Liquid Chromatography-Mass Spectrometry-Based Metabolomics for Understanding the Compositional Changes Induced by Oxidative or Anoxic Storage of Red Wines.

The aim of this work was to study the physicochemical changes of eight red wines stored under conditions differing in O2 exposure and temperature and time under anoxia. The methods used to analyze the wines included the measurement of volatile sulfur compounds, color, tannin (T) polymerization, and liquid chromatography-mass spectrometry untargeted metabolomic fingerprint. After 3 months, the color of the oxidized samples evolved 4-5 times more intensively than in wines stored under anoxia. The major metabolomic differences between oxidative and anoxic conditions were linked to reactions of acetaldehyde (favored in oxidative) and SO2 (favored in anoxia). In the presence of oxygen, the C-4 carbocation of flavanols delivered ethyl-linked tannin-anthocyanin (T-A) and tannin-tannin (T-T) adducts, pyranoanthocyanins, and sulfonated indoles, while under reduction, the C-4 carbocation delivered direct linked T-A adducts, rearranged T-T adducts, and sulfonated tannins. Some of these last reactions could be related to the accumulation of reduced species, eventually ending with reductive off-odors.

Food intake biomarkers for berries and grapes.

Grapes and berries are two types of widely consumed fruits characterized by a high content in different phytochemicals. However, their accurate dietary assessment is particularly arduous, because of the already wide recognized bias associated with self-reporting methods, combined with the large range of species and cultivars and the fact that these fruits are popularly consumed not only in fresh and frozen forms but also as processed and derived products, including dried and canned fruits, beverages, jams, and jellies. Reporting precise type and/or quantity of grape and berries in FFQ or diaries can obviously be affected by errors. Recently, biomarkers of food intake (BFIs) rose as a promising tool to provide accurate information indicating consumption of certain food items. Protocols for performing systematic reviews in this field, as well as for assessing the validity of candidate BFIs have been developed within the Food Biomarker Alliance (FoodBAll) Project. This paper aims to evaluate the putative BIFs for blueberries, strawberries, raspberries, blackberries, cranberries, blackcurrant, and grapes. Candidate BFIs for grapes were resveratrol metabolites and tartaric acid. The metabolites considered as putative BFI for berries consumption were mostly anthocyanins derivatives together with several metabolites of ellagitannins and some aroma compounds. However, identification of BFIs for single berry types encountered more difficulties. In the absence of highly specific metabolites reported to date, we suggested some multi-metabolite panels that may be further investigated as putative biomarkers for some berry fruits.

Biomarkers of intake for tropical fruits.

Consumption of fruit and vegetable is a key component of a healthy and sustainable diet. However, their accurate dietary assessment remains a challenge. Due to errors in self-reporting methods, the available dietary information is usually biased. Biomarkers of intake constitute objective tools to better reflect the usual or recent consumption of different foods, including fruits and vegetables. Partners of The Food Biomarker Alliance (FoodBall) Project have undertaken the task of reviewing the available literature on putative biomarkers of tropical fruit intake. The identified candidate biomarkers were subject to validation evaluation using eight biological and chemical criteria. This publication presents the current knowledge on intake biomarkers for 17 tropical fruits including banana, mango, and avocado as the most widely consumed ones. Candidate biomarkers were found only for banana, avocado, and watermelon. An array of banana-derived metabolites has been reported in human biofluids, among which 5-hydroxyindole-acetic acid, dopamine sulfate, methoxyeugenol glucuronide, salsolinol sulfate, 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-ß-carboline-sulfate, and other catecholamine metabolites. Their validation is still at an early stage, with insufficient data on dose-response relationship. Perseitol and mannoheptulose have recently been reported as candidate biomarkers for avocado intake, while the amino acid citrulline has been associated with watermelon intake. Additionally, the examination of food composition data revealed some highly specific phytochemicals, which metabolites after absorption may be further studied as putative BFI for one or several tropical fruits. To make the field move forward, untargeted metabolomics, as a data-driven explorative approach, will have to be applied in both intervention and observational studies to discover putative BFIs, while their full validation and the establishment of dose-response calibration curves will require quantification methods at a later stage.

