Pesticide residues in grapes, wine, and their processing products

In this review the results obtained in the 1990s from research on the behavior of pesticide residues on grapes, from treatment to harvest, and their fate in drying, wine-making, and alcoholic beverage processing are reported. The fungicide residues on grapes (cyproconazole, hexaconazole, kresoxim-methyl, myclobutanil, penconazole, tetraconazole, and triadimenol), the application rates of which were of a few tens of grams per hectare, were very low after treatment and were not detectable at harvest. Pyrimethanil residues were constant up to harvest, whereas fluazinam, cyprodinil, mepanipyrim, azoxystrobin, and fludioxonil showed different disappearance rates (t(1/2) = 4.3, 12, 12.8, 15.2, and 24 days, respectively). The decay rate of the organophosphorus insecticides was very fast with t(1/2) ranging between 0.97 and 3.84 days. The drying process determined a fruit concentration of 4 times. Despite this, the residue levels of benalaxyl, phosalone, metalaxyl, and procymidone on sun-dried grapes equalled those on the fresh grape, whereas they were higher for iprodione (1.6 times) and lower for vinclozolin and dimethoate (one-third and one-fifth, respectively). In the oven-drying process, benalaxyl, metalaxyl, and vinclozolin showed the same residue value in the fresh and dried fruit, whereas iprodione and procymidone resides were lower in raisins than in the fresh fruit. The wine-making process begins with the pressing of grapes. From this moment onward, because the pesticide on the grape surface comes into contact with the must, it is in a biphasic system, made up of a liquid phase (the must) and a solid phase (cake and lees), and will be apportioned between the two phases. The new fungicides have shown no effect on alcoholic or malolactic fermentation. In some cases the presence of pesticides has also stimulated the yeasts, especially Kloeckera apiculata, to produce more alcohol. After fermentation, pesticide residues in wine were always smaller than those on the grapes and in the must, except for those pesticides that did not have a preferential partition between liquid and solid phase (azoxystrobin, dimethoate, and pyrimethanil) and were present in wine at the same concentration as on the grapes. In some cases (mepanipyrim, fluazinam, and chlorpyrifos) no detectable residues were found in the wines at the end of fermentation. From a comparison of residues in wine obtained by vinification with and without skins, it can be seen that their values were generally not different. Among the clarifying substances commonly used in wine (bentonite, charcoal, gelatin, polyvinylpolypyrrolidone, potassium caseinate, and colloidal silicon dioxide), charcoal allowed the complete elimination of most pesticides, especially at low levels, whereas the other clarifying substances were ineffective. Wine and its byproducts (cake and lees) are used in the industry to produce alcohol and alcoholic beverages. Fenthion, quinalphos, and vinclozolin pass into the distillate from the lees only if present at very high concentrations, but with a very low transfer percantage (2, 1, and 0.1%, respectively). No residue passed from the cake into the distillate, whereas fenthion and vinclozolin pass from the wine, but only at low transfer percentages (13 and 5%, respectively).     

Antioxidant activity of tomato products as studied by model reactions using xanthine oxidase, myeloperoxidase, and copper-induced lipid peroxidation

The antioxidant content and activity of commercial tomato products differing in variety and processing were studied. Two procedures for extracting hydrophilic and lipophilic antioxidants, namely, two-step 0.1 M phosphate buffer (pH 3.0 and 7.4) extraction and tetrahydrofuran extraction followed by petroleum ether fractionation, were developed. Carotenoids (lycopene, beta-carotene, and lutein) and ascorbic acid were analyzed by HPLC with spectrophotometric and electrochemical detectors, respectively. Total phenolics were determined by using the Folin-Ciocalteu reagent. The antioxidant activity was studied by the following three model systems: (a) the xanthine oxidase (XOD)/xanthine system, which generates superoxide radical and hydrogen peroxide; (b) the myeloperoxidase (MPO)/NaCl/H(2)O(2) system, which produces hypochloric acid; and (c) the linoleic acid/CuSO(4) system, which promotes lipid peroxidation. Results showed that the hydrophilic and lipophilic fractions of all tomato products were able to affect model reactions, whatever reactive oxygen species and catalysts were used to drive oxidation. In the XOD/xanthine system both the hydrophilic and lipophilic fractions displayed an inhibitory activity. The hydrophilic fractions were more effective (I(50) ranging from 680 to 3200 microg, dry weight) than the lipophilic fractions (I(50) ranging from 4000 to 7750 microg, dry weight). In the MPO/NaCl/H(2)O(2) system the hydrophilic fractions inhibited oxidation (I(50) ranging from 2300 to 2900 microg, dry weight), whereas the lipophilic fractions had a lower inhibitory effect at the same concentration. Conversely, in the copper-catalyzed lipid peroxidation only the lipophilic fractions were effective (I(50) ranging from 1030 to 2100 microg, dry weight), whereas the hydrophilic fractions had a pro-oxidant effect in the same concentration range. The extent of inhibition varied according to the tomato sample in the superoxide and hydrogen peroxide generating system and in lipid peroxidation, but was substantially the same in the HClO generating system. Fresh tomato varieties differed considerably in the antioxidant activities of their hydrophilic and lipophilic fractions. Processed tomatoes showed a significantly lower antioxidant activity than fresh tomatoes in their hydrophilic fractions but had a high antioxidant activity in their lipophilic fractions. Because the oxidative reactions produced by the above-mentioned model systems are also involved in the pathogenesis of several chronic diseases, the antioxidant activity of tomato fractions might be related to their in vivo activity. Hence, these measurements may be used for optimizing tomato technologies.     

