FT-Raman Spectra of Unsoaked and NaOH-Soaked Wheat Kernels, Bran, and Ferulic Acid

Cereal Chem. 80(2):188–192 The sodium hydroxide (NaOH) test for determining wheat color class depends on the observation that on soaking in NaOH, red wheat turns a darker red and white wheat turns straw yellow. To understand the mechanism of this test, Raman spectra of wheat bran, wheat starch, ferulic acid, and whole kernels of wheat, before and after NaOH soak, were studied. The major observable components in the whole kernel were that of starch, protein, and ferulic acid, perhaps esterified to arabinoxylan and sterols. When kernels are soaked in NaOH, spectral bands due to ferulic acid shift to lower energy and show a slightly reduced intensity that is consistent with deprotonation of the phenolic group and extraction of a portion of the ferulic acid into solution. Other phenolic acids, alkyl resorcinols, and flavonoids observed in the NaOH extracts of wheat by HPLC were not observed in the Raman spectra. Wheat bran accounts for most of the ferulic acid in the whole kernel, as indicated by the increased intensity of the doublet at 1,631 and 1,600 cm in the bran. The intense starch band at 480 cm in whole kernel wheat was nearly absent in the wheat bran. The sodium hydroxide (NaOH) test for determination of wheat color class depends on the observation that on soaking in NaOH, genetically red wheat turns a darker red and genetically white wheat turns straw yellow. This test works for all cultivars of wheat, including red wheat that appears visually white and white wheat that appears visually red (Ram et al 2002). The mechanism of this reaction is not understood. It is possible that NaOH deprotonates the phenolic compounds in wheat bran to cause the color changes. Wheat contains several phenolic acids such as ferulic (4hydroxy-3-methoxycinnamic), isoferulic (3-hydroxy-4-methoxycinnamic), coumaric (4-hydroxycinnamic ), vanillic (4-hydroxy-3methoxybenzoic), syringic (3,5-dimethoxy-4-hydroxybenzoic), caffeic (3,4-dihydroxycinnamic), and sinapinic (3,5-dimethoxy-4-hydroxycinnamic) acids (Rybka et al 1993; Hatcher and Kruger 1997). Ferulic acid, the main phenolic acid in wheat occurs in a concentration of 200 g/g in wheat flour and 2,000 g/g in bran (Pussayanawin et al 1988; Rybka et al 1993). Ferulic acid was determined a cell wall constituent by UV microscopy (Akin 1995), by HPLC analysis of bran extracts (Collins and D’Attilio 1996), and by fluorescence microscopy of wheat (Fulcher and Wong 1980). Wheat flour contains 2–3% pentosans (Geissmann and Neukom 1973; Petit-Benvegnen et al 1998). These are composed of arabinoxylans with a linear backbone of -1,4-linked xyloses. The phenolic acids are generally esterified to arabinosyl residues at 2-O or 3-O branches of the xylan backbone (Izydorczyk and Billiaderis 1995). Phenolic acids esterified to plant sterols also have been reported (Seitz 1989). Arabinoxylan chains are cross-linked in the cell wall through diferulic bridges (Peyron et al 2001). Polymerized ferulic acid bound by ether bonds in lignin are alkali-resistant (Scalbert et al 1985). Other phenolic compounds in wheat bran include apigenin and other flavonoids (Collins and D’Attilio 1996; Feng et al 1988; Feng and McDonald 1989). Miyamoto and Everson (1958) identified catechin and catechin tannin as the precursors of brown pigment and showed a correlation between kernel color. McCallum and Walker (1990) suggested that trace levels of proanthocyanidins in the bran contributed to seed coat color in wheat. Wheat also contains a major group of 5-n-alkylresorcinols (Seitz and Love 1987; Al-Ruqaie and Lorenz 1992) with odd-number side chains of C15-C25, 5-(2-oxoalkyl), and 5-(2-oxoalkenyl) resorcinols (Seitz 1992). NIR spectroscopy has been used to distinguish red from white wheat before NaOH soak (Dowell 1998) and after NaOH soak (Dowell 1997; Ram et al 2002). However, identification of individual components is difficult and many times impossible. Aromatic compounds, particularly the cinnamic acid derivatives, exhibit characteristic and intense Raman spectra. Generally, the intensity of Raman bands for nonpolar or slightly polar groups is higher than for polar groups, and intensity of stretching vibrations is higher than that for deformation vibrations. Raman intensity is also usually higher for symmetric vibrations than for antisymmetric vibrations and enhanced for stretching vibrations of multiple bonds, such as that of C=C (Baranska et al 1987; Grasseli and Bulkin 1991; Lewis and Edwards 2001). Raman spectroscopy has been applied to determine protein and apparent amylose contents of milled rice (Himmelsbach et al 2001), amylose content in maize (Phillips et al 1999a), benzyl and acetyl modification of starches (Phillips et al 1999b, 2001), and for monitoring a bioprocess for ethanol production (Sivakesava et al 2001). Ma and Phillips (2002) have reviewed the applications of FT-Raman spectroscopy to cereal science. Wheat sections have been studied by confocal Raman microspectroscopy, and the spatial distribution of protein and phenolic compounds has been described (Piot et al 2000, 2002). The objective of the research described here was to study the reaction of wheat soaked in NaOH by Raman spectroscopy. MATERIALS AND METHODS Samples. Hard red (R) and hard white (W) wheat samples were available from a previous study (Ram et al 2002). Raman spectra were obtained for whole kernels of eight samples of Heyne (W), seven samples of Tam 107 (R), six samples each of White Eagle (W) and NuPlains (W), five samples each of Larned (R) and Scout 66 (R), four samples each of Arlin (W) and Big Dawg (R), two samples each of Trego (W), 2174 (R), Ike ( R), and Quantum (R), and one sample each of Betty (W), Oro Blanco (W), Rio Blanco (W) , Jagger (R), Custer (R), and Akron (R). Hot and cold watersoaked samples were prepared by soaking kernels in 60°C water for 20 min and water at ambient temperature ( 22°C) for 30 min. Spectra of Betty (W), Rio Blanco (W), Scout66 (R), and Tam107 (R) soaked in hot and cold water were obtained. NaOH-soaked wheat was obtained by soaking whole wheat kernels according to 1 Engineering Research Unit, USDA-ARS, Grain Marketing and Production Research Center, Manhattan, KS 66502. 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, Manhattan, KS 66502. Publication no. C-2003-0213-02R. 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., 2003.