Wheat analysis, HPLC

Figure 8 Statistical analysis of bread wheat RP-HPLC data (A) chromatograms of 12 hard red spring wheats (B) PCA weights for data from (A) and (C) PLS weights for loaf volume. (From Ref. 87.)...

Eor wheat grain, dissolve the residue in an appropriate volume of acetonitrile prior to HPLC analysis. 

HPLC has many applications in the food industry. Two of interest in the bakery sector are the analysis of triglycerides and wheat proteins. 

Radionuelides can be also used to study the accumulation and degradation of organic pollutants. In our experiments we have followed the uptake and degradation of labelled TNT by wetland plants (Nepovim et al., 2005), and showed that about 63% of the localized in the roots of Ph. australis was bound (Fig. 6) and the remainder was acetone-extractable. An HPLC analysis of the acetone extract failed to detect any TNT, showing that all TNT had been chemically transformed. Thus TNT was not adsorbed on the root surface. In similar experiments performed in wheat (Triticum aestivum). Sens et al. (1999) found that 57% of the taken up was bound... 

Figure 2.13 HPLC analysis of aldolase-mediated DCL templated by wheat germ agglutinin (WGA. (a) Blank DCL and (b) DCL composition in the presence of WGA. Reprodnced from Reference 36 with permission of Wiley-VCH Verlag GmbH Co. KGaA. Copyright Wiley-VCH Verlag GmbH Co. KGaA.

Most of the applications of HPLC for protein analysis deal with the storage proteins in cereals (wheat, corn, rice, oat, barley) and beans (pea, soybeans). HPLC has proved useful for cultivar identihcation, protein separation, and characterization to detect adulterations (illegal addition of common wheat flour to durum wheat flour) [107]. Recently Losso et al. [146] have reported a rapid method for rice prolamin separation by perfusion chromatography on a RP POROS RH/2 column (UV detection at 230nm), sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and molecular size determination by MALDl-MS. DuPont et al. [147] used a combination of RP-HPLC and SDS-PAGE to determine the composition of wheat flour proteins previously fractionated by sequential extraction. 

Since the introduction of HPLC in the field of protein analysis, this technique has become very popular because of its versatility. The development of improved or new packing materials has often resulted in a decrease in the analysis time. However, with regard to the analysis of food proteins, the application of HPLC has remained limited mainly to wheat proteins and milk proteins. A quick survey of recent literature confirms this observation, and most publications about the... 

Enhancing the Se levels in crops can be achieved by adding organic amendments (manure of Se-supplemented farmed animals) or inorganic Se to mineral fertilizers [116, 117]. The use of sodium selenate-enriched fertilizers in Finland resulted in increased Se levels in different foods and, consequently, the average serum Se in the population improved over the period 1984 D1988. ICP-MS was used to study the feasibility of wheat enrichment by selenate addition to soil fertilizers [118]. AE-HPLC-ICP-MS was optimized for the separation of selenite, selenate, selenocysteine, and selenomethionine. Total Se determination and speciation analysis were performed in water extracts and in enzymatic digests of wheat samples. It was shown that a major part of the selenate taken up by cereals was converted to selenomethionine. 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Detection methods for T-2 toxin and other Fusarium toxins have been recently reviewed (Krska et al., 2007 Ler et al., 2006). Trichothecene analysis can be done by screening methods such as thin layer chromatography (TLC) and ELISA or analytical methods such as gas chromatography (GC) and high performance hquid chromatography (HPLC). GC instrumentation has been the most frequently used method for experimental work with trichothecenes. Newer methodologies, such as GC-MS and LC-MS, have an excellent lowest level of detection (LOD) of 5 ng/g for T-2 toxin in cereals and food, and wheat flour respectively (Ler et al., 2006). Improved sensitivity for... 

