Part I. Polyphenols composition and antioxidant potential during 'Blaufränkisch' grape maceration and red wine maturation, and the effects of trans-resveratrol addition.

The effects of extended maceration (13 days) were investigated for extraction of trans-resveratrol and other phenolics from grapes of cultivar 'Blaufränkisch', and then during the subsequent maturation of the wine (250 days). Total phenolics and three subgroups of phenolics were followed. The concentrations of total phenolics, total flavonoids, and total anthocyanins, but not of total nonflavonoids, increased with extended maceration, and then decreased after 250 days of maturation. Trans- and cis-resveratrol concentrations increased following extended maceration and maturation (6.5, 2.9 mg/L, respectively). The maximum polydatin concentration was reached after only 6 days of maceration (10.9 mg/L). The antioxidant potential of the must increased following extended maceration (12.3 mmol DPPH2/L), and then remained unchanged for the red wine after maturation. Addition of trans-resveratrol to the red wine and into model solutions showed increased solubility and stability of trans-resveratrol in the red wines over the model solutions. Minor increases in antioxidant potential and better stability of malvidin-3-glucoside were also seen.

Part II. Influence of trans-resveratrol addition on the sensory properties of 'Blaufränkisch' red wine.

Trans-resveratrol was added to 'Blaufränkisch' red wine to determine the changes in total phenolics, total anthocyanins, and antioxidant potential. The results of this first investigation were presented in Part I; the results of the sensory analyses are included here, in Part II. Addition of increasing concentrations of trans-resveratrol to the 'Blaufränkisch' red wine improved the intensity of color by ~7%. and also the stability of malvidin-3-glucoside. Surprisingly, these additions of trans-resveratrol affected the odor of the red wine. Correlations among the sensory attributes showed Pearson's coefficients for positive correlations among these parameters. The higher additions of trans-resveratrol to the red wine (150, 200 mg/L) showed significantly improved sensory scores for color and odor, compared to the no-addition control (p ≤ 0.05). The highest positive correlation was between flavor and overall acceptance (0.876), which reached statistical significance (p ≤ 0.01); the lowest Pearson's coefficient was between clarity and flavor (0.061). This study shows that trans-resveratrol increases the stability of the enriched 'Blaufränkisch' red wine, and improves the sensory acceptance.

White Hop Shoot Production in Slovenia: Total Phenolic, Microelement and Pesticide Residue Content in Five Commercial Cultivars.

Harvesting of white hop shoots might be justified if they can be shown to be beneficial to human health. The aim of the present study is to determine the effects of hop cultivars and year of production on total phenolics, antioxidant potential, microelements and pesticide residues. Biomass per plant was highly variable across the cultivars (3.1-7.1 g dry mass per plant) and depended on hop cultivar and year (2009-2011). Total phenolics as chlorogenic acid equivalents (CAE) on dry mass basis varied from 0.60 to 1.80 mg/g, and showed significant effects across hop cultivar and year. The radical scavenging activities of the samples collected in years 2010-2012 ranged from 11 to 19 µg CAE. Ferric reducing activity was <0.01, with significantly different effects across hop cultivars (pC≤0.05) and year (py≤0.05) observed only in 2012. Traces of microelements and potentially active compounds from the use of pesticides in white hop shoots of Humulus lupulus 'Dana' were analysed. The content of zinc in the hop shoots on dry mass basis was very low (4 mg/kg), and it was below the limit of detection in the soil. The content of copper in the hop shoots was also very low (2.3 mg/kg), while in the soil it was below the critical emission (100 vs 300 mg/kg, respectively). All 182 active ingredients from the residues of the previously used pesticides were below the limits of detection. It can be concluded that these white hop shoots are better antioxidants than hop cones and hop leaves, and that they do not contain any pesticide residues.

Effects of industrial and home-made spread processing on bilberry phenolics.

Bilberries processed into spreads represent an important source of anthocyanins if these remain rich in the final product. The effects of thermal processing were studied with non-ground and ground bilberries processed into spreads according to industrial and home-made procedures. Samples were analysed by LC-DAD-MS/MS and LC-MS. The spreads had 28-60% less total phenolics, 4-62% less anthocyanins, and 1-fold to 2-fold more phenolic acids and total flavonols than the bilberries, but approximately equal flavanols. The home-made spread from ground bilberries had ca. 26% higher antioxidant activity. Delphinidin 3-glucoside and cyanidin 3-glucoside were taken through the two spread procedures, with their degradation to gallic acid (38-57%), protocatechuic acid (1-2%) and 2,4,6-trihydroxybenzaldehyde determined. The amounts of gallic and protocatechuic acids did not reflect well for anthocyanin degradation. The industrial spread procedure with non-ground bilberries is a more suitable procedure to maintain the final content of anthocyanins.

