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This work reports a new approach to extract the maximum chemical information from the absorption spectrum of extra virgin olive oils (EVOOs) in the 390-720 nm spectral range, where "oil pigments" dominate the light absorption. Four most important pigments, i.e., two carotenoids (lutein and β-carotene) and two chlorophylls (pheophytin-a and pheophytin-b), are chosen as reference oil pigments, being present in all the reported analytical data regarding pigments of EVOOs. The method allows the quantification of the concentration values of these four pigments directly from the deconvolution of the measured absorption spectrum of EVOOs. Advantages and limits of the method and the reliability of the pigment family quantification are discussed. The main point of this work is the description of a fast and simple method to extract of such information in less than a minute, through the mathematical analysis of the UV-vis spectrum of untreated samples of oil.

The hen egg white contains proteins able to strongly bind, with a definite stoichiometry, small molecules such as biotin and riboflavin, or ions such as Cu2+ or Fe3+. The complexation process modifies the spectral properties of these low-molecular-weight species. On the basis of these changes, it is possible, in principle, to measure the quantity of the binding protein and to evaluate the protein-substrate interactions. Here, we present a method to determine the concentration of both the apo and holo forms of the riboflavin-binding protein (RFBP) present in avian egg white, by measuring the circular dichroism (CD) related to the controlled addition of riboflavin (or vitamin B2) to the egg white. At the same time, front-face fluorescence is used to confirm the concentration of apo-RFBP obtained from CD data. The method is based on data only from spectroscopy, and no process involving either extraction, chromatography, electrophoresis, or mass spectrometry is involved. We study the egg whites from four different avian species, reporting and comparing the concentration of the apo- and holo-RFBP and the molar circular dichroism spectra (Deltaepsilon) of riboflavin in the RFBP binding site. Finally, egg whites from different hen individuals are analyzed, and a surprising variation of the RFBP concentration is found.

Unlabelled: Methods of analysis of vitamin B2 in foods generally consist of the extraction of the sample, followed by enzymatic hydrolysis and quantitative measurement of the analyte, typically through RP-HPLC. The scope of our work here is to present a soft method to measure the free riboflavin content of a nontransparent and nonhomogeneous matrix such as milk, avoiding any extraction and separation of phases that are required in any published method for determination of the free RBF content in foods. We combine the front-face (FF) measurement of the light emission of milk with the ability of the apo-form of the riboflavin-binding protein (RBP) from chicken egg white to quench the riboflavin fluorescence. Thus, we titrate the RBF present in milk by gradually adding a solution of RBP to the milk sample and measuring, upon each addition, the FF residual emission due to uncomplexed RBF. The RBP binding capability has been measured in the same matrix of the analyte. Our results indicate a concentration of free RBF practically co-incident with the certified value for total B2 vitamin content in reference milk CRM 421., Keywords: Front-face fluorescence; riboflavin; apo-riboflavin-binding protein; milk fluorescence.

Fluorescence spectra of undiluted extra virgin olive oil obtained with the traditional setup (right-angle fluorescence) show considerable artifacts and deformations due to self-absorption phenomena, even when the spectra are corrected for inner filter effects. On the other side, front-face fluorescence spectra are much less affected by self-absorption. Front-face fluorescence of native olive oil reveals the presence of different fluorophores and can provide information about their amount. From the intense emission at ca. 315-330 nm, it is possible to detect fluorescent polyphenols and pherols and to evaluate their overall content. Low-intensity emission bands at 350-600 nm are correlated to vitamins and other important molecules. Among them, the fluorescence of the riboflavin fluorophore can be used to evaluate its concentration. The intense emission of chlorophyll derivatives, measured in the 640-800 nm spectral region, can provide information on their concentration.

Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

Front-face emission spectra of powders can be recorded with a commercial spectrofluorometer. By combining the emissions of a scatterer powder and of a wheat flour sample, the scattering contribution to the front-face emission spectra of flour is removed, and the fluorescence of the flour is isolated. The fluorescence depends on the concentration of the fluorophores. By choosing convenient measurement parameters and by measuring the emission spectra of flour samples suitably enriched with riboflavin, the fluorescence of riboflavin could be isolated from that of other substances present in flours and the concentration of vitamin B(2) in native substrates could be determined. This method is particularly apt for the measurement of vitamin B(2) in low riboflavin-containing powders such as wheat flours, which are usually analyzed through complex chemical and microbiological methods. The method is essentially phenomenological, in view of the interpretation difficulties connected to the origin of the fluorescence resulting from the absorption of multiply scattered photons.

