A preliminary characterization of Aglianico (Vitis vinifera L. cv.) grape proanthocyanidins and evaluation of their reactivity towards salivary proteins.

The flavan-3-ol and proanthocyanidin composition of Aglianico seeds and skins were for the first time determined by HPLC-MS in comparison with the international grapes Merlot and Cabernet Sauvignon. Monomers [(+)-catechin C, (-)-epicatechin EC, (-)-epicatechin-3-O-gallate, ECG] and oligomers [B1, B2, B3, B4 dimers and trimer C1] were identified and quantified in grape extracts. In order to evaluate the reactivity towards salivary proteins of model wine solutions of seeds and skins monomeric/oligomeric and polymeric fractions, the Saliva Precipitation Index (SPI) was carried out. Fractions were also analyzed for their mean degree of polymerization (mDP), percentage of galloylation (%G) and of prodelphinidin (%P) by phloroglucinolysis. Aglianico was the most effective in precipitating proteins than Merlot and Cabernet Sauvignon, mainly for the high percentage of galloylation of grape fractions. The mDP and the percentage of ECG in terminal units resulted to significantly contribute to the precipitation of salivary proteins by grape proanthocyanidins.

Dealcoholated red wine induces autophagic and apoptotic cell death in an osteosarcoma cell line.

Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 µg/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells.

Effect of winemaking practices on color indexes and selected bioactive phenolics of Aglianico wine.

Phenolic compounds are responsible for the sensory properties of wine as well as the properties beneficial to human health. The objective of this study was to establish the effect of the use of SO2 and pectolitic enzymes in the prefermentative phase, maceration time, and oak aging on color, anthocyanins, tannins, (+)-catechin, (-)-epicatechin, rutin, trans-resveratrol, and quercetin content of Aglianico wine. Color indexes and phenolics were analyzed by HPLC and spectrophotometric methods. The addition of SO2 and pectolitic enzymes before fermentation caused an increase in color intensity, color stability, total phenolics, anthocyanins, (+)-catechin, (-)-epicatechin rutin, trans-resveratrol, and quercetin content in Aglianico wine. Longer maceration times gave wines richer in total phenolics and with better chromatic characteristics. Storage in oak caused a decrease in anthocyanins, (+)-catechin, (-)-epicatechin, trans-resveratrol, and quercetin content but an increase in total phenolic content, and a stabilizing effect on color also occurred.

A beta-glucosidase gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses.

AIMS: Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative beta-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. METHODS AND RESULTS: The coding region of the putative beta-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oenibeta-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61.19 kDa. Furthermore, the expression of the beta-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. CONCLUSIONS: The beta-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of temperature and ethanol on the L. plantarumbeta-glucosidase gene may be useful to explain the differences found in beta-glucosidase activity reported in wines by several authors.

Relationships between flavoring capabilities, bacterial composition, and geographical origin of natural whey cultures used for traditional water-buffalo mozzarella cheese manufacture.

Natural whey cultures (NWC) (n = 29) used for traditional water-buffalo Mozzarella cheese manufacture and arising from different geographical areas of production were characterized and grouped on the basis of their capability to develop neutral volatile compounds and according to their microbial diversity as revealed by molecular analysis. The flavoring properties of NWC were studied in dairy microcosms resembling the specific technological procedure used in the traditional water-buffalo Mozzarella cheese-making. Neutral volatile compounds were identified by high-resolution gas chromatography (HRGC)-mass spectrometry analysis while information on the microbial diversity occurring in the NWC was retrieved by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA after direct DNA extraction. Neoformation volatile substances (n = 27) were found; 23 were identified and some of them recognized as odor-conferring molecules. Eight different bands, referable to eight microbial species, were obtained by PCR-DGGE analysis of the NWC. Statistical analyses were applied to PCR-DGGE and HRGC data. Interestingly, the flavoring capabilities and the microbial diversity of the NWC proved to be closely linked and both related to the geographical origin of the NWC. These results suggested a possible use of the molecular characterization of the dairy products to support the traceability criteria of typical dairy products like water-buffalo Mozzarella cheese.

