Repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is not toxic

The water-soluble ellagitanin punicalagin has been reported to be toxic to cattle. Taking into account that this antioxidant polyphenol is very abundant in pomegranate juice (> or =2 g/L), the present study evaluated the possible toxic effect of punicalagin in Sprague-Dawley rats upon repeated oral administration of a 6% punicalagin-containing diet for 37 days. Punicalagin and related metabolites were identified by HPLC-DAD-MS-MS in plasma, liver, and kidney. Five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic acid, 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide, and 3,8,10-trihydroxy-6H-dibenzo[b,d]pyran-6-one. Feedstuff intake, food utility index, and growth rate were lower in treated rats during the first 15 days without significant adverse effects, which could be due to the lower nutritional value of the punicalagin-enriched diet together with a decrease in its palatability (lower food intake). No significant differences were found in treated rats in any blood parameter analyzed (including the antioxidant enzymes gluthatione peroxidase and superoxide dismutase) with the exception of urea and triglycerides, which remained at low values throughout the experiment. Although the reason for the decrease is unclear, it could be due to the lower nutritional value of the punicalagin-enriched diet with respect to the standard rat food. Histopathological analysis of liver and kidney corroborated the absence of toxicity. In principle, the results reported here, together with the large safety margin considered, indicate the lack of toxic effect of punicalagin in rats during the 37 day period investigated. However, taking into account the high punicalagin content of pomegranate-derived foodstuffs, safety evaluation should be also carried out in humans with a lower dose and during a longer period of intake.     

Role of olive seed in the biogenesis of virgin olive oil aroma

Results obtained in a set of experiments point to an effective participation of olive seeds in the biosynthesis of olive oil aroma through the lipoxygenase pathway during the extraction process to produce virgin olive oil. Data showed that olive seeds should contain enzymatic activities metabolizing 13-hydroperoxides other than hydroperoxide lyase, giving rise to a net decrease in the content of C6 unsaturated aldehydes during the olive oil extraction process. Olive seeds seem also to supply this process with alcohol dehydrogenase activity, being more specific for saturated C6 aldehydes and not acting on C5 alcohols. Moreover, olive seeds would be responsible for the biosynthesis of 30-50% esters during the olive oil extraction process of intact fruits. Thus, olive seeds would afford a load of alcohol acyltransferase activity that might be quite unspecific in terms of substrate, producing any kind of esters.     

Enzymatic and fungal treatments on sugarcane bagasse for the production of mechanical pulps

Crude ligninolytic enzyme extracts from Phanerochaete chrysosporium fungi were applied to sugarcane bagasse, prior to thermomechanical (TMP) and chemithermomechanical pulping (CTMP), and their properties were compared with the normal TMP and CTMP and also with TMP and CTMP pretreated with Ceriporiopsis subvermispora and P. chrysosporium fungi. The sugarcane bagasse was impregnated with the crude enzyme extract containing lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac). The results show that pretreatment with enzyme crude extract is an advantageous way to produce TMP and CTMP from sugarcane bagasse, as compared with only fungal pretreatment. Enzymatic pretreatments need only hours to enhance pulping and paper properties, compared with the weeks necessary for fungal treatments. Higher pulp yields were obtained compared with the fungal pretreatments. Enzymatic pretreatment reduced the energy consumption in a proportion similar to that of C. subvermispora fungal pretreatment and increased the pulp tensile index compared with the normal TMP and CTMP pulps, although the tensile strength was somewhat lower than that for pulps from C. subvermispora fungal pretreatment before CTMP processing. An advantage of enzymatic pretreatment is that brightness is increased compared with normal TMP and CTMP processes, whereas fungal pretreatments reduce the brightness.     

Tequila volatile characterization and ethyl ester determination by solid phase microextraction gas chromatography/mass spectrometry analysis

Solid phase microextraction (SPME) and gas chromatography were used for tequila volatile characterization and ethyl ester quantitation. Several factors determined the differences in tequila volatile profiles obtained by the SPME technique, namely, sampling mode, fiber coating, and fiber exposure time. Each of these factors determined the most suitable conditions for the analysis of volatile profiles in tequila. Volatile extraction consisted of placing 40 mL of tequila in a sealed vial kept at 40 degrees C. A poly(dimethylsiloxane) fiber was immersed in the liquid for 60 min and desorbed for 5 min into the gas chromatograph. The identified volatiles by mass spectrometry were mainly alcohols, esters, and ketones. The calibration curves for ethyl hexanoate, octanoate, and decanoate followed linear relationships with highly significant (p < 0.001) determination coefficients (R2 = 0.99). The coefficients of variation of less than 10% for ethyl ester concentrations indicated that the technique was reproducible. The limits of quantitation for ethyl esters were 0.05 parts per million, which were below the concentration range (0.27-15.03 ppm) found for different tequila samples. Quantitative differences in ethyl esters were found for the four most commonly known tequila types: silver, gold, aged, and extra-aged.     

