**3. Results**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

and control flasks was incubated without shaking at 25°C.

filtrates were used as crude preparation.

**2.4 Ammonium sulfate fractionation**

Pfueller and Elliott [132] and Xiao et al. [133].

flasks were assayed for amylase activity.

fied methods of Pfueller and Elliott [132] and Xiao et al. [133].

mately 6 × 104

**2.5 Dialysis**

**2.6 Enzyme assay**

*2.6.1 α-Amylase*

121°C. Experimental Conical flasks (250 ml) containing 100 ml of the rice medium was inoculated with 1 ml of an aqueous spore suspension containing approxi-

medium not inoculated with aqueous spore suspension of the isolate. Experimental

The crude enzymes were treated with ammonium sulfate (analytical grade) within the limits of 40–90% saturation. Precipitation was allowed to continue at 4°C for 24 h. The mixtures were then centrifuged 10,000 g for 30 min at 4°C using a high speed cold centrifuge (Optima LE-80 K Ultracentrifuge, Beckman, USA). The supernatant was discarded. The precipitate was re-dissolved in 0.2 M citrate phosphate buffer, pH 6.0. The protein contents were determined using the Lowry et al. [131] method while amylase activity was determined using the modified methods of

Using acetylated dialysis tubings (Visking dialysis tubings, Sigma) [134] and a multiple dialyser (Pope Scientific Inc. Model 220, USA), the enzyme preparations were dialysed under several changes of 0.2 M citrate phosphate buffer pH 6.0 at 4°C for 24 h. The protein contents of the dialysed enzymes were determined using the Lowry et al*.* [131] method while amylase activity was determined using the modi-

Both experimental (fungal isolate inoculated) and control (un-inoculated)

α-Amylase activity was determined using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133]. The reaction mixtures consisted 2 ml of 0.1% (w/v) starch (Sigma) in 0.2 M citrate phosphate buffer, pH 6.0 as substrate and 0.5 ml of enzyme. These were the experimentals in the assay procedure. The controls in the assay procedure consisted only 2 ml of the prepared substrate. The contents of both experimental and control tubes were incubated at 35°C for 30 min. The reactions were terminated with 3 ml of 1 N HCl. Enzyme (0.5 ml) was added to the contents of each control. About 2 ml of the mixture from each of the sets of experimentals and controls was transferred into new sets of clean test tubes. About 3 ml of 0.1 N HCl was added into the contents of each test tube after which 0.1 ml of iodine solution was added. Optical density readings were taken spectrophotometrically at 620 nm. Enzyme activ-

ity was defined in units and specific activity as enzyme units per mg protein.

One unit of α-amylase activity was defined as the amount of enzyme which produced 0.1% reduction in the intensity of the blue color of starch-iodine complex

On a daily basis, the contents of each flask was filtered through glass fiber filter paper (Whatman GF/A). The protein content of the filtrates was determined using the method of Lowry et al. [131]. The filtrates were analyzed for amylase activity using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133]. The

spores per ml of each isolate. Control flasks contained sterilized rice

**60**

under conditions of the assay.

### **3.1 Amylase activities of isolates on growth media**

Toxigenic strains of *Aspergillus flavus* (A1), *Aspergillus parasiticus* (A2), *Penicillium citrinum* (P1) and *Penicillium rubrum* (P2) grew and exhibited amylase activities, varyingly, in modified growth medium used for this research.

Using different carbon sources (rice, starch, maltose, sucrose, lactose, glucose and galactose) in the growth medium, amylase activity expressed by each isolate on the tenth day of incubation is shown in **Table 1**.

With different sources of nitrogen (NH4Cl, urea, KNO3, ammonium sulfate, glycine, sodium nitrate, tryptone and peptone) in the growth medium, amylase activity expressed varyingly by each isolate on the tenth day of incubation is shown in **Table 2**.

Toxigenic *P. citrinum* (P1) produced active α-amylase (0.75 ± 0.01 Units) and this was when potassium nitrate was nitrogen source with maltose as carbon source of the defined growth medium. Toxigenic *A. parasiticus* (A2) also expressed an α-amylase activity value of 0.72 ± 0.04 Units when rice was both carbon and nitrogen source of medium for fungal growth (**Table 1**).


