Evaluation of the Composition of Vine Shoots and Oak Chips forOenological Purposes by Superheated Liquid Extraction and High-Resolution Liquid ChromatographyminusTime-of-FlightMassSpectrometry AnalysisM Pilar Delgado de la Torre Feliciano Priego-Capote and Mariacutea Dolores Luque de Castro

Department of Analytical Chemistry Annex Marie Curie Building Campus of Rabanales and Institute of Biomedical ResearchMaimonides (IMIBIC) Reina Sofiacutea Hospital University of Cordoba Cordoba E-14071 Spain

S Supporting Information

ABSTRACT Vine shoots are characterized in this research and compared to oak chips frequently employed in the aging ofwine or spirits For this purpose liquid chromatographyminusdiode array detection and liquid chromatographyminustime-of-flightmassspectrometry (LC-TOFMS) analyses of hydroalcoholic extracts from vine shoots pertaining to 18 different vine varieties andfrom five varieties of oak chips have been carried out The concentrations of a representative panel of interesting compoundsfrom an oenological point of view have been compared in the extracts finding similarity patterns for many of them The analysisby LC-TOFMS in high accuracy mode has led to the identification of numerous compounds in the hydroalcoholic extracts Thestatistical analysis has enabled identification of the vine-shoot varieties providing extracts with more similar composition to thatgiven by extracts from oak chips Therefore these vine-shoots varieties are suitable to be presented as an alternative to the use ofoak barrels or oak chips in the aging process of wine and spirits

KEYWORDS vine-shoots extracts oak wood extracts superheated liquid extraction chemometric tools metabolic profile oenology

INTRODUCTIONSpain is the country with the largest area in the world dedicatedto vineyards where approximately 15 million tons of vineshoots are produced every year This fact has led to a growinginterest on exploitation of this agricultural residue with the aimof turning it into a valuable product Most research about vineshoots has been focused on the production of paper pulp1

ethanol lactic acid23 methanol fuels biomass biosurfactants2

and activated carbon for wine treatment4 extraction of volatilecompounds phenols56 and ferulic and coumaric acids7 amongothersPhenols can be obtained from vine shoots in two compatible

ways namely (a) by extracting the phenols that are notconstituents of lignin and (b) by degrading lignin to obtain lowmolecular weight phenols The composition of vine shoots ischaracterized by a lignin content around 20 (dry weight) Aslignin can be hydrolyzed to release aromatic phenoliccompounds such as low molecular mass alcohols aldehydesketones or acids vine shoots are suitable to be used as aphenols source In addition they could play a key role in theoenological or spirits field to improve quality acting similarly towine-aging agents either in contact with oak chips or in barrels8

In fact lignin oligomers or intermediate and high molecularmass phenols from lignin are considered the main phenols inold spirits and wine67

Traditionally oak barrels have been used for centuries tostore and age wine and other beverages since the sensorycomplexity of the substrate subjected to aging is increased asthe wood transfers to the liquid phase a series of aromaticcompounds9 Wine aging involves changes in color and

organoleptical properties which are highly appreciated by theconsumers10

Extraction of compounds of interest from oak barrelsdepends on the quantity of potentially extractable compoundsoriginally present in the barrel and on time-related factorsparticularly the contact time between the beverage subjected toaging and wood11minus13 During barrel aging wine or spirits arepartially enriched with lignin-derivative compounds formingcomplexes that are hydrolyzed to release aromatic aldehydeswhich undergo oxidation reactions generating aromatic acidsThus the most important phenolic acids and phenolicaldehydes present in these beverages come exclusively fromthe barrel woodEconomic grounds have led to the use of alternatives to oak

barrels This is the case of segments staves and morecommonly oak chips1214minus17 which provide results similar tothose obtained by barrel aging for several years9 The finalproduct is determined not only by the different characteristicsof the added oak wood portions such as their origin18 size andtoasting process but also by the dosage period of contact withwine and possible oxygenation of the aging product12

Superheated liquid extraction (SHLE) can be an attractiveindustrial alternative for isolation of these compounds as itpossesses two fundamental advantages over conventionalextraction The first one is ascribed to the fact that raisingthe temperature above the boiling point of the extractant (but

Received December 27 2011Revised March 13 2012Accepted March 14 2012Published March 14 2012

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keeping it under liquid state by increasing the pressure asrequired) increases the diffusion rate solubility and masstransfer of the compounds and decreases the viscosity andsurface tension of the extractant These changes improve thecontact of the compounds with the extractant and enhancemass transfer which can then be achieved more rapidly andwith less solvent consumption as compared with conventionalindustrial extraction methods Second the absence of light andair significantly reduces both degradation and oxidation of thesecompounds during extraction19

At this point and once the importance of wood in generaland lignin in particular is discussed in the aging of wine thecomparison of extracts obtained from oak wood and vineshoots would provide information about the possibilities ofusing vine shoots in the aging process either as a complementto oak barrels or as a cheaper and available alternative To thisend the different chemometric tools currently available play afundamental role in multivariate analysis to treat the largeamount of information provided by analytical techniques suchas liquid chromatographyminustime-of-flightmass spectrometry(LC-TOFMS) This powerful analytical arrangement actuallyprovides effective validation of the information obtained byother less complex techniques such as LCminusdiode arraydetection (DAD) and in addition it would also provide newinformation such as identification of unknown compoundsOn the basis of this background the objective of this study wasto compare the metabolic profile of extracts from vine shootsand oak chips and similaritydissimilarity patterns amongdifferent varieties of vine shoot and oak wood with the final aimof using these vineyard residues in the aging step

MATERIALS AND METHODSSamples Vine shoots from different Vitis vinifera cultivars were

sampled from Sierra de Segura (Spain) The studied cultivars were asfollows Airen Baladiacute Bobal Cabernet Franc Cabernet SauvignonChardonnay Garnacha Tinta Garnacha Tintorera Malbec MazueloMerlot Montepila Moscatel Pedro Ximenez Petit Verdot SauvignonBlanc Syrah and Tempranillo Five varieties of oak chips were studiedAmerican Blend (toasted) American Fresh French Sweet (toasted)French Spice and French Intense (toasted) All species were dried for72 h at 35 degC and then milled to get a homogeneous 40 mesh particlesize (less than 042 mm diameter)Reagents Ethanol (96 vv) PA from Panreac (Barcelona Spain)

and distilled water were used to prepare the extractant solutionMethanol (LC-MS grade) and formic acid (MS grade) (both suppliedby Panreac) were used to prepare the mobile phases Deionized water(18 MΩ cm) was obtained from a Millipore (Bedford MA) Milli-Qplus system and n-hexane (LiChrosolv Merck Darmstadt Germany)was used for liquidminusliquid extractionCalibration curves were constructed for the following phenols

