Detection and localization of oxidized proteins in muscle cells by fluorescence microscopy

In meat, no detailed studies on the intracellular distribution of oxidized proteins during oxidative stress have been performed, to our knowledge. Therefore, we used fluorescence microscopy to detect and locate protein carbonyls, oxidation products of basic amino acids, generated in bovine M. Rectus abdominis during either exposition to a chemical free radical generating system, or refrigerated storage, or cooking. The technique consisted of an immunohistochemical detection of carbonyls by reaction with the specific probe DNPH (2,4-dinitrophenylhydrazine) followed by the sequential addition of a first antibody against DNPH-carbonylated proteins and a CY3-labeled secondary antibody. The fluorescence of the CY3 probe increased regularly with level of free radical generating system and storage time. Moreover, an important heterogeneity of carbonyl distribution was observed, with a higher oxidation level at the periphery than inside the muscle cells. Cooking induced fluorescence increase only at the periphery of cells. Specific coloration of collagen by Sirius red showed that collagen was not involved in fluorescence. We can deduce that accumulation of oxidized proteins observed in the cell periphery was linked to membrane protein oxidation and not to connective tissue oxidation. Biochemical assays were performed in parallel on membrane and myofibrillar proteins to provide complementary quantitative data on level of oxidized proteins.     

Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis

The combination of (1)H NMR fingerprinting of lipids from gilthead sea bream (Sparus aurata) with nonsupervised and supervised multivariate analysis was applied to differentiate wild and farmed fish and to classify farmed specimen according to their areas of production belonging to the Mediterranean basin. Principal component analysis (PCA) applied on processed (1)H NMR profiles made a clear distinction between wild and farmed samples. Linear discriminant analysis (LDA) allowed classification of samples according to the geographic origin, as well as for the wild and farmed status using both PCA scores and NMR data as variables. Variable selection for LDA was achieved with forward selection (stepwise) with a predefined 5% error level. The methods allowed the classification of 100% of the samples according to their wild and farmed status and 85-97% to geographic origin. Probabilistic neural network (PNN) analyses provided complementary means for the successful discrimination among classes investigated.     

Inheritance of Aluminum Tolerance and its Effects on Grain Yield and Grain Quality in Oats (Avena Sativa L.)

Summary Aluminum toxicity due to the cation Al⁺³ is a major factor limiting yields in acid soils. Wide genetic variability to aluminum tolerance is found in oat genotypes. The objectives of this study were to determine the number of genes controlling aluminum tolerance in oats and to verify if any detrimental effects were present of the aluminum tolerance genes on grain yield and grain quality in Al⁺³free soils. Aluminum tolerance was estimated as the average regrowth of the main root after exposure to toxic levels of Al⁺³ in a hydroponic solution under controlled conditions. The number of genes controlling that trait was estimated from the distribution of the average root regrowth frequencies in a population of 333 recombinant inbred lines (RIL's) in generations F_5:6 and F_5:7. The effects on grain yield and grain quality were assessed in a subpopulation of 162 RIL's chosen based on their aluminum tolerance response. Aluminum tolerance in the evaluated population was controlled by one dominant major gene with the tolerant genotypes carying Al _a Al _a and the sensitive ones al _a al _a alleles. No detrimental effects of the Al _a allele on grain yield or grain quality were detected.Part of the Master of Science dissertation of the first author     

Carbon-14 biolabeling of (+)-catechin and proanthocyanidin oligomers in willow tree cuttings.

Proanthocyanidin polymers, oligomers, and the structurally related monomer (+)-catechin were labeled by incorporation of radioactive precursors in shoots of willow tree (Salix caprea L.). [1-(14)C]-Acetate and [U-(14)C]-phenylalanine precursors were fed through the cut stems or petioles of leaves. Optimization of several parameters such as the nature and origin of the plant material, leaf maturity, nature, and quantity of radioactive precursor applied and the duration of metabolism led to incorporation yields of 3.2% and to specific activities of 500 microCi/g. Detailed characterization of the products (polymerization degree, procyanidin/prodelphinidin ratio, specific activities) and purification by chromatography are reported. Some sugars bound to radiolabeled proanthocyanidin polymers were removed by enzymic treatment with a mixture of glycosidases. A radioactive purity close to 100% and specific activities suitable for bioavailability studies were obtained.     

1-methylcyclopropene increases storability and shelf life in climacteric and nonclimacteric plums

The effect of 1-methylcyclopropene (1-MCP) at three different doses (0.25, 0.50, and 0.75 microL L(-1)) on the ripening processes of a climacteric, cv. Santa Rosa, and a suppressed climacteric type, cv. Golden Japan, plum was studied. For both cultivars, positive effects were observed in terms of inhibition of ethylene production and delays of the physical, chemical, and biochemical changes associated with ripening. 1-MCP-treated plums were firmer with lower weight loss, reduced degrees Brix/titratable acidity ratios, and lower color changes during cold storage and subsequent shelf life at 20 degrees C than controls. For most factors, the effectiveness of 1-MCP was dose-dependent in Santa Rosa but dose-independent for Golden Japan.     