Towards an open grapevine information system.

Viticulture, like other fields of agriculture, is currently facing important challenges that will be addressed only through sustained, dedicated and coordinated research. Although the methods used in biology have evolved tremendously in recent years and now involve the routine production of large data sets of varied nature, in many domains of study, including grapevine research, there is a need to improve the findability, accessibility, interoperability and reusability (FAIR-ness) of these data. Considering the heterogeneous nature of the data produced, the transnational nature of the scientific community and the experience gained elsewhere, we have formed an open working group, in the framework of the International Grapevine Genome Program (www.vitaceae.org), to construct a coordinated federation of information systems holding grapevine data distributed around the world, providing an integrated set of interfaces supporting advanced data modeling, rich semantic integration and the next generation of data mining tools. To achieve this goal, it will be critical to develop, implement and adopt appropriate standards for data annotation and formatting. The development of this system, the GrapeIS, linking genotypes to phenotypes, and scientific research to agronomical and oeneological data, should provide new insights into grape biology, and allow the development of new varieties to meet the challenges of biotic and abiotic stress, environmental change, and consumer demand.

Antioxidant activity of phenolic acids and their metabolites: synthesis and antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic acid.

The main metabolites of caffeic and ferulic acids (ferulic acid-4'-O-sulfate, caffeic acid-4'-O-sulfate, and caffeic acid-3'-O-sulfate), the most representative phenolic acids in fruits and vegetables, and the acyl glucuronide of ferulic acid were synthesized, purified, and tested for their antioxidant activity in comparison with those of their parent compounds and other related phenolics. Both the ferric reducing antioxidant power (FRAP) assay and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging method were used. Ferulic acid-4'-O-sulfate and ferulic acid-4'-O-glucuronide exhibited very low antioxidant activity, while the monosulfate derivatives of caffeic acid were 4-fold less efficient as the antioxidant than caffeic acid. The acyl glucuronide of ferulic acid showed strong antioxidant action. The antioxidant activity of caffeic acid-3'-O-glucuronide and caffeic acid-4'-O-glucuronide was also studied. Our results demonstrate that some of the products of phenolic acid metabolism still retain strong antioxidant properties. Moreover, we first demonstrate the ex vivo synthesis of the acyl glucuronide of ferulic acid by mouse liver microsomes, in addition to the phenyl glucuronide.

Red wine prevents the postprandial increase in plasma cholesterol oxidation products: a pilot study.

Moderate wine consumption has been shown to lower cardiovascular risk. One of the mechanisms could involve the control of postprandial hyperlipaemia, a well-defined risk factor for atherosclerosis, reasonably by reducing the absorption of lipid oxidised species from the meal. The objective of the present study was to investigate whether wine consumption with the meal is able to reduce the postprandial increase in plasma lipid hydroperoxides and cholesterol oxidation products, in human subjects. In two different study sessions, twelve healthy volunteers consumed the same test meal rich in oxidised and oxidisable lipids (a double cheeseburger), with 300 ml of water (control) or with 300 ml of red wine (wine). The postprandial plasma concentration of cholesterol oxidation products was measured by GC-MS. The control meal induced a significant increase in the plasma concentration of lipid hydroperoxides and of two cholesterol oxidation products, 7-ß-hydroxycholesterol and 7-ketocholesterol. The postprandial increase in lipid hydroperoxides and cholesterol oxidation products was fully prevented by wine when consumed with the meal. In conclusion, the present study provides evidence that consumption of wine with the meal could prevent the postprandial increase in plasma cholesterol oxidation products.

Effective analysis of rotundone at below-threshold levels in red and white wines using solid-phase microextraction gas chromatography/tandem mass spectrometry.

