agar plates and were incubated at 37 0 C for 48 hr and the average number of colonies appearing in the petriplates was used to calculate the increase in the number of viable cells to be expressed as the log 10 colony forming units (CFU) per milliliter (log 10 units). The values were expressed as mean of three values±sd. A negative control of orange juice was always maintained in order to corroborate that the starting food material did not contain Salmonella typhimureum, Staphylococcus aureus and Bacillus cereus. Preparation of paneer: For the preparation of paneer, pasteurized milk was boiled in an open pan and it was allowed to cool to 90 0 C in a laminar flow hood. Citric acid solution (1%) maintained at 90 0 C was added to the milk and was stirred. The milk was allowed to coagulate and it was strained through double folded muslin cloth to remove the whey. The strained coagulant was pressed by keeping it under 2 kg weight in a laminar flow hood. Bacteriocin treatment: The freshly prepared paneer was cut in to 2.0 cm 3 aseptically. They were spiked with 1.71, 1.83, 1.85 log 10 CFU/ml of Staphylococcus aureus, Bacillus cereus and Salmonellatyphimureum respectively and were again dipped in a desired concentration of bacteriocin samples with 1660, 2490 AU/ml in the case of Pediococcus sp.dfr6, Pediococcus sp.dfr8 and 1527, 2290 AU/ml for Lactobacillus fermentum DFR13. The paneer samples were in the bacteriocin solution till they absorbed completely and transferred to fresh sterile petriplates for incubation at ambient temperature (25-30 0 C) 125
for 10days. Controls were maintained without treatment of bacteriocin Total plate count (TPC) of bacteria was measured by using nutrient agar as the culture medium and TPC of mold and fungi was enumerated by using potato dextrose agar (PDA). The samples were withdrawn periodically at every 48 hr interval. The paneer was homogenized in mortar and pestle with 10 ml of sterile saline and it was centrifuged aseptically and 1.0ml of supernatant was plated on to nutrient agar to take total plate count of bacteria and another 1.0 ml was plated on to potato dextrose agar to take TPC of molds and fungi. The nutrient agar plates were incubated at 37 0 C for 48 hr and PDA plates were incubated at room temperature for 72 hr. Treatment of cell free supernatants with activated charcoal Activated charcoal was used at various concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 gm) to remove colour from 50 ml of cell free supernatant. Activated charcoal is well known for its ability to remove certain impurities without causing a reduction of bigger protein molecules such as bacteriocins. After passing the supernatant through charcoal the obtained filtrate was concentrated to 2.5ml (1/20 th ) by using rotary flash evaporator. The obtained concentrated sample was used to check the antibacterial activity by agar well diffusion assay using an indicator organism, Staphylococcus aureus. 126
Results and Discussion Inhibition of pathogens in orange juice by bacteriocins produced by Pediococcus sp. DFR6, Pediococcus sp. DFR8 and Lactobacillus fermentum DFR13: The bacteriocins were active on Staphylococcus aureus, Salmonella typhimureum and Bacillus cereus, in orange juice incubated at 30 0 C and the bacteriocin effect was more pronounced and the efficacy was proportional to the added bacteriocin concentration. When inoculated with Bacillus cereus, the initial count was 4.39 log 10 CFU/ml and the number of viable cells was reduced below the detection limits with 75 μl (124.5 AU/ml) of bacteriocin, after 24 hr of incubation and no viable cells were detected after 36hr when inoculation with the bacteriocin produced by Pediococcus sp. DFR6, Pediococcus sp. DFR8 (Fig. 5.1 and 5.2), where as the effect of bacteriocins produced by Lactobacillus fermentum DFR13 was comparatively less effective towards Bacillus cereus in orange juice. In the control the count was increased up to 6 log cycles after 24 hr of incubation (Fig. 5.3) in the absence of bacteriocin supplementation. 127
log CFU/ml 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 41.5 AU/ml 83 AU/ml 124.5 AU/ml 166.0 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.1.Effect of bacteriocin from Pediococcus sp. DFR6 on growth of Bacillus cereus in orange juice 128
log CFU/ml 8 7 6 5 4 3 2 1 0 0 6 12 24 36 48 60 72 Time (hr) Control 41.5 AU/ml 83 AU/ml 124.5 AU/ml 166.0 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.2 Effect of bacteriocin from Pediococcus sp. DFR8 on growth of Bacillus cereus in orange juice 129