(+)-catechin 5-hydroxymethylfurfural (5-hydroxymethyl-2-furancar-boxaldehyde) C6 phenols pyrogallol (123-trihydroxybenzene) andpyrocatechol (12-dihydroxybenzene) C6minusC1 phenols acetovanil-lone [1-(4-hydroxy-3-methoxyphenyl)-ethanone] vanillin (4-hydroxy-3-methoxybenzaldehyde) guaiacol (2-methoxyphenol) and gallic(345-trihydroxybenzoic acid) protocatechuic (34-dihydroxybenzoicacid) p-hydroxybenzoic vanillic (4-hydroxy-3-methoxybezoic acid)and syringic (4-hydroxy-35-dimethoxybenzoic acid) acids C6minusC3phenols coniferaldehyde (4-hydroxy-3-methoxycinnamaldehyde) si-napaldehyde (4-hydroxy-35-dimethoxycinnamaldehyde) and p-cou-maric (4-hydroxycinnamic acid) ferulic (4-hydroxy-3-methoxycin-namic acid) and sinapic (4-hydroxy-35-dimethoxycinnamic acid)acids and p-cresol (1-hydroxy-4-methylbenzene) used as externalstandard were from Sigma-Aldrich (St Louis MO)Apparatus Vine shoots were milled with a ball grinder (Restch

MM301 Haan Germany) SHLEs were performed by a laboratory-

made dynamic extractor11 consisting of the following units (a) anextractant supply (b) a high-pressure pump (Shimadzu LD-AC10)that propels the extractant through the system (c) a switching valveplaced next to the pump to develop static extractions (d) a stainlesssteel cylindrical extraction chamber (550 mm times 10 mm id 43 mLinternal volume) where the sample is placed This chamber is closed atboth ends with screws whose caps contain cotton-made filters toensure that the sample is not carried away by the extractant (e) arestriction valve to maintain the desired pressure in the system (f) acooler made of a stainless steel tube (1 m length 04 mm id) andrefrigerated with water and (g) a gas chromatograph oven (KonixCromatix KNK-2000) where the extraction chamber is placed andheated

Shaking and centrifugation of the extracts were carried out by anMS2 minishaker (IKA Germany) Vortex and a Mixtasel (SelectaBarcelona Spain) centrifuge respectively An R-220 rotary evaporatorfrom Buchi (Flawil Switzerland) working with a 50 mL balloon flaskwas used to concentrate the liquid extracts

Individual separation of phenolic compounds and carbohydratederivatives was carried out by an LC consisting of a ProStar 410autosampler equipped with a 05 mL sample loop (Varian Palo AltoCA) connected online with an LC pump (Varian 240 pump) A 330Varian PDA detector was used to monitor the chromatographic eluateat the optimal wavelength for each analyte Data processing was carriedout using the Star Chromatography Workstation version 552 softwarerunning on a personal computer

The Polyview-2000 software (Varian) was used for both character-ization of the spectra and assessment of peak purity This softwareallows examination and analysis of spectra including plots of purityparameter setting of absorbance ratios and determination ofmaximum absorbance Determination of the purity of chromatographicpeaks and recalculation of the peak at different wavelengths andintegration parameters which allow exchange signal-to-noise ratio in adiode array data file are also provided by this software

Extraction of Phenolic Compounds and Treatment ofExtracts Extracts of vine shoots and oak wood were performed bySHLE as described in ref 15 where the extractant used was 8020 (vv) ethanolminuswater at pH 3 with an extraction time of 1 h and anextraction temperature of 180 degC The extracts were dried in a rotaryevaporator and then reconstituted in 5 mL of methanol Thereconstituted solutions were subjected to liquidminusliquid extractionwith n-hexane (10 mL 5 min of shaking and 6 min of centrifugation at855g) to remove nonpolar compounds which could complicate thechromatographic separation Preconcentration of the methanolic phasewas attained by evaporation of 2 mL of it to a final volume of 200 μLFinally this fraction was taken up to 650 μL with milli-Q water andfiltered using a 045 μm pore size filter before injection into thechromatograph

LC-DAD Analysis Separation of the analytes was performed on anInertsil ODS-2 column (250 mm times 46 mm id 5 μm particle AnalisisViacutenicos Tomelloso Ciudad Real Spain) using an injection volume of20 μL and a flow rate of 1 mLmin A mobile phase A consisting of02 (vv) phosphoric acid aqueous solution and a mobile phase Bconsisting of methanol were used The gradient method was asfollows from 96 to 82 mobile phase A in 20 min held for 20 minfrom 82 to 74 mobile phase A in 24 min and from 74 to 50 mobilephase B in 9 min The analytes were identified by comparing boththeir retention times (RTs) and UV spectra with those of thecorresponding standards The absorption wavelengths were set at 260nm for monitoring ellagic acid at 280 nm for hydroxybenzoic acidscatechin and phenolic aldehydes at 320 nm for hydroxycinnamicacids and at 360 nm for hydroxycinnamic aldehydes

LC-TOFMS Confirmatory Analysis The analyses conductedwith a view to confirming the identity of the studied compounds wereperformed in an Agilent 1200 Series LC system (consisting of a binarypump a vacuum degasser an autosampler and a thermostattedcolumn compartment) interfaced to an Agilent 6540 UHD Accurate-Mass LC-TOFMS detector (Palo Alto CA) equipped with anAgilent Jet Stream Technology electrospray ion source operating inthe negative and positive ion mode

