Isolation, identification, characterization of potential marine bacteria and extraction of bacteriocins

Introduction

Marine habitats harbour diverse bacterial communities that produce bioactive molecules capable of inhibiting several human pathogens. Among these, bacteriocins are ribosomally synthesized peptides with potent antimicrobial effects. Their applications range from food preservation to medical therapeutics, offering an eco-friendly alternative to chemical preservatives and antibiotics. The study focuses on isolating marine bacterial strains from seawater samples collected along the coastal region of cuddalore district. Bacteriocins are proteinaceous compounds produced by bacteria that inhibit the growth of closely related or even unrelated bacterial strains (Zhao et al., 2016) [8] . Unlike traditional antibiotics, they are biodegradable, non-toxic, and can be easily degraded by digestive enzymes, making them safe for use in food and medical applications (Wadekar, 2018) [7] . Moreover, bacteriocins from marine bacteria exhibit superior stability under a wide range of temperature, pH, and salinity conditions compared to terrestrial counterparts, highlighting their potential industrial significance (Balakrishnan and Subramanian, 2020) [1] . Marine ecosystems represent one of the most diverse habitats on Earth, containing a wide variety of microorganisms that possess unique metabolic and adaptive capabilities (Rahman et al., 2023) [5] . These marine microbes thrive under extreme environmental conditions such as high salinity, pressure, and low nutrient availability, which often lead to the synthesis of novel bioactive metabolites (Saha and Ghosh, 2022) [6] . Among these metabolites, bacteriocins ribosomally synthesized antimicrobial peptides-have attracted considerable attention due to their natural origin, safety, and potential as alternatives to chemical preservatives and antibiotics (Cotter et al., 2005) [2] . Bacteriocins are proteinaceous compounds produced by bacteria that inhibit the growth of closely related or even unrelated bacterial strains (Zhao et al., 2016) [8] . Unlike traditional antibiotics, they are biodegradable, non-toxic, and can be easily degraded by digestive enzymes, making them safe for use in food and medical applications (Wadekar, 2018) [7] . Moreover, bacteriocins from marine bacteria exhibit superior stability under a wide range of temperature, pH, and salinity conditions compared to terrestrial counterparts, highlighting their potential industrial significance (Balakrishnan and Subramanian, 2020) [1] . The discovery of marine-derived bacteriocin producers, particularly from genera such as Bacillus, Lactobacillus, and Pseudomonas, has opened new avenues for developing natural antimicrobial agents (Kim et al., 2021) [4] . Several studies have reported Bacillus species from marine sediments and seawater producing thermostable bacteriocins effective against foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus (Zheng et al., 2020) [9] . These findings emphasize the importance of marine bacteria as an untapped source of new bacteriocins for application in food preservation, aquaculture, and pharmaceutical formulations (Dawood et al., 2022) [3] . The discovery of marine-derived bacteriocin producers, particularly from genera such as Bacillus, Lactobacillus, and Pseudomonas, has opened new avenues for developing natural antimicrobial agents (Kim et al., 2021) [4] . Several studies have reported Bacillus species from marine sediments and seawater producing thermostable bacteriocins effective against foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus (Zheng et al., 2020) [9] . These findings emphasize the importance of marine bacteria as an untapped source of new bacteriocins for application in food preservation, aquaculture, and pharmaceutical formulations (Dawood et al., 2022) [3] . These isolates were identified using classical and molecular techniques. Their potential to produce bacteriocins was screened using agar well diffusion assays, and the bacteriocins were extracted, purified, and characterized for antimicrobial properties.

Materials and Methods

Sample Collection

Seawater samples were collected from three coastal sites at cuddalore district viz, parangipettai, Pichavaram and killai at a depth of 1 meter using sterile glass bottles. Samples were transported to the laboratory and processed within 4 hours.

Isolation of Marine Bacteria

Serial dilution was performed, and aliquots were plated on marine agar supplemented with 2% NaCl. Plates were incubated at 28 °C for 48 hours. Morphologically distinct colonies were selected and purified through repeated streaking.

Identification of Isolates

Morphological characters such as Gram staining and colony characteristics were studied based on the standard procedure. The Biochemical tests such as Catalase, oxidase and carbohydrate utilization carried out based on Cappuccino, J. G., and Sherman, N. (2014). The Molecular identification 16S rRNA gene sequencing was performed on selected isolates.

Screening for Bacteriocin Production

Screening was done by using agar well diffusion assay was used. The Pathogens tested were Listeria monocytogenes, Staphylococcus aureus, Escherichia coli. The Inhibition zones were measured in millimetres after 24 hour incubation at 37 °C.

Extraction and Purification of Bacteriocins

The Culture supernatant was obtained by centrifugation at 10,000 rpm for 15 min. Filtered through 0.22 µm membrane. Ammonium sulfate precipitation (80% saturation) was used to concentrate the proteins. Dialysis against phosphate buffer to remove salts.

Experimental Results

The biochemical characterization of the marine bacterial isolate MB 1 revealed that the organism was Gram-positive and rod-shaped. It shows positive for catalase and oxidase activities, indicating its aerobic nature and ability to degrade hydrogen peroxide. The isolate was negative for indole and methyl red tests, suggesting the absence of tryptophan degradation and stable acid production from glucose fermentation. It showed a positive Voges-Proskauer reaction, The isolate exhibits positive citrate utilization. The results were presented in Table 1. The bacteriocin activity of the marine bacterial isolate MB1 was evaluated against major dairy pathogens using the agar well diffusion assay. The isolate produced clear inhibition zones of 18 mm against Listeria monocytogenes, 15 mm against Staphylococcus aureus, and 12 mm against Escherichia coli after incubation at 37 °C for 24 hours. These results indicate that the bacteriocin produced by MB1 is effective against both Gram-positive and Gram-negative bacteria, showing the strongest activity against Listeria monocytogenes. The results were presented in Table -2.

References

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