Paper of Aflatoxin with Nanis Essay Example

is an important alternative to synthetic chemicals for combating pests or plant pathogens(Siddiquietal., 2005; Nourozianetal., 2006).

Biological control is increasingly being considered by the scientific community as a reliable alternative to pesticide usage in the field and after harvest. This approach is highly desired for reducing agrichemical residues in grapes, wine, and related products by controlling fungal growth on grapes (Cabras and Angioni, 2000). Actinomycetes are filamentous, Gram-positive bacteria that are known for their ability to produce antibiotics. In fact, they generate 75% of all known antibiotic products (Williams et al. 1993). These mesophilic, saprophytic, aerobic organisms naturally reside in the soil and several members of Actinomycetes produce essential secondary metabolites like antibiotics, herbicides, and growth-promoting substances (Connell, 2001). Streptomyces is a genus within Actinomycetes that consists mostly of soil saprophytes with over 400 species. Interestingly, Streptomyces has provided more than half of isolated antibiotics (Embley, 1994). One specific gram-positive bacterium called S. noursei ATCC 11455 is well-known for producing a complex mixture of polyene macrolides called nystatins which act as antifungal agents.

The aim of this research is to use a biological approach to hinder the growth of A. niger, which produces ochratoxin. The main focus is on studying the potential impact of Actinomycete as an antagonist against this organism. The bacteria used in this study, including True and lactic acid bacteria (LAB), were obtained from Tanta University's Faculty of Science Microbiology unit at the Bacteriology Laboratory. The Actinomycetes used in this investigation were isolated from soil samples collected from various locations in Egypt, such as Tanta, El-Mansoura, Kafr El-Zayat, and El-Mehala El-Kobra.

Soil samples were collected by removing approximately 5 cm of

the surface and storing them in clean plastic bags. Subsequently, 0.2 ml samples of the soil dilution were spread on starch nitrate agar plates using a sterilized glass rod. The plates were then incubated at a temperature of 28 ± 2°C for a duration of 7 days. In terms of biological control, various bacterial and Actinomycete isolates were cultivated in their respective appropriate liquid media. True bacteria were grown in Nutrient Broth, LAB in de Man, Rogosa and Sharpe (MRS) media, and Actinomycetes in starch nitrate media, with incubation periods of 24 hours and 7 days respectively.

Aliquots (100 ? l) of each cell free extract, obtained by milling the cells in sterile saline, were applied on the holes using the agar diffusion method. After 5 days of incubation, the diameter of the inhibition zones was measured. To fully identify the Actinomycete isolate number 3 and determine its antagonistic effects on fungi and ochratoxin, various physical, morphological, and biochemical properties were analyzed.

The identification process followed the criteria provided by Kuster (1972), Nonomura (1974), Szabo et al. (1975), and Szabo and Csortos (1975). The Actinomycetes were characterized using the JEOL JSM-5200 LV model at Tanta University's Electron Microscope Unit, with the assistance of light and scanning electron microscopes. Bergey's Manual of Systematic Bacteriology authored by Williams et al. (1989) and Holt et al. (1994) was relied upon for the identification process.

Determination of diaminopimelic acid (DAP)-isomer was conducted by adapting Becker's (1964) method. The Actinomycete isolate 3 was cultivated in nutrient yeast extract broth at 28°C under shake culture conditions. Once the cells reached maximum growth, 1 mg of the dried bacterial

cells were hydrolyzed with 1 ml 6 N HCl in a sealed Pyrex tube, which was heated at 100°C for 18 hours. After cooling, the sample was filtered using Whatman no. 1 filter paper, and subsequently washed with 1 ml water.

The residual solution was dried multiple times on a rotary evaporator with reduced pressure at 40°C in order to remove most of the HCl. The remaining substance was dissolved in 0.3 ml of water, and then 5 ?l of this solution was applied onto a thin layer macrocrystalline cellulose plate (Art 5577 Dc- Plastikfolien cellulose 20 ? 20 cm, layer thickness 0.1 mm, Merck). To separate the amino acids, a solvent mixture of Methanol-water-10 N HCl-Pyridine (80:17.5:2.5:10, by volume) was used. Detection of the amino acids was achieved by spraying acetonic ninhydrin (0.1, w/v) and subsequently heating for 2 min at 100°C.

