Cassava starch has relatively higher enzyme susceptibility than other starches². Cassava starch is more susceptible to α-amylase and glucoamylase attack than sweet potato starch and potato starch ^{3, 4}. In a SSF process, cassava not only supplies the nutrients to the microbial culture growing in it but also serves as an anchorage for the cells. The substrate that provides all the needed nutrients to the microorganisms growing in it should be considered as the ideal substrate. Amylases are enzymes, which hydrolyze starch. The hydrolysis of starch with amylase results just in the production of short chain polymers called dextrin, then the disaccharide maltose and finally glucose⁵. Highest amylase activities by the molds of cassava tuber were recorded between pH 6 – 7 and at 25°C temperature⁶. The study sought to assess the potential utilization of fermented cassava as a source of industrial amylase by solid state fermentation using various extracellular amylase secreting non pathogenic, harmlessness and safe microorganisms such as Bacillus cereus, Saccharomyces cerevisae and Aspergillus niger. Since these microorganisms are the most significant and convenient sources of commercial enzymes and can also be made to produce abundant quantities of enzymes under suitable growth conditions, they are widely studied.

MATERIALS AND METHODS:

Cassava tubers were obtained from agricultural market Koyambedu, Chennai, Tamil Nadu, India. Tubers of eight months maturity were selected for the work. Pure cultures of extracellular amylase secreting Bacillus cereus, Saccharomyces cerevisae and Aspergillus niger were used for the study. Aspergillus niger and Saccharomyces cerevisae were maintained on Potato- Dextrose – Agar (PDA) and Bacillus cereus was maintained on Nutrient Agar slants. The slants of Aspergillus niger, Saccharomyces cerevisae and Bacillus cereus were grown at 30°C/37°C and stored at 4 °C. These were subcultured fortnightly.

Preparation of inoculum:

Ten ml of distilled water containing 0.1% Tween 80 was transferred to a sporulated (7 days old) PDA slant culture. The spores were dislodged using the inoculation needle under aseptic conditions and the suspension with appropriate dilution was used as inoculum for Aspergillus niger. A loopful of Bacillus cereus and Saccharomyces cerevisae cultures were taken and inoculated in to nutrient broth and potato dextrose broth respectively and kept in shaker incubator, these were used as inoculum.

Solid–state fermentation:

100 g of grated cassava was taken in to each 250ml Erlenmeyer flasks. Distilled water was added to a salt solution (5ml) containing (g/ml) KH2PO4 – 0.2g, NH4NO3 – 0.5g, NaCl-0.1g, MgSO4.7H2O – 0.1 g to adjust the required moisture level. Three flasks each were inoculated with 5% inoculums of Bacillus cereus, Saccharomyces cerevisae and Aspergillus niger respectively. The contents of the flasks were mixed thoroughly, kept for solid state fermentation for five days. Unfermented cassava tubers served as control.

Enzyme extraction:

Crude enzyme was extracted by mixing a known quantity of fermented matter with distilled water on a rotary shaker (180rpm) for one hour. The suspension was then centrifuged at 7000 x g at 4 °C for 10 minutes and the supernatant was used for enzyme assay.

Iodine Test:

Two ml of Iodine was added to 0.5ml of unfermented and fermented enzyme extract and the colour changes were observed.

Estimation of Protein content:

Soluble protein concentrations were determined in the aqueous extract of fermented matter using Bovine Serum Albumin as standard⁷. 50μl of the enzyme was taken and made up to 1ml with distilled water. Then it was mixed with alkaline copper reagent followed by Folin’s reagent. Then it was read at 660nm.

Estimation of Reducing Sugar and Amylase assay:

Reducing sugar was estimated by dinitrosalicylic acid method (DNS) using maltose as standard. Amylase activity was determined⁸ . The reaction mixture consists of enzyme extract in 1% soluble starch and 100mM Citric acid buffer (pH– 4.5). After 10 minutes of incubation at 30° C, the liberated reducing sugars were estimated by the dinitrosalicylic acid (DNS) method. The color developed was read at 540 nm using a Shimadzu – 1601 UV Spectrophotometer. Maltose was used as the standard. The blank consist of 0.8ml of Citric acid buffer (PH-4.5), 1ml of 1% starch solution and 1ml of distilled water. One unit (IU) of Amylase activity was defined as the amount of enzyme in one ml releasing one mg of reducing sugar under the assay conditions. The unit and specific activity can be calculated as follows:

Characterization of enzyme

The amylase from fermented cassava tubers was characterized by studying the effect of temperature on amylase at 30°C, 40°C, 50°C, 60°C and 70°C; the effect of pH at 4.5, 5.5, 6.5, 7.5 and 8.5; at various substrate concentrations of 1 – 5 % starch solutions.

