Rice Chaff a Novel Substrate
for Fibrinolytic Enzyme Production by Solid State
Fermentation Using Penicillium chrysogenum SGAD12
S.M. Gopinath*,
Suneetha T.B. Ashwini Patil G.M
Department of Biotechnology, Acharya Institute of Technology, Soldevanhalli,
Bengaluru-560090, Karnataka, India
ABSTRACT:
Rice chaff - a polished substance extracted from Oryza sativa L. cv. Devamallige
was used as a novel substrate for the production of the fibrinolytic
enzyme. This vital enzyme is used in thrombolytic therapy, as a clot buster.
The production was done by solid state fermentation of rice chaff by Penicillium chrysogenum
SGAD12, locally isolated from vegetable markets. Of the 28 strains isolated and
screened, Penicillium chrysogenum
SGAD12 was found to give an inhibition zone greater than 2 mm. Hence it was
identified as the potential organism showing maximum fibrinolytic
activity under specified culture conditions. Activity optimization was done
under the parameters: Time, Inoculum ratio, Moisture
content, Substrate particle size. The
fibrinolytic activity was favourably
maximized at 104 hrs, 7% (v/v) inoculum ratio, 35-45
% (v/w) moisture content and 500µm particle size.
KEYWORDS: Rice chaff, Fibrinolytic
enzyme, Solid state fermentation, Penicillium chrysogenum SGAD12.
INTRODUCTION:
Now-a-days thrombolytic diseases are responsible for
significant incapacitation and morbidity. Fibrinolytic
enzymes have apparent significance in thrombolytic therapy in human being
(Haber et al, 1989). Despite most of their wide spread use, the currently
available fibrinolytic enzymes have a number of
significant limitations (Collen and Lijnen, 1991).
Therefore great attention has been directed towards a search of
thrombolytic agents of various origins, particularly through microbial
activity.
Fibrinolytic enzymes occur in bacteria, earthworm, snake venom,
from fermented foods (Collen and Lejnin,
1993)(Sumi et al, 1987) ,but upto now there have been very few
evidences for production of fibrinolytic enzyme from fungi ( S. A. El-Aassar et al, 1990)( Sun Tao et al, 1997). Solid state
fermentation (SSF) process indicates significant difference in comparision with submerged state fermentation (SMF). Its
main advantage is that it is a simple technique, it utilizes less amount of
water, and it has a low operating cost and high productivity. Fungi have been
widely utilized by SSF in the production of enzyme (Lonsane
et al, 1992) (Pandey, 1992). This study reports the
production of fibrinolytic enzyme by Penicillin chrysogenum
SGAD12, locally isolated from vegetable markets and new substrate rice chaff
extracted from Oryza sativa L. Cv. Devamallige
collected from local regions of Karnataka, India.
MATERIALS
AND METHODS:
Penicillium chrysogenum SGAD12, a fibrinolytic enzyme producer was
isolated from soil of vegetable market and was identified. It was found to
produce fibrinolytic enzyme on fibrin plate.
This potential trait was used for further production on
solid state fermentation. Culture
was maintained on Czepek Dox
Agar at 4°C and sub cultured fortnightly. The basic medium contained rice chaff
(variety: Devamallige) (20g), KH2PO4
(0.5g), MgSO4.7H2O (0.5g), MnSO4.7H2O
(0.001g), ZnSO4.7H2O (0.002g), FeSO4.7H2O
(0.0005g) and 9 ml water. After autoclaving at 121˚C for 30 minutes, the
medium was adjusted to a moisture content of 45% (v/w) and inoculum
size of 7% (v/v). It was then inoculated with spore suspension of 106
spores/ml and incubated at 28˚C.
Enzyme Extraction:
The fresh moldy pith in each flask was soaked in
distilled water and incubated in a rotary shaker at 130 r.p.m
at 28 ˚C for 1 hour. The extracts
were obtained by filtering through filter paper. For 1 g of dry substrate taken
2.8 ml of filtrate was recovered.