log CFU/ml 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 38.17 AU/ml 76.34 AU/ml 114.5 AU/ml 152.6 AU/ml values represent mean values of three different experiments ±SD Fig. 5.3 Effect of bacteriocin from Lactobacillus fermentum DFR13 on growth of Bacillus cereus in orange juice The count was drastically reduced from 5.27 log 10 CFU/ml to 2 log 10 CFU/ml after 36 hr of incubation in the case of Staphylococcus aureus and with 75 µl of bacteriocin (124.5 AU/ml in the case of Pediococcus sp. DFR6, DFR8 and 114.5 AU/ml for Lactobacillus fermentum) The effectiveness of bacteriocins produced by all the isolates showed almost same effectiveness in inhibiting Staphylococcus aureus. The count did not 130
log CFU/ml become zero even after 72 hr of incubation and it remained same (Fig. 5.4; 5.5 and 5.6). 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 41.5AU/ml 83.0 AU/ml 124.5 AU/ml 166.0 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.4. Effect of bacteriocin from Pediococcus sp. DFR6 on the growth of Staphylococcus aureus in orange juice 131
log CFU/ml 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 41.5 AU/ml 83.0 AU/ml 124.5 AU/ml 166.0 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.5 Effect of bacteriocin from Pediococcus sp. DFR8 on Staphylococcus aureus in orange juice 132
log CFU/ml 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 38.17 AU/ml 76.34 AU/ml 114.5 AU/ml 152.6 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.6. Effect of bacteriocin from Lactobacillus fermentum DFR13 on growth of Staphylococcus aureus in orange juice 133
In the case of Salmonella typhimureum the count was reached to 7.93 log CFU/ml in the control sample after 24 hr of incubation, the initial inoculum was 5.76 log CFU/ml. When the juice was spiked with 75 µl of bacteriocin (124.5AU/ml) produced by Pediococcus isolates DFR6, DFR8 the count bacteria were not detected after 60 hr of incubation and the count was almost become zero after 48h of incubation (fig. 5.7 and 5.8) when the juice was inoculated with the bacteriocin produced by Lactobacillus fermentum DFR13 (114.5 AU/ml). In this case the bacteriocins produced by Pediococcus isolates are seems to be more effective in controlling the growth of Bacillus cereus in comparision to the bacteriocins produced by Lactobacillus fermentum DFR13. But in inhibiting the growth of Salmonella typhimureum the bacteriocin produced by Lactobacillus fermentum DFR13 was found to be more effective (Fig. 5.9) and It was finally dependent on the bacteriocin concentration. 134
log CFU/ml 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time(hr) Control 41.5AU/ml 83AU/ml 124.5AU/ml 166AU/ml Values represent mean values of three different experiments ±SD Fig. 5.7 Effect of bacteriocin from Pediococcus sp. DFR6 on growth of Salmonella typhimureum in orange juice 135
log CFU/ml 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 41.5AU/ml 83.0AU/ml 124.5AU/ml 166AU/ml Values represent mean values of three different experiments ±SD Fig. 5.8. Effect of bacteriocin from Pediococcus sp. DFR8 on growth of Salmonella typhimureum in orange juice 136
logcfu/ml 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 6 12 24 36 48 60 72 Time (hr) Control 38.17AU/ml 76.34AU/ml 114.5AU/ml 152.6AU/ml Values represent mean values of three different experiments ±SD Fig. 5.9 Effect of bacteriocin from Lactobacillus fermentum DFR13 on growth of Salmonella typhimureum in orange juice 137
Effect of bacteriocins on the bacterial growth in paneer After dipping the paneer pieces in bacteriocin at various concentrations the plates were incubated at ambient temperature (25-30 0 C) In the control sample the colour as well as the texture change was observed (Fig. 5.10), the bacterial count was increased to 8.58 log 10 CFU/ml after 3 days of incubation. In the bacteriocin treated samples the initial count was 6.94 log 10 CFU/ml where as after 72 h of incubation the count was reduced to 2log 10 CFU/ml and the count remained same when the paneer was treated with the bacteriocins produced by Pediococcus sp.isolates DFR6, Pediococcus sp.dfr8. The results are shown in Fig. 5.11 In the paneer sample treated with the bacteriocin produced by Lactobacillus fermentum DFR13 the count didn t come down beyond 3 log cycles and in the control the count has been increased to 8 log 10 CFU/ml. (Fig. 5.12 and 5.13) C: Control C 1. Bacteriocin produced by Pediococcus sp. DFR 6 2. Bacteriocin produced by Pediococcus sp.dfr 8 3. Bacteriocin produced by Lactobacillus fermentum DFR 13 1 3 2 Fig. 5.10 Effect of bacteriocins in paneer 138
