Current Research - Kennesaw State University



CURRENT RESEARCH

Molecular and Biochemical Characterization of Aflatoxin producing fungi, Aspergillus flavus and

A. parasiticus in Peanuts from Georgia.

Introduction:

Georgia produces 37 percent of U.S. peanuts. Peanut is the edible seed of the plant, Arachis hypogaea. Peanuts are also known as Groundnuts. Poor storage of peanuts can lead to an infection by the mould fungus Aspergillus flavus and A. parasiticus releasing the toxic substance aflatoxin. The major producers/exporters of peanuts are the United States, Argentina, Sudan, Senegal, and Brazil. These five countries account for 71 percent of total world exports. In recent years, the United States has been the leading exporter of peanut .Of greatest concern is possible contamination of damaged or spoiled seeds with the teratogenic, carcinogenic aflatoxins. Aflatoxins are polyketide secondary metabolites produced by important food contaminating species A. flavus and A. parasiticus. The four main aflatoxins produced, aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2) and aflatoxin G2 (AFG2) are furanocoumarin derivatives and potent liver carcinogens for a wide variety of animal species including humans (Smith et al. 1994). Isolates of A. parasiticus produce both B and G aflatoxins, while most North American A. flavus isolates produce only B aflatoxins and West African isolates may also produce AFG1 (Cottony and Cardwell, 1991).

It has been established by previous studies that the occurrence of aflatoxins is influenced by favorable conditions which include high moisture content and high temperature. Hence, the extent of contamination will vary with geographic location, agricultural and agronomic practices, and the susceptibility of the peanuts to fungus before they are harvested, and during storage, and processing periods under favorable temperature and humidity conditions these fungi grow on stored peanuts. The toxins are particularly carcinogenic in humans and eating contaminated peanuts often results in liver cancer, amongst other diseases

A. flavus and A. parasiticus, can invade groundnut seeds in the field before harvest, during post harvest drying and curing, and in storage and transportation. Aflatoxin contamination can be minimized by adopting certain cultural, produce handling, and storage practices. However, these practices are not widely adopted particularly by the small farmers in the developing countries, which contribute about 60% to the world groundnut production. Since spores of both the species can survive for a long period in the air and can get disseminated from one place to another, storage malpractices and mishandling of peanuts in commercial outlets in Georgia and all peanut growing States may trigger infection.

Humans are exposed to aflatoxins by consuming foods contaminated with products of fungal growth. Such exposure is difficult to avoid because fungal growth in foods is not easy to prevent. Even though heavily contaminated food supplies are not permitted in the market place in developed countries, including US, concern still remains for the possible adverse effects resulting from long-term exposure to low levels of aflatoxins in the food supply. Moreover, liver cancer takes time to develop and the aflatoxins also act an immuno-suppressant so that affected individuals become susceptible to a wide range of diseases. Acute liver damage can occur in humans from ingestion of high amounts of aflatoxin. Aflatoxins have received greater attention than any other mycotoxins because they clearly have a potent carcinogenic effect in laboratory rats and their acute poisonous effects in humans.

In spite of all the precautionary measures taken during harvest, storage and transportation of peanuts and despite all the regulatory measures, both the species exist at large at peanuts in commercial outlets. There are several ongoing research programs here, and in other peanut growing States to minimize loss due to preharvest infection of peanuts by the two species of Aspergilli, however, very little is known about post infection during storage.

It is known (Keith Ingram, chair of the Georgia Envirotron committee) that it does not take many seeds to cause major health problems. Current investigation aims at detecting A. flavus and A. parasitus in commercial peanuts during storage and on shelf at commercial outlets in Georgia. Both conventional and molecular methods will be used for detection. Novel approach will be used to differentiate the toxin and the non-toxin producing strains of the Aspergilli sps. The effect of changes in the environmental conditions such as temperature, moisture and relative humidity on growth of A. flavus and A. parasiticus during storage will be determined. A relation between percentage of infection and aflatoxin production by the two Aspergilli sps will be established. Moreover, the biochemical activities related to aflatoxin ingested peanuts will be studied using cell lines or experimental models. Because aflatoxins, especially aflatoxin B1, are potent carcinogens in some animals, there is interest in the effects of long-term exposure to low levels of these important mycotoxins on humans. The long-term exposure to low levels of this important Aflatoxin from contaminated peanuts, using cell lines or experimental models also forms part of the current investigation.

Objectives of the Research:

1. To detect presence of Aspergillus flavus and A. parasiticus in commercial peanuts at storage and on shelf from commercial outlets in Georgia using conventional methods.

