April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 3 (2) 172-180, <strong>April</strong> 2009. ISSN 0973-8916<br />
100 g/l <strong>of</strong> NaCl, purified on the same medium<br />
<strong>and</strong> maintained on 1% malt extract agar slant at<br />
4 0 C.<br />
Preparation <strong>of</strong> substrates<br />
Different agricultural byproducts, wheat<br />
bran (WB), wheat straw (WS), rye straw (RS)<br />
<strong>and</strong> corncob leaf (CL) were obtained from local<br />
market. These waste materials were washed first<br />
with tap water followed by distilled water to<br />
remove the adhered surface dust particles. Then<br />
bleaching operation was carried out by immersing<br />
them in hot water (75-80 0 C) for 20 minutes<br />
followed by oven drying at 45 0 C. The dried<br />
material was grinded in a mixer grinder (Remi)<br />
then sterilized at 121 0 C, 15 psi pressure for 15<br />
minutes <strong>and</strong> stored at 4 0 C before further use.<br />
Inoculum preparation<br />
Actively growing <strong>and</strong> heavily sporulating<br />
(ten days) old malt agar slant culture was added<br />
to 10 ml sterile distilled water. The spores were<br />
gently scraped <strong>of</strong>f with the help <strong>of</strong> a sterile needle<br />
<strong>and</strong> contents were passed through glass wool so<br />
as to obtain spore inoculums free from mycelia<br />
bits (31-33). A volume <strong>of</strong> one ml <strong>of</strong> spore<br />
suspension contained more than 10 6 spores. They<br />
were cultured in a medium containing soluble<br />
starch 5 g., yeast extract 2 g., KH 2<br />
PO 4<br />
1 g.,<br />
MgSO 4<br />
. 7H 2<br />
O 0.5 g., distilled water 1000 ml. The<br />
medium was autoclaved (121 0 C for 15 minutes),<br />
allowed to cool, then aseptically added to sterile<br />
500 ml Erlenmeyer flasks (100 ml added per<br />
flask).These flasks with 100 ml liquid medium<br />
were incubated with 2 ml spore suspension with<br />
autoclaved distilled water <strong>and</strong> incubated at 50 ±<br />
2 0 C <strong>and</strong> 120 rpm for 2-3 days in the preliminary<br />
experiments <strong>and</strong> only two days in all subsequent<br />
experiments. All chemicals used were <strong>of</strong> reagent<br />
grade.<br />
Solid state fermentation<br />
Four agriculture byproducts; wheat bran,<br />
corncob leaf, rye straw, wheat straw was<br />
174<br />
considered as substrate. Five gm <strong>of</strong> each substrate<br />
was taken into 250 ml conical flasks. To adjust<br />
percentage moisture levels (w/v), 0.1M acetate<br />
buffer, pH = 6.0 was added. The content <strong>of</strong> the<br />
flasks were mixed thoroughly <strong>and</strong> sterilized in the<br />
autoclave at 121 0 C temperature <strong>and</strong> 1<br />
atmospheric pressure for 20 min <strong>and</strong> then cooled<br />
at room temperature. Each flask was incubated<br />
with one ml <strong>of</strong> inoculum <strong>and</strong> subsequently rotated<br />
in a rotary incubator shaker at 50 0 C.<br />
Optimization <strong>of</strong> process parameters<br />
Various process parameters affecting á-<br />
amylase production in solid state fermentation<br />
(SSF) are optimized. The strategy was to optimize<br />
each parameter independently <strong>and</strong> subsequently<br />
optimum conditions were employed in each<br />
experimental run. The best solid substrate was<br />
selected for optimum production <strong>of</strong> á-amylase<br />
production <strong>and</strong> the suitable solid substrate was<br />
used in subsequent experiments. The tested<br />
process parameters in this study were initial<br />
moisture content (20, 30, 40, 50, 60, 70, 80, 90,<br />
100, 110%, w/v), inoculum concentration (5, 10,<br />
15, 20, 25, 30%, v/w), incubation time (24, 48, 72,<br />
96, 120, 144, 168, 192 h), incubation temperature<br />
(30, 40, 50, 60, 70, 80 0 C), initial pH (4, 5, 6, 7, 8),<br />
<strong>and</strong> supplementary carbon sources (soluble<br />
starch, sucrose, maltose, glucose). On the basis<br />
<strong>of</strong> experimental data wheat bran was found to be<br />
the best solid substrate in solid state fermentation<br />
(SSF) process (details are mentioned in result <strong>and</strong><br />
discussion section).<br />
Determination <strong>of</strong> dry weight <strong>of</strong> substrate<br />
All four substrates are not available in<br />
completely dried form. They generally contain<br />
moisture. Prior to utilize them in bioprocess, it is<br />
necessary to dry these solid substrates. Therefore,<br />
in the present study the amount <strong>of</strong> wet solid<br />
substrate was kept in the oven at 70 0 C for 24 h<br />
to remove the moisture from the solid substrate.<br />
After drying, the mass <strong>of</strong> solid substrate was<br />
measured.<br />
Production <strong>of</strong> á-amylase