L. A. Forato

Microwave-assisted digestion procedures for biological samples with diluted nitric acid: identification of reaction products.

Microwave-assisted sample preparation using diluted nitric acid solutions is an alternative procedure for digesting organic samples. The efficiency of this procedure depends on the chemical properties of the samples and in this work it was evaluated by the determination of crude protein amount, fat and original carbon. Soybeans grains, bovine blood, bovine muscle and bovine viscera were digested in a cavity-microwave oven using oxidant mixtures in different acid concentrations. The digestion efficiency was evaluated based on the determination of residual carbon content and element recoveries using inductively coupled plasma optical emission spectrometry (ICP OES). In order to determine the main residual organic compounds, the digests were characterized by nuclear magnetic resonance ((1)H NMR). Subsequently, studies concerning separation of nitrobenzoic acid isomers were performed by ion pair reversed phase liquid chromatography using a C18 stationary phase, water:acetonitrile:methanol (75:20:5, v/v/v)+0.05% (v/v) TFA as mobile phase and ultraviolet detection at 254 nm. Sample preparation based on diluted acids proved to be feasible and a recommendable alternative for organic sample digestion, reducing both the reagent volumes and the variability of the residues as a result of the process of decomposition. It was shown that biological matrices containing amino acids, proteins and lipids in their composition produced nitrobenzoic acid isomers and other organic compounds after cleavage of chemical bonds.

Conformation of the Z19 prolamin by FTIR, NMR, and SAXS.

The alpha zein, the maize storage prolamin, is a mixture of several homologous polypeptides that shows two bands in SDS-PAGE, called Z19 and Z22. The conformation studies carried out by several authors in this mixture are conflicting. To elucidate these inconsistencies, we analyzed the conformation of the Z19 fraction, extracted from BR451 maize variety by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and small-angle X-ray scattering. The infrared results show that Z19 has 46% of alpha helix and 22% of beta sheet. The fast N-H to N-D exchange measured by (1)H NMR spectroscopy showed that Z19 is not a compact structure. The scattering measurements indicated an extended structure with 12 by 130 A. With these data, we have modeled the Z19 structure as a hairpin, composed of helical, sheet, turns, and secondary structures, folded back on itself.

High-throughput non-destructive nuclear magnetic resonance method to measure intramuscular fat content in beef

High intake of saturated fat from meats has been associated with cardiovascular disease, cancer, diabetes, and others diseases. In this paper, we are introducing a simple, high-throughput, and non-destructive low-resolution nuclear magnetic resonance method that has the potential to analyze the intramuscular fat content (IMF) in more than 1,000 beef portions per hour. The results can be used in nutritional fact labels, replacing the currently used average value. The method is based on longitudinal (T1) and transverse (T2) relaxation time information obtained by a continuous wave-free precession (CWFP) sequence. CWFP yields a higher correlation coefficient (r = 0.9) than the conventional Carr-Purcell-Meiboom-Gill (CPMG) method (r = −0.25) for IMF in beef and is just as fast and a simpler pulse sequence than CPMG. The method can also be applied to other meat products.

Fast and Simple Nuclear Magnetic Resonance Method To Measure Conjugated Linoleic Acid in Beef

Conjugated linoleic acids (CLAs) are a group of linoleic acid isomers that are naturally found in food products originating from ruminants (meat and dairy). These acids have received special attention in recent years due to their potential human health benefits. Research efforts have been proposed to increase the CLA content in beef to improve public health. However, because there are more than 30 million beef cattle used each year by the American food industry, it will be necessary to ensure their content in a large number of samples. Therefore, it is important to have an inexpensive and rapid analytical method to measure CLA content in food products. Because gas chromatography (GC), a current popular method for measuring CLAs, is slow, this paper describes a nuclear magnetic resonance spectroscopy ((1)H NMR) method that is potentially >10 times faster than the GC method. Analyses show a correlation coefficient of 0.97, indicating the capacity of NMR to quantify the CLA content in beef samples. Furthermore, the method proposed herein is simple and does not require sophisticated sample preparation.

Estudo de métodos de aumento de resolução de espectros de FTIR para análise de estruturas secundárias de proteínas

The FTIR spectroscopy has been used to quantify the secondary structures of proteins, using amide I band (1600 - 1700 cm-1). The resolution enhancement methods have been used to resolve the individual components of this band that correlate to the secondary structure. In this paper we discuss the methods of derivative, Fourier deconvolution and fitting with simulated spectra. The results shows that they have serious problems and can be used only as a qualitative or semiquatitative method.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Propriedades mecânicas e molhabilidade de filmes de zeínas extraídas de glúten de milho

Zeins comprise a group of maize storage proteins (prolamines) that can be extracted via solubization in alcoholic media. Such proteins are highly hydrophobic with elevated degree of polymerization. In the present study, zein type a (molecular mass between 19 and 22 kDa) was extracted from corn gluten meal and characterized concerning the main spectroscopy absorption bands in the infrared region. Cast films were processed by blending zein and oleic acid (OA) as plasticizer. Films with no plasticizer were extremely brittle, and thus impossible to handle or used for mechanical analysis. The addition of 1 or 2% wt OA resulted in significant increase in the elongation length, according to DMA analysis. The presence of the plasticizer led to microscopic surface irregularities and to a reduction in the original hydrophobic characteristics.

Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS.

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

Quantification of protein secondary structure by 13 C solid-state NMR

High-resolution 13C solid-state NMR stands out as one of the most promising techniques to solve the structure of insoluble proteins featuring biological and technological importance. The simplest nuclear magnetic resonance (NMR) spectroscopy method to quantify the secondary structure of proteins uses the areas of carbonyl and alpha carbon peaks. The quantification obtained by fitting procedures depends on the assignment of the peaks to the structure, type of line shape, number of peaks to be used, and other parameters that are set by the operator. In this paper, we demonstrate that the analysis of 13C NMR spectra by a pattern recognition method—based on the singular value decomposition (SVD) regression, which does not depend on the operator—shows higher correlation coefficients for α-helix and β-sheet (0.96 and 0.91, respectively) than Fourier transform infrared spectroscopy (FTIR) method. Therefore, the use of 13C solid-state NMR spectra and SVD is a simple and reliable method for quantifying the secondary structures of insoluble proteins in solid-state.

Microstructure and thermal and functional properties of biodegradable films produced using zein

Research is being conducted in an attempt to produce biodegradable packaging to replace plastic products, thereby reducing solid waste disposal. In this work, zein films were produced from vegetable oils (macadamia, olive and buriti) and from pure oleic acid. The surface of zein-based films made using oleic acid has a good lipid distribution. The high content of oleic acid produced a film with the greatest elongation at break (8.08 ± 2.71%) due to the greater homogeneity of the protein matrix. The different oils did not affect the glass transition temperature (Tg). Tg curves of films with fatty acids showed a reduction in mass at between 50 and 120 °C due to water evaporation. At 120 °C the weight loss was 3-5% and above this temperature further weight loss was observed with the highest loss being seen in the film made using pure oleic acid. In conclusion, although biodegradable films were produced using the four different oils, the film made from pure oleic acid has the best characteristics.