#### **Table 1.**

*Effect of carbon sources on activity of amylase produced by isolates.*


#### **Table 2.**

*Effect of nitrogen sources on activity of amylase produced by isolates.*

Toxigenic *A. flavus* (A1) produced the most active α-amylase (3.25 ± 0.15 Units) and this was when ammonium sulfate was nitrogen source with starch as carbon source of the defined growth medium. Toxigenic *A. flavus* (A1) also expressed an α-amylase activity value of 3.02 ± 0.18 Units when starch was carbon source and ammonium chloride was nitrogen source of the defined fungal growth medium (**Table 2**).

### **4. Discussion**

The results of this investigation show that the toxigenic strains of *A. flavus* (A1), *A. parasiticus* (A2), *P. citrinum* (P1) and *P. rubrum* (P2) grew in a synthetic medium with varying carbon and nitrogen sources exhibiting α-amylase activities. α**-**Amylase activities were detected in the extracts of growth medium with rice as carbon source, infected with the toxigenic strains of *A. flavus* (A1), *A.* 

**63**

*α-Amylase Production by Toxigenic Strains of* Aspergillus *and* Penicillium

*parasiticus* (A2), *P. citrinum* (P1) and *P. rubrum* (P2). When the carbon source was varied, potassium nitrate was the nitrogen source. When the nitrogen source was varied, starch was the carbon source for fungal growth. According to Olutiola [135], *Aspergillus chevalieri* from moldy maize produced extracellular amylase when grown in a liquid medium containing starch as carbon source. According to Barnett and Fergus [136], increasing the amount of starch-yeast extract medium increased the extracellular amylase produced by *Humicola lanuginosa*. Studies carried out by Okafor et al. [137] revealed that *Lactobacillus delbrueckii*, *Lactobacillus coryniformis* and *Saccharomyces* sp., isolated from cassava processing environments were high amylase producers. Among a series of starch sources of carbon, wheat and soluble starch were inducers of a thermostable amylase by a yeast strain isolated from starchy soil [138]. According to Bluhm and Woloshuk [139], amylopectin, an important constituent of starch, induces fumonisin B(1) production in *Fusarium verticillioides* during colonization of maize. According to Coleman [140], extracellular α**-**amylase was secreted by *Bacillus subtilis* in a complex medium containing maltose, starch, glycerol or glucose as carbon source; the general characteristics of secretion indicated a low but definite production of exoenzyme from the moment the cells of the organism started to grow until the end of the logarithmic phase after which, the rate of increase in cell mass decreased, the rate of enzyme secretion increased to a high linear value which was

Aflatoxin B1-producing-toxigenic strains of *Aspergillus flavus*, *Aspergillus parasiticus*, *Penicillium citrinum* and *Penicillium rubrum* can be explored industrially for α-amylase production using the specific growth medium and the rice medium used in this investigation. Varying the specific C and N source of this growth medium is

The genetic make-ups of these aflatoxin B1-producing-α-amylase-producing fungal strains are important in their ability to produce the enzyme α-amylase. Specific genes are necessary and important in the production of this enzyme. Mutant strains lacking the specific genes for α-amylase production will not be ideal in the exploration for production of the enzyme. More so, there seems to be a significant relationship between the ability to produce α-amylase and aflatoxin B1

The toxigenic strains of *A. flavus* (A1), *A. parasiticus* (A2), *P. citrinum* (P1) and *P.* 

Authors are thankful to the British Mycology Society (BMS), United Kingdom

*rubrum* (P2) can be explored in the industrial production of α-amylases.

*DOI: http://dx.doi.org/10.5772/intechopen.86637*

maintained even in the stationary phase.

of upmost significance in such an exploration.

production in mycotoxigenic fungi from literature.

**4.1 Significance of study**

**4.2 Limitations**

**5. Conclusion**

**Acknowledgements**

for Grant supports.

*α-Amylase Production by Toxigenic Strains of* Aspergillus *and* Penicillium *DOI: http://dx.doi.org/10.5772/intechopen.86637*