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Chromatographic separation was performed using an Inertsil ODS-2 column (250 mm times 46 mm id 5 μm particle Analisis ViacutenicosTomelloso Ciudad Real Spain) kept at a temperature of 25 degCMobile phases were water (phase A) and acetonitrile (phase B) bothLC-MSMS grade and with 01 formic acid as the ionization agentThe LC pump was programmed with a flow rate of 1 mLmin and thefollowing gradient elution was carried out from 4 to 18 mobilephase B in 20 min held for 20 min from 18 to 26 mobile phase B in44 min from 26 to 50 mobile phase B in 26 min and from 50 to100 phase B in 30 min The injection volume was 10 μL and theinjector needle was rinsed five times with 70 methanol Furthermorethe needle seat back was flushed for 12 s at a flow rate of 4 mLminwith 70 methanol to clean itThe operating conditions of the mass spectrometer were as follows

gas temperature 350 degC drying gas nitrogen at 10 Lmin nebulizerpressure 35 psi sheath gas temperature 380 degC sheath gas flownitrogen at 10 Lmin capillary voltage 3250 V skimmer 65 Voctopole radiofrequency voltage 750 V and focusing voltage 90 VData acquisition (25 Hz) in both the centroid and the profile modeswas governed via the Agilent MassHunter Workstation software Theinstrument was operated in MS-high accuracy mode The mass rangeand detection window were set at mz 100minus1100 and 100 ppmrespectively The instrument was calibrated and tuned according toprocedures recommended by the manufacturer To ensure the desiredmass accuracy of recorded ions continuous internal calibration wasperformed during analyses with the use of signals at mz 1210509(protonated purine) and mz 9220098 [protonated hexakis (1H 1H3H-tetrafluoropropoxy)phosphazine or HP-921] in positive ion modein negative ion mode ions with mz 1190362 (proton abstractedpurine) and mz 966000725 (formate adduct of HP-921) were usedData Processing and Statistical Analysis MassHunter Work-

station software (version 301 Qualitative Analysis Agilent Tech-nologies Santa Clara CA) was used for processing all data obtainedwith LC-TOFMS in full single MS mode The feature extractionalgorithm took into account all ions exceeding 5000 counts with acharge state equal or above to one and a feature had to be composedof two or more ions to be valid (eg two ions in the isotope cluster)Within the algorithm employed for full single MS data ions withidentical elution profiles and related mz values (representing differentadducts or isotopes of the same compound) were extracted asmolecular features (MFs) or entities characterized by RT intensity inapex of chromatographic peak and accurate mass Various intensitythreshold settings ranging from 3000 to 15000 counts per second(cps) were tested for the MFs extraction in the whole RT range Filesin compound exchange format (cef files) were created for each sampleand exported into the Mass Profiler Professional (MPP) softwarepackage (version 20 Agilent Technologies) for further processing Inthe next step alignment of RT and mz values was carried out acrossthe sample set using a tolerance window of 02 min in RT and 5 ppmmass accuracy Baseline correction eliminated the contribution ofbackground noise Stepwise reduction of MFs number was performedbased on frequency of occurrence abundance of the respective MFs inclasses and PCA results of the data MPP software also enabled one-way analysis of variance (ANOVA) The data were mean centered as a

data pretreatment to lower relatively large differences among therespective MFs abundances

RESULTS AND DISCUSSION

Composition of Extracts from Different Varieties ofVine Shoot and Oak Wood by LC-DAD Analysis Eighteenvine-shoot varieties and five types of oak chips were subjectedto SHLE under optimum operation conditions to evaluate thecontent of interesting compounds from an oenological point ofview Oak samples could be grouped as follows intense oaksweet oak and spice oak for the French variety and blend oakand fresh oak for the American variety Also three of them weretoasted oak chips particularly American Blend French Sweetand French Intense Table 1 lists the concentrations of a panelof representative compounds in extracts from vine shootswhich are in common with those in oak wood chips used inoenologyFirst hydroxymethylfurfural a degradation product from

hexoses was found in all extracts which reported similar levelsof this furanic aldehyde responsible for light creamy toast andtoffee flavors in vine shoots and oak chips as can be seen inTable 1 Taking into account the content of cellulose andhemicellulose in vine shoots furanic compounds are a family ofcompounds to be taken into account The extractiontemperature 180 degC foreseeably enhances the degradation ofsugars released from vine-shoots wood and promotes theformation of furans20 There is a controversy about the interestof these compounds The contribution of two furaniccompounds such as furfural and hydroxymethylfurfural toflavoring in processed food by heating is well-known21

However several international organisms such as the Foodand Drug Administration in United States (US FDA) haveexamined furans not only as flavor compounds but also asnovel harmful substances in foods that undergo a thermaltreatment The European Food Safety Authority (EFSA) hasarticulated that furan is obviously carcinogenic in rats and miceprobably due to the combination of a genotoxic mechanism2223

and hepatotoxicity24 From a safety perspective and for foodquality assurance the EC Regulation No 149399 sets up amaximum limit for hydroxymethylfurfural of 25 mgkg inconcentrated rectified grape must With these premises furanscontents in extracts should be controlled for a properexploitation of the latter Anyway attending to the concen-trations of the extracts (Table 1) an enrichment onhydroxymethylfurfural that surpasses legal established limits inspirits or wine would not be a shortcomingOne of the most characteristic phenolic compounds found in

extracts from all varieties of vine shoots was gallic acid a finalproduct from the hydrolysis of ellagitannins responsible for the

Table 1 Concentrations of a Panel of Interesting Compounds in Extracts by SHLE from Oak Wood Chips and the AveragedConcentration Found for Each Compound in the Different Varieties of Vine Shoots Expressed as μgg Vine Shootsa

Pg GAc Hf Py PAc HAc C VAc G V SAc Av CAc FAc Cf SAc S

French Intense oak 2618 121 126 81 61 66 154 340 ND 259 505 ND 282 52 130 ND 3597French Sweet oak 5951 275 94 55 82 166 643 298 66 163 316 ND 232 ND 282 ND 7390American Blend oak 999 45 92 29 79 16 1436 143 D 214 178 ND 236 19 143 ND 4017American Fresh oak 8489 393 227 77 176 ND 2163 102 8 87 48 ND 41 34 43 4911 953French Spice oak 6680 309 144 89 38 138 599 58 ND ND 133 171 83 Nd 202 Nd 2896vine-shoots media 6756 313 385 121 171 41 3936 78 86 30 45 21 22 269 26 1155 239aD detected ND not detected Pg pyrogallol GAc gallic acid Hf hydroxymethylfurfural Py pyrocatechol PAc protocatechuic acid HAchydroxybenzoic acid C catechin VAc vanillic acid G guaiacol V vanillin SAc syringic acid Av acetovanillone CAc coumaric acid FAc ferulicacid Cf coniferaldehyde SAc sinapic acid and S sinapaldehyde

