4.2.1. Determination of protease activity produced from the isolates
Four selected strains of A.oryzae were cultured in Potato Dextrose media by shaking at 200rpm at 30oC for 4 days. After 4 days of culturing, a large
number of fungal pellets were appeared in the medium flasks. The cultures were collected, extracted and centrifuged for crude enzyme.
Figure 4.2. Aspergillus oryzae in 4-day PD broth culture
Screening fungal strains producing protease was conducted by well diffusion on casein agar plates. After 48-72 hour incubation at 30oC, the clear distinct zones were observed and quantification of the protease activity was indicated as the following table.
Table 4.3. Diameter of clear zones of protease produced from isolates
No Isolates Diameter of clear zones (mm)
Enzyme activity (u/l)
1 TB1 17.0 49.26
2 TB2 9.5 13.73
3 M1 7.5 15.49
4 G2 11.0 29.10
Figure 4.3. The clear distinct zones of proteases on the casein agar plates flooded with BCG reagent after 3 day incubation
The clear zones were observed on the BCG casein agar plates after being incubated at 30oC in 3 days for protease degradation of casein. The zones were distinct and the surrounding was greenish blue in color, but the color of the plates strongly depended on the pH value of the agar medium. In this experiment, pH of the culture medium was maintained at 8.0, so the plates appeared as blue color.
The proteolytic activity was determined as the clear zone where as the rest of the plate was greenish blue.
Table 4.3 showed that the protease produced from the isolate TB1 was the highest with 49.26 u/l of activity, which incorporated with 17 mm of clear zone in the casein agar plate.
The strain G2 has the second largest casein hydrolysis. Protease created a clear zone of 11mm and the protease activity was also high at 29.10u/l.
For isolate TB2, although clear zone appeared with a large diameter (9.5 mm) but the activity of the protease generated was low with only 13.73u/l. In contrast, protease from isolate M1 created a smaller zone 7.5 mm but the the enzymatic activity was higher with 15.49 u/l.
The higher the proteolytic activity, the stronger the fungi produced enzyme. The strains TB1 and G2 with the highest enzymatic activity were selected as their representative for enzyme characterization. These results were compatible with the protease activity reported by Rodarte et al.(2011) that the highest proteolytic activities at pH 7.0 to 9.0 obtained from the filamentous fungi as Aspergillus sp. was in range of 23.53 u/l to 48.75 u/l.
4.2.2. Growth rate of the fungi on the different media
Fungi differ not only in morphology but also in their rate of growth and development in different culture media. To determine the growth rate of the isolated fungi, the fungi of interest were cultured on PDA and CYA plates within 5 days of incubation, then colony diameter were monitored. The results of monitoring growth rate of fungi were shown in Table 4.4.
Table 4.4. Diameter (mm) of colony on PDA and CYA
STT Isolates
Colony diameter (mm)
1 day 2 days 4 days 5 days
PDA CYA PDA CYA PDA CYA PDA CYA
1 G2 6.0 4.0 17.5 14.0 24.5 24.0 27.0 26.0
2 TB1 5.0 3.5 11.5 11.0 23.0 19.5 24.5 20.5
3 TB2 5.0 3.0 11.0 10.0 18.5 17.5 20.0 18.5
4 M1 6.5 6.0 19.5 18.5 34.0 32.5 35.5 33.5
After the first day, the colony size of the isolates was small. On PDA, TB1 and TB2 emerged with a same small size of 5.0 mm, and also still grew slowly on the CYA medium with 3.5mm and 3.0 mm, respectively. M1 grew fastest with the highest size of 6.5 mm, followed by G2 with 6.0 mm.
On the day 2nd, the growth rate of fungal strains increased sharply. Two isolates TB1 and TB2 grew up to 11.5 mm and 11.0 mm, respectively. That was the same growth rate as two largest colonies G2 and M1, up to 19.5 mm and 17.5 mm on the PDA, 14.0 mm and 18.5 mm on CYA medium.
The growth rate of fungi increased significantly on the day 4th and 5th. On the fifth day, the maximum colony sizes were 35.5 mm for M1, 27.0mm for G2 isolate on PDA, 33.5mm and 26.0mm on CYA correspondingly. TB1 and TB2 were the smallest, 24.5mm and 20.0mm on PDA, 20.5mm and 18.5mm on CYA, respectively.
The table 4.4 showed that fungal growth in PDA medium was stronger than that in CYA medium (the size of colonies on PDA were larger than that in CYA). Furthermore, PDA is a cheaper and easier medium to operate, so PDA was selected as the main culture medium for further experiments.
Figure 4.4. The growth rate of fungus TB1 on CYA and PDA after 2 days and 5 days
The fungal colonies grown with high nutrient medium as PDA (various glucose and agar) became thick and compact. In addition, at high concentration of glucose, thick colonies covered the medium homogenously, and the colony growth was higher.
Therefore, from the description of macroscopic characteristics in table 4.1 in comparison with the yellow green color of reference colonies and growth rate of fungi on PDA medium in table 4.4 as well as the high protease activity in table 4.3, the strains G2, TB1 were subjected to further identification.