Fluorescence excitation-emission features of aflatoxin and related secondary metabolites and their application for rapid detection of mycotoxins

Authors: HRUSKA, ZUZANA - Mississippi State University; YAO, HAIBO - Mississippi State University; KINCAID, RUSSELL - Mississippi State University; Brown, Robert; Cleveland, Thomas; Bhatnagar, Deepak

Submitted to: Food and Bioprocess Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2014
Publication Date: 4/1/2014
Citation: Hruska, Z., Yao, H., Kincaid, R., Brown, R.L., Cleveland, T.E., Bhatnagar, D. 2014. Fluorescence excitation-emission features of aflatoxin and related secondary metabolites and their application for rapid detection of mycotoxins. Food and Bioprocess Technology. 7:1195-1201.

Interpretive Summary: Aflatoxins are poisons produced by the fungus Aspergillus flavus after it infects agricultural commodities such as corn. Since aflatoxins in food and feed are regulated, enhanced ability to detect and measure fungal growth and aflatoxin contamination of corn could contribute significantly towards the separation of contaminated from healthy grain. A collaboration between ARS-SRRC, Food and Feed Safety Research Unit and Mississippi State University, Stennis Space Center, MS, is exploring the use of hyperspectral imaging non-destructive technology to detect aflatoxins in grain products. The present study compared aflatoxin fluorescence-based identification data obtained with a fluorescence spectrophotometer to image data of aflatoxin acquired with a hyperspectral sensor to evaluate the potential of image-based technology for detecting aflatoxin in grain. Other secondary metabolites of Aspergillus flavus often occurring simultaneously with aflatoxin, such as kojic acid, BGYF and a kojic acid mixture where the fluorescence was induced by peroxidase and hydrogen peroxide were also studied to determine potentially confounding peak overlaps of these substances with aflatoxin. Results showed both systems to be comparable, since all fluorescence peaks were in the blue range thus validating the feasibility of image based technology for non-destructive detection of aflatoxin in corn. Also additional peaks were revealed for aflatoxin in another excitation range not present in the kojic acid mixture, allowing for the separation of the aflatoxin signature from those of other secondary metabolites with potentially confounding overlaps occurring in the blue and blue-green spectral range. Further experiments may lead to this technology being used to rapidly and accurately detect/measure aflatoxin levels in corn without destruction of healthy grain. This could provide a useful tool to both growers and buyers in the corn industry that could enhance protection of food and feed as well as increase profits.

Technical Abstract: The persistent occurrence of aflatoxins in food and feed remains a problem for producers of commodities subject to colonization with toxigenic molds. Aflatoxins are secondary metabolites of fungi of the Aspergillus spp. associated with deleterious health effects. Because current screening methods for these toxins are lengthy, destructive, and costly, there is a continuous search for a more rapid, non-invasive and cost effective technology. The present study utilized a fluorescence excitation-emission matrix of aflatoxin measured with a fluorescence spectrophotometer (a spectrofluorophotometer) and compared it to image data of aflatoxin acquired with a hyperspectral sensor in order to evaluate the potential of image-based technology for detecting aflatoxin in grain. Other secondary metabolites of Aspergillus flavus often occuring simultaneously with aflatoxin are kojic acid and BGYF. EEM was also generated of a kojic acid mixture where the fluorescence was induced by peroxidase and hydrogen peroxide, to simulate the BGYF compound in order to determine confounding peak overlaps of these substances with aflatoxin. The spectrofluorophotometer is an instrument that provides fluorescence excitation-emission relationship information of materials exhibiting natural fluorescence. Generally, the UV excitation light source is a tunable Xenon lamp in the range of 200-400 nm with emission wavelengths between 400 and 700 nm. The hyperspectral instrument used in this study is effective in the 400-900 nm range of the electromagnetic spectrum. If a given target is excited with a specific UV light source, the instrument can be used for fluorescence emission measurement within that range. The excitation-emission matrix of aflatoxin B1 standard produced overlapping peaks at 340-400 nm excitation range centered at 365 and emitting in the blue range at around 450 nm. The spectral signature extracted from the hyperspectral image was also in the blue range, emitting blue fluorescence. Because the results from both systems were comparable, where all fluorescence peaks were in the blue range, the present study validates the feasibility of image based technology for non-destructive detection of aflatoxin in corn. Additional peaks were revealed in the aflatoxin EEM in the 260 nm excitation range that were not present in the kojic acid mixture. This new information allows for the separation of the aflatoxin signature from the confounding overlap of the other secondary metabolites occurring in the blue and blue-green spectral range.