Rotundone is an oxygenated sesquiterpene belonging to the family of guaianes, giving the 'peppery' aroma to white and black pepper and to red wines. Here we describe a novel, convenient protocol for the synthesis of rotundone, starting from a commercially available compound and requiring only two reaction steps, and an improved, faster method of GC separation (30 min) with selective quantisation of rotundone using tandem mass spectrometry in multiple reaction monitoring (MRM) mode with d(5)-rotundone as internal standard. With limits of detection (LODs) of 1.5 ng/L in white wine and 2.0 ng/L in red wine, intraday repeatability CV values of 6% and 5% at 50 ng/L and 500 ng/L and interday repeatability CV values of 13% and 6% at 50 ng/L and 500 ng/L, respectively, the improved protocol provides the desired sensitivity and selectivity for routine analysis of rotundone in both white and red wines. Initial application of this method highlighted the presence of unexpectedly high concentrations of rotundone, thus explaining the origin of the distinctive peppery aroma in Schioppettino and Vespolina red wines and in Gruener Veltliner white wines.

Stable free radicals as ubiquitous components of red wines.

A stable ESR signal, centred at g = 2.0037 +/- 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5-82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.

High-performance liquid chromatographic determination of the riboflavin concentration in white wines for predicting their resistance to light.

This research paper describes a new application in the field of quality control of white wine. A high-performance liquid chromatography analysis of riboflavin was used together with a simple sensorial test to produce a classification of wines, rating their susceptibility to exposure to light. Based on a wide survey covering 85 commercial white wines of different varieties in three countries (Italy, Spain and Slovenia), an average value of riboflavin of 98.63 microg/l with a rather high standard deviation of 41.91 microg/l, and a normal distribution was obtained. Our statistical study of the frequency distribution of the sensorial scores of light-exposed wines by means of the Expectation-Maximization algorithm demonstrated that a large majority (71%) of these products were susceptible to the light, 31% of them belonging to the most severely affected group. Content of riboflavin was correlated with severity of appearance of the off-flavor in light-exposed white wines. These methods are directly applicable in the quality control of wine, being a valuable aid for oenologists in choosing appropriate fining protocols to reduce the appearance of the undesired "sunlight flavor" in bottled products.

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.

Determination of indole-3-acetic acid, tryptophan and other indoles in must and wine by high-performance liquid chromatography with fluorescence detection.

The development of a robust method to analyse the content of tryptophan and of indole-3-acetic acid at the microgram per litre level in must and wine is necessary in order to study the formation of 2-aminoacetophenone and of other indole compounds causing the 'untypical ageing off-flavour'. The present paper discusses the development and validation of a reversed-phase high-performance liquid chromatography method with fluorescence detection for the analysis of indole-3-acetic acid, tryptophan, tryptophol, indole and skatole in must and white wine. The required selectivity and sensitivity was gained through the solid-phase extraction on a polystyrene-based polymer column.

Determination of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection.

A new direct HPLC method with fluorescence detection has been developed for the routine analysis of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide, in wines and other beverages. These compounds are the main agents responsible for the "taste of light" that some white wines and other beverages develop when they are exposed to the light, due to the formation of sulfur compounds that produce an anion/garlic odor. A Hewlett-Packard 1100 gradient liquid chromatograph with 1046A fluorescence detector was used. To improve the selectivity, each compound was monitored to fit the best lambda excitation/lambda emission (265/525 nm). A 500 nm cut-off filter was used. The column was a Hypersil C18 ODS, 200 x 2.1 mm, 5 microns particle size. The volume injected was 20 microliters. A constant flow-rate of 0.6 ml/min was used with two solvents: solvent A, 0.05 M buffer NaH2PO4 at pH = 3.0 with H3PO4 and solvent B, acetonitrile. The precision, linearity and sensitivity of this method have been established.

Solid phase extraction of trans-resveratrol from wines for HPLC analysis.

Some recent studies have put forward the hypothesis that the presence of trans-resveratrol (trans-3,5,4'-trihydroxystilbene) in red wine may be related to some of its therapeutic properties. A fundamental step in view of this evaluation is the development of a method for the quick, accurate and precise analysis of this compound. Sample enrichment and purification can be obtained by solid-phase extraction using reverse-phase C18 cartridges. HPLC analysis carried out by means of a photodiode-array detector, with an internal standard method, allows the detection of up to 10 micrograms/L in wine, with a linear range between 0.6 and 300 ng injected and a precision of 3.3%. The results of the first analyses show that the concentrations of trans-resveratrol in wines might be much higher than so far reported in the literature.