log CFU/ml 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of Days Control 1660AU/ml 2490AU/ml Values represent mean values of three different experiments ±SD Fig. 5.11 Effect of bacteriocin from Pediococcus sp. DFR6 on TPC of bacteria in paneer 139
log CFU/ml 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of days Control 1660AU/ml 2490AU/ml Values represent mean values of three different experiments ±SD Fig. 5.12 Effect of bacteriocin from Pediococcus sp. DFR8 on TPC of bacteria in paneer 140
log CFU/ml 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of days Control 1527AU/ml 2290AU/ml Values represent mean values of three different experiments ±SD Fig. 5.13. Effect of bacteriocin from Lactobacillus fermentum DFR13 on TPC of bacteria in paneer 141
log CFU/ml Effect of bacteriocins on fungi and mold growth in paneer The bacteriocins produced by the three LAB isolates did not show any effect in reducing the TPC of mold and fungus. (Fig. 5.14; 5.15 and 5.16). 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of days Control 1660 AU/ml 2490 AU/ml Values represent mean values of three different experiments ±SD Fig. 5.14 Effect of bacteriocin from Pediococcus sp. DFR6 on TPC of yeast and mold in paneer 142
log CFU/ml 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of days Control 1660AU/ml 2490AU/ml Values represent mean values of three different experiments ±SD Fig. 5.15 Effect of bacteriocin from Pediococcus sp. DFR8 on TPC of yeast and mold in paneer 143
log CFU/ml 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 No.of days Control 1527AU/ml 2290AU/ml Values represent mean values of three different experiments ±SD Fig. 5.16 Effect of bacteriocin from Lactobacillus fermentum DFR13 on TPC of yeast and mold in paneer 144
Antibacterial activity of charcoal treated cell free supernatant: It was found that 2.5gm of charcoal could effectively remove the colour from 50ml of cell free supernatant. The concentrated samples were used to determine the antibacterial activity by agar well diffusion assay. Controls were maintained without any treatment. It was found that after passing through charcoal the samples did not loose the bacteriocin activity in comparision to the control samples as measuring the zone of inhibition. It was found that the activated charcoal did not adsorb any bacteriocin from the culture supernatant (Fig. 5.17). Only higher molecular weight proteins occurring as contaminants were removed by the charcoal through adsorption. Fig. 5.17 Antibacterial activity of charcoal treated cell free supernatant of Pedioococcus DFR 6 Consumers as well as modern food and beverage manufacturing practices demand complementary methods to solve the problems of food spoilage. To utilize bacteriocins as complementary agents of preservation different studies must be conducted to 145
approximate their efficacy under practical situations. Bacteriocin addition to orange juice as well as in paneer was found to inhibit the growth of various indicator bacteria although higher bacteriocin concentrations were required for effective inhibition of pathogens. Results from the present study indicated that application of purified bacteriocins can reduce viable counts of Staphylococcus aureus, Bacillus cereus, Salmonella typhimureum in orange juice and total plate count of bacteria in paneer without inhibitory effect on the total plate count of yeast and mold in paneer. However, at a lower bacteriocin concentration it was possible to reduce the bacterial count to certain extent. The use of lactic acid bacteria as biopreservatives to control undesirable microorganisms in foods at refrigerated or abuse temperatures was proposed by several researchers (Garver & Muriana, 1993; Okereke & Montville, 1991) and has been a topic of increasing interest over the last few years. This study emphasizes the protective action against in the orange juice as well as paneer. Homeostatic mechanisms in microbial cells maintain essential physiological and biochemical characteristics such as intracellular ph, membrane potential, ATP concentration, cytoplasmic osmolarity and water content within the physiological range permitting normal growth. Hence, a key factor in achieving additive or synergistic preservative effect is attributed to the interference on bacterial homeostasis at certain target sites such as the bacterial cell membrane. As for many bacteriocins of lactic acid bacteria, the production is related to the growth phase, and highest bacteriocin was noticed at the end of the exponential phase with 146
