Biomedical Engineering Reference
In-Depth Information
and the effects of pH and temperature on the stability of amylase produced by
A. oryzae
strain
FQB-01were also investigated. Because
A. oryzae
strains are also capable of growing on solid
substrates (Murado et al., 1997; Francis et al., 2002), the feasibility of sugar cane bagasse as a
support material for the production of amylase by
A. oryzae
FQB-01 was also studied in SSF.
To optimize the enzyme production in SSF, the effects of particle size, pH, incubation
temperature, moisture content, inoculum size and initial starch levels on the production of
amylase were studied.
Materials and Methods
Microorganism
Aspergillus oryzae
strain
FQB-01, was acquired from the Biotechnology Center of the
University of Oriente (Santiago de Cuba, Cuba) and it was maintained on potato dextrose
agar slants at 4ºC (Ellaiah et al., 2002; Salas et al., 2006).
Inoculum Preparation
Inocula were prepared by transferring 2-mL of 60-h old slant culture in 50 mL of medium
(250 mL Erlenmeyer) composed by (g/L): glucose, 20; (NH
4
)
2
SO
4
, 6.6; KH
2
PO
4
, 3.5;
FeSO
4
·7H
2
O, 0.15; MgSO
4
·7H
2
O, 0.10; MnCl
2
·2H
2
O, 0.45 and mycological peptone, 3.0; at
pH 6.8. The culture was incubated at 30ºC/48 h in an orbital shaker at 200 rpm (Salas et al.,
2006).
Submerged Fermentations
Brewery (BW) and meat processing (MPW) wastewaters, which were used as a base of
the culture media, were obtained from a local brewery and a local meat processing plant. Both
wastes
were centrifuged at 15000 rpm/15 min to remove the solids in suspension. The
supernatant obtained from BW contained (g/L): COD, 3.40; total sugars, 1.98; reducing
sugars, 1.46; total nitrogen, 0.095; total phosphorous, 0.034. The composition of the
supernatant obtained from MPW (g/L) was: COD, 3.00; total sugars, 1.82; reducing sugars,
0.99; total nitrogen, 0.172; total phosphorous, 0.028 (Salas et al., 2006).
After being used as culture media, both supernatants were supplemented with the
following nutrients (g/L): mycological peptone, 3.0; (NH
4
)
2
SO
4
, 6.6; CaCO
3
, 8.0; NaCl, 5;
KH
2
PO
4
, 3.5; FeSO
4
·7H
2
O, 0.15; MgSO
4
·7H
2
O, 0.10. To study the influence of the initial
concentration of starch on amylase production, the media were supplemented with soluble
potato starch to obtain initial starch concentrations of 10, 20, 30 and 40 g/L. Media without
starch were used as controls. The media were adjusted at pH 6.0, sterilized (121ºC/15 min),
inoculated with a 2% inoculum level (3 × 10
7
spores/mL) and incubated at 30ºC/96 h. All
submerged cultures were carried out in 250-mL Erlenmeyer flasks with 50 mL of production
media in an orbital shaker (200 rpm), using eight flasks in each fermentation serie (Salas et
al., 2006).