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astringency character of wines and was found in aconcentration similar to that in extracts from oak woodPyrogallol formed from gallic acid decarboxylation wasdetected in all extracts with similar concentrations There wasa similarity between concentrations of pyrogallol and gallic acidamong vine-shoots varieties demonstrating that both com-pounds are connected through a biochemical pathway Thesame behavior was not found in the case of protocatechuic acidand pyrocatechol (decarboxylated products of protocatechuicacid) The pyrocatecholprotocatechuic acid pair was moreconcentrated in extracts from vine shoots than in those fromany oak wood variety except for the American Fresh oakvariety The same situation was found for the guaiacolvanillicacid pair The concentration of vanillic acid in oak extracts washigher than that found in any vine-shoot variety except for theFrench Spice oak class Guaiacol was exclusively detected intwo oak varieties French Sweet oak and American Fresh oakThe same behavior as guaiacol was found for vanillin whichwas slightly more concentrated in oak wood (except FrenchSpice oak) than in extracts from vine shoots (Table 1) Thiscompound is of special interest because of its contribution toflavorCatechin the building block for tannins synthesis was found

at significant concentrations which is indicative of an importanteffect of the extraction process on the hydrolysis of tanninsThe maximum concentration of catechin in extracts from oakwood was found in the American class with 14 and 21 mggfor fresh oak and blend oak woods respectivelyThe rest of phenolic acids reported similar concentrations in

extracts from vine shoots and those from oak wood thereforeno discrimination was possible attending to the concentrationof these compounds Acetovanillone was detected in allvarieties of vine shoots while this compoundwith a highflavor contributionwas only detected in extract from FrenchSpice oak chips Other compounds with organolepticalincidence were coniferaldehyde and sinapaldehyde which

were detected at lower concentrations in extracts from vineshoots particularly in the case of sinapaldehyde as can be seenin Table 1

LC-TOFMS Analysis of Vine-Shoot Extracts The LC-TOFMS analysis provided a global profile of polar or midpolarcompounds present in extracts by SHLE that represent acharacteristic fingerprinting of each variety Comparisonbetween them enables one to evaluate the similaritydissimilarity patterns among cultivars considering the completedata set representative of the extracts composition Addition-ally the identification of characteristic compounds for eachvariety is allowed which is not accessible by LC-DAD Thisinformation could lead to the selection of a panel ofcompounds with interest from the oenological point of viewwhich would enable one to select the cultivar varieties forcollection of vine shoots to extract the target compounds Inthis study nontargeted analysis of extracts was performed Thereverse-phase chromatographic method selected for thisresearch enabled separation of a wide range of compoundspresent in the extracts A representative example is given inFigure 1 that illustrates the base peak chromatograms (BPCs)corresponding to the analysis in positive and negativeionization modes of extract isolated from Garnacha Tintoreravine shootsAs described in the Materials and Methods MFs or entities

were extracted to compare vine-shoot cultivars in bothionization modes The range of MFs extracted for the differentvarieties encompasses from 90 to 422 for positive ionizationmode and from 310 to 1138 for negative ionization modeIdentification of MFs in both ionization modes was supportedon online searching of monoisotopic masses corresponding tomolecular entities found in the analyses to PlantCyc databaseMonoisotopic masses included in this database were searchedin the raw data files obtained by the analysis of extracts fromvine-shoot varieties Search parameters were mass accuracycutoff below 10 ppm minimum peaks height of 2500 counts on

Figure 1 BPCs in positive (A) and negative (B) ionization modes by LC-TOFMS analysis of an extract by SHLE from Garnacha Tintorera vineshoot

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the profile and centroid spectra and a peak spacing tolerance of00025 mz plus 7 ppm Table 1 in the Supporting Informationlists the compounds identified in the extracts from GarnachaTintorera Merlot and Syrah vine-shoot cultivars thusdemonstrating their different composition

Qualitative Comparison of LC-TOFMS FingerprintsCorresponding to Vine-Shoot Extracts Attending to thecomplexity of the obtained data sets a strategy was designed tosimplify the dimensionality of the multivariate matrix The firstalgorithm employed for this purpose was the application of a

Figure 2 PCA scores plot of the metabolite profiles obtained for different vine-shoot varieties in positive (A) and negative (B) ionization modesThe original data set was filtered by frequency eliminating MFs not detected in at least 80 of the samples pertaining to each class Baladiacute CabernetFranc Montepila Syrah Merlot Bobal Pedro-Ximenez Garnacha Tintorera and Petit Verdot (B CF MP S M BO PX G and PV)

Figure 3 PCA scores plot resulting from the data set obtained in negative ionization mode after one-way ANOVA with (A) 985 and (B) 99confidence level Moscatel Chardonnay Syrah Pedro-Ximenez Merlot Garnacha Tintorera Baladiacute and Petit Verdot (MG CH S PX MP M GB and PV)

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frequency filter which involved the elimination of those MFsabsent in at least 20 of the samples In this way comparabilityof vine-shoot extracts is based on two fractions one of themcommon to all extracts and another one that could becharacteristic of each variety This data mining involved thereduction of the MFs to 68 in the negative mode and 190 in thepositive mode as the pretreated data set After this pretreat-ment the data matrix was mean-centered prior to statisticalanalysis which was initiated by nonsupervised analysis usingPCA both to evaluate the distribution of the samples and todetect clusters in the new space defined by principalcomponents Figure 2 shows the scores plots obtained fordata matrices acquired in the positive and negative ionizationmodes The variability explained by PC1 PC2 and PC3 was8968 and 8750 in positive and negative ionization modesrespectively As can be seen there is a random distribution ofthe vine-shoot varieties but some trends can be observedThere is a main cluster grouping most of the varieties but alsosome extracts present differences in their composition Thus inthe positive ionization mode extracts from Bobal MontepilaSyrah Merlot and Cabernet Franc (B MP S M and CF)provided significant composition differences whereas thenegative ionization mode revealed differences in thecomposition of extracts from Montepila Merlot SyrahBobal and Pedro-Ximenez (MP M S BO and PX) vine

shoots The BPCs obtained from these extracts (and theirreplicates) do not reveal anomalous behaviors that could justifytheir labeling as laboratory outliers and for this reason thediscrimination should be linked to their compositionAfter this preliminary statistical analysis the next objective

was to apply an ANOVA test to identify MFs with the highestcontribution to explain the variability observed in PCA graphsThe MannminusWhitney test against zero was employed forestimation the p value computation was asymptotic and nomultiple testing correction was performed A key parameter tobe defined was the confidence level to set the number offeatures Thus the higher the confidence level the lowernumber of MFs A 95 confidence level involved a reductionfrom 68 and 190 to 47 and 27 MFs for the positive andnegative ionization modes respectively A 985 confidencelevel filtered MFs to 10 and 11 in the negative and positiveionization modes (Figure 3A) while a 99 confidence levelreduced them to 8 in global terms (Figure 3B)This test allowed labeling of the most significant features

contributing to explain the variability visualized for compositionof extracts from vine-shoot varieties Figure 3 shows the PCAscores plots resulting from the data set obtained after ANOVAwith a 985 confidence level which limited the number ofMFs to 10 Attending to this panel of compounds a maincluster was observed with other two additional groupings one