A new approach to examine the relationships between sensory and gas chromatography-olfactometry data using Generalized Procrustes analysis applied to six French Chardonnay wines.

Six French Chardonnay wines were submitted to both sensory and combined headspace/gas chromatography-olfactometry analyses. The detection frequencies allowed five hierarchical levels to be distinguished: P25, the odorant areas (OAs) having a detection frequency > or =25% (the complete olfactogram without the odor noise); P40, > or =40%; P55, > or =55%; P70, > or =70%; and P85, > or =85%. Moreover, the detection frequencies were analyzed to distinguish 21 discriminative OAs. Wines tested by sensory analysis and the headspace samples analyzed by gas chromatography-olfactometry (GC-O) were described by a heterogeneous vocabulary distributed into nine overall classes of descriptors. The new statistical treatment to examine hierarchical or discriminative OA categories with respect to sensory data used Generalized Procrustes analysis (GPA) from coordinate tables provided by correspondence analysis (CA). The successive data sets supplied by CA were subjected to GPA to yield consensus method maps. The more selective levels of detection frequency (P70 and P85) were responsible for incomplete or distorted information with respect to sensory data. The most appropriate segmentation of the OA distribution (olfactogram) to represent the sensory profile of the six samples would correspond to the intermediate pattern (P40 and P55). The other interest was to study the reasons of distortion due to the dynamic headspace extraction. The highest proportions of the variance were at all times related to the same classes: spicy, herbaceous, and, to a lesser degree, microbiological. This would indicate that the dynamic headspace analysis induces a distortion with respect to sensory data, which systematically affected the perception of both spicy and herbaceous characters of wines.

Influence of flavor on goat feeding preferences.

Goat feeding preferences for straw pellets flavored with ryegrass (Lolium perenne, cv. Belida) or white clover (Trifolium repens, cv. Huia) aromatic extracts, obtained by means of a cold aromatic extraction method, were assessed with cafeteria trials. Prior to the trials, odor differences between the two plant species, the two aromatic extracts, and the straw pellets sprayed with the two aromatic extracts were verified using sensory analysis performed by 30 human panelists. Since odor differences observed among fresh samples were still detectable in aromatic extracts and moistened pellets, the extraction method was considered effective in reproducing plant odors. Straw pellets sprayed with either distilled water (W) or ryegrass (R) or clover (C) aromatic extracts were used to assess flavor preferences of 12 female Rossa Mediterranea goats. Sprayed pellet preference was evaluated in two sessions conducted in two consecutive weeks. Each session consisted of three two-choice presentations performed on three consecutive days. In both sessions, food intake, proportion of food intake, time spent feeding, and proportion of time spent feeding were significantly affected by pellet type (0.05 > P > 0.001). In particular, straw pellets sprayed with ryegrass extract were highly selected compared to those sprayed with clover (0.01 > P > 0.001) or water (0.01 > P > 0.001). In addition, in the second session, the clover extract was preferred to distilled water (0.05 > P > 0.01). The results of this study gave two main indications: first, goat selectivity for rye-grass against clover was consistent even when straw pellets sprayed with odors of these plants were offered, and secondly, the addition of aromatic extracts to straw pellets increased the preference for pellets.

Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey.

AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Odour-impact compounds of Gorgonzola cheese.

Volatile concentrates were obtained by vacuum distillation from both natural and creamy Gorgonzola cheese and isolated by continuous liquid-liquid extraction. Both were analysed by high resolution gas chromatography (HRGC), HRGC-mass spectrometry and HRGC-olfactometry. A total of 63 components were identified in the neutral extract of the natural type (21 esters, 13 ketones, 14 alcohols, 5 aldehydes, 1 sulphur compound, 7 aromatic compounds and 2 terpenes) and 52 in the creamy type (17 esters, 12 ketones, 10 alcohols, 5 aldehydes, 1 sulphur compound, 5 aromatic compounds and 2 terpenes). Ketones, whose major components were 2-nonanone and 2-heptanone, were the predominant constituents of the neutral fraction. By olfactometric analysis of the neutral extracts, 23 odour-impact compounds were found in the natural and 21 in the creamy Gorgonzola cheese. 1-Octen-3-ol, ethyl hexanoate, 2-nonanone, 2-heptanone, 2-heptanol, ethyl butanoate, 2-nonanol and 4-methylanisole were the key odorants of the natural cheese, whereas 2-heptanone, 2-heptanol, ethyl butanoate, 3-methyl thiopropanal and an unidentified constituent with a fruity odour were characteristic of the creamy Gorgonzola cheese. On the basis of high odour unity values, 2-nonanone, 1-octen-3-ol, 2-heptanol, ethyl hexanoate, methylanisole and 2-heptanone were the most important odorants of natural and creamy Gorgonzola cheese aroma.

