Detection and Removal of Single Mycotoxin Contaminated Maize Grains Following Harvest
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Microorganisms, Mycotoxins, and Other Biological Contaminants KPS2-1 Detection and removal of single mycotoxin contaminated maize grains following harvest D.T. Wicklow1,*, T.C. Pearson2 Abstract corn hybrids. Protective endophytes, including mycoparasites that live asymptomatically in Grains highly contaminated by aflatoxin and maize, are not readily distinguished from fumonisin are unevenly distributed in a grain lot uninfected grains and represent confounding and may be concentrated in a very small variables in maize variety trials for fungus- percentage of the product. Near-infrared (NIR) mycotoxin resistance. reflectance spectra (500-1,700 nm) were analyzed to select the pair of absorbance bands (filters) Key words: aflatoxin, classification, corn, giving the lowest classification error rate for endophyte, endosperm, fumonisin, kernel-rot, removing whole yellow maize grains contaminated maize pathogens, near-infrared, neural network, with aflatoxin (750 and 1,200 nm) or white maize sorting. grains contaminated with fumonisin (500 and 1200 nm) in a single pass through a commercial high speed sorter (@ 7,000 kg/hr). Our research Introduction also seeks to classify individual grains infected with different fungal species and to distinguish Fungi can reduce yield, quality, and nutritional resistance and susceptibility reactions among value of the grain, while also contaminating it corn varieties. Neural networks are being trained with fungal-derived chemicals, some of which to classify grains by fungal species using are recognized as mycotoxins because of their principle components of the full reflectance deleterious biological effects in animals and spectra. Spectra of single maize grains can be humans (Richard and Payne, 2003). Aflatoxins measured automatically and grains with multiple produced by Aspergillus flavus Link and symptoms and mycotoxins can be sorted into fumonisins produced by Fusarium verticillioides different fungal species categories at rates of (Sacc.) Nirenb. are prominent among the about 1 per second using commercial mycotoxins associated with economic losses to instruments. Our initial work has shown that maize (Zea mays L.) growers, grain handlers, classification accuracies for severely discolored livestock and poultry producers, and food and grains infected with Aspergillus flavus, feed processors. No commercial corn hybrid is Stenocarpella maydis, Fusarium graminearum, able to escape mycotoxin contamination when Fusarium verticillioides, and Trichoderma viride grown in environments conducive to outbreaks averaged 92.1 % and 94.8 % for two commercial of aflatoxin or fumonisin. While conventional 1 USDA-ARS, National Center for Agricultural Utilization Research. 815 N. University Street. Peoria, Illinois, 61604. U.S.A. 2 USDA-ARS, Grain Marketing Research and Production Research Center. 1515 College Avenue. Manhattan, Kansas, 66502. U.S.A. * Corresponding author: fax 309-681-6686; e-mail: [email protected] 109 9th International Working Conference on Stored Product Protection breeding has produced commercial maize hybrids contaminated kernels in a single pass through a with substantial resistance to Fusarium commercial optical sorter. High speed sorting graminearum Schwabe, which produces the equipment can process corn at rates of mycotoxins deoxynivalenol and zearalenone, approximately 7,000 kg/hr. Most commercial efforts to produce hybrids with adequate sorting machines are able to only measure one resistance to A. flavus and F. verticillioides have spectral band of light while some machines can proven more difficult, and effective practical measure two bands. Only two-way sorts (i.e. control practices are lacking. As ‘gatekeepers’ “accept” or “reject”) can be performed at high to food safety, the food and feed industry must speed. In the past, red optical filters were used continue to rely on convenient, accurate and to separate mold-contaminated products using sensitive methods for detection of the major mono-chromatic sorters (Dickens and Whitaker, mycotoxins in grain. Seeds highly contaminated by 1975). At present, bi-chromatic color sorters have aflatoxin and fumonisin are unevenly distributed near-infrared detecting capability in addition to in a seed lot and may be concentrated in a very visible light capability, which extend their small percentage of the product (Whitaker and usefulness for detecting mold-infected seeds Dickens, 1983; Desjardins et al., 1998; Whittaker based on both color and bio-chemical et al., 1998; Pearson et al., 2001; Whittaker et composition. Near-infrared transmittance (NIRT) al., 2001; Wicklow, 1994; Wicklow, 1999). and near-infrared reflectance (NIRR) Therefore, removing a small