{'fraction_non_alphanumeric': 0.026253521126760562, 'fraction_numerical': 0.021971830985915493, 'mean_word_length': 5.255109231853418, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Isolation and Identification of a Bacteriocinic Substance Producing Bacteria from Various Sources

Bacteriocins are generally ribosomally synthesised low molecular weight antimicrobial peptides that are not lethal to the host cells (Law, 2005). It was first discovered by Gratia in 1925 (Gratia, 2000) and they find their application in gastrointestinal disorders, biotechnological, food and agro-industries (Jack et al., 1995). The antimicrobial effect of bacteriocins (Sahu et al., 2008) have a potential use as natural food preservatives (Riley, 2009). Prevention of spoilage and pathogenic microorganisms will be more efficient if bacteriocinic activity is increased. Bacteriocins of Grampositive bacteria are abundantly found and are more diverse than those found in Gram negative bacteria and they resemble many of the antimicrobial peptides produced by eukaryotes. They are generally non lethal cationic, amphiphilic and membranepermeabilizing peptides (Sahl et al., 1998). They can provide both broad-spectrum killing of many microbes or can target on individual bacterial species.


Introduction
Bacteriocins are generally ribosomally synthesised low molecular weight antimicrobial peptides that are not lethal to the host cells (Law, 2005). It was first discovered by Gratia in 1925(Gratia, 2000 and they find their application in gastrointestinal disorders, biotechnological, food and agro-industries (Jack et al., 1995). The antimicrobial effect of bacteriocins (Sahu et al., 2008) have a potential use as natural food preservatives (Riley, 2009). Prevention of spoilage and pathogenic microorganisms will be more efficient if bacteriocinic activity is increased. Bacteriocins of Gram-positive bacteria are abundantly found and are more diverse than those found in Gramnegative bacteria and they resemble many of the antimicrobial peptides produced by eukaryotes. They are generally non lethal cationic, amphiphilic and membranepermeabilizing peptides (Sahl et al., 1998). They can provide both broad-spectrum killing of many microbes or can target on individual bacterial species.
Besides these applications, they can also be used in combination with other antibiotics for therapeutic use which finds $22 to $24 Bacteriocins are low molecular proteinaceous antimicrobial compounds secreted by bacteria to inhibit the growth of similar or closely related bacterial strains. In the present study, an attempt was made to isolate bacteria from various sources and screen for bacteriocinic activity. A total of 609 bacteria were isolated from different sources out of which 303 gram positive bacteria were screened for antimicrobial activity by primary streak method. Among these, 51 positive isolates were studied for colony and cell morphology which exhibited good antimicrobial activity. Thirty two isolates were streamlined for determination of bacteriocinic activity by agar well diffusion assay against four pathogens. Further three isolates IB23, AH4 and BrMk4 revealed significantly better antimicrobial activity against S. aureus, P. aeruginosa and E. coli. very scarce antimicrobial activity was observed against L. monocytogenes. Based on the biochemical tests, the organism was identified to be Staphylococcus aureus, Bacillus cereus and Lactobaccilus sp. respectively. billion globally at 2-3% increase per annum (Malini Maria, 2012). Their use in food and dairy industry lies in preservation by increasing the shelf-life of the products and are also used as additives, flavour enhancers which replaces harmful chemicals. They also find their application in livestock by feeding on bacteriocin producing bacteria and as probiotics in aquaculture.

Isolation of the Culture
A total of 51 different samples, as shown in table 1, were collected in sterile containers. All samples were appropriately labelled and transported to the lab and stored at 10 º C for further investigations. Different procedures were followed for different samples based on the load of the microbes as shown in table 1. Serial dilutions of the collected sample were carried out and 0.1 ml of each diluents was transferred to 0.9 ml of sterile distilled water.
Correspondingly dilutions were prepared from 10 -2 to 10 -4 according to the sample. The dilution of 10 -3 and 10 -4 were used for isolation of bacteria. The inoculum was spread on Nutrient agar (NA) plates and the plates were incubated at 37 º C for 24 hours. Morphological appearances of the inoculated plates were observed after 24 hours of incubation (Fig 1.) and distinct colonies were sub-cultured to obtain pure isolates which were then subcultured on NA slants.
The pure bacterial isolates (Fig 2.

