Natural Fluorescence of Red and White Wheat Kernels

Cereal Chem. 81(2):244–248 Red and white wheats must be segregated for marketing purposes because they have different end uses. Identification of wheat color is not straightforward, and currently there is interest in characterizing red and white wheats using spectroscopic methods and chemical tests. The kernels of both red and white wheats exhibit natural fluorescence that can be readily viewed under UV light, although it is not possible to differentiate the fluorescence spectra of red and white wheats by visual inspection only. Fluorescence emission spectra in the wavelength range of 370– 670 nm for 91 wheat samples consisting of 48 red (from 30 cultivars) and 43 white (from 18 cultivars) were analyzed by partial least squares (PLS) and neural networks analyses (NNA). Samples included cultivars that were difficult to classify visually as well as wheat harvested after rainfall. Classification accuracies were ≈85% for calibration and ≈72% for the validation samples by both analyses. A plot of β-coefficient vs. wavelength in PLS analysis indicated that fluorescence of red wheat cultivars was greater than that for white wheat cultivars at 425 (±20) nm wavelength. Fluorescence of white wheat cultivars was greater than that for red cultivars at 587 (±35) nm. Fluorescence emission at ≈450 nm from wheat samples increased in intensity after treatment with NaOH. The increase was greater for red than for white wheat. Wheat harvested after rainfall also exhibited a slight increase in fluorescence. Red and white wheats must be segregated for marketing purposes. Mixtures are usually discounted and some of the wheats are intended for different end uses. Both hard red and hard white wheat cultivars may be used for bread, but some white wheats are also used for Asian noodles. Identification of wheat color is not straightforward, and grain handlers and farmers are interested in characterizing red and white wheat by using rapid spectroscopic or chemical tests (Dowell 1997, 1998; Ram et al 2002a). Intrinsic fluorescence has been suggested for quantification of botanical components in wheat (Jensen et al 1982; Jensen and Martens 1983; Symons and Dexter 1993). Fluorescence microscopic studies indicated a correlation of the autofluorescence with wheat hardness (Irving et al 1989), but these studies did not include samples of hard white wheat. Intense blue fluorescence of the aleurone cell walls of wheat is due to high concentrations of ferulic acid (Fulcher et al 1972; Fulcher and Wong 1980). Other fluorescence and UV absorption studies also indicate the presence of ferulic acid in cell wall fluorescence (Pussayanawin et al 1988; Akin 1995; Collins and D’Attilio 1996). McKeehen et al (1999) identified a potential association between phenolic acid concentrations and Fusarium resistance. Several other compounds in wheat were identified in these studies, but none were described as contributing to autofluorescence. Three emission bands in fluorescence from cereal flours have been reported (Zandomeneghi 1999). The most intense at ≈330 nm (excitation 280 nm) was attributed to amino acids. The band at 430 nm (excitation 330 nm) was ascribed to tocopherol and related compounds, and the band at 540 nm (excitation 445 nm) was assigned to a xanthophyll. These results have not been corroborated. Flavonoids have been reported in wheat germ milling fractions (Barnes et al 1987; Barnes and Tester 1987). The fluorescence of whole wheat kernels had not been examined previously. We were interested in examining differences in the surface emissions from red and white wheat kernels. Some other grains are segregated by fluorescence in breeding programs. For example, white and yellow oats are viewed in UV boxes by oat breeders, and annual and perennial rye grasses are segregated on the basis of fluorescence of the primary root during the early stages of germination. Also, neural network analysis (NNA) has been used recently to classify genetics of barleys based on phenolic finger prints (Gorodkin et al 2001). MATERIALS AND METHODS Wheat samples used in this study are listed in Table I (red wheat cultivars) and Table II (white wheat cultivars). Most of these samples were used in previous studies (Dowell 1997, 1998; Ram et al 2002a); the other wheat cultivars were harvested in 2000-2002 and were obtained from Joe Martin, Kansas Agricultural Experimental Station, Hays, KS. The color classes of wheat samples that were not obviously red or white were determined using the NaOH soak test (Ram et al 2002a). The sample set contained 11 samples that were difficult to color classify. Samples were stored at 4°C to avoid mold and insect infestation. Fluorescence Wheat kernels were viewed under long-wavelength UV light (360–400 nm), referred to as blacklight, in a Spectroline CX-20 UV cabinet. For magnified viewing, a laboratory microscope with up to 50× magnification was used with a blacklight (model B100A, Ultraviolet Products, San Gabriel, CA) excitation. Fluorescence emission spectra were obtained from bulk samples using a FluoroMax-2 spectrofluorometer (Jobin Yvon Spex, Edison, NJ), with 1-nm resolution and 1.0-sec integration time. The instrument had one monochromator for excitation and one for emission. Instrument control and data acquisition were computercontrolled and spectra were saved in Grams/32 (Thermo Galactic, Salem, NH). Fluorescence spectra of whole wheat kernels were obtained using a 10-mm path-length quartz spectrophotometric cuvette that held ≈30 kernels. No special adaptation to increase the signal from solids such as described by Zandomeneghi (1999) was used. Emission spectra (370–670 nm) were obtained with 350 and 300 nm excitation. Broken kernels, straw, and chaff were removed, and only whole kernels were used. Most cultivars were scanned once, but more than one sample was used for a few cultivars, as noted in Tables I and II. PLS Analysis Fluorescence data of red and white wheat were analyzed using partial least squares (PLS) analysis (Martens and Naes 1989). All 1 Engineering Research Unit, USDA-ARS, Grain Marketing and Production Research Center, 1515 College Ave, Manhattan, KS 66502. Names are necessary to report factually on available data: however, the USDA neither guarantees not warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be available. 2 Corresponding author. Phone: 785-776-2761. Fax: 785-537-5534. E-mail: [email protected] 3 Grain Quality and Structure Research Unit, USDA-ARS, Grain Marketing and Production Research Center, 1515 College Ave, Manhattan, KS 66502. Publication no. C-2004-0202-01R. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 2004.