Figure 4 PCA scores plot for positive ionization mode using the original data set after filtration of those MFs not present in all samples belonging toeach class vine shoots or oak wood As can be seen there is a clear discrimination between extracts from oak wood (diamonds) and vine shoots(circles)

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formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

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Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

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Chromatographic separation was performed using an Inertsil ODS-2 column (250 mm times 46 mm id 5 μm particle Analisis ViacutenicosTomelloso Ciudad Real Spain) kept at a temperature of 25 degCMobile phases were water (phase A) and acetonitrile (phase B) bothLC-MSMS grade and with 01 formic acid as the ionization agentThe LC pump was programmed with a flow rate of 1 mLmin and thefollowing gradient elution was carried out from 4 to 18 mobilephase B in 20 min held for 20 min from 18 to 26 mobile phase B in44 min from 26 to 50 mobile phase B in 26 min and from 50 to100 phase B in 30 min The injection volume was 10 μL and theinjector needle was rinsed five times with 70 methanol Furthermorethe needle seat back was flushed for 12 s at a flow rate of 4 mLminwith 70 methanol to clean itThe operating conditions of the mass spectrometer were as follows

gas temperature 350 degC drying gas nitrogen at 10 Lmin nebulizerpressure 35 psi sheath gas temperature 380 degC sheath gas flownitrogen at 10 Lmin capillary voltage 3250 V skimmer 65 Voctopole radiofrequency voltage 750 V and focusing voltage 90 VData acquisition (25 Hz) in both the centroid and the profile modeswas governed via the Agilent MassHunter Workstation software Theinstrument was operated in MS-high accuracy mode The mass rangeand detection window were set at mz 100minus1100 and 100 ppmrespectively The instrument was calibrated and tuned according toprocedures recommended by the manufacturer To ensure the desiredmass accuracy of recorded ions continuous internal calibration wasperformed during analyses with the use of signals at mz 1210509(protonated purine) and mz 9220098 [protonated hexakis (1H 1H3H-tetrafluoropropoxy)phosphazine or HP-921] in positive ion modein negative ion mode ions with mz 1190362 (proton abstractedpurine) and mz 966000725 (formate adduct of HP-921) were usedData Processing and Statistical Analysis MassHunter Work-

station software (version 301 Qualitative Analysis Agilent Tech-nologies Santa Clara CA) was used for processing all data obtainedwith LC-TOFMS in full single MS mode The feature extractionalgorithm took into account all ions exceeding 5000 counts with acharge state equal or above to one and a feature had to be composedof two or more ions to be valid (eg two ions in the isotope cluster)Within the algorithm employed for full single MS data ions withidentical elution profiles and related mz values (representing differentadducts or isotopes of the same compound) were extracted asmolecular features (MFs) or entities characterized by RT intensity inapex of chromatographic peak and accurate mass Various intensitythreshold settings ranging from 3000 to 15000 counts per second(cps) were tested for the MFs extraction in the whole RT range Filesin compound exchange format (cef files) were created for each sampleand exported into the Mass Profiler Professional (MPP) softwarepackage (version 20 Agilent Technologies) for further processing Inthe next step alignment of RT and mz values was carried out acrossthe sample set using a tolerance window of 02 min in RT and 5 ppmmass accuracy Baseline correction eliminated the contribution ofbackground noise Stepwise reduction of MFs number was performedbased on frequency of occurrence abundance of the respective MFs inclasses and PCA results of the data MPP software also enabled one-way analysis of variance (ANOVA) The data were mean centered as a

data pretreatment to lower relatively large differences among therespective MFs abundances

RESULTS AND DISCUSSION

Composition of Extracts from Different Varieties ofVine Shoot and Oak Wood by LC-DAD Analysis Eighteenvine-shoot varieties and five types of oak chips were subjectedto SHLE under optimum operation conditions to evaluate thecontent of interesting compounds from an oenological point ofview Oak samples could be grouped as follows intense oaksweet oak and spice oak for the French variety and blend oakand fresh oak for the American variety Also three of them weretoasted oak chips particularly American Blend French Sweetand French Intense Table 1 lists the concentrations of a panelof representative compounds in extracts from vine shootswhich are in common with those in oak wood chips used inoenologyFirst hydroxymethylfurfural a degradation product from

hexoses was found in all extracts which reported similar levelsof this furanic aldehyde responsible for light creamy toast andtoffee flavors in vine shoots and oak chips as can be seen inTable 1 Taking into account the content of cellulose andhemicellulose in vine shoots furanic compounds are a family ofcompounds to be taken into account The extractiontemperature 180 degC foreseeably enhances the degradation ofsugars released from vine-shoots wood and promotes theformation of furans20 There is a controversy about the interestof these compounds The contribution of two furaniccompounds such as furfural and hydroxymethylfurfural toflavoring in processed food by heating is well-known21

However several international organisms such as the Foodand Drug Administration in United States (US FDA) haveexamined furans not only as flavor compounds but also asnovel harmful substances in foods that undergo a thermaltreatment The European Food Safety Authority (EFSA) hasarticulated that furan is obviously carcinogenic in rats and miceprobably due to the combination of a genotoxic mechanism2223

and hepatotoxicity24 From a safety perspective and for foodquality assurance the EC Regulation No 149399 sets up amaximum limit for hydroxymethylfurfural of 25 mgkg inconcentrated rectified grape must With these premises furanscontents in extracts should be controlled for a properexploitation of the latter Anyway attending to the concen-trations of the extracts (Table 1) an enrichment onhydroxymethylfurfural that surpasses legal established limits inspirits or wine would not be a shortcomingOne of the most characteristic phenolic compounds found in

extracts from all varieties of vine shoots was gallic acid a finalproduct from the hydrolysis of ellagitannins responsible for the