The olive-green DAP spots gradually turned yellow, while the other amine acids produced purple spots. We investigated various physical and nutritional factors that can affect the growth and antifungal activity of Streptomyces sp. Our goal was to determine the optimal conditions for the growth and antifungal activity of the most effective antagonist. Therefore, we examined different media, pH levels, temperature variations, as well as carbon and nitrogen sources. To isolate and purify the antifungal substance(s) produced by the experimental Actinomycete isolate, we used a modified technique based on Jakoby's 1971 method. This involved fractionation through salting out with ammonium sulfate.

The study investigated different concentrations of ammonium sulfate (25%, 50%, 75%, and 90% w/v). The mixture of the supernatant with ammonium sulfate was chilled at 4°C for thirty minutes, then centrifuged

to separate it. Subsequently, the resulting precipitate was put in a dialysis bag along with buffer solution and stored overnight in a refrigerator at 4°C. This process was repeated until the protein inside the bag reached saturation point. To analyze the protein, native electrophoresis method described by Stegemann et al. (1985) was used. Gel electrophoresis was conducted using a dissociating polyacrylamide gel based on Laemmli's protocol (1970).

The molecular weight of proteins dissolved or extracted in the presence of sodium dodecyl sulfate (SDS) was determined using Weber and Osborne's method from 1969. The mode of action of the antifungal substance was evaluated by testing different concentrations (0.10, 0.25, and 0.50%) of the active protein extracted from table 1 against A. niger, and measuring the antifungal activity of cell-free extracts from various microorganisms.

The table presented below provides information on the quantity and category of bacterial isolates, as well as their inhibition zone diameters (in mm):
- Actinomycetes: 1 2 3 4 5 6 7
- True bacteria: 8 9 10 Bacillus pumilus
- Lactic acid bacteria: 11 12 13 Lactobacillus plantarum Lactobacillus acidophilus Lactobacillus bulgaricus
According to Table 1, there were a total of seven Actinomycete isolates, four true bacteria isolates, and six lactic acid bacteria isolates. Among these bacterial types, isolate number three of Actinomycete (Photo1) demonstrated the highest antifungal activity against A. niger with an inhibition zone diameter measuring precisely thirty-three millimeters.

The growth and antifungal activity of isolate 3 were influenced by various factors. Figures 1, 2, 3, 4, and 5 depict the impact of different conditions on the growth of this isolate. Optimal growth was observed at a temperature of 30°C and

a pH level of 7 when using starch nitrate medium with starch as the carbon source and potassium nitrate as the nitrogen source. This particular isolate exhibited the highest inhibitory activity against ochratoxin A-producing A. niger among all seven Actinomycetes isolates tested, leading to its selection for further characterization and identification.

Light microscopic examination revealed that the branches were arranged in whorls and spiral spore chain (Photo 2). Scanning electron microscope (Photo 3) illustrated the presence of spiny spore surface. The results from the previous characterization program (Table 2) indicated that isolate no 3 belonged to the genus Streptomyces. This conclusion was supported by the observation of aerial mycelia color, LL-DAP in the cell wall, spore form, and physiological and biochemical characteristics. Based on these findings, this isolate was found to be similar to S. noursei.

In summary, the Actinomycete isolate no 3 is likely related to S. noursei and can be named S. noursei. The study focused on extracting and semi-purifying the antifungal substance from the culture broth of the tested S. noursei. Fractionation through salting out with ammonium sulphate was the most effective method in separating the antifungal substance and exhibited the highest antifungal activity. The process also involved characterizing and semi-purifying the antifungal substance.

Actinomycete isolate 3 was incubated with A. niger on Czapex's Dox media. The changes in the morphology of fungi were captured through photographs taken by a Scanning Electron Microscope (model JEOL, JSM-5200 LV) at Tanta University's Electron Microscope Unit. Statistical analysis involved One-way analysis of variance (ANOVA). Duncan multiple range test (LSR) was used to distinguish between different means, and simple linear correlation analysis (r) was conducted

using SAS (1985) software for windows version (6.2). All experiments and analytical determinations were replicated at least three times.