Assay method:
Fibrin plate analysis method:
The original fibrin plate method (Astrup
and Mullertz, 1952) with slight modification was used
for measurement of the fibrinolytic activity of the
test preparation along with streptokinase. Petri dishes containing 9 ml of 0.2%
fibrinogen solution (pH 7.8) were placed on a horizontal glass plate. To each
of these Petri dishes, 0.2 ml of plasminogen (10
units) was also added and mixed well. Clotting was induced by the addition of
0.2 ml of thrombin solution (20 units). In order to speed up the clotting
process, the plates were incubated
at 28 ˚C for 20 mins. Plates were prepared afresh every time. Known quantities of the enzyme
solution and standards were placed as small droplets on the surface of the
fibrin clot. The plates were incubated
at 28 ˚C for 2 h and visually inspected for liquefaction. The area of the
digested fibrin was considered as a quantitative measure of the fibrinolytic activity of the enzyme.
RESULTS
AND DISCUSSION:
Enzyme production profile:
Time course:
Good fungal growth was supported by rice chaff. The
spores germinated within 20 h, giving rise to mycelium formation, whose density
was seen to increase with time. Visual examination showed that the substrate
was fully impregnated with mycelium in about 40 h while the uninnoculated
control plate showed no detectable change. During initial 24 h no fibrinolytic activity was seen, thereafter the enzyme activity
increased reaching a maximum at 104 h. With further incubation the enzyme
activity decreased.
Effect of Inoculum
Ratio:
While optimization for inoculum
ratio it was found that 7% to 14% (v/v) (based on the volume of mineral
solution) elicited the best enzyme activity, with 7 %(v/v),as adopted for this
experiment, giving the maximum result
(fig 2). Any inoculum size beyond the optimal range
showed lower activity. An inference can
be drawn that larger inoculum sizes containing more
amount of water led to decrease in the enzyme activity.
Fig.
1.
Time course of enzyme production by Penicillium chrysogenum SGAD12 on rice chaff (variety: Devamallige). With fermentation temperature 28 ˚C,
Inoculums size: 7% v/v
Moisture level: 45%, Particle
size: mixture of sizes
Fig.
2. Effect
of inoculum size on production by Penicillium chrysogenum SGAD12 on rice chaff.
(variety: Devamallige) Fermentation time: 104 hours.
Temperature: 28 ˚C. Particle size: Mixture of different sizes.
Effect of moisture level:
Water has a profound effect on productivity and hence
used in limited amount in solid state fermentation (Lonsane
et al, 1992). Moisture level between 35% - 45% (v/w) (fig3) results in the
maximum enzyme production. Any value beyond this was unable to give increase in
production. A conclusion can be drawn
that lower moisture level leads to dry culture, sparse growth and hence reduced
production. Higher moisture concentration also creates an unsuitable
environment for solid mycelium growth and hyphal
diffusion, leading to less production of enzyme.
Fig.
3. Effect of moisture level on the productivity
by Penicillium chrysogenum
SGAD12on Rice chaff (variety: Devamallige).
Fermentation time: 104 hours. Temperature: 28 ˚C. Particle size: Mixture
of all sizes.
Effect of substrate particle
size:
The effect of specific surface area is of high
importance in solid state fermentation. Maximum production was obtained at a
particle size of 500µm (fig4). Through visual observation it was inferred that
larger particle size provided less surface area hence productivity was less.
Whereas significantly smaller particles though gave large surface area but the
porosity was decreased to an extent that the filamentous fungi could not reach
deep inside to the substrate particles leading to decrease in production.
Fig.
4. Effect
of substrate particle size on production
by Penicillium chrysogenum SGAD12.
Fermentation time: 104 hours, temperature:
inoculum size-7% (v/v), moisture content 45 %(
v/w)
CONCLUSION:
The above work indicates that Rice chaff (variety: Devamallige) can be used as a potential substrate for
production of economically important fibrinolytic enzyme with Penicillium chrysogenum SGAD12 . This substrate is
easily available and economically feasible. Penicillium chrysogenum SGAD12 was identified as the
potential organism showing maximum fibrinolytic
activity under specified culture conditions. Time, Inoculum
ratio, Moisture content, Particle size; were found to be ideal parameters for
the activity optimization. The fibrinolytic activity was favourably
maximized at 104 hrs, 7% (v/v) inoculum ratio, 35-45
% (v/w) moisture content and 500µm particle size.
ACKNOWLEDGEMENT:
The authors are thankful to Visveswaraya
Technological University, Belgaum for their financial assistance; and Acharya Institute of Technology for all the facilities
graciously extended to carry out the project.
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Received on 09.05.2011
Modified on 18.05.2011
Accepted on 21.05.2011
© A&V Publication all right reserved
Research J.
Science and Tech. 3(5): Sept.-Oct. 2011: 261-263