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.     

Effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion

The effect of some polyphenols (resveratrol, catechin, epigallocatechin-3-gallate, and quercetin) and beverages (red wine and green tea) on the enzymatic activity of pepsin during the digestion of three different substrates (pork meat, insoluble azocasein, and denatured hemoglobin) has been investigated. The tested polyphenols and beverages increase the initial velocity of the reaction, and the activating effect is concentration dependent. The order of effectiveness of polyphenols in increasing the initial velocity of the reaction is resveratrol > or = quercetin > epigallocatechin-3-gallate > catechin. The kinetic data obtained with soluble denatured hemoglobin show that the K(m) for the substrate is not changed, whereas the V(max) of the reaction is increased. Pepsin activity follows a simple Michaelis-Menten kinetic suggesting that k(3) is increased by polyphenols. To the authors' knowledge, the present study is the first demonstration that some polyphenols and related beverages are able to enhance the enzymatic activity of pepsin.     

GISQ, a multifunctional indicator of soil quality

We present here an indicator of soil quality that evaluates soil ecosystem services through a set of 5 subindicators, and further combines them into a single general Indicator of Soil Quality (GISQ). We used information derived from 54 properties commonly used to describe the multifaceted aspects of soil quality. The design and calculation of the indicators were based on sequences of multivariate analyses. Subindicators evaluated the physical quality, chemical fertility, organic matter stocks, aggregation and morphology of the upper 5 cm of soil and the biodiversity of soil macrofauna. A GISQ combined the different subindicators providing a global assessment of soil quality.Research was conducted in two hillside regions of Colombia and Nicaragua, with similar types of land use and socio-economic context. However, soil and climatic conditions differed significantly. In Nicaragua, soil quality was assessed at 61 points regularly distributed 200 m apart on a regular grid across the landscape. In Colombia, 8 plots representing different types of land use were arbitrarily chosen in the landscape and intensively sampled. Indicators that were designed in the Nicaragua site were further applied to the Colombian site to test for their applicability.In Nicaragua, coffee plantations, fallows, pastures and forest had the highest values of GISQ (1.00; 0.80; 0.78 and 0.77, respectively) while maize crops and eroded soils (0.19 and 0.10) had the lowest values. Examination of subindicator values allowed the separate evaluation of different aspects of soil quality: subindicators of organic matter, aggregation and morphology and biodiversity of macrofauna had the maximum values in coffee plantations (0.89; 0.72 and 0.56, respectively on average) while eroded soils had the lowest values for these indicators (0.10; 0.31 and 0.33, respectively).Indicator formulae derived from information gained at the Nicaraguan sites were not applicable to the Colombian situation and site-specific constants were calculated.This indicator allows the evaluation of soil quality and facilitates the identification of problem areas through the individual values of each subindicator. It allows monitoring of change through time and can guide the implementation of soil restoration technologies. Although GISQ formulae computed on a set of data were only valid at a regional scale, the methodology used to create these indices can be applied everywhere.     

Drought pretreatment increases the salinity resistance of tomato plants

Agricultural productivity is worldwide subjected to increasing salinity problems. Various strategies are applied to overcome the deleterious effects of salinity on plants. This study was conducted in order to determine whether drought pretreatment of seedlings or seed pretreatment with NaCl increases the long‐term salinity resistance of tomato (Solanum lycopersicum L.) and whether the adaptive response to salinity is accompanied by physiological changes throughout the plant‐growth cycle. When plants were pretreated at the five‐leaf growth stage, the plant dry weight was significantly higher in drought‐pretreated than in non‐pretreated plants after 50 d of salt treatment. The positive effect of drought pretreatment applied at the five‐leaf stage was maintained throughout the entire growth cycle, as fruit yield of drought‐pretreated plants was 40% higher than that of non‐pretreated plants at the end of the harvest period (150 d of 70 mM NaCl treatment). Moreover, the most productive plants maintained lower Na⁺ and Cl^– accumulation in their leaves until the end of the growth cycle, which shows that adaptation is a long‐term response during which the plants adjust their physiology to the environmental conditions. Salt resistance was also improved through seed pretreatment with NaCl. In conclusion, drought pretreatment applied at the five‐leaf stage or seed pretreatment with NaCl provide an alternative way to enhance salt resistance in tomato, and the increase in yield is associated with physiological changes throughout the plant‐growth cycle.     

Biochemical and immunochemical characterization of different varieties of amaranth (Amaranthus L. ssp.) as a safe ingredient for gluten-free products

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.     

Analysis of the effect of temperature changes combined with different alkaline pH on the β-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS

Temperature and pH influence the conformation of the whey protein β-lactoglobulin (β-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on β-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of β-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.     

Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana)

A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.     

Countercurrent supercritical fluid extraction and fractionation of high-added-value compounds from a hexane extract of olive leaves

Countercurrent supercritical fluid extraction (CC-SFE) at a pilot scale plant was used for fractionation of high-added-value products from a raw extract of olive leaves in hexane. Compounds found in the raw extract were waxes, hydrocarbons, squalene, beta-carotene, triglycerides, alpha-tocopherol, beta-sitosterol, and alcohols. The CC-SFE extraction process was investigated according to a 2(3) full factorial experimental design using the following variables and ranges: extraction pressure, 75-200 bar; extraction temperature, 35-50 degrees C; and ethanol as modifier, 0-10%. Data were analyzed in terms of extraction yield, enrichment, recovery, and selectivity. Higher extraction yields were attained at 200 bar. For most of the compounds analyzed enrichment was attained at the same conditions, that is, 75 bar, 35 degrees C, and 10% ethanol. Hydrocarbons were usually recovered in the separators, whereas waxes and alpha-tocopherol remain in the raffinate. Selectivity data reveal that alpha-tocopherol is the most easily separable compound. The influence of the experimental factors on the recovery of all the compounds was studied by means of regression models. The best fitted model was attained for beta-sitosterol, with R2 = 99.25%.     

Soil bacterial communities under slash and burn in Mozambique as revealed by a metataxonomic approach

The slash-and-burn system is a subsistence agronomical practice widespread in tropical areas worldwide. This system has been extensively studied,especially for its impacts on agronomical aspects and soil physicochemical properties; however, knowledge of soil microbial diversity under slash and bum is scarce. In this study, for the first time, soil bacterial diversity of three locations from Central Mozambique, where slash and burn has been practiced for different durations of the forest fallow p...     

Organic acid contents in onion cultivars (Allium cepa L.)

The following organic acids (glutamic, oxalic, pyruvic, malic, tartaric, citric, and fumaric), pungency, Brix degree, acidity, and pH were determined in onion cultivars (Texas, Guayonje, San Juan de la Rambla, Carrizal Alto, Carrizal Bajo, and Masca) harvested in the same agroclimatic conditions. Glutamic acid was the most abundant organic acid (325 +/- 133 mg/100 g) followed by citric acid (48.5 +/- 24.1 mg/100 g) and malic acid (43.6 +/- 10.4 mg/100 g). There were significant differences between the onion cultivars in the mean concentrations of all of the analyzed parameters. The San Juan de la Rambla and Masca cultivars presented, in general, higher concentrations of the organic acids than the other cultivars. Significant differences in most of the analyzed parameters were observed between the two seed origins for the Masca and San Juan de la Rambla cultivars. The onion samples tended to be classified according to the cultivar and, in the case of San Juan de la Rambla cultivar, according to the precedence of the seeds after applying discriminant analysis.