Effect of flavonoid structure on the fluidity of model lipid membranes.

We investigated how the structural properties of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG) and butylated hydroxytoluene (BHT) correlate with structural changes of phosphatidylcholine plus sphingomyelin (2.4:1) model lipid membranes. Changes were measured by fluorescence anisotropy, electron paramagnetic resonance, and differential scanning calorimetry. Two fluorophores and two spin probes were used to monitor membrane characteristics close to water-lipid interface and in the middle of the bilayer. The data obtained were correlated to the amount of bounded compounds, the number of H-bonds, and the topological polar surface area (TPSA) of the compounds. These correlations reflect the behaviours of (+)-catechin, (-)-epicatechin, EGC, EGCG and BHT. Our results confirm that phenolics studied here are bounded to a membrane surface predominantly via hydrogen bonds, while BTH is inserted into the lipid bilayer.

Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells.

Berry anthocyanins have pronounced health effects, even though they have a low bioavailability. The common mechanism underlying health protection is believed to relate to antioxidant activity. Berry extracts, chemically characterised for their phenolic content, were prepared from bilberries (Vaccinium myrtillusL.) and blueberries (Vaccinium corymbosumL.); the bilberry extract was further purified to obtain the anthocyanin fraction. The antioxidant activity of each extract was examined at the cellular level. For this purpose a specific assay, known as cellular antioxidant activity assay (CAA), was implemented in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). Here we show for the first time that anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 µg/l; nM range), thereby providing a long-sought rationale for their health protecting effects in spite of their unfavorable pharmacokinetic properties.

Phenolics in Slovenian bilberries ( Vaccinium myrtillus L.) and blueberries ( Vaccinium corymbosum L.).

Phenolics from bilberries ( Vaccinium myrtillus L.) sampled from seven different locations and highbush blueberries ( Vaccinium corymbosum L.) from one location in Slovenia were analyzed. In samples of both species 15 anthocyanins were identified by LC-MS/MS. Their contents were expressed as cyanidin 3-glucoside equivalents (C3GE); bilberries contained 1210.3 ± 111.5 mg C3GE/100 g fw and blueberries 212.4 ± 14.1 mg C3GE/100 g fw. Glycosides of delphinidin and cyanidin were predominant (488.5 vs 363.6 mg C3GE/100 g fw) in the bilberries and glycosides of malvidin (108.0 vs 100.8 mg C3GE/100 g fw) in the blueberries, whereas the contents of peonidin were lowest (74.5 vs 4.8 mg C3GE/100 g fw) in both berries. The contents of flavanols, flavonols, phenolic acids, and stilbenes were determined by LC-MS. For the first time, rutin was identified (bilberries, 0.2 ± 0.0 mg/100 g fw; blueberries, 3.1 ± 0.1 mg/100 g fw). Chlorogenic acid (as 3-caffeoylquinic acid) was the most abundant among the phenolic acids (23.1 ± 1.0 mg/100 g fw in bilberries and 70.0 ± 3.4 mg/100 g fw in blueberries). Statistical analysis shows that the content of 27 individual flavonoids, phenolic acids, and stilbenes can be used to identify the picking region of these Slovenian bilberries.

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products.

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.

Antioxidant activity of Sempervivum tectorum and its components.

The antioxidant properties of components of leaf extracts of the evergreen plant, Sempervivum tectorum (ST), have been evaluated using UV irradiated liposomal systems containing the spin trap 5-(diethoxyphosphoryl)-5-methyl-pyrroline-N-oxide. Decreases in free radical activity in the liposomal systems as measured by electron paramagnetic resonance (EPR) spectroscopy demonstrate that the lipophilic ST juice components, kaempferol (KA) and kaempferol-3-glucoside (KG) contribute significantly to the antioxidant properties of the juice. EPR spectral simulation established the presence of oxygen and carbon centered free radical adducts. The mixtures with low pH, citric and malic acid, and ST juice reveal increased EPR signals from oxygen centered radicals in comparison to the control, pointing to the important role of pH in oxygen radical formation. Parallel assays that measured thiobarbituric acid related substances confirm the antioxidant effects of KA and KG and explain the results of spin trapping experiments complicated by low pH's.