Antimicrobial and physicochemical properties of chitosan-HPMC-based films

To prepare composite films from biopolymers with anti-listerial activity and moisture barrier properties, the antimicrobial efficiency of chitosan-hydroxy propyl methyl cellulose (HPMC) films, chitosan-HPMC films associated with lipid, and chitosan-HPMC films chemically modified by cross-linking were evaluated. In addition, the physicochemical properties of composite films were evaluated to determine their potential for food applications. The incorporation of stearic acid into the composite chitosan-HPMC film formulation decreased water sensitivity such as initial solubility in water and water drop angle. Thus, cross-linking of composite chitosan-HPMC, using citric acid as the cross-linking agent, led to a 40% reduction in solubility in water. The water vapor transfer rate of HPMC film, approximately 270 g x m(-2) x day(-1) x atm(-1), was improved by incorporating chitosan and was further reduced 40% by the addition of stearic acid and/or cross-linking. Anti-listerial activity of films was determined on solid medium by a numeration technique. Chitosan-HPMC-based films, with and without stearic acid, inhibited the growth of Listeria monocytogenes completely. On the other hand, a loss of antimicrobial activity after chemical cross-linking modification was observed. FTIR and 13C NMR analyses were then conducted in order to study a potential chemical modification of biopolymers such as a chemical reaction with the amino group of chitosan. To complete the study, the mechanical properties of composite films were determined from tensile strength assays.     

Comparison of mass spectrometry-based electronic nose and solid phase microextraction gas chromatography-mass spectrometry technique to assess infant formula oxidation

Two headspace techniques based on mass spectrometry detection (MS), electronic nose, and solid phase microextraction coupled to gas chromatography-mass spectrometry (SPME-GC/MS) were evaluated for their ability to differentiate various infant formula powders based on changes of their volatiles upon storage. The electronic nose gave unresolved MS fingerprints of the samples gas phases that were further submitted to principal component analysis (PCA). Such direct MS recording combined to multivariate treatment enabled a rapid differentiation of the infant formulas over a 4 week storage test. Although MS-based electronic nose advantages are its easy-to-use aspect and its meaningful data interpretation obtained with a high throughput (100 samples per 24 h), its greatest disadvantage is that the present compounds could not be identified and quantified. For these reasons, a SPME-GC/MS measurement was also investigated. This technique allowed the identification of saturated aldehydes as the main volatiles present in the headspace of infant milk powders. An isotope dilution assay was further developed to quantitate hexanal as a potential indicator of infant milk powder oxidation. Thus, hexanal content was found to vary from roughly 500 and 3500 microg/kg for relatively non-oxidized and oxidized infant formulas, respectively.     

Phenolic composition of champagnes from Chardonnay and Pinot Noir vintages

Nineteen phenolic compounds including hydroxybenzoic acids, hydroxycinnamic acids, flavonoids, phenolic alcohols, and phenolic aldehydes have been identified and quantified in two monovarietal champagnes, Chardonnay and Pinot Noir, by using a reverse-phase high-performance liquid chromatography (HPLC) system coupled with diode array detection. The identification of four hydroxycinnamic tartaric esters (caftaric, coutaric, fertaric, and 2-S-glutathionylcaftaric acids), two flavanonols (astilbin and engeletin), and some other compounds was confirmed by HPLC coupled with mass spectrometry. Caftaric acid and tyrosol were the major phenols. Hydroxybenzoic acids and flavonoids were present at low concentrations. The phenolic compositions of 2000 and 2001 Chardonnay and Pinot Noir vary quantitatively according to the year and the variety, but the chemical natures of the molecules are the same. The total phenolic content determined by colorimetric measurement ranges from 176 to 195 mg/L of gallic acid equivalent and is similar to that described in white wines.     

Direct analysis of pesticide residues in olive oil by on-line reversed phase liquid chromatography-gas chromatography using an automated through oven transfer adsorption desorption (TOTAD) interface

A fully automated on-line reversed phase liquid chromatography-gas chromatography system is described. The system uses a prototype of the automated through oven transfer adsorption desorption interface. The system is demonstrated by presenting a new rapid method for the determination of pesticide residue in olive oil, which is injected directly with no sample pretreatment step other than filtration. Methanol:water is used as the eluent in the LC preseparation step, while the LC fraction containing the pesticide is automatically transferred to the gas chromatograph. Detection limits of pesticides varied from 0.18 to 0.44 mg/L when a flame ionization detector was used. As an example, relative standard deviation and linear calibration are presented for terbutryne.     

Effects of pig slurry application on soils and soil humic acids

The effect of three annually consecutive additions of pig slurry at two rates (90 and 150 m3 x ha(-1) x year(-1) on soils and soil humic acids (HAs) was investigated in a field experiment under semiarid conditions. Soils and pig slurries were analyzed by standard methods. The HAs were isolated from soils and pig slurry by a conventional procedure based on alkaline extraction, acidic precipitation to pH 1, purification by repeated alkaline dissolutions and acidic precipitations, water washing, dialysis, and final freeze-drying. The HAs obtained were analyzed for elemental (C, H, N, S, and O) and acidic functional group (carboxylic and phenolic) composition, and by UV-vis, FT-IR, fluorescence, and ESR spectroscopies. With respect to the control soil, the pig slurry amended soils had greater pH and electrical conductivity, slightly larger total N content, and smaller values of C/N ratio. A decrease of total organic C was observed only in soils amended for 2 and 3 years at the higher slurry rate. With respect to control soil HA, pig slurry HA was characterized by larger contents of S- and N-containing groups, smaller acidic functional group and organic free radical contents, a prevalent aliphatic character, extended molecular heterogeneity, and smaller aromatic polycondensation and humification degrees. Amendment with pig slurry HA determines a number of modifications in soil HAs, including increase of C, S, and COOH contents, C/N ratios, and aliphaticity and decrease of extraction yields and N, O, phenolic OH, and organic free radical contents. These effects are generally more evident after the first year of slurry application and tend to disappear with increasing number of treatments. Most probably, over the years the slightly humified slurry HA is mineralized through extended microbial oxidation, whereas only the most recalcitrant components, such as S-containing, phenolic, and aliphatic structures, are partially accumulated by incorporation into soil HA.