Nine spice and aromatic herb samples (i.e., basil, bird pepper, black pepper, cinnamon, nutmeg, oregano, parsley, rosemary, and sage) were gamma-irradiated at a dose of 10 kGy according to commercial practices. The effects of the disinfection treatment on the content of organic radicals and some nutrients (namely, vitamin C and carotenoids) in the samples were investigated by chromatographic and spectroscopic techniques. Irradiation resulted in a general increase of quinone radical content in all of the investigated samples, as revealed by electron paramagnetic resonance spectroscopy. The fate of these radicals after storage for 3 months was also investigated. The cellulose radical was clearly observed in a few samples. Significant losses of total ascorbate were found for black pepper, cinnamon, nutmeg, oregano, and sage, whereas a significant decrease of carotenoids content was observed for cinnamon, oregano, parsley, rosemary, bird pepper, and sage.

Accelerated aging was performed by incubation of wheat seeds at 40 degrees C and 100% relative humidity for 3, 4, 6, 10, and 14 days. The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activity) and gluten (free radical content and flexibility) were evaluated. A decrease of germinability was found during aging, the germination being completely inhibited after 14 days. The lutein content decreased gradually, without going to zero, while that of free radicals increased. A reduction of soluble proteins and a degradation of glutenins and gliadins were observed, associated with a substantial increase of protease activity and a decrease in gluten flexibility. The results were discussed in reference to those previously obtained by natural aging of wheat seeds of the same species and cultivar.

Complementary information on the structure and dynamics of high molecular weight glutenin subunits (HMW-GS) of durum wheat (Triticum durum) was obtained by means of two spectroscopic techniques. Electron paramagnetic resonance was used to investigate the dynamics of the HMW-GS hydrated with two 2-propanol/water mixtures at temperatures between 268 and 308 K by specific spin labeling of their cysteine residues. Spectra were of a composite type, resulting from two populations of spin labels differing in molecular mobility, both undergoing isotropic rotational diffusion. Diffusional coefficients and populations of the fast- and slow-moving spin labels, determined by an accurate spectral line shape analysis, are discussed as a function of temperature and water content in the solvent systems. Far-UV circular dichroism was employed to provide information on the secondary structure of the HMW-GS in three different solvents [aqueous 50% (v/v) 2-propanol, aqueous 0.1% (v/v) trifluoroacetic acid, and trifluoroethanol]. For the first one, the influence of temperature on HMW-GS structure was also investigated.

Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.

Front-surface absorbance spectra of wheat flours in the 250-650 nm region can be obtained by measuring reflectance spectra with a conventional spectrofluorometer suitably set to detect light scattered from powder samples. The spectra recorded on flour samples, obtained from seeds of four bread and five durum wheats, show high-intensity absorption bands due to aromatic amino acids of wheat proteins and low-intensity bands due to chromophores bound to low-molecular-weight compounds. The intensity of these last bands is proportional to the concentration of the corresponding chromophores present in the flour; thus, it can be used to measure the content of the compounds containing the chromophore(s). In particular, a quantitative determination of the carotenoids actually present in the flours is made, obtaining information on the original content of the seeds. This determination is important, as, for example, xanthophylls are well-known antioxidants and free-radical scavengers involved in aging processes of seeds. Reflectance measurements on powder samples are far more economic in terms of time and materials consumption than methods such as extraction and HPLC analysis of extracts and, in addition, give an evaluation of the overall content of carotenoids with absorption bands in the spectral range 450-500 nm. Application of the technique to other food powders with low-intensity absorption bands in the near-UV and vis region is possible.

Free radical oxidative attack is considered a major cause of disruption and deteriorative changes observed in aged seeds. Antioxidant defense mechanisms may remove potentially damaging molecular species, and carotenoids may act as radical scavengers. The content of lutein, the major carotenoid in wheat seeds, was determined in the flours. It showed a rapid decrease during seed aging. In addition, the content of free radicals in glutens made from flours of wheat seeds after long-term storage was studied. The concentration of radicals appeared to be age dependent, because the highest content of radicals was detected between 13 and 15 years of aging over 36 years of storage. Specific spin labeling of the sulfhydryl groups of gluten proteins enabled comparative EPR studies of the rigidity of the protein chains. A progressive stiffening of polymeric gluten with seed storage was found.

Front-surface fluorescence spectra of cereal flours are easily measured with a commercial spectrofluorometer and a simple cell. The emission spectra are intense in the range 290-600 nm in which three emission bands are active. The most intense is due to the aromatic amino acid residues present in the proteins of flours. At least two other fluorophores are active in the above spectral range and compete for light with the chromophores present in the flours. Four absorption bands are revealed in the same spectral region by reflectance spectra, the most intense being due to the amino acidic fluorophores. Thus, the measured emission is the result of absorption, scattering, emission, and reabsorption processes. Information on the microenvironment of some fluorophores can be obtained. The possibility to recognize different species of cereals, even of different cultivars, is shown.