Table 1 Concentrations of a Panel of Interesting Compounds in Extracts by SHLE from Oak Wood Chips and the AveragedConcentration Found for Each Compound in the Different Varieties of Vine Shoots Expressed as μgg Vine Shootsa

Pg GAc Hf Py PAc HAc C VAc G V SAc Av CAc FAc Cf SAc S

French Intense oak 2618 121 126 81 61 66 154 340 ND 259 505 ND 282 52 130 ND 3597French Sweet oak 5951 275 94 55 82 166 643 298 66 163 316 ND 232 ND 282 ND 7390American Blend oak 999 45 92 29 79 16 1436 143 D 214 178 ND 236 19 143 ND 4017American Fresh oak 8489 393 227 77 176 ND 2163 102 8 87 48 ND 41 34 43 4911 953French Spice oak 6680 309 144 89 38 138 599 58 ND ND 133 171 83 Nd 202 Nd 2896vine-shoots media 6756 313 385 121 171 41 3936 78 86 30 45 21 22 269 26 1155 239aD detected ND not detected Pg pyrogallol GAc gallic acid Hf hydroxymethylfurfural Py pyrocatechol PAc protocatechuic acid HAchydroxybenzoic acid C catechin VAc vanillic acid G guaiacol V vanillin SAc syringic acid Av acetovanillone CAc coumaric acid FAc ferulicacid Cf coniferaldehyde SAc sinapic acid and S sinapaldehyde

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astringency character of wines and was found in aconcentration similar to that in extracts from oak woodPyrogallol formed from gallic acid decarboxylation wasdetected in all extracts with similar concentrations There wasa similarity between concentrations of pyrogallol and gallic acidamong vine-shoots varieties demonstrating that both com-pounds are connected through a biochemical pathway Thesame behavior was not found in the case of protocatechuic acidand pyrocatechol (decarboxylated products of protocatechuicacid) The pyrocatecholprotocatechuic acid pair was moreconcentrated in extracts from vine shoots than in those fromany oak wood variety except for the American Fresh oakvariety The same situation was found for the guaiacolvanillicacid pair The concentration of vanillic acid in oak extracts washigher than that found in any vine-shoot variety except for theFrench Spice oak class Guaiacol was exclusively detected intwo oak varieties French Sweet oak and American Fresh oakThe same behavior as guaiacol was found for vanillin whichwas slightly more concentrated in oak wood (except FrenchSpice oak) than in extracts from vine shoots (Table 1) Thiscompound is of special interest because of its contribution toflavorCatechin the building block for tannins synthesis was found

at significant concentrations which is indicative of an importanteffect of the extraction process on the hydrolysis of tanninsThe maximum concentration of catechin in extracts from oakwood was found in the American class with 14 and 21 mggfor fresh oak and blend oak woods respectivelyThe rest of phenolic acids reported similar concentrations in

extracts from vine shoots and those from oak wood thereforeno discrimination was possible attending to the concentrationof these compounds Acetovanillone was detected in allvarieties of vine shoots while this compoundwith a highflavor contributionwas only detected in extract from FrenchSpice oak chips Other compounds with organolepticalincidence were coniferaldehyde and sinapaldehyde which

were detected at lower concentrations in extracts from vineshoots particularly in the case of sinapaldehyde as can be seenin Table 1

LC-TOFMS Analysis of Vine-Shoot Extracts The LC-TOFMS analysis provided a global profile of polar or midpolarcompounds present in extracts by SHLE that represent acharacteristic fingerprinting of each variety Comparisonbetween them enables one to evaluate the similaritydissimilarity patterns among cultivars considering the completedata set representative of the extracts composition Addition-ally the identification of characteristic compounds for eachvariety is allowed which is not accessible by LC-DAD Thisinformation could lead to the selection of a panel ofcompounds with interest from the oenological point of viewwhich would enable one to select the cultivar varieties forcollection of vine shoots to extract the target compounds Inthis study nontargeted analysis of extracts was performed Thereverse-phase chromatographic method selected for thisresearch enabled separation of a wide range of compoundspresent in the extracts A representative example is given inFigure 1 that illustrates the base peak chromatograms (BPCs)corresponding to the analysis in positive and negativeionization modes of extract isolated from Garnacha Tintoreravine shootsAs described in the Materials and Methods MFs or entities

were extracted to compare vine-shoot cultivars in bothionization modes The range of MFs extracted for the differentvarieties encompasses from 90 to 422 for positive ionizationmode and from 310 to 1138 for negative ionization modeIdentification of MFs in both ionization modes was supportedon online searching of monoisotopic masses corresponding tomolecular entities found in the analyses to PlantCyc databaseMonoisotopic masses included in this database were searchedin the raw data files obtained by the analysis of extracts fromvine-shoot varieties Search parameters were mass accuracycutoff below 10 ppm minimum peaks height of 2500 counts on

Figure 1 BPCs in positive (A) and negative (B) ionization modes by LC-TOFMS analysis of an extract by SHLE from Garnacha Tintorera vineshoot

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the profile and centroid spectra and a peak spacing tolerance of00025 mz plus 7 ppm Table 1 in the Supporting Informationlists the compounds identified in the extracts from GarnachaTintorera Merlot and Syrah vine-shoot cultivars thusdemonstrating their different composition

Qualitative Comparison of LC-TOFMS FingerprintsCorresponding to Vine-Shoot Extracts Attending to thecomplexity of the obtained data sets a strategy was designed tosimplify the dimensionality of the multivariate matrix The firstalgorithm employed for this purpose was the application of a

Figure 2 PCA scores plot of the metabolite profiles obtained for different vine-shoot varieties in positive (A) and negative (B) ionization modesThe original data set was filtered by frequency eliminating MFs not detected in at least 80 of the samples pertaining to each class Baladiacute CabernetFranc Montepila Syrah Merlot Bobal Pedro-Ximenez Garnacha Tintorera and Petit Verdot (B CF MP S M BO PX G and PV)