To assess the impact of various bacteria on ochratoxin-producing A. niger growth, control by antagonistic microorganisms was examined. A. niger, which is a common fungal producer of ochratoxin found in herbs and medicinal plants in Egypt, was previously isolated and identified. The well diffusion method was employed to evaluate the antifungal activity of different bacterial isolates.

Allam et al. [669] utilized a dialysis bag to perform dialysis and extract the antifungal substance. Photo 1 shows the impact of Actinomycete isolate 3 on the growth of Aspergillus niger, a known producer of ochratoxin. After dialysis to remove excess ammonium sulfate, the supernatant of isolate 3 in (b) showed reduced growth compared to the control (a). The obtained antifungal substance was then subjected to gel electrophoresis using protein polyacrylamide gel and SDS-PAGE for demonstration of its subunit structure and confirmation of its purity.

The native polyacrylamide gel showed one thick band (Photo 4) when analyzed. The eluted proteins were then subjected to SDS-PAGE using the method of Laemmli (1970), and four bands were observed in the sample (Photo 5). The molecular weights ranged from 670 on the Afr. J. Biotechnol. scale. Figure 1 illustrates the impact of different media on the growth and antifungal activity of isolate no 3.

Figure 2 demonstrates how the growth and antifungal activity of isolate no 3 were examined at different temperatures. The measurement of the diameter of the inhibition zone revealed that the active substance contains protein subunits with sizes approximately 12, 35, 41, and 150 kDa. This indicates that

a group consisting of four compounds is responsible for the antifungal activity. To investigate its mechanism, various concentrations (0.10%, 0.5%, and 0.50%) of the antifungal substance were added to A. niger fungus growth media and incubated for five days.

The results of Photos 6, 7, and 8 demonstrate that increasing the concentration of the semi-purified antifungal substance produced by S. noursei has inhibitory effects on spore formation, as well as thinning and deformation of conidiophores, strigmata, and conidia. The objective of this study was to control contamination of ochratoxin A (Allam et al., 671).

Figure 3 shows the impact of different pH values on the growth and antifungal activity of isolate 3. Figure 4 illustrates the influence of various carbon sources on the growth and antifungal activity of isolate 3. Meanwhile, Figure 5 demonstrates the effect of diverse nitrogen sources on the growth and antifungal activity of isolate 3 (Afr. J. Biotechnol., Photo 2).

The presence of A. niger in common medicinal and herbal plants used in Egypt was discovered by Allam et al. (2008). The discovery was made through a light micrograph of isolate (3) grown on different starch nitrate media for 7 days at 30°C, with magnifications of x40, x25, and x100. In their study, fungal contamination was found in 22 samples of herbal and medicinal plants from Egypt. These samples included species from 5 genera: Aspergillus, Penicillium, Fusarium, Botrytis, and Cladosporium. The majority of the contamination came from the genus Aspergillus. Among the Aspergillus species identified, A. niger was the most prevalent one responsible for producing ochratoxin A. To address this issue, a screening program using liquid cultures was

conducted to implement biological control against ochratoxin A production by A. niger. This involved testing antagonist bacteria and Actinomycetes as potential solutions. Out of all the organisms tested, Actinomycete isolate no 3 exhibited the highest antifungal activity. Further studies were then carried out to characterize and identify this potent organism.

The strain that was chosen was found to thrive on a medium that contains starch nitrate. It is a bacterium that requires oxygen, forms spores, and belongs to the Gram-positive category. It was originally discovered in soil. The mass of spores had a gray color. The aerial mycelium consisted of long filaments that were straight and had evenly spaced spindles along them. The vegetative mycelium produced branched mycelium and contained LL-DAP in the cell wall. All these characteristics confirm that the selected strain is part of the Streptomyces genus. When viewed under a microscope, the spores had a surface with spikes and were arranged in a spiral chain.

