Application of Fourier Transform Infrared (FTIR) Spectroscopy for Rapid Detection of Fumonisin B2 in Raisins

Introduction
Spergillus niger is a well-known black aspergilli that causes rot in a variety of fruits and vegetables, including grapes, corn, and onions (1–3). A. niger is also responsible for the production of mycotoxins in plantderived foods and beverages. Because A. niger is a commonly found species in vineyards (4), it could be a major postharvest fumonisin producer in grapes. In fact, fumonisin B2 (FB2) and FB4 have been detected in A. niger-infected grapes, raisins, grape juice, and wine (5–7). A few strains of this mold are also known to be ochratoxin A (OTA) producers (8); however, no OTA was detected in grapes and raisins inoculated with A. niger (6). Before A. niger was discovered to be a fumonisin producer, only Fusarium spp. were known as the major fumonisinproducing molds. Fusarium verticillioides and F. proliferatum are considered to be the most important global sources of fumonisin contamination in food and feed (9, 10). Fumonisin A, B, C, and P series are sphingosine-analog mycotoxins among the 28 analogs of fumonisins (11, 12). Of the fumonisins, the B-series analogs are the most abundant and most toxic (13). Fumonisins are carcinogenic mycotoxins, inhibit ceramide synthase, and cause accumulation of bioactive intermediates of sphingolipid metabolism (14). Fumonisins have been associated with an increased incidence of human esophageal cancer and increased neural tube defects in some parts of the world (15, 16). Among the various methods used for the determination of mycotoxins in foods and feeds, spectroscopic methods are useful. IR spectroscopy is based on the overtones and vibrations of the atoms of molecules in a sample subjected to IR radiation (17).

Mid-IR (MIR; 4000–400 cm−1 ) and near-IR (NIR; 13 333–4000 cm−1 ) spectroscopies are widely used to determine macro and micro molecules in agricultural products. Exhibiting absorption at characteristic wavelengths in the IR spectrum, various functional groups give a detectable chemical fingerprint with which unknown substances can be identified. Recent FTIR instrumentation, combined with multivariate data analysis methods, enables rapid screening and characterization of minor food components down to parts per billion (micrograms per kilogram) levels (18).

Experimental
Materials and Reagents

In this study, a pure mold isolate was produced in our laboratory from raisins and was identified as “A. niger EG16” (accession No. HQ014697). DNA was sequenced by polymerase chain reaction and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal RNA (rRNA). The isolate was identified by comparing partial 18S rRNA and 28S rRNA sequences with known ribosomal sequences of A. niger using the National Center for Biotechnology Information Basic Local Alignment Search Tool. A. niger EG16 was used to inoculate sultana raisin (golden yellow, medium sized, seedless) extract agar, black raisin (large, with seeds) extract agar, and malt extract agar (MEA; Merck, Darmstadt, Germany). The pure isolate was maintained on slant MEA at −18°C for future uses.

Preparation of Raisin Extract Agar and MEA

(a) Preparation of raisin extract agar (either sultana or black).—1 L distilled water was added to 150 g raisins in a blender jar and blended at high speed for 2 min. The mixture was mixed and boiled for 45 min. After filtering, 20 g agar was added and the resulting mixture was autoclaved at 115°C for 30 min. (b) Preparation of MEA.—30 g malt extract, 3 g peptone from soymeal, and 15 g agar in 1 L distilled water were homogenized and then autoclaved at 121°C for 15 min. (c) Preparation of agar plate.—15 mL sultana raisin extract agar, black raisin extract agar, or MEA was poured into respective sets of 100 × 15 mm Petri dishes.

Preparation of Spore Suspension and Inoculation

A. niger was cultivated in test tubes containing 5 mL MEA for 7 days at 25°C. Sterile Tween 80 was added, and spores were harvested using sterile 0.1% peptone water. Spores were counted using a Thoma cell counting chamber (Marienfeld, Germany) and a suspension containing 106 spores/mL was prepared. Raisin extract agar plates and the MEA plate were inoculated at a single point with 2 μL aliquots of the spore suspension, at a concentration of 106 spores/mL. The plates were then incubated for 9 days in darkness at 25°C, for fumonisin analysis. Each experiment was performed in duplicate.

Equipment and Sample Preparation for FTIR
FTIR spectra of samples were recorded at wave numbers ranging from 1800 to 800 cm−1 , with a resolution of 4 cm−1 . Spectra were obtained at intervals of approximately 2 cm−1 and a sample and background scan time of 24 scans/min by an ALPHA-T spectrometer (Bruker Corp., Ettlingen, Germany). The internal reflectance system of this instrument is equipped with a platinum optical sampling probe that incorporates the ATR technique. Test samples were analyzed by FTIR 3–9 days after incubation. Separate plates were used for each respective day. The plate was turned over, and the agar plug (diameter, 6 mm; weight, 0.2 g) was cut out below the center of the colony from a layer that included only the agar (no mold spores). Special care was given to take a plug without any fungal cellular material in the test samples. The agar plug was placed directly on the ATR cap and covered the crystal surface completely.

Conclusions

The production and the increase in the amount of fumonisin can be monitored by FTIR, with the increase in the absorbance of characteristic peaks corresponding to functional groups. The signals obtained from samples may be complex because each functional group in a molecule forms a peak in the spectrum. However, samples cleaned of fungal cellular materials, as were used in this study, produce fewer relevant vibrational modes, facilitating band assignment and identification of the functional groups of the mycotoxin of interest. The formation of new ester bonds in the sample showed an increase in the absorbance of the characteristic bands at 1733–1736 cm−1 and 1708 cm−1 in raisins contaminated with A. niger, which contributed to the increase in the concentration of ester bonds. Amine production is one of the most important indicators of fumonisin accumulation in contaminated products. Esterification was also important to follow fumonisin formation. Thus, FTIR spectroscopic technique has potential for determining fumonisin and other mycotoxins in agricultural commodities. Although the cost of large-scale sampling of grains and analysis for mycotoxins may overshadow health concerns, the noninvasive or nondestructive nature of FTIR offers a partial solution to this problem.