Figure 3 PCA scores plot resulting from the data set obtained in negative ionization mode after one-way ANOVA with (A) 985 and (B) 99confidence level Moscatel Chardonnay Syrah Pedro-Ximenez Merlot Garnacha Tintorera Baladiacute and Petit Verdot (MG CH S PX MP M GB and PV)

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frequency filter which involved the elimination of those MFsabsent in at least 20 of the samples In this way comparabilityof vine-shoot extracts is based on two fractions one of themcommon to all extracts and another one that could becharacteristic of each variety This data mining involved thereduction of the MFs to 68 in the negative mode and 190 in thepositive mode as the pretreated data set After this pretreat-ment the data matrix was mean-centered prior to statisticalanalysis which was initiated by nonsupervised analysis usingPCA both to evaluate the distribution of the samples and todetect clusters in the new space defined by principalcomponents Figure 2 shows the scores plots obtained fordata matrices acquired in the positive and negative ionizationmodes The variability explained by PC1 PC2 and PC3 was8968 and 8750 in positive and negative ionization modesrespectively As can be seen there is a random distribution ofthe vine-shoot varieties but some trends can be observedThere is a main cluster grouping most of the varieties but alsosome extracts present differences in their composition Thus inthe positive ionization mode extracts from Bobal MontepilaSyrah Merlot and Cabernet Franc (B MP S M and CF)provided significant composition differences whereas thenegative ionization mode revealed differences in thecomposition of extracts from Montepila Merlot SyrahBobal and Pedro-Ximenez (MP M S BO and PX) vine

shoots The BPCs obtained from these extracts (and theirreplicates) do not reveal anomalous behaviors that could justifytheir labeling as laboratory outliers and for this reason thediscrimination should be linked to their compositionAfter this preliminary statistical analysis the next objective

was to apply an ANOVA test to identify MFs with the highestcontribution to explain the variability observed in PCA graphsThe MannminusWhitney test against zero was employed forestimation the p value computation was asymptotic and nomultiple testing correction was performed A key parameter tobe defined was the confidence level to set the number offeatures Thus the higher the confidence level the lowernumber of MFs A 95 confidence level involved a reductionfrom 68 and 190 to 47 and 27 MFs for the positive andnegative ionization modes respectively A 985 confidencelevel filtered MFs to 10 and 11 in the negative and positiveionization modes (Figure 3A) while a 99 confidence levelreduced them to 8 in global terms (Figure 3B)This test allowed labeling of the most significant features

contributing to explain the variability visualized for compositionof extracts from vine-shoot varieties Figure 3 shows the PCAscores plots resulting from the data set obtained after ANOVAwith a 985 confidence level which limited the number ofMFs to 10 Attending to this panel of compounds a maincluster was observed with other two additional groupings one

Figure 4 PCA scores plot for positive ionization mode using the original data set after filtration of those MFs not present in all samples belonging toeach class vine shoots or oak wood As can be seen there is a clear discrimination between extracts from oak wood (diamonds) and vine shoots(circles)

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formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

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Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

astringency character of wines and was found in aconcentration similar to that in extracts from oak woodPyrogallol formed from gallic acid decarboxylation wasdetected in all extracts with similar concentrations There wasa similarity between concentrations of pyrogallol and gallic acidamong vine-shoots varieties demonstrating that both com-pounds are connected through a biochemical pathway Thesame behavior was not found in the case of protocatechuic acidand pyrocatechol (decarboxylated products of protocatechuicacid) The pyrocatecholprotocatechuic acid pair was moreconcentrated in extracts from vine shoots than in those fromany oak wood variety except for the American Fresh oakvariety The same situation was found for the guaiacolvanillicacid pair The concentration of vanillic acid in oak extracts washigher than that found in any vine-shoot variety except for theFrench Spice oak class Guaiacol was exclusively detected intwo oak varieties French Sweet oak and American Fresh oakThe same behavior as guaiacol was found for vanillin whichwas slightly more concentrated in oak wood (except FrenchSpice oak) than in extracts from vine shoots (Table 1) Thiscompound is of special interest because of its contribution toflavorCatechin the building block for tannins synthesis was found

at significant concentrations which is indicative of an importanteffect of the extraction process on the hydrolysis of tanninsThe maximum concentration of catechin in extracts from oakwood was found in the American class with 14 and 21 mggfor fresh oak and blend oak woods respectivelyThe rest of phenolic acids reported similar concentrations in

extracts from vine shoots and those from oak wood thereforeno discrimination was possible attending to the concentrationof these compounds Acetovanillone was detected in allvarieties of vine shoots while this compoundwith a highflavor contributionwas only detected in extract from FrenchSpice oak chips Other compounds with organolepticalincidence were coniferaldehyde and sinapaldehyde which

were detected at lower concentrations in extracts from vineshoots particularly in the case of sinapaldehyde as can be seenin Table 1

LC-TOFMS Analysis of Vine-Shoot Extracts The LC-TOFMS analysis provided a global profile of polar or midpolarcompounds present in extracts by SHLE that represent acharacteristic fingerprinting of each variety Comparisonbetween them enables one to evaluate the similaritydissimilarity patterns among cultivars considering the completedata set representative of the extracts composition Addition-ally the identification of characteristic compounds for eachvariety is allowed which is not accessible by LC-DAD Thisinformation could lead to the selection of a panel ofcompounds with interest from the oenological point of viewwhich would enable one to select the cultivar varieties forcollection of vine shoots to extract the target compounds Inthis study nontargeted analysis of extracts was performed Thereverse-phase chromatographic method selected for thisresearch enabled separation of a wide range of compoundspresent in the extracts A representative example is given inFigure 1 that illustrates the base peak chromatograms (BPCs)corresponding to the analysis in positive and negativeionization modes of extract isolated from Garnacha Tintoreravine shootsAs described in the Materials and Methods MFs or entities

were extracted to compare vine-shoot cultivars in bothionization modes The range of MFs extracted for the differentvarieties encompasses from 90 to 422 for positive ionizationmode and from 310 to 1138 for negative ionization modeIdentification of MFs in both ionization modes was supportedon online searching of monoisotopic masses corresponding tomolecular entities found in the analyses to PlantCyc databaseMonoisotopic masses included in this database were searchedin the raw data files obtained by the analysis of extracts fromvine-shoot varieties Search parameters were mass accuracycutoff below 10 ppm minimum peaks height of 2500 counts on