Polyacrylamide Gel Electrophoresis:

Native (non – denaturing) Polyacrylamide gel electrophoresis of the enzymes was done in 1mm thick vertical slab of 7% W/V polyacrylamide gel ⁹. The enzymes used for polyacrylamide gel electrophoresis were crude extract of cassava tuber fermented with various microorganisms such as Bacillus cereus, Saccharomyces cerevisae and Aspergillus niger. Amylase (Hi Media) was used as the marker. All samples and standards were mixed with Sucrose and Tracking dye. The tracking dye used was Bromophenol blue. The gel solutions were taken out of fridge and brought to room temperature. The spacer was kept between clean, dry glass plates and clipped well on both sides. It was then placed on the stand and fixed tightly using screws. Now 7% resolving gel was prepared by mixing 2ml of acrylamide bis acrylamide with 1ml of resolving gel buffer (Tris HCl, buffer PH 8.9), 1ml distilled water, 4ml of APS from 400mg in 4ml distilled water stock and 6ml TEMED. APS and TEMED were added at the last to prevent polymerization before adding to glass slides. When the resolving gel was ready, it was poured into the glass plate carefully through the sides to prevent bubble formation.

Then it was layered carefully with water for getting an even gel surface without disturbing the surface of the gel. Then the gel was allowed to stand for 30 – 40 minutes to polymerize. Once the gel has polymerized the water layer was removed and gel surface was washed gently with spacer gel buffer. Now the Stacking gel or Spacer gel was prepared by mixing 2ml of acryl amide bisacrylamide, 1ml of stacking gel buffer, pH 6.7, 4ml of 40% sucrose and 1ml of APS and 6ml of TEMED. This was poured over the resolving gel. Comb was gently inserted between the plates and it was kept about 30 minutes to polymerize. Once the stacking gel has polymerized, the comb was taken out gently. The wells were washed well with distilled water and then with stacking gel buffer. Then the enzyme sample and standards were loaded into the wells. (Unused wells were filled with sample buffer pH 6.7). It was then inserted into the PAGE tank and electrode buffer was poured into it. The current was initially maintained at 30mA and 100V and the temperature at 4°C. Once the dye has entered the resolving gel the current was raised to 60mA and 150V and temperature still at 4°C. When the dye has reached an appropriate distance, the glass plates were taken out by removing the spacers. The gel was transferred carefully into a tray containing soluble starch 0.1mol 1-1 for 1 hour at 25°C. The gel was then kept in the same buffer, followed by staining with Iodine solution (0.05g Iodine and 0.5g KI in 100ml of distilled water) for 5 minutes. The solution was then pipetted out and the gel was then photographed using a gel documentation system.

RESULTS AND DISCUSSIONS:

The protein content and reducing sugar of the enzyme extracts were determined (Table 1) High protein content of 18.4mg / ml was found in sample inoculated fermented with Aspergillus niger. Intermediate protein content was obtained in sample fermented with Bacillus cereus which was found to be 16.4 mg/ ml. Low protein content was obtained in sample fermented with Saccharomyces cerevisae, and it was 14mg / ml. There was slight increase in protein content and reducing sugar than unfermented sample after fermentation. The increase in protein content could be attributed to possible secretion of some extracellular enzymes such as amylases, linamerase and cellulase into cassava mash by fermenting organisms in an attempt to make use of cassava starch as a source of carbon^10,11. These also indicate the potential of experimental micro organisms in protein enrichment of cassava by solid state fermentation. The reducing sugar content was found to be highest in sample fermented with Saccharomyces cerevisae (1.55 mg / ml) and intermediate in Bacillus cereus (0.55 mg /ml), Aspergillus niger (0.45mg/ml). Fermented samples showed increase in reducing sugar content when compared with unfermented. Fermented samples showed yellow colour on addition of iodine indicating the presence of lower dextrin. Amylase activity of the enzyme extracts were done and results of unit and specific activity were tabulated in (Table 2).

Table 1: Protein content and reducing sugar in crude extract from fermented cassava tubers.

Sample	Protein content (mg / ml)	Reducing sugar (mg / ml)
Unfermented	15.2	0.35
Inoculated fermented
a) Bacillus cereus	16.4	0.55
b)Saccharomyces cerevisae	14	1.55
c) Aspergillus niger	18.4	0.45

Table 2: Unit activity and specific activity of amylase obtained from crude extract of fermented Cassava tubers.