In vitro antioxidant and ex vivo protective activities of green and roasted coffee

The antioxidant properties of green and roasted coffee, in relation to species (Coffea arabica and Coffea robusta) and degree of roasting (light, medium, dark), were investigated. These properties were evaluated by determining the reducing substances (RS) of coffee and its antioxidant activity (AA) in vitro (model system beta-carotene-linoleic acid) and ex vivo as protective activity (PA) against rat liver cell microsome lipid peroxidation measured as TBA-reacting substances. RS of C. robustasamples were found to be significantly higher when compared to those of C. arabica samples (p < 0.001). AA for green coffee samples were slightly higher than for the corresponding roasted samples while PA was significantly lower in green coffee compared to that of all roasted samples (p < 0.001). Extraction with three different organic solvents (ethyl acetate, ethyl ether, and dichloromethane) showed that the most protective compounds are extracted from acidified dark roasted coffee solutions with ethyl acetate. The analysis of acidic extract by gel filtration chromatography (GFC) gave five fractions. Higher molecular mass fractions were found to possess antioxidant activity while the lower molecular mass fractions showed protective activity. The small amounts of these acidic, low molecular mass protective fractions isolated indicate that they contain very strong protective agents.     

Contribution of proanthocyanidins to the peroxy radical scavenging capacity of some Italian red wines

Highly reactive radicals, ROO(*), were generated from 2, 2'-azobis[2-(2-imidazolin-2-yl)propane] and linoleic acid. The ROO(*) scavenging capacity of some Italian red wines was evaluated following the changes in oxygen consumption. Under the experimental conditions the time course of oxygen consumption shows two typical behaviors: trolox-like (class I) and gallic acid-like (class II). Usually the time course of wine was similar to that of gallic acid. The rate of oxygen consumption was found to decrease exponentially with the amount of wine or gallic acid added to the test solution. On this basis the capacity of red wines to scavenge peroxy radicals was expressed as content of gallic acid (S(GA)). The S(GA) values were found to be correlated to the amount of total proanthocyanidins and total polyphenols of some Italian red wines (p < 0.01). The proanthocyanidins extracted from seeds were shown to make a major contribution to the peroxy radical scavenging capacity of red wines, whereas, interestingly, the chemical class of the low molecular weight tannins reactive to vanillin did not correlate with the S(GA) values.     

Analysis of glycosidically bound aroma precursors in tea leaves. 1. Qualitative and quantitative analyses of glycosides with aglycons as aroma compounds

Twenty-six synthetic glycosides constituting aglycons of the main tea aroma compounds ((Z)-3-hexenol, benzyl alcohol, 2-phenylethanol, methyl salicylate, geraniol, linalool, and four isomers of linalool oxides) were synthesized in our laboratory as authentic compounds. Those compounds were used to carry out a direct qualitative and quantitative determination of the glycosides as aroma precursors in different tea cultivars by capillary gas chromatographic-mass spectrometric (GC-MS) analyses after trifluoroacetyl conversion of the tea glycosidic fractions. Eleven beta-D-glucopyranosides, 10 beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) with aglycons as the above alcohols, and geranyl beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) were identified (tentatively identified in the case of methyl salicylate beta-primeveroside) in fresh tea leaves and quantified on the basis of calibration curves that had been established by using the synthetic compounds. Primeverosides were more abundant than glucosides in each cultivar we investigated for making green tea, oolong tea, and black tea. Separation of the diastereoisomers of linalool and four isomers of linalool oxides by GC analyses is also discussed.     