*parasiticus* (A2), *P. citrinum* (P1) and *P. rubrum* (P2). When the carbon source was varied, potassium nitrate was the nitrogen source. When the nitrogen source was varied, starch was the carbon source for fungal growth. According to Olutiola [135], *Aspergillus chevalieri* from moldy maize produced extracellular amylase when grown in a liquid medium containing starch as carbon source. According to Barnett and Fergus [136], increasing the amount of starch-yeast extract medium increased the extracellular amylase produced by *Humicola lanuginosa*. Studies carried out by Okafor et al. [137] revealed that *Lactobacillus delbrueckii*, *Lactobacillus coryniformis* and *Saccharomyces* sp., isolated from cassava processing environments were high amylase producers. Among a series of starch sources of carbon, wheat and soluble starch were inducers of a thermostable amylase by a yeast strain isolated from starchy soil [138]. According to Bluhm and Woloshuk [139], amylopectin, an important constituent of starch, induces fumonisin B(1) production in *Fusarium verticillioides* during colonization of maize. According to Coleman [140], extracellular α**-**amylase was secreted by *Bacillus subtilis* in a complex medium containing maltose, starch, glycerol or glucose as carbon source; the general characteristics of secretion indicated a low but definite production of exoenzyme from the moment the cells of the organism started to grow until the end of the logarithmic phase after which, the rate of increase in cell mass decreased, the rate of enzyme secretion increased to a high linear value which was maintained even in the stationary phase.

#### **4.1 Significance of study**

Aflatoxin B1-producing-toxigenic strains of *Aspergillus flavus*, *Aspergillus parasiticus*, *Penicillium citrinum* and *Penicillium rubrum* can be explored industrially for α-amylase production using the specific growth medium and the rice medium used in this investigation. Varying the specific C and N source of this growth medium is of upmost significance in such an exploration.

#### **4.2 Limitations**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

Ammonium sulfate *Aspergillus flavus* (A1)

Glycine *Aspergillus flavus* (A1)

Potassium nitrate *Aspergillus flavus* (A1)

Ammonium chloride *Aspergillus flavus* (A1)

Peptone *Aspergillus flavus* (A1)

Sodium nitrate *Aspergillus flavus* (A1)

Tryptone *Aspergillus flavus* (A1)

Urea *Aspergillus flavus* (A1)

*Each value represents the mean of three replicates with standard error.*

*Effect of nitrogen sources on activity of amylase produced by isolates.*

**Nitrogen source Isolate Amylase activity (Units)**

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

*Aspergillus parasiticus* (A2) *Penicillium citrinum* (P1) *Penicillium rubrum* (P2)

3.25 ± 0.15 0.05 ± 0.00 0.38 ± 0.13 0.13 ± 0.03

0.38 ± 0.13 2.48 ± 0.03 0.00 ± 0.00 1.53 ± 0.48

0.50 ± 0.00 1.30 ± 0.10 0.25 ± 0.25 0.68 ± 0.03

3.02 ± 0.18 0.05 ± 0.05 0.13 ± 0.13 0.13 ± 0.03

0.25 ± 0.00 1.50 ± 0.15 0.13 ± 0.13 2.48 ± 0.03

0.38 ± 0.13 1.48 ± 0.13 0.25 ± 0.00 0.93 ± 0.73

0.25 ± 0.00 0.20 ± 0.05 0.38 ± 0.13 2.40 ± 0.10

0.15 ± 0.00 2.32 ± 0.03 0.00 ± 0.00 2.33 ± 0.08

Toxigenic *A. flavus* (A1) produced the most active α-amylase (3.25 ± 0.15 Units) and this was when ammonium sulfate was nitrogen source with starch as carbon source of the defined growth medium. Toxigenic *A. flavus* (A1) also expressed an α-amylase activity value of 3.02 ± 0.18 Units when starch was carbon source and ammonium chloride was nitrogen source of the defined fungal

The results of this investigation show that the toxigenic strains of *A. flavus* (A1), *A. parasiticus* (A2), *P. citrinum* (P1) and *P. rubrum* (P2) grew in a synthetic medium with varying carbon and nitrogen sources exhibiting α-amylase activities. α**-**Amylase activities were detected in the extracts of growth medium with rice as carbon source, infected with the toxigenic strains of *A. flavus* (A1), *A.* 

**62**

growth medium (**Table 2**).

**4. Discussion**

**Table 2.**

The genetic make-ups of these aflatoxin B1-producing-α-amylase-producing fungal strains are important in their ability to produce the enzyme α-amylase. Specific genes are necessary and important in the production of this enzyme. Mutant strains lacking the specific genes for α-amylase production will not be ideal in the exploration for production of the enzyme. More so, there seems to be a significant relationship between the ability to produce α-amylase and aflatoxin B1 production in mycotoxigenic fungi from literature.

#### **5. Conclusion**

The toxigenic strains of *A. flavus* (A1), *A. parasiticus* (A2), *P. citrinum* (P1) and *P. rubrum* (P2) can be explored in the industrial production of α-amylases.

#### **Acknowledgements**

Authors are thankful to the British Mycology Society (BMS), United Kingdom for Grant supports.
