Analytical and sensorial characterization of the aroma of wines produced with sour rotten grapes using GC-O and GC-MS: identification of key aroma compounds

In the present work, the aroma profiles of wines elaborated from sound and sour rot-infected grapes as raw material have been studied by sensory analysis, gas chromatography-olfactometry (GC-O), and gas chromatography-mass spectrometry (GC-MS), with the aim of determining the odor volatiles most likely associated with this disease. The effect of sour rot was tested in monovarietal wines produced with the Portuguese red grape variety Trincadeira and in blends of Cabernet Sauvignon and sour rotten Trincadeira grapes. Wines produced from damaged berries exhibited clear honey-like notes not evoked by healthy samples. Ethyl phenylacetate (EPhA) and phenylacetic acid (PAA), both exhibiting sweet honey-like aromas, emerged as key aroma compounds of sour rotten wines. Their levels were 1 order of magnitude above those found in controls and reached 304 and 1668 μg L(-1) of EPhA and PAA, respectively, well above the corresponding odor thresholds. Levels of γ-nonalactone also increased by a factor 3 in sour rot samples. Results also suggest that sour rot exerts a great effect on the secondary metabolism of yeast, decreasing the levels of volatiles related to fatty acids and amino acid synthesis. The highest levels of γ-decalactone of up to 405 μg L(-1) were also found in all of the samples, suggesting that this could be a relevant aroma compound in Trincadeira wine aroma.     

Using BCG,MPT-51 and Ag85 as antigens in an indirect ELISA for the diagnosis of bovine tuberculosis

This study evaluated the Mycobacterium tuberculosis protein antigen MPT-51, the trimeric antigen 85 (Ag85) complex, and Bacillus Calmette-Guérin (BCG) in an indirect ELISA to diagnose bovine tuberculosis (TB) from serum samples. Serum was collected from 208 intra-dermal tuberculin test (ITT)-positive and 54 ITT-negative animals from a region where bovine TB is endemic. Using the Ag85 and BCG antigens, the indirect ELISA was able to discriminate ITT-positive from ITT-negative animals. This level of discrimination was not achieved when using the MPT-51 antigen. The highest sensitivity (Se) and specificity (Sp) of the test was found when BCG was used (Se, 82%; Sp, 91%). Further work in different epidemiological settings and with larger numbers of animals will be required to validate these findings.     

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.     

Rapid liquid chromatography tandem mass spectrometry assay to quantify plasma (-)-epicatechin metabolites after ingestion of a standard portion of cocoa beverage in humans

A rapid liquid chromatography electrospray ionization tandem mass spectrometry with negative ion detection method was developed and validated to determine cocoa flavonoid metabolites in human plasma and urine after the intake of a standard portion of a cocoa beverage. A chromatographic run time of only 9 min provided clear separation of all metabolites and internal standards. Samples were analyzed in a product-ion scan of m/z 289, 369, and 465 to identify the metabolites and in multiple reaction monitoring acquisition mode to quantify (-)-epicatechin ((-)-Ec) (289/ 245), (-)-epicatechin-glucuronide ((-)-EcG) (465/289), and (-)-epicatechin-sulfate ((-)-EcS) (369/289). One (-)-Ec-G and three (-)-Ec-S were identified and confirmed in urine as the major metabolites, and one (-)-Ec-G was the only metabolite present in plasma volunteers (n = 5) at a mean concentration of 625.7 +/- 198.3 nmol/L at 2 h after consumption of a cocoa beverage containing 54.4 mg of (-)-Ec.     

Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.     

Discrimination of recombinant and pituitary-derived bovine and porcine growth hormones by peptide mass mapping

Somatotropins, which are used in cattle for growth and lactating performances, are difficult to reliably detect because no direct method exists. Reversed-phase high-performance liquid chromatography (RP-HLC) coupled to electrospray ionization quadrupole mass spectrometry (ESI/MS) has been developed to separate and characterize the N-terminal peptides resulting from tryptic cleavage of natural and recombinant growth hormones from different species (bovine, porcine, and human) and suppliers. Conditions for tryptic digestion were optimized. This technique was found to be optimal to cleave efficiently the N-terminal peptide of the proteins without releasing too much noise from the matrix. Characterization of the peptides through ESI(+)-MS allowed natural and recombinant growth hormones from bovine and porcine species with N-terminal amino acid sequences differing from one amino acid residue to be discriminated. However, the studied human growth hormones had similar primary sequences that did not permit any discrimination between recombinant and natural forms, thus confirming the known identity of these hormones. Protein digestions with pepsin and chymotrypsin were also compared but were not conclusive due to the too small N-terminal peptides released after proteolysis.     

Comparative study of methods for DNA preparation from olive oil samples to identify cultivar SSR alleles in commercial oil samples: possible forensic applications

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.     

Dynamic oxygen mapping in the root zone by fluorescence dye imaging combined with neutron radiography

Purpose  

The rooted zone of a soil, more precisely the rhizosphere, is a very dynamic system. Some of the key processes are water uptake and root respiration. We have developed a novel method for measuring the real-time distribution of water and oxygen concentration in the rhizosphere as a biogeochemical interface in soil. This enables understanding where and when roots are active in respect to root respiration and water uptake and how the soil responds to it.