Production of bioavailable flavonoid glucosides in fruit juices and green tea by use of fungal alpha-L-rhamnosidases

Flavonoid glucosides have been reported to be more bioavailable than their rutinoside counterparts. The aim of this study is to describe a first step in the use of alpha-L-rhamnosidases (RhaA and RhaB) from Aspergillus aculeatus as a way to produce functional beverages based on their potentially increased flavonoid bioavailability. Blackcurrant juice (BCJ), orange juice (OJ), and green tea infusion (GT) were incubated with either RhaA or RhaB at 30 degrees C for 10 h. Aliquots of controls and enzyme-treated samples were taken at different time points and analyzed by high-performance liquid chromatography-photodiode-array detector-mass spectrometry of daughter fragments (HPLC-DAD-MS-MS). Both RhaA and RhaB selectively catalyze in situ the removal of terminal rhamnosyl groups in the three beverages despite the heterogeneity of assay conditions such as different rutinosides and pH. Incubation of the three beverages with the two rhamnosidases resulted in a hyperbolic decrease in the flavonoid rutinosides (anthocyanins in BCJ, flavanones in OJ, and flavonols in GT) and a concomitant increase in their flavonoid glucoside counterparts. The time required for conversion of 50% of the rutinoside into the corresponding flavonoid glucoside ranged from 30 min (RhaB-rutin in GT) to 6 h (RhaB-delphinidin 3-rutinoside in BCJ). The results presented in this paper are a step forward in the use of enzyme-treated beverages as a source of bioavailable flavonoid glucosides.     

Phenolic compounds and fatty acids from acorns (Quercus spp.), the main dietary constituent of free-ranged Iberian pigs

The aim of the present work was to identify and quantify the phenolic compounds and fatty acids in acorns from Quercus ilex, Quercus rotundifolia, and Quercus suber. The concentration of oleic acid was >63% of total fatty acids in all cases, followed by palmitic and linoleic acids at similar concentrations (12-20%). The concentrations of alpha-tocopherol in Q. rotundifolia, Q. ilex, and Q. suber were 19, 31, and 38 mg/kg of dry matter (DM), respectively, whereas the concentrations of gamma-tocopherol were 113, 66, and 74 mg/kg of DM, respectively. Thirty-two different phenolic compounds were distinguished. All of them were gallic acid derivatives, in the form of either galloyl esters of glucose, combinations of galloyl and hexahydroxydiphenoyl esters of glucose, tergallic O- or C-glucosides, or ellagic acid derivatives. Several tergallic acid C-glucosides were also present in the extracts obtained from Q. suber. Acorns from Q. ilex and Q. rotundifolia showed similar polyphenol patterns mainly with gallic acid-like spectra. Chromatograms of Q. suber showed mainly polyphenols with ellagic acid-like spectra. Valoneic acid dilactone was especially abundant in Q. suber skin. The contribution of skin to the total phenolics of the acorn was relatively small in Q. rotundifolia and Q. ilex but relatively high in Q. suber. Skin extracts from Q. suber, Q. rotundifolia, and Q. ilex showed 1.3, 1.4, and 1.0 antioxidant efficiencies, respectively (compared to that of butylhydroxyanisole). Endosperm extracts showed lower capacity to prevent lipid peroxidation than skin extracts.     

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).     

Density and microviscosity studies of palm oil/water emulsions

Electron paramagnetic resonance (EPR) and densitometry were used to measure the temperature- and rate-dependent formation of fat crystals in emulsion droplets in hardened palm kernel oil in water emulsions. The solid fat content in emulsions can be critical for the functionality of the emulsions in a wide variety of applications. Therefore, new and accessible methods are needed to monitor solid fat content in order to control the functional properties of these emulsions. EPR measurements showed that the microviscosity within the oil droplets could be measured as a function of temperature and that the storage temperature below the main fat melting point is crucial for an increase in the microviscosity, due to fat solidification. The microviscosity of the oil droplets could be an important parameter for defining functional performance (e.g., rheology and partial coalescence). Density measurements provided a simple and accurate method for monitoring changes in phase state of the oil. The phase behavior agreed well with the EPR results, demonstrating that accurate density measurements could prove to be a valuable tool for monitoring fat crystallization processes.     

Immunochemical screening and liquid chromatographic-tandem mass spectrometric confirmation of drug residues in edible tissues of calves injected with a therapeutic dose of the synthetic glucocorticoids dexamethasone and flumethasone

A field study was performed to assess the drug residue level in edible tissues after a therapeutic application of the synthetic glucocorticoids dexamethasone and flumethasone. Three diseased calves were injected intramuscularly with a commercial batch of dexamethasone esters and slaughtered 72 h after treatment. Another three calves were injected intramuscularly with an aqueous flumethasone preparation and slaughtered 24 h later. Residues of synthetic glucocorticoids in liver, muscle, kidney, and urine were assessed by competitive enzyme immunoassay. All dexamethasone concentrations exceeded the maximal residue level of 0.75 microg/kg in muscle and kidney and 2 microg/kg in the liver. The presence of both dexamethasone and flumethasone in the liver was confirmed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These results indicate that liver tissue provides a suitable matrix to monitor the presence of illegal residues of synthetic glucocorticoids in slaughtered animals.     

Characterization of white wine mannoproteins

Mannoproteins, Saccharomyces cerevisiae yeast polysaccharides, play a major role in wine processing and characteristics. A systematic characterization of these polymers in terms of chemical composition and molecular structure is addressed in this study. Mannoproteins were isolated from white wine through a sequence of operations that consisted of nanofiltration for concentration of macromolecules, polysaccharide precipitation with ethanol, and affinity chromatography on concanavalin A. The whole wine mannoproteins present a very broad molecular mass distribution with several populations. Two major populations with very different compositions were separated by size exclusion chromatography. The mannoproteins with higher molecular mass are a mannose homopolymer containing 10.3% protein. The mannoproteins with lower molecular mass consisted of 87.5% of mannose and some other residues and a protein content of 2.5%. The highest molecular weight mannoprotein structure was examined by (1)H and (13)C NMR spectroscopic techniques such as 1-D TOCSY, 2-D COSY, and 2-D HMQC.