Physicochemical characteristics of onion (Allium cepa L.) tissues

The structure and mechanical properties of onions are important factors affecting their textural quality. The onion bulb consists of several layers of pigmented, papery scales surrounding fleshy storage scales that comprise an upper epidermis, an intermediate parenchyma tissue, and a lower epidermis. The purpose of this study was to examine the chemical composition of cell walls from the papery scales and outer fleshy scales of onion (Allium cepa L. cv. Sturon) in relation to their mechanical properties. Cell-wall material (CWM) was prepared from the component tissues and analyzed for its carbohydrate and phenolic composition. The CWMs were rich in uronic acid and glucose, with smaller quantities of arabinose, galactose, and xylose. In the fleshy scales, the lower epidermis contained relatively more galactose-rich pectic polysaccharides, whereas the upper epidermis and the papery scales contained virtually no galactose. Analysis of mechanical properties showed that the order of strength of the tissues was papery scales > fleshy scales, which were in the order lower epidermis > upper epidermis > intermediate parenchyma. The upper epidermis of fleshy scales was stronger in the vertical than the horizontal direction, and both orientations showed negligible notch sensitivity. Cyclohexane-trans-1,2-diaminetetraacetate-induced vortex-induced cell separation of the intermediate layer of fleshy scales indicated that calcium cross-linking may play an important role in cell-cell adhesion. A small but significant amount of ferulic acid was found in the walls, predominantly in the thick cuticle of the lower epidermis of fleshy scales. Alkali-labile wall-bound flavonoids were also detected.     

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.     

Synthesis and resistance to in vitro proteolysis of transglutaminase cross-linked phaseolin, the major storage protein from Phaseolus vulgaris

The ability of phaseolin to act as an acyl donor and acceptor substrate of transglutaminase was studied by using an enzyme isolated from Streptoverticillium mobarense. Phaseolin, a trimeric storage protein from Phaseolus vulgaris L., was shown to possess both glutamine and lysine residues reactive for the enzyme. The extent of transglutaminase-catalyzed cross-linking has been studied in function of both incubation time and enzyme concentration. Native- and SDS-PAGE demonstrated that phaseolin is intra- and intermolecularly cross-linked by transglutaminase and gives rise to different polymers as well as to modified forms of the protein having a similar molecular weight but lower Stokes radius if compared to unmodified phaseolin. Cross-linked phaseolin was found to be more resistant to proteolytic cleavage than the unmodified counterpart, as demonstrated by in vitro trypsin and pepsin digestion experiments. This behavior could suggest novel possible uses of the transglutaminase-modified phaseolin.     

Interactions between a non glycosylated human proline-rich protein and flavan-3-ols are affected by protein concentration and polyphenol/protein ratio

Interactions between salivary proline-rich proteins and tannins are involved in astringency, which is one of the most important organoleptic sensations perceived when drinking wine or tea. This work aimed to study interactions between a recombinant human salivary proline-rich protein, IB-5, and a flavan-3-ol monomer, epigallocatechin gallate (EGCG). IB-5 presented the characteristics of natively unfolded proteins. Interactions were studied by dynamic light scattering, isothermal titration microcalorimetry, and circular dichroism. The interaction mechanism was dependent on protein concentration. At low concentrations, a three-stage mechanism was evidenced. Saturation of the interaction sites (first stage) was followed by protein aggregation into metastable colloids at higher EGCG/protein ratios (second stage). Further increasing this ratio led to haze formation (third stage). At low ratios, a disorder-to-order transition of IB-5 structure upon binding was evidenced. At high protein concentrations, direct bridging between proteins and EGCG was observed, resulting in significantly lower aggregation and turbidity thresholds.     

Inhibition of tumorigenesis in ApcMin/+ mice by a combination of (-)-epigallocatechin-3-gallate and fish oil

The effect of a combination of (-)-epigallocatechin-3-gallate (EGCG) with fish oil on intestinal tumorigenesis in Apc (Min/+) mice fed a high-fat diet was investigated in the present study. The combined treatment of EGCG and fish oil for 9 weeks reduced the tumor number by 53% as compared to controls while neither agent alone had a significant effect. Apoptosis was significantly increased in all treatment groups. beta-Catenin nuclear positivity in adenomas from the combination group was lower than control mice, implicating the modulation of Wnt signaling by the combination. Fish oil and the combination significantly reduced prostaglandin E 2 (PGE 2) levels in small intestinal tumors as compared to controls, suggesting modulation of aberrant arachidonic acid metabolism by fish oil. Akt phosphorylation in adenomas was significantly reduced in all treatment groups, which may have contributed to the observed increase in apoptosis. The results indicate that a combination of low doses of EGCG and fish oil can inhibit tumor multiplicity in Apc (Min/+) mice.