Sample	Unit Activity (Unit / ml)	Specific activity (Unit / min / mg)
Unfermented	1.75	0.11
Inoculated fermented
a) Bacillus cereus	2.75	0.16
b)Saccharomyces cerevisae	7.75	0.55
c) Aspergillus niger	2.25	0.12

The sample fermented with Saccharomyces cerevisae showed the maximum unit and specific activity (7.75 unit/ml and0.55 unit/min/mg) unlike fermenting with Bacillus cereus and Aspergillus niger. This specific activity was higher than the specific activity reported earlier¹²on the activity of crude beta amylase from sweet potatoes. The effect of temperature on amylase activity in crude extract of cassava fermented with Aspergillus niger, Bacillus cereus and Saccharomyces cerevisae were determined (Fig 1). There was a gradual increase in the enzyme activity fermented with Saccharomyces cerevisae and Bacillus cereus from 30° - 60°C and 40° - 50°C. In case of Saccharomyces cerevisae there was a sudden decline in enzyme activity from 60°C to 70°C. For Bacillus cereus there was decline in enzyme activity from 50°C and at 60°C and 70°C it was constant. In Aspergillus niger and there was slow decline in enzyme activity from 40 - 70°C. The enzyme had its maximum activity at 60°C, 40°C and 30°C respectively. Unfermented sample showed its optimum activity at 40°C and there was decline in enzyme activity constantly. The maximum activity of the Saccharomyces cerevisae fermented tuber was at 60°C is the same with that of beta amylase from C.thermocellum SS8, B.circulans and B. megaterium ^13-15.The result also agrees with the temperature for optimal activity reported for A.flavus and M.pusillus ¹⁶ and for S. cerevisae with L.delbruckii and L.coryneformi ¹⁷.

The effect of pH on enzyme activity fermented with different organisms was given in (Fig 2). The result indicated that there was a gradual increase in enzyme activity from acidic to neutral ranges. Amylase is active at pH 7.5 for Bacillus cereus. It showed gradual increase in the enzyme activity for pH ranging from 4.5 to 7.5 and then decrease from 7.5.

The optimum pH obtained in this study also agrees with earlier investigations regarding Bacillus cereus ¹⁸. For Saccharomyces cerevisae, it showed gradual increase from 4.5 to 6.5, and the maximum activity was at 6.5 and then gradual decrease from 6.5 to 8.5. For Aspergillus niger it showed activity at 5.5 and then a sudden decline at 6.5 and again increase in activity at 7.5 and 8.5. This shows that except for Aspergillus niger rest all samples looses enzyme activity at alkaline region.

The effect of substrate concentration on amylase activity showed that the increase in substrate concentration increases enzyme activity. The sample fermented with various fermenting organisms such as Bacillus cereus, Saccharomyces cerevisae and Aspergillus niger showed its maximum activity at 5% starch concentration as shown in (Fig.3). There was a drastic increase in the enzyme activity with increase in starch concentration from 1 to 3% and a minimal increase from 3 to 5% .This result is in agreement with earlier reports on the effect of substrate concentration on some fungal (A. niger, A. flavus, R. oryzae and M. pusillus) amylase activities, which indicated that increase in substrate concentration from 1 to 3% led to progressive increase in amylase activity. This indicates that for optimal utilization of resources, the use of the amylase from this fermented cassava tubers, should be correlated with the starch to be hydrolyzed at 3% substrate concentration level. Amylase activity was localized by running the enzyme in a Native PAGE. The gel was immersed in 0.1 mol 1^-1 soluble starches for 1 hour at 25°C. The gel was then kept in the same buffer, followed by staining with Iodine solution (0.05g Iodine and 0.5g KI in 100 ml of distilled water) for 5 minutes. Activity staining of amylase showed pale yellow band in the gel which confirmed the enzyme activity.

CONCLUSION:

The crude extract containing amylase from fermented cassava tubers is active at wide ranges of temperature, pH and substrate concentration. Conversion of readily and cheaply available raw cassava by microbial enzymes eliminated the cooking steps and saved much needed energy. Cassava being an inexpensive source of starch and biological matter for production of commercially valuable microbial metabolites, it can be used by starch industry for the production of value added products and it could also be employed as a source of amylase for Industrial applications.

ACKNOWLEDGEMENTS:

Authors are grateful to the Management Vel’s Educational Trust, Pallavaram, Chennai, India for providing all the facilities to carry out this research work in the Department of Biotechnology, Vels University, Chennai.

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Received on 27.02.2011

Modified on 02.03.2011

Accepted on 06.03.2011

Research J. Science and Tech. 3(4): July-August. 2011: 192-196