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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1283 Micromonospora and Actinoplanes (4). Although Candida spp. occur among normal body flora in mouth and gastrointestinal tract, they can be responsible for many types of diseases in both humans and animals ranging from superficial mucocutaneous illness to deep life-threatening invasive, systemic diseases that can occur in different organs thus, causing different signs and symptoms. There are more than 20 species of Candida, of which Candida albicans is the most common. Yet, all Candida spp. cause the same spectrum of diseases however, differences in disease severity and susceptibility to antifungal agents were reported. Among the increase in the incidence of nosocomial fungal infections, those caused by Candida albicans, has increased proportionally. This problem appears to be a global one; in large, university-affiliated hospitals (5). Therefore, new sources and strategies are required to find new broad-spectrum antifungal molecules to combat these pathogens. For this reason, intensive program searching for new antibiotics is running worldwide mainly by screening for members of the clade actinomycetes as being a rich, unrivaled source of antibiotics. Impressed by these facts, our work aims to investigate the potentialities of some locally isolated actinomycetes to produce bioactive compounds acting against Candida albicans pathogen. The optimal fermentation conditions for bioactive compound production by the most active actinomycetes isolate have been investigated. In addition, trials to identify the active substances were reported. Materials and Methods Isolation of actinomycetes : Stone samples were crushed to a fine powder. Stone powder was dispersed in sterile 1/4 strength Ringer’s solution supplemented with 0.001 % Tween 80 and El-Refai et al shaken vigorously for up to one hour (6). Suspensions were then serially diluted in phosphate buffer. Spread plate method was applied for actinomycetes isolation; 0.1 ml of the appropriate dilution was spread evenly over the corresponding overnight dried medium plates. Five different isolation media known to support the growth of actinomycetes were used namely; starch casein medium (7), arginine glycerol salts medium (9), starch nitrate medium, M3 medium (9) and ISP medium 5 (Glycerol- Asparagine Agar) (10). After incubation, representatives of each colony form were picked off and streaked to obtain a pure culture. They were finally stored on starch casein slopes at 4 °C, and as spore suspensions in 20 % glycerol at 20 °C (11). Target organisms : The isolated strain activities were tested against: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27953 and Alcaligenes faecalis NRRL B-170 as examples for Gram negative pathogens, Staphylococcus aureus ATCC 29213, Bacillus subtilis NRRL B-4219 and Micrococcus luteus NRRL B-287 as examples for Gram positive pathogens, Aspergillus niger NRRL 599, Aspergillus flavus NRRL 1957 and Trichophyton mentagrophytes ATCC 9533 as examples for fungal pathogens and Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC 10275 as examples for yeasts. The challenged fungi were maintained and cultured on Dox’s agar medium that has the following composition (g/l): sucrose, 20; NaNO 3 , 2; MgSO 4 .7H 2 O, 0.5; KCl, 0.5; FeSO 4 .7H 2 O, 0.1; agar, 20. The pH of the medium was adjusted to 7.0-7.2 before sterilization. The tested yeasts were maintained and cultured on malt yeast medium that has the following composition (g/l): glucose, 4; yeast extract, 4; malt extract, 20; agar, 20. The pH of
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1284 the medium was adjusted to 7.0-7.2 before sterilization. The used bacteria were maintained and cultured on nutrient agar medium that has the following composition (g/l): glucose, 20; peptone, 10 and agar, 20. The pH of the medium was adjusted to 7.0-7.2 before sterilization. Screening of actinomycetes isolates for antimicrobial activity : Five days old slants were harvested by scratching with sterile distilled water and poured into 250 ml Erlenmeyer flasks containing 50 ml sterile starch casein nitrate broth. The production flasks were incubated in a rotary shaker at 150 rpm and 30 °C ± 2 °C for 7 days. At the end of the incubation period, the cultured broths were filtered and assayed for their activity against the target organisms using agar well diffusion method. 0.1 ml of each culture filtrate was placed in holes (0.9 cm diameter) made in seeded agar plates. The agar plates were incubated at 30 °C for 24 hours for bacteria and for 48 hours for fungi and yeasts. The diameters of the resulting inhibition zones were then measured and considered to estimate the isolates antimicrobial potentialities. Characterization of actinomycete isolate of choice : Only the actinomycetes isolate of the highest activity was subjected to identification at the genus and species level. A wide range of morphological, physiological and biochemical criteria were used to characterize this isolate. These criteria include morphological features, pigmentation, utilization of organic compounds and resistance to antibiotics as well as diaminopimelic acid in whole cell hydrolysate. Cultivation factors affecting the production of antifungal compound(s) : Unless otherwise stated, the basal fermentation conditions were: 4 ml of distilled water was added to a 5 day old slant and a spore suspension in 250 ml Erlenmeyer flask containing 50 ml of starch casein nitrate medium. The flask was incubated in a rotary shaker at 150 rpm, 30 °C± 2 for 2 days. 2 ml was used to inoculate 50 ml of starch casein nitrate medium in 250 ml Erlenmeyer flasks. The flasks were incubated on an incubatory shaker at 30 °C at 150 rpm for 7 days. At the end of the fermentation process, the antifungal activity was tested against Candida albicans using agar well diffusion method. Also, the cell dry weight in the fermentation broth was determined by filtering the flask content on a preweighed filter paper (Whatman no.1). The filter papers were dried at 60-70 °C until a constant weight. The difference between the weights of the filter papers after and before filtration should correspond to the cell dry weight. Effect of fermentation period, inoculum age, size : The effect of prolongation of incubation for maximum growth and antibiotic production was observed up to 12 days of incubation. To determine the effect of the age of inocula, 2 ml of the inoculum broth were taken at time intervals 2, 3, 4, 5, 6, 7 days to inoculate separate flasks of the fermentation media. In addition, different inoculum volumes (ranging from 2-8 % v/v) were used to inoculate the fermentation media to determine the most suitable volume for producing antifungal compound(s). Carbon and nitrogen supplements : Six carbon sources namely: starch, fructose, maltose, glucose, mannose, and sucrose were tested; each at 1 % level. After selection of the best source, different concentrations (5, 10, 15, 20 gm/l) were tested. Different organic and inorganic nitrogen sources were individually added as sole nitrogen sources in fermentation media to replace KNO 3 and casein. These are: peptone, yeast extract, corn steep liquor, sodium nitrate and ammonium chloride. They were added according to nitrogen Studies on the production of actinomycin by Nocardioides luteus
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(December 7-9, 2011) Venue: Karunya
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