further incubation results in a decrease in the bacteriocin activity during the stationary phase which either due to a proteolytic degradation or a ph mediated inactivation or adsorption of the bacteriocin to the producer cells (Villani et al., 1995). The inhibition of growth and elimination of pathogenic and spoilage bacteria by bacteriocins during processing and storage of foods result from the interactions of bacteriocins with food matrices and microorganisms (Ganzle et al., 1999). However, the application of bacteriocins in foods can be limited by properties, such as solubility of the peptide, the interaction of bacteriocins with food components, food ph and inactivation by proteases (Ganzle et al., 1999). The application of bacteriocins in food preservation can also be affected by adaptation or selection of resistant mutants in sensitive populations (Cotter et al., 2005; Mazzotta et al., 1997). Several studies have demonstrated that sensitive bacterial cells can become resistant to bacteriocins, such as nisin and pediocin PA-1 (Crandall & Montville, 1998; Mantovani & Russel 2001). Although many bacteriocins have been purified and characterized, to date, the only commercially produced bacteriocins are nisin, and to a lesser extent, the pediocin PA-1 (Cotter et al., 2005) because nisin is shown to be ineffective in some food matrices (eg. Meat). Considering the potential emergence of nisin-resistant populations, it is essential attractive to explore the use of other bacteriocins to prevent microbial growth in food products. Reported differences in bacteriocin effectiveness against various indicator organisms tested might be partly explained by variations in inoculum size and experimental conditions (ph, culture media, physiological state of sensitive bacteria and incubation temperatures). Pucci et al. (1988) showed the effect of ph on pediocin activity against L 147
monocytogenes. At low ph values, the activity of bacteriocins produced by Pediococcus sp. isolates and Lactobacillus fermentum in orange juice was effective after 48 hr of incubation. According to Liu and Hansen (1990), solubility and consequently nisin activity decrease at ph values. Other factors might also contribute to the variations in bacteriocin sensitivities of various pathogenic organisms. The inhibition of pathogens is dose-dependent at bacteriocin concentrations exceeding MIC (Christensen & Hutkins, 1992; Huang et al., 1994) Activated charcoal is a material that is treated with oxygen to open up millions of tiny pores between the carbon atoms in the charcoal. Most adsorbents are highly porous materials and adsorption takes place primarily on the walls of the pores or at specific sites inside the particle. The pores are generally very small with the internal surface area being in the order of magnitude greater than the external area. Separation by this material occurs because of differences in molecular weight, shape or polarity, causing some molecules to be held more strongly on the surface, because the pores are too small to admit the larger molecules. Once all the binding sites are filled, activated charcoal stops further adsorption. The decolourization process therefore, solid activated charcoal was employed as adsorbent, for the removal of coloured complexes from the cell free supernatant. Adsorption is a separation process in which certain components of a fluid phase are transferred to the surface of a solid adsorbent. In many cases, the adsorbing component is held strongly enough to permit complete removal of that component from the fluid with very little adsorption of the other component. The amount of material removed depends on the capacity of the activated carbon (charcoal) as well as the affinity of the material for the charcoal. The main problem associated with the use 148
