Production of
Neomycin Using Immobilized Cells of Streptomyces marinensis and It’s Antimicrobial Activity
YM Subbarao, Madhukar
Vajanepalli, Dupaguntla Suresh, KL Senthil Kumar* and S Pratheep
Padmavathi College of Pharmacy, Periyanahalli, Dharmapuri- 635
205, Tamilnadu
ABSTRACT
The use of
immobilized whole cell is promising and advantageous in several cases. The
mutant strain of Streptomyces marinensis was
grown on Potato Agar Medium. Among various supporting matrices studied so far for
whole-cell immobilization of S marinensis, calcium alginate was found to be a better
entrapment matrix for neomycin production. The effect of several parameters,
such as the effect of alginate concentration, the effect of cation, the
concentration of cation, and the curing times, on neomycin production and bead
stability were studied. The Neomycin production
started after 24 hours fermentation and reached a minimum level by 120 hours
(1245 mg/1). On further incubation, there was no appreciable change in Neomycin
liter. Finally, Minimum Inhibitory Concentrations (MICs) of Ihe
produced Neomycin was assayed against major Bacterial groups. The MICs for E.coli at a high inoculum
density was 64 /µg/ml under aerobic conditions and 128 /µg/ml under anaerobic
conditions. The latter value was higher than those of other intestinal
organisms.
KEYWORDS: Streptomyces marinensis, Neomycin, immobilization
INTRODUCTION:
Streptomycetes are
Gram-positive, soil-dwelling bacteria with a complex life cycle. Streptomyces is a genus of Actinobacteria,
a group of Gram-positive and generally high Guanine-Cytosine content
(GC-content) bacteria. Streptomycetes are found predominantly in soil and in
decaying vegetation, and most produce spores. Streptomycetes
are noted for their distinct "earthy" odor which results from
production of a volatile metabolite, geosmin. Streptomycetes are characterized by a complex secondary
metabolism. They produce a large number of antibiotics that are in clinical use
(Neomycin, Chloramphenicol). (Madigan M et al 2005)
Neomycin,
which is a broad-spectrum antibiotic, is effective against both gram positive,
gram-negative bacteria & mycobacteria and is produced
by different species of Streptomyces such
as Streptomyces fradiae,
Streptomyces marinensis, and
other microbial sp. It interferes with protein synthesis in the sensitive
bacterial cells such as species of proteus and
staphylococcus. Neomycin is mainly used topically in the treatment of skin and
mucous membrane infections, wounds and burns. Although it is also used
systematically, it is highly toxic.
Structure of Neomycin:-
Neomycin is used to treat superficial infections in humans and is
given orally to cattle, sheep, pigs, goats and poultry for bacterial
gastrointestinal infections and by intramammary
administration to treat mastitis. It is an amino glycoside and is active
against bacteria that grow aerobically. Neomycin is produced by Streptomyces sps. Preparations
are complexes consisting of Neomycin A, Neomycin B and Neomycin C, generally
containing more than 90% Neomycin B, the remainder being mainly Neomycin C. Neomycins B and C both contain three amino sugars attached
by glycosidic linkage to the central hexose. Neomycin A, more appropriately referred to as neamine, is a hydrolysis product of either Neomycin B or
Neomycin C and usually comprises less than 1 % of the mixture.
AIM AND OBJECTIVE:
Ø
To immobilize the whole cells of Streptomyces
marinensis in Calcium alginate
Ø
To produce Neomycin from immobilized cells.
Ø
To determine the Antibacterial activity of the produced Neomycin.
Ø UV
spectral study of produced Neomycin.
MATERIALS AND METHODS:
Of basic importance to the study
of antibiotic production by microorganism in a chemically defined medium in
which the various nutrients are present. All chemicals and medium
constituents used in this study were procured from Hi-Media.
Microorganism:
A mutant strain of Streptomyces
marinensis used in this study was procured from
MTCC,
The strain was grown on potato agar (pH 7.0-7.5)
The culture was maintained in culture media, Jowar
starch agar media.
Culture Media:
Composition g/L
Jowar Starch 20
Corn steep liquor 5.0
Ammonium Sulphate 5.0
Sodium Chloride 5.0
Calcium Carbonate 5.0
Agar - Agar 20
Distilled Water 1000
ml
PH 6.5
Immobilization of Bacterial Cells:
The Cells were immobilized using sodium alginate by the ionotrophic method and procedure has given below:
Antibacterial activity (FILTER PAPER DISC METHOD):
The antibacterial activity of the various extracts was evaluated
by the filter paper disc method. This method is based on the diffusion of an
antibiotic from a filter paper disc through the solidified culture media of a
Petri dish used for study. Growth of inoculated micro organism is inhibited
entirely in a circular area Zone around the filter paper disc organization
containing a solution of the antibiotic and the plant extract.
Anti
Bacterial Study:
CUP
PLATE METHOD:
Nutrient broth was prepared and inoculated
with different species of bacteria and incubated at 37C overnight.From
this overnight culture 1 % stock culture was prepared (99 ml of sterile
nutrient broth + 1ml of overnight culture).Nutrient agar was prepared and 25 ml
was poured in sterile Petri plates and allowed to cool.Each
agar plates were inoculated with 0.2ml of 1 % bacterial culture and spreaded by spreader. Using a sterile cark borer, 6mm
diameter of holes were made in the solidified agar
plates containing respective bacterial culture (1 %). A total volume of 0.02ml
of Neomycin was poured into the wells with the concentration as 250µmg /ml. One
well was poured with antibiotic and incubated at 37 C for 24 hrs. After 24Hrs
of incubation Zone of Inhibition was measured in millimeter.
RESULTS AND DISCUSSION:
Estimation of Neomycin Titer: -
Studies were carried out to investigate the growth and antibiotic
production profiles of the immobilized cells of Streptomyces
marinensis in Calcium Alginate solution. The
fermentation was carried out for 144 hours in batch mode.
The Neomycin content was quantitatively determined by
microbiological assay using staphylococcus epidermis as test organism.
Time course Profiles of Neomycin production with Immobilized cells
of Streptomyces marinensis
in Calcium Alginate
TIME (Hours)
From the results, it was observed that the Neomycin production
started after 24 hours fermentation and reached a minimum level by 120 hours (1245
mg/1). On further incubation, there was no appreciable change in Neomycin
titer.
At 144 hours fermentation, the disintegration began due to
accumulation of secondary metabolites and the change in the surrounding
environment of the microbial cells.
Antimicrobial Susceptibility Test (CUP PLATE METHOD)
|
S. NO |
BACTERIA |
Neomycin(250
µg/ml) |
|
1 |
Gram
negative 1.Pseudomonas aeruginosa 2.Escherichia
coli |
17mm 19mm |
|
2 |
Gram
positive 1.Staphylococcus aureus 2.Streptococcus aureus |
20mm 22mm |
CONCLUSION:
Neomycin having antibacterial
activity against Gram negative and Gram positive bacteria. When
compared to gram negative the neomycin having more antibacterial activity
against gram positive bacteria.
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Received on 03.08.2009
Accepted on 10.10.2009
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all right reserved
Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 12-14