Assuming that the lignin which is insoluble is poorly affected by the hydrolysis process, the lignin quantity (in weight) stays constant from the initial wheat straw to the LCFo i. Cellulose and hemicellulose contents have been decreased under the hydrolysis treatment. Around 40% of the total cellulose and 85% of the hemicellulose have been eliminated during the process and collected mostly in the soluble fraction (see Table 17.1). We can notice that the process yield of hemicellulose sugars recovery is not total. By HPLC analysis, the hemicellulose composition can be given. Hemicellulose is composed with 96% of xylose, 3% of arabinose, and 1% of mannose. 

Fig. 3 Flow field-flow fractionation (flow FFF) analysis of size-exclusion HPLC sublractions from Katepwa wheat flour, a, SE-HPLC subfractions Ci g b, flow FFF profiles of SE-HPLC subfractions ci. ...

Source From A simplified dilute acetic acid based extraction procedure for the preparation of polymeric wheat flourprotein for SE-HPLC and flow FFF analysis, in Cereal Chem. " ... 

Burnouf and Bietz [19] first explored methods for solubilization, stabilization, and RP-HPLC analysis of wheat glutenin. For optimal separations, glutenin was extracted with solutions containing 8M urea or 6M guanidine hydrochloride and 5% 2-mercaptoethanol or 0.1% DTT resulting thiol groups were stabilized by alkylation with 4-vinylpyridine acidified reaction mixtures could then be analyzed directly by RP-HPLC. 

Use of Standard Analytical Methods. To compare samples between laboratories and develop standard methods, agreement about optimal extraction and analysis methods would be desirable. The diverse aims and nonstandard nature of most separations, however, combined with inevitable changes in columns and optimization of procedures, make such a goal elusive. A collaborative study to determine optimal RP-HPLC analytical conditions for wheat... 

Water-autoclave extractions were also much more efficient than neutral EDTA extractions in recovering sorbed/fixed phenolic acids from plant tissues/residues (Blum et al. 1992). For example HPLC analysis of EDTA extracts for wheat stubble collected after a wheat harvest contained no detectable phenolic acid peaks. Water-autoclave extracts of this wheat stubble had 11 distinct peaks. Concentrations for ferulic acid, vanillic acid, p-coumaric acid, and p-hydroxybenzoic acid were 33, 22, 1034, and 47 p,g/g dry weight, respectively (Fig. 3.8). On the other hand water and... 

So what were the concentrations of phenolic acids in the Cecil A soil wheat stubble (Triticum aestivum L. Coker 916 )/soybean (Glycine maxL. Deltapine417 ) systems Subsamples taken from wheat stubble/soybean (no-till), wheat stubble tilled under/soybean (conventional-till), and fallow/soybean soil (conventional-till) cores were extracted by the water-autoclave procedure and analyzed for 7 common phenolic acids (ferulic, caffeic, p-coumaric, p-hydroxybenzoic, sinapic, syringinc, and vanillic) and total phenolic acid (Blum et al. 1991). With minor exception, individual phenolic acids were correlated with each other, with the sum of the 7 phenolic acids identified by HPLC analysis, and total phenolic acid as determined by the Folin Ciocalteu s phenol reagent method. 

Toukairin-Oda reported an isocratic, reversed-phase HPLC method for determination of all six nutritionally active Bg vitamers in food (96). PLP fluorescence was enhanced by precolumn potassium cyanide treatment to convert PLP to the highly fluorescent 4-pyridoxic acid-5 -phosphate. However, potassium cyanide causes oxidation of PL to 4-pyridoxic acid lactone, which shows little fluorescence at the acid pH (pH = 3.5) of the mobile phase used (Table 2). This problem is circumvented by duplicate analysis (1) without prior potassium cyanide treatment to determine all the B vitamers except PLP, and (2) after potassium cyanide treatment to determine PLP as 4-pyridoxic acid-5-phosphate. This method has been applied to fruit juices, wheat flour, cream cheese, eggs, and baker s yeast. 

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