The identification of Streptomyces species involves observing their growth on synthetic media with different carbon and nitrogen sources, as well as other characteristics. Several international keys have been used to describe Streptomyces species, including those provided by Kuster (1972), Nonomura (1974), Szabo et al. (1975), and Szabo and Csotros (1975). For this particular isolate, Williams et al. (1989) and Holt et al. (1994) determined it to belong to S. noursei, thus assigning the name S. noursei to it. S. noursei is well-known for its production of the polyene macrolide antibiotic called nystatin, which is a significant antifungal agent.The structure of the nystatin molecule consists of a 38-membered macrolactone ring attached to the deoxysugar mycosamine through

a polyketide moiety.Scientists are interested in cloning and characterizing the genes responsible for nystatin biosynthesis because this knowledge could potentially lead to the development of novel antifungal antibiotics(Brautaset et al.,2000).

This study suggests that the ability of biological control organisms to inhibit fungal growth and reduce ochratoxin contamination in herbs may depend on the specific chemical composition of each herb. In order to test this idea, researchers investigated how different environmental factors affect the growth and antifungal activity of a bacteria strain called S. noursei. They used fractional precipitation with varying concentrations of ammonium sulphate to isolate the active antifungal compound produced by S. noursei. The findings revealed that a concentration of 50% ammonium sulphate exhibited the highest antifungal activity. Similar protein extraction methods using ammonium sulphate have been employed in previous studies involving other bacterial strains such as Streptomyces megasporus, Shizophyllum commune BL23, B. subtilis DC33, and B. subtilis LD-8547 (Chitte and Dey, 2000; Patcharaporn et al., 2008; Cheng et al., 2006; Wang et al., 2007). The specific characteristics and identification of the isolate used in this study are detailed in Table 2 (3) of the original research source (Afr. J. Biotechnol., 674).

The text below describes various morphological, physiological, and biochemical characteristics of a specific organism. These characteristics include aerial mycelium, branching, substrate (vegetative) mycelium, spore mass, spore surface, gram reaction, motility, pH, temperature, cell wall hydrolysis (diaminopimelic acid), production of melanin on peptone-yeast extract iron agar, tyrosine agar medium, tryptone yeast extract broth, nitrate reduction, H2S production, hydrolysis of starch, protein, lipid, cellulose, casein, and pectin, degradation of xanthin, esculin, and tyrosine, as well as utilization of carbon sources such as D-Xylose,

D-Glucose, D-Galactose, Sucrose, L-Rhamnose, Mannitol, L-Arabinose, Raffinose, Meso-Inositol, and D-Fructose.

The mycelium in the air has a fluffy appearance and is arranged in whorls. It consists of long straight filaments. At the top, spiral spore chains are produced by the mycelium through branches called verticile. The vegetative mycelium is gray and branched. The spores have a surface covered in spines and cannot move on their own. Their diameter measures 0.9-1.2 ?m. The fungus thrives best at temperatures between 26-32°C for growth.

Nitrogen is obtained from sources like L-Asparagine, L-Tryptophane, and L-Glutamic acid to support its growth.

Antibiosis occurs when interacting with Aspergillus niger and Candida albicans but not Bacillus subtilis.

Two pictures are provided: one confirms the presence of the purified protein on a native polyacrylamide gel while another displays an SDS-PAGE photograph of the purified protein alongside a molecular weight marker.

The study found that using native polyacrylamide gel results in one thick band. However, four proteinaceous compounds within the active substance are responsible for its effectiveness against A. niger. The SDS-polyacrylamide gel electrophoresis determined that these compounds have molecular masses of 12, 35, 41, and 150 kDa respectively. The partially purified substance inhibits spore formation, thinning of conidiophore, and deformation of both strigmata and conidial head. Increasing the concentration of the protein substance leads to greater changes and deformation in the fungus. Therefore, it can be concluded that the protein substance extracted from S. noursei causes desporulation and deformation in A. niger.

According to Taechowisan et al. (2005), S. noursei can effectively control the growth and production of ochratoxin A by A. niger as a biological control method. In addition, the

antifungal substance obtained from S. noursei inhibits A. niger, as shown in , which presents a scanning electron microscope image at a magnification of x750 of the organism responsible for ochratoxin production. The impact of the antifungal substance on A. niger can be observed in and , displaying scanning electron microscope images at a magnification of x750 that reveal effects such as suppression of spore formation, thinning of conidiophore, and deformation of both strigmata and conidial head for doses of 0.25% and 0.50%, respectively.