percentage of spectroscopy have been used to evaluate internal contaminated kernels, instead of discarding the quality on many whole nuts and grains. We have entire lot is a reasonable approach for reducing applied NIRR and NIT to optimizing the filter aflatoxin or fumonisin contamination to satisfy selection for bi-chromatic sorters and have shown statutory levels. Corn kernels infested with fungi that only a few absorbance bands in the visible are more friable and may have reduced densities and near infrared spectrum can detect whole (Shotwell et al., 1974). However, standard post yellow corn kernels highly contaminated in the harvest cleaning operations (aspiration, gravity field with aflatoxin (Pearson et al., 2001) and table separation, grain scouring, and wet fumonisin (Dowell et al., 2002). cleaning) have not been shown to be entirely For high speed sorting operations, whole effective for reducing aflatoxin or fumonisin spectra cannot be acquired at throughput rates levels in commercially harvested corn (Brekke that are economically feasible. Near-infrared et al., 1975; Pearson et al., 2004). Regional (NIR) reflectance spectra (500 - 1,700 nm) are aflatoxin outbreaks are commonly accompanied analyzed to select the optimal pair of optical by outbreaks in fumonisin (Mubatanhema et al., filters that can be used to detect and remove 2002) and therefore, aflatoxin and fumonisin can mycotoxin contaminated white or yellow maize be present at unacceptable levels in the same grains through using high speed, high volume grain samples at harvest (Chamberlain et al., optical grain sorters. Sorter performance is 1993, Chu and Li, 1994, Yoshizawa et al., 1996, verified with naturally fungus-infested grain Shetty and Bhat, 1997, Ali et al., 1998, Medina- samples from different hybrids, years and Martinez and Martinez, 2000, Ono et al., 2001). locations. The first phase involves inoculating corn ears in the field with either A. flavus or F. verticillioides, recording reflectance spectra of Detection and sorting grain for food individual kernels, then chemically measuring processors and industrial aflatoxin or fumonisin in each kernel so that the applications most discriminating pair of absorbance bands could be chosen to separate between mycotoxin- Our research seeks to simultaneously contaminated and uncontaminated kernels eliminate both aflatoxin- and fumonisin- (Dowell et al., 2002; Pearson et al., 2004). 110 Microorganisms, Mycotoxins, and Other Biological Contaminants Whole-kernel reflectance spectra from 500 to source (Figure 1). Spectra are first collected from 1,700 nm are measured using a diode-array near- all kernels oriented at the germ-down position infrared spectrometer (DA7000, Perten (germ facing the optical fiber bundle), then a Instruments, Springfield, IL). Kernels are second set for kernels oriented germ-up. All manually placed on a bifurcated interactance spectra are stored on a hard disk for subsequent probe attached to the spectrometer and light analysis. Figure 1. Bifurcated interactance probe attached to the spectrometer and light source to collect reflectance spectra of individual kernels. The viewing area was 17 mm in diameter and 10 mm above the termination of the illumination and reflectance fibers. The next step is to select the optimal filters for independently, then linked back to the spectra from discriminating contaminated or fungal infested the opposite side of the kernel using binary logic. In kernels from non-contaminated or un-infested other words, a kernel can be classified as kernels. The procedure we developed tests all contaminated if the spectra from only one side of combinations of two spectral bands within the NIRR the kernel indicates contamination or the decision spectra collected from each kernel. The spectra rule can be set to classify kernels as contaminated from both sides of the kernel is taken into account only if the spectra from both sides of the kernel 111 9th International Working Conference on Stored Product Protection indicate contamination. All sorting machines inspect fumonisin below 10 ppm have been found to be at least two sides of kernels so this logic needs to more similar to uncontaminated kernels (Pearson be taken into account when selecting filters. The et al., 2001; Dowell et al., 2002). Further classification methods include both discriminant research, using additional sources of aflatoxin analysis and nearest neighbor schemes. Usually and fumonisin contaminated corn and guided by these two methods yield similar results, however, full spectrum neural network classification, is when the distribution of the data is abnormal, needed to improve sorting of corn with low levels the nearest neighbor method tends to work better. of aflatoxin and fumonisin (Pearson et al., 2004). Single-kernel mycotoxin levels are Near infrared