of purified bacteriocins in food systems is cost of the production as well as the yield. The yield of the purified bacteriocins usually very low and the use of concentrated form of cell free supernatant containing bacteriocins in food systems is not advisable because of the brown colour, occuring due to Millard reaction of carbohydrates with media proteins during autoclaving. To overcome these problems associated with the colour as well as yield. These charcoal-treated cell free supernatant were found directly applicable in food systems. There is now considerable interest in extending the range of foods containing probiotic organisms from dairy foods to baby foods, fruit juices, cereal-based products and pharmaceuticals. Traditional probiotic dairy strains of LAB have a long history of safe use and most strains are considered commensal with no pathogenic potential. The pediocin like bacteriocins can play a significant potential as biopreservatives in foods because of their antilisterial effectiveness. As bacteriocins are proteins their presence in foods would be, in general safe for consumers because they would be inactivated by pancreatic or gastric enzymes. Preliminary results indicated that bacteriocins produced by Pediococcus sp. DFR6, Pediococcus sp DFR8 and Lactobacillus fermentum DFR13 are remarkably effective in reducing the survival of pathogenic bacteria viz; Staphylococcus aureus, Bacillus cereus and Salmonella typhimureum in orange juice and reduced the total plate count (TPC) of bacteria in paneer and without any effect on yeast and mold. 149
Summary and Conclusion The isolates were identified to species level by ribotyping method. The use of 16s rrna sequence analysis had facilitated the accurate identification of dairy cultures.the DFR6 and DFR8 isolates were identified as pediococcus L0 4 sp. and DFR13 was Lactobacillus fermentum. Bacteriocins produced by these isolates displayed a wide spectrum of inhibitory activity against food borne pathogens, food spoilage bacteria and other lactic acid bacteria selected as test strains comprising Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimureum and Lactococcuscremoris. Higher amount of bacteriocin production was noticed in MRS broth as growth medium, supplemented with 6.0% glucose as carbon source in the presence of 2.0% bacteriological peptone as nitrogen source.the addition of sodium acetate, magnesium sulphate and manganese sulphate did not effect the bacteriocin production in the lactic acid bacterial isolates. The cell adsorption-desorption method used for the purification of the bacteriocins preparations were fairly pure however of low yield. There was further loss of yield in gel permeation chromatograph (GPC) purified preparation with further enhancement in the specific activity from 2.49 to 392.0 in the case of Pediococcus sp.l04 DFR 6, 2.6 to 402.4 in the case of Pediococcus sp.l04and 2.9 to 401.8 in the case of Lactobacillus fermentum DFR 13.Though the yield was more the specific activity of the bacteriocinand bacteriocin activity (AU/ml) was increased from 701.7 to 1650.6 in the case of Pediococcus sp.l04 DFR 6 and Pediococcus sp. L04 DFR 8 and 614.0 to 1527.7 for DFR 13. Though the yield (55.6 %) was more the specific activity (46.5)of the 150
bacteriocin obtained by cold acetone precipitation method was less. Molecular size of the bacteriocins was determined by Tris-tricine SDS-PAGE. The molecular weight of the bacteriocins is 3.75 kda for Lactobacillus fermentum DFR 13 and 5.67 and 5.98 for Pediococcus sp. L04 DFR 6 and DFR 8. Theactivity of the bacteriocins was confirmed by haif gel activity assay against Staphylococcus aureus. The bacteriocinactivity was more pronounced and the efficacy was proportional to the added bacteriocin concentration. In controlling the growth of food borne pathogens in orange juice and paneer the bacteriocins produced by the three lactic acid bacterial isolates were promising butit was dependent on the bacteriocin concentration. The concentrations used in the study were 124.5 and 166.0 AU/ml in the case of Pediococcus sp. L04 DFR 6 and DFR 8 and 114.5 and 152.6 AU/ml for Lactobacillusfermentum DFR 13.The bacteriocins produced by the three LAB isolates did not show any effect in reducing the total plate count (TPC) of molds. Treatment of the bacteriocin samples (CFSC) with charcoal was used to remove the brown colour imparting media components without affecting the antibacterial activity of the bacteriocin samples. Further studies are required to optimize the bacterial growth process and bacteriocin production in the presence of various media components and other physical parameters for large scale production of these bacteriocins and their application to enhance the shelf life of foods. 151