Sodium propionate

Sample collectionSamples were collected from the sediments of Caspian Sea at the depths of 5-10 m by Van vein grab (0.2 m²). Two sampling stations were located along of the Caspian Sea with the following latitudes 36°43'N and 36°44'N. The surface of each grab sample was aseptically collected and processed within 30 minutes.
Sample treatmentThe samples were subjected to physical pretreatment method in order to facilitate the isolation of actinomycetes. Heat treatments were performed by holding sediment samples in a water bath (Memmert) at 50 °C for 60 minutes. All samples were diluted with sterile 0.9 % saline prior to inoculation in triplicate onto isolation plates (11 (link)).
Isolation of actinomycetesActinomycetes were isolated by serial dilution method from sediments (17 ). Stock solution was prepared by diluting 1 g of sediment in 9 ml of sterile saline water and shaking well by using a vortex mixer (IKA). From the stock solution, 1 ml was used to prepare the final volume of 10^-2 and 10³ by serial dilution method. Samples were inoculated on Starch Casein Agar (SCA) (composition: soluble starch: 10 g, K₂HPO₄: 2 g, KNO₃: 2 g, casein: 0.3 g, MgSO₄.7H₂O: 0.05 g, CaCO₃: 0.02 g, FeSO₄.7H₂O: 0.01 g, agar: 15 g, filtered sea water: 1000 ml and pH: 7.0±0.1), Yeast Extract Malt Extract Agar (ISP2) (Composition: yeast extract: 4 g, malt extract: 10 g, dextrose: 4 g, agar: 15 g, filtered sea water: 1000 ml and pH: 7.3) and Kuster's Agar (composition: glycerol: 10 g, casein: 0.3 g, KNO₃: 2 g, K₂HPO₄: 2 g, soluble starch: 0.5 g, asparagine: 0.1 g, FeSO₄.7H₂O: 0.01 g, CaCO₃: 0.02 g, MgSO₄.7H₂O: 0.05 g, agar: 15 g, filtered sea water: 1000 ml and pH: 7.0±0.1). Each medium was supplemented with 25 µg ml⁻¹ nystatin to minimize contamination with fungi and 10 μg ml⁻¹ nalidixic acid to minimize contaminant growth (11 (link), 18 (link)). Plates were incubated for 7 to 20 days at 28 °C. Then the colonies with a tough or powdery texture, dry or folded appearance and branching filaments with or without aerial mycelia were sub-cultured on slants SCA (19 (link)). Until further use, the slants were kept in cold room at 4 °C (6 ).
Preliminary screening for antibacterial activity using cross-streak methodIsolated strains MN1 to MN44 were inoculated onto nutrient agar plates by streak in the center. The plates were incubated at 28 °C for 3 days. Six bacteria including Staphylococcus aureus ATCC 25923, Bacillus subtilis PTCC 1156, Escherichia coli PTCC 1533, Pseudomonasaeruginosa PTCC 1074, Salmonellatyphi PTCC 1609 and Klebsiellapneumonia were used as test organisms. A pure colony of test bacteria was transferred into fresh nutrient broth and incubated at 37 °C for 24 hours until the visible turbidity and density equal to that of 0.5 McFarland. After adjusting the turbidity, sterile cotton swab was dipped into the bacterial suspension and streaked perpendicular to the antagonist on the agar medium. The plates were incubated at 37 °C for 24 hours. The microbial inhibitions were observed by determining the diameter of the inhibition zones.
Extraction of antimicrobial compoundsThe selected antagonistic actinomycetes were inoculated into 100 ml of actinomycete isolation broth (composition: glycerol: 5.0 g, sodium propionate: 4.0 g, sodium caseinate: 2.0 g, K₂HPO₄: 0.5 g, asparagine: 0.1 g, MgSO₄·7H₂O: 0.1 g, FeSO₄·7H₂O: 1.0 mg, water: 1000 ml and pH: 8.0±0.1) and incubated in orbital shaker at 28 °C and 190 rpm for 7 days. To extract the antimicrobial compounds, the cultures were filtered then centrifuged (Sigma) at 6000 rpm, 10 minutes (1 (link)). The supernatant was transferred aseptically into a screw capped bottle and stored at 4 °C for further assay.
Secondary screening of antibacterial activity using disk diffusion methodThe antimicrobial activities of those extracts were tested against different test organisms by using agar disc diffusion method as described by Kirby-Bauer with modification (20 (link)).
Late exponential phase of the test bacteria were prepared by inoculating 1% (v/v) of the cultures into the fresh Muller-Hinton broth (Merck) and incubating on an orbital shaker at 37 °C and 100 rpm overnight. Before using the cultures, they were standardized with a final cell density of approximately 10⁸ cfu ml^-1. Muller-Hinton agar (Merck) were prepared and inoculated from the standardized cultures of the test organisms then spread as uniformly as possible throughout the entire media. Sterile paper discs (6 mm diameter, Padtan, Iran) were impregnated with 30 µl of the extracts then allowed to dry. The impregnated disc was introduced on the upper layer of the seeded agar plate and incubated at 37 °C for 24 hours.
The antibacterial activities of the extracts were compared with known antibiotic tetracycline (30 µg/disc) as positive control and ethyl acetate (30 µl/disc) as negative control. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (mm) on the surface of plates and the results were reported as Mean ± SD after three repeats (21 ). The potential actinomycetes isolates were selected from the primary and secondary screening then characterized by morphological and physiological methods for further studies.
Exoenzymatic assayThe potential actinomycetes isolates were screened for hydrolytic exoenzymatic activities including amylase and protease. These tests were conducted on Yeast Extract Malt Extract Agar (ISP2) containing 1% soluble starch for amylolytic activity and 1% skimmed milk for proteolytic activity.The presence of amylase was visualized by decolorized halo around the culture due to starch digestion. Proteolytic activity was observed by clearing of the milk around the colony (6 ).