2. To detect the presence of Aspergillus sps by molecular methods and to survey the genetic variation among isolates.

3. To screen peanuts for presence of aflatoxin and determine the effect of environmental factors on the growth of A. flavus and A. parasiticus and production of toxin.

4. To differentiate Aflatoxin producing and non-producing strains of A. flavus and A. parasiticus

5. To develop a novel and rapid technique to detect and differentiate Aspergillus sps. and corresponding aflatoxin contaminants.

6. To quantify the production of aflatoxin at optimum growth of the Aspergillus sps. at varying environmental conditions.

7. To isolate and purify specific aflatoxins from contaminated peanuts.

8. To determine the toxicity of aflatoxins using conventional cell culture techniques.

9. To involve and train students in various molecular, analytical techniques and statistical methods.

Key questions to be answered:

1. What is the percentage of infection by Aspergillus sp. in commercial nuts distributed by commercial outlets in the State of Georgia?

2. Which of the isolates collected in Georgia are potential producers of Aflatoxin? What are the environmental conditions governing the production of the toxin?

3. What are the optimum environmental conditions for the production of toxin?

4. What are the parameters that influence the stability of the pure toxin?

5. What are the biochemical changes in the presence of the pure toxin in experimental models using cell lines?

6. Which detection methods tested against presence of Aspergilli sps and presence of aflatoxin is more sensitive and reliable and can be used for easy and quick detection?

Relevance to Directed Studies at the Department of Biological and Physical Sciences.

A. Students development under Directed Studies

There are libraries of information on peanut research and aflatoxin production in Georgia and outside the state. However, this investigation uniquely looks at training undergraduate at KSU through a specific area of research in peanuts. The broader impact of current research is to expose these students to several analytical and molecular techniques. Ultimately students will be able to conduct independent research. Study of toxin forms a large component of industrial, agricultural and medical biotechnology. Students will gain challenging knowledge in this field of biotechnology and will be able to operate several state-of-the-art equipments using cutting edge technologies.

Miniprojects are given to selected students for the course entitled “Directed Studies”. Several research proposals using detection methods, both conventional and molecular methods to detect Aspergilli sps. in peanuts are being used. Molecular methods such as PCR, RAPD, RFLP and AFLP have been incorporated in the research titles for detection. Microscopy from light to Electron to study the host-pathogen interaction at cellular and sub cellular level forms part of the various techniques. Determination of molecular relatedness among Aspergilli sps using microsatellites is sought to be a reliable technique to study population genetic. Detection of aflatoxin using kits forms a basis and a routine method to test presence of aflatoxin in peanuts in the present study. More effectively students at the completion of their research will be able to compare conventional from molecular methods such as multiplex PCR or RT-PCR with regards to reliability, sensitivity and cost effectiveness. Miniprojects such as protein determination or differentiate one strain from another based on protein type can be challenging specially with regards to novel approach such as SELDI. The analytical phase of current research offers numerous miniprojects for students from detection, isolation, quantification and purification of aflatoxin. A wide range of instruments have been incorporated to achieve several goals of the proposal. They range from TLC, HPLC, GC, Mass Spectrophotometry, and Fluorometry to several other instrumentation techniques which may not have been necessarily mentioned in the methodologies in the proposal.

Above all, the very basic of aseptic techniques while handling microorganisms are taught to students. Supervision also includes teaching of safety methods (ISO). Culturing and sub culturing of Aspergilli sps has become an art which we envisage transferring to our young budding scientists.

SALT project student is currently working on the detecting the Aspergilli sps in peanuts from various locations in Georgia.

B. Marketability of students

Students trained under this project, having learnt combinations of techniques in the field of Molecular biology, Biotechnology and Analytical chemistry and having presented research findings at conferences are highly marketable in the eyes of employers in these fields. The research will have a significant impact on undergraduate students who are either in search of employment or pursue graduate studies. The various techniques mentioned above will equip KSU students to take up a career in the field of Biotechnology, an emerging and expanding field in the State of Georgia.

C. Biotechnology course and Directed Studies at KSU

Students involve in this project will be very comfortable in taking the new Biotechnology course offered by the Department of Biological and Physical Sciences at KSU because of the several molecular and analytical techniques incorporated in the project. Students assistants involved in this project will also be more confident to register for Directed Studies under the supervision of participating Faculties.

Participating Faculties

Dr Premila N Achar - Principal Investigator, Dept of Biological & Physical Sciences, KSU

Dr Thomas McElroy- Co Investigator, Dept of Biological & Physical Sciences, KSU

Dr Msimanga Huggins- Co Investigator, Dept of Chemistry and Biochemistry, KSU

Dr Daniel J. Schoeffner - Field Research Scientist, Ciphergen Biosystems, Inc, Atlanta

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