The isolation and characterization of aflatoxin-producing fungi in spices is the focus of this proposed project for a Bachelor of Science degree. The research will be conducted under the supervision of the Department of Microbiology in the Faculty of Life Sciences at Ahmadu Bello University in Zaria, starting in February 2023.

Aflatoxins are a type of mycotoxin produced by certain Aspergillus species, including Aspergillus flavus and Aspergillus parasiticus. While the presence of Aspergillus mold does not always indicate the presence of aflatoxin, the risk is increased. Aflatoxins are highly toxic, carcinogenic, teratogenic, hepatotoxic, and mutagenic, making them a significant threat to human health.

Spices are widely consumed in Nigeria, with many growing in the wild, particularly in the Southern rainforest zone of the country. Nigerian farmers cultivate a variety of spices, including curry, sweet basil, bush tea, ginger, guinea pepper, African black pepper, and turmeric. These spices are used not only for culinary purposes but also in medicine, religious rituals, cosmetics, and perfume production.

Aflatoxins can be found on a variety of agricultural crops, including maize, peanuts, cottonseed, spices, and tree nuts. They are primarily produced by Aspergillus flavus and Aspergillus parasiticus, with other species also capable of producing them. There are nearly 20 different forms of aflatoxins, with the most important being aflatoxin B₁, B₂, G₁, G₂, M₁, and M₂.

Aflatoxins are produced under atmospheric conditions that favor fungal growth, including high moisture during harvest, dry weather near crop maturity, and inadequate drying and storage of crops. Post-harvest conditions such as transport, storage (excess heat and moisture, pest-related damage, long periods of storage), and food processing can also influence the production of aflatoxins.

In summary, this proposed project aims to investigate the isolation and characterization of aflatoxin-producing fungi in spices, with the ultimate goal of reducing the risk of aflatoxin contamination in Nigeria's spice industry.

The process of post-harvesting of tropical spices is often insufficiently controlled with respect to food hygiene due to the lack of knowledge of handling methods and equipment of farmers. This leads to the spices being susceptible to infestation by storage fungi, particularly during the drying process, transportation, and storage. Furthermore, the increase of spice moisture content due to an infection of xerophilic fungi or physical damage by insect infestation during storage can accelerate the growth of other fungal species that are able to produce aflatoxin. Aflatoxin contamination is a major concern in many countries where agricultural practices are not strictly controlled, and can lead to human and animal exposure to mycotoxins with adverse health effects such as an impaired immune system, cancer, and stunted growth in infants.

In this research project, we will investigate the conditions under which aflatoxin is produced, the primary sources of aflatoxin contamination, whether sunlight can destroy aflatoxin, methods to detect aflatoxin, and ways to remove it from spices. The spices that will be specifically studied in this project are black pepper, chili, and turmeric. A consensus in previous studies has shown that black pepper has one of the highest concentrations of aflatoxin compared to other spices. Chili has also been shown to have a high probability of aflatoxin contamination. Turmeric, on the other hand, has a medium risk of aflatoxin contamination.

The significance of this study lies in its potential to improve the food hygiene practices of farmers, collectors, and exporters of tropical spices, particularly in countries where agricultural practices are not strictly regulated. By identifying the conditions that support aflatoxin production, the primary sources of contamination, and methods to detect and remove it from spices, we can help prevent adverse health effects, economic losses, and ultimately enhance food security in affected areas.

GOALS

This study aims to isolate and characterize fungi that produce aflatoxin in spices. The specific objectives are as follows:

1. To determine the level of aflatoxin in spices obtained from Zaria Market in Nigeria.

2. To identify the strains of filamentous fungi (moulds) present in the spices using microbial culture techniques.

3. To verify the identity of the strains that produce aflatoxin in the spice samples.

METHODS AND MATERIALS

SAMPLE COLLECTION

A total of 50 spices samples were collected from various markets in Zaria for this study. The samples were brought to the laboratory and stored at 4˚C until they were used.

MAJOR SOURCE OF AFLATOXIN

Fungi such as A. flavus, A. parasiticus, and A. nomius are the main sources of aflatoxins, although they are also produced by other species of Aspergillus and by Emericella spp. There are over 20 known aflatoxins, but the main ones are AFB1, AFB2, AFG1, and AFG2; meanwhile, hydroxylated metabolites of AFB1 and AFB2, AFM1 and AFM2, respectively, are produced (Giray et al., 2007; Hussain and Anwar, 2008).

ISOLATION AND CHARACTERIZATION OF AFLATOXIN-PRODUCING FUNGI

Fungi that fluoresced upon exposure to UV radiation and had pink reverse when exposed to ammonia vapors were isolated and cultured using PDA and coconut milk PDA media. Colonies with positive reactions in Aspergillus differentiation agar were selected and cultured onto PDA plates and slants. Microscopic observations were made using a Leica DME binocular microscope to determine fungal morphology. The characteristics and morphology of these strains were compared to known fungal identification manuals, and they were identified.

ISOLATION AND CHARACTERIZATION OF AFLATOXIN-PRODUCING FUNGI FROM THE SPICE SAMPLES

Fungi that produced aflatoxin were isolated by repeatedly transferring them to PDA plates and slants. Isolates with differing colony characteristics were considered to be unique. Table 6 presents the morphological characterization and identification of each isolate, while Figs. 4 to 6 depict photomicrographs of the different isolates observed under the light microscope. Subsequently, the aflatoxin producers identified via the screening methodologies were isolated and pure cultured onto potato dextrose agar. Based on their cultural and morphological characteristics, A. parasiticus or A. flavus were identified as the species present in all the isolates, with A. flavus being found in all five spice samples and A. parasiticus in only one.