Screening of Antimicrobial activity
The pure isolates were further screened for bacteriocinic activity by single streak method against 4 pathogenic bacteria procured from Culture collection centers (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC-27853, Staphylococcus aureus ATCC 13709, and Listeria monocytogenes MTCC 1143) as shown in Table 2. Mueller Hinton Agar medium was prepared and aseptically poured into Petriplates. After solidification, lawn culture of indicator microorganisms were made on the agar surface by using sterile cotton swabs. The plates were incubated for 15 minutes in room temperature inside the laminar air flow. After incubation the isolated cultures were streaked in single line using a sterile inoculating loop. The plates were incubated at 37°C for 24 hours (Fig 3.). After 24 hours of incubation period, microorganisms displaying clear zones of inhibition against the pathogens were observed.

Secondary Screening of Antimicrobial Activity
Antimicrobial activity was analysed against various pathogens mentioned above by agar well diffusion method. The pathogens were incubated in Brain-Heart infusion broth at 37°C for different hours and adjusted according to 0.5 McFarland standards was used. The pathogens were lawn cultured using sterile cotton swab. Agar well was made using sterile cork borer and 50μl of isolate supernatants grown in MRS media (centrifuged at 10,000 g for 15 minutes) was incorporated into the wells and incubated for 24 hours at 37°C. After incubation, zone of inhibition was measured (in mm) as shown in table 2. (Fig 4.).

Results and Discussion
A total of 609 bacteria were isolated from various sources, among them 303 gram positive bacteria were screened for antimicrobial activity by primary streak method. Out of these, 51 positive isolates were studied for colony morphology (colour, shape, margin, elevation and surface) and cell morphology (shape, arrangement, and Gram's staining) which exhibited good antimicrobial activity. Further, after secondary screening, 32 positive isolates were selected as they exhibited zone of inhibition against all the four pathogens as shown in the Table 2. On secondary screening three isolates IB23, AH4 and BrMk 4 revealed significantly better antimicrobial activity against S. aureus, P. aeruginosa and E. coli. However, very scarce antimicrobial activity was observed against L. monocytogenes. According to the biochemical tests as shown in Table 3 and the microscopic observation revealed IB23 as S. aureus, the organism AH 4 was Bacillus cereus and BrMk 4 belonged to Lactobaccilus sp. Ashok et al., 2014 reported better antimicrobial activity of isolates from milk and curds against S .aureus, K .pneumoniae E.coli, however P. aeruginosa was resistant against these isolates. In the present study 32 isolates showed activity against P. aeruginosa. Similar to the bacteriocinic activity from Lactobacillus sp. isolated from breast milk, Abdulla et al., 2014, also quoted antimicrobial compounds from Lactobacillus acidophilus. Our study on antimicrobial activity of Lactobacillus sp. were similar to the reports of L. plantarum of Das et al., 2014. However Gaamouche et al., 2014, reported better antimicrobial activity of lactic acid bacteria against Listeria monocytogenes.
Bacteriocins of Gram-positive bacteria are abundant and diverse than that of Gramnegative bacteria and also differ in ecological and evolutionary aspects. The spectrum of antimicrobial activity is broader in gram-positive bacteria, than that of gram negative species and also less toxic for preservation. In addition, the gram-positive bacteria have relatively higher-molecularweight, heat-labile bacteriocin-like substances.
In conclusion, the present study on isolation and identification of a bacteriocinic substance producing organisms from various sources provides an overview of the diversity of the ability of microbes to produce antimicrobial substances which could be commercially exploited by the food, dairy, medical industries. It is also evident that the bacteriocin like products of gram-positive bacteria revealed broader antibacterial spectrum and will continue to be an active area of applied research.