Antioxidant effect of red wine polyphenols on red blood cells.

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.

Odorous constituents of ovine milk in relationship to diet.

The neutral volatile compounds of ovine milk from ewes fed on natural pasture, grass meadow, and on mixed grain rations were isolated by distillation under vacuum and then collected in traps that were cooled with liquid nitrogen. The distillate was extracted with dichloromethane, and the extract obtained was analyzed using high resolution gas chromatography and high resolution gas chromatography coupled to mass spectrometry. Seventy compounds were identified and quantitatively determined. The volatile compounds obtained were mainly esters, aldehydes, alcohols, ketones, nitrogen compounds, sulfur compounds, aromatic hydrocarbons, and lactones. The olfactometric indices and olfactory properties of 16 compounds were determined by gas chromatography and olfactometry. All of the substances were present in all three milk types with the exception of two sesquiterpenes, which were not detected in the milk from ewes fed the mixed grain ration. The two sesquiterpenes were absent also in ovine cheese obtained from milk from ewes fed the mixed grain ration, but the two sesquiterpenes were identified in cheese produced from milk of sheep fed on natural pasture. The presence of these constituents in the milk can be significant because of their potential role in determining milk and cheese flavor. Accordingly, cheese obtained during summer may be identified using sesquiterpenes as chemical markers of the milk used to make cheese.

New alpha s2-casein variant from caprine milk.

A new alpha s2-casein variant was isolated from goat milk by means of covalent chromatography. With gel electrophoresis at alkaline pH, the alpha s2-casein variant showed a double band with the highest anodic mobility amongst the casein components. The mobility of the isolated alpha s2-casein variant was similar to that occurring in three individual samples of whole casein. Two dimensional electrophoresis revealed a more pronounced heterogeneity of this protein. Developing the two dimensional plate with polyclonal antibodies obtained against the four main bovine casein fractions, we demonstrated that the variant in question belongs to the alpha s2-casein family. Since the alpha s2-casein fractions, immunospecifically developed, spread along two different zones on the gel, it was concluded that a heterozygous alpha s2-casein form was present in the sample.

Determination of ovine casein heterogeneity using gel electrophoresis and immunochemical techniques.

Discontinuous PAGE at alkaline and acid pH, polyacrylamide gel isoelectric focusing, two dimensional electrophoresis and immunoblotting have been used to study the heterogeneity of sheep caseins. Three main phenotypes were selected either because of mobility at alkaline and acid pH of the individual alpha s components or because of their relative intensity. On electrophoresis at alkaline pH, one phenotype showed two distinct bands of lower electrophoretic mobility than beta 1- and beta 2-caseins. A detailed study of these components using immunospecific detection with beta-casein antiserum showed that these minor components of ovine casein may have a polypeptide chain similar to that of beta 1- and beta 2-caseins. Complete electrophoretic patterns of the casein components in some individual milks are also presented.

Fast isoelectric focusing of milk proteins on small ultrathin polyacrylamide gels containing urea.

The preparation of 0.45 mm thin polyacrylamide gels, containing urea, for horizontal micro isoelectric focusing of milk proteins with PhastSystem is described. Isoelectric focusing in the small gels, stained either with Coomassie Brilliant Blue R-250 or with the more sensitive silver stain, affords a fast and sensitive procedure for an analysis of milk and cheese proteins. The procedure can be effectively exploited in detecting adulteration in ovine cheese with bovine milk.