full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
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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