**2. Materials and methods**

#### **2.1 Sources and identification of isolates**

The isolates of aflatoxin B1-producing-toxigenic strains of *Aspergillus flavus* (A1), *Aspergillus parasiticus* (A2), *Penicillium citrinum* (P1) and *Penicillium rubrum* (P2) for this research were from deteriorated rice and identified at the Seed Health Unit of the International Institute for Tropical Agriculture, Ibadan, Nigeria using techniques contained in the illustrated Handbook of fungi [127, 128].The identification was done by observing cultural and morphological characteristics. Each isolate was cultured on Potato Dextrose agar. The nature of growth, rate of growth, colony color and sporulation patterns were carefully observed. Sporulating mature cultures was used in microscopic examination. Fungal samples were taken from advancing margins and centers of the growth regions with the aid of sterile inoculating needle. The samples were smeared on glass slides and stained with lactophenol cotton blue. After placing the cover slips, macroscopic and microscopic morphological characteristics like arrangement and shape of spores, type of sporangia, type of hyphae, presence or absence of septa on hyphae was examined under the high power objective of a compound binocular microscope.

#### **2.2 Culture conditions and preparation of inocula**

The isolates were subcultured and maintained on Potato Dextrose agar plates and slants. Each fungus was further subcultured into test tubes of the same medium and incubated at 25°C. A 96-hr-old culture of toxigenic strains of *Aspergillus flavus* (A1), *Aspergillus parasiticus* (A2) and *Penicillium rubrum* (P2) and 120-h-old culture of *Penicillium citrinum* (P1) was used as inocula. According to the modified method of Olutiola and Ayres [129], cultures was grown in a defined medium of the underlisted composition: MgSO4.7H20, K2HPO4, KH2PO4, L-cysteine, biotin, thiamine and FeSO4.7H20 with added carbon and nitrogen sources (Sigma). Conical flasks (250 ml) containing 100 ml growth medium will be inoculated with 1 ml of an aqueous spore suspension containing approximately 6 × 104 spores per ml of each isolate. Experimental and control flasks was incubated without shaking at 25°C [130].

#### **2.3 Rice as a source of carbon**

Rice (Caprice) from Spain was bought at the main market, Bodija, Ibadan, Nigeria. The rice was added to distilled water (1% w/v) and autoclaved at 15Ib/in<sup>2</sup> at 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 approximately 6 × 104 spores per ml of each isolate. Control flasks contained sterilized rice medium not inoculated with aqueous spore suspension of the isolate. Experimental and control flasks was incubated without shaking at 25°C.

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 filtrates were used as crude preparation.

#### **2.4 Ammonium sulfate fractionation**

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

#### **2.5 Dialysis**

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

#### **2.6 Enzyme assay**

Both experimental (fungal isolate inoculated) and control (un-inoculated) flasks were assayed for amylase activity.

#### *2.6.1 α-Amylase*

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 activity 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 under conditions of the assay.

**61**

**Table 1.**

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

Toxigenic strains of *Aspergillus flavus* (A1), *Aspergillus parasiticus* (A2), *Penicillium citrinum* (P1) and *Penicillium rubrum* (P2) grew and exhibited amylase

Using different carbon sources (rice, starch, maltose, sucrose, lactose, glucose and galactose) in the growth medium, amylase activity expressed by each isolate on

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

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

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

0.54 ± 0.01 0.72 ± 0.04 0.43 ± 0.23 0.62 ± 0.06

0.06 ± 0.01 0.53 ± 0.13 0.36 ± 0.05 0.09 ± 0.04

0.50 ± 0.04 0.63 ± 0.08 0.44 ± 0.08 0.32 ± 0.11

0.10 ± 0.00 0.66 ± 0.10 0.40 ± 0.17 0.37 ± 0.08

0.52 ± 0.03 0.68 ± 0.04 0.75 ± 0.01 0.58 ± 0.12

0.45 ± 0.04 0.57 ± 0.12 0.68 ± 0.03 0.60 ± 0.14

0.46 ± 0.05 0.69 ± 0.03 0.59 ± 0.13 0.39 ± 0.06

*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)

activities, varyingly, in modified growth medium used for this research.

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

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

the tenth day of incubation is shown in **Table 1**.

nitrogen source of medium for fungal growth (**Table 1**).

Rice *Aspergillus flavus* (A1)

Galactose *Aspergillus flavus* (A1)

Glucose *Aspergillus flavus* (A1)

Lactose *Aspergillus flavus* (A1)

Maltose *Aspergillus flavus* (A1)

Starch *Aspergillus flavus* (A1)

Sucrose *Aspergillus flavus* (A1)

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

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

**3. Results**

in **Table 2**.

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