Figure 1 BPCs in positive (A) and negative (B) ionization modes by LC-TOFMS analysis of an extract by SHLE from Garnacha Tintorera vineshoot

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173412

the profile and centroid spectra and a peak spacing tolerance of00025 mz plus 7 ppm Table 1 in the Supporting Informationlists the compounds identified in the extracts from GarnachaTintorera Merlot and Syrah vine-shoot cultivars thusdemonstrating their different composition

Qualitative Comparison of LC-TOFMS FingerprintsCorresponding to Vine-Shoot Extracts Attending to thecomplexity of the obtained data sets a strategy was designed tosimplify the dimensionality of the multivariate matrix The firstalgorithm employed for this purpose was the application of a

Figure 2 PCA scores plot of the metabolite profiles obtained for different vine-shoot varieties in positive (A) and negative (B) ionization modesThe original data set was filtered by frequency eliminating MFs not detected in at least 80 of the samples pertaining to each class Baladiacute CabernetFranc Montepila Syrah Merlot Bobal Pedro-Ximenez Garnacha Tintorera and Petit Verdot (B CF MP S M BO PX G and PV)

Figure 3 PCA scores plot resulting from the data set obtained in negative ionization mode after one-way ANOVA with (A) 985 and (B) 99confidence level Moscatel Chardonnay Syrah Pedro-Ximenez Merlot Garnacha Tintorera Baladiacute and Petit Verdot (MG CH S PX MP M GB and PV)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173413

frequency filter which involved the elimination of those MFsabsent in at least 20 of the samples In this way comparabilityof vine-shoot extracts is based on two fractions one of themcommon to all extracts and another one that could becharacteristic of each variety This data mining involved thereduction of the MFs to 68 in the negative mode and 190 in thepositive mode as the pretreated data set After this pretreat-ment the data matrix was mean-centered prior to statisticalanalysis which was initiated by nonsupervised analysis usingPCA both to evaluate the distribution of the samples and todetect clusters in the new space defined by principalcomponents Figure 2 shows the scores plots obtained fordata matrices acquired in the positive and negative ionizationmodes The variability explained by PC1 PC2 and PC3 was8968 and 8750 in positive and negative ionization modesrespectively As can be seen there is a random distribution ofthe vine-shoot varieties but some trends can be observedThere is a main cluster grouping most of the varieties but alsosome extracts present differences in their composition Thus inthe positive ionization mode extracts from Bobal MontepilaSyrah Merlot and Cabernet Franc (B MP S M and CF)provided significant composition differences whereas thenegative ionization mode revealed differences in thecomposition of extracts from Montepila Merlot SyrahBobal and Pedro-Ximenez (MP M S BO and PX) vine

shoots The BPCs obtained from these extracts (and theirreplicates) do not reveal anomalous behaviors that could justifytheir labeling as laboratory outliers and for this reason thediscrimination should be linked to their compositionAfter this preliminary statistical analysis the next objective

was to apply an ANOVA test to identify MFs with the highestcontribution to explain the variability observed in PCA graphsThe MannminusWhitney test against zero was employed forestimation the p value computation was asymptotic and nomultiple testing correction was performed A key parameter tobe defined was the confidence level to set the number offeatures Thus the higher the confidence level the lowernumber of MFs A 95 confidence level involved a reductionfrom 68 and 190 to 47 and 27 MFs for the positive andnegative ionization modes respectively A 985 confidencelevel filtered MFs to 10 and 11 in the negative and positiveionization modes (Figure 3A) while a 99 confidence levelreduced them to 8 in global terms (Figure 3B)This test allowed labeling of the most significant features

contributing to explain the variability visualized for compositionof extracts from vine-shoot varieties Figure 3 shows the PCAscores plots resulting from the data set obtained after ANOVAwith a 985 confidence level which limited the number ofMFs to 10 Attending to this panel of compounds a maincluster was observed with other two additional groupings one

Figure 4 PCA scores plot for positive ionization mode using the original data set after filtration of those MFs not present in all samples belonging toeach class vine shoots or oak wood As can be seen there is a clear discrimination between extracts from oak wood (diamonds) and vine shoots(circles)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173414

formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173415

Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

the profile and centroid spectra and a peak spacing tolerance of00025 mz plus 7 ppm Table 1 in the Supporting Informationlists the compounds identified in the extracts from GarnachaTintorera Merlot and Syrah vine-shoot cultivars thusdemonstrating their different composition

Qualitative Comparison of LC-TOFMS FingerprintsCorresponding to Vine-Shoot Extracts Attending to thecomplexity of the obtained data sets a strategy was designed tosimplify the dimensionality of the multivariate matrix The firstalgorithm employed for this purpose was the application of a

Figure 2 PCA scores plot of the metabolite profiles obtained for different vine-shoot varieties in positive (A) and negative (B) ionization modesThe original data set was filtered by frequency eliminating MFs not detected in at least 80 of the samples pertaining to each class Baladiacute CabernetFranc Montepila Syrah Merlot Bobal Pedro-Ximenez Garnacha Tintorera and Petit Verdot (B CF MP S M BO PX G and PV)

Figure 3 PCA scores plot resulting from the data set obtained in negative ionization mode after one-way ANOVA with (A) 985 and (B) 99confidence level Moscatel Chardonnay Syrah Pedro-Ximenez Merlot Garnacha Tintorera Baladiacute and Petit Verdot (MG CH S PX MP M GB and PV)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173413

frequency filter which involved the elimination of those MFsabsent in at least 20 of the samples In this way comparabilityof vine-shoot extracts is based on two fractions one of themcommon to all extracts and another one that could becharacteristic of each variety This data mining involved thereduction of the MFs to 68 in the negative mode and 190 in thepositive mode as the pretreated data set After this pretreat-ment the data matrix was mean-centered prior to statisticalanalysis which was initiated by nonsupervised analysis usingPCA both to evaluate the distribution of the samples and todetect clusters in the new space defined by principalcomponents Figure 2 shows the scores plots obtained fordata matrices acquired in the positive and negative ionizationmodes The variability explained by PC1 PC2 and PC3 was8968 and 8750 in positive and negative ionization modesrespectively As can be seen there is a random distribution ofthe vine-shoot varieties but some trends can be observedThere is a main cluster grouping most of the varieties but alsosome extracts present differences in their composition Thus inthe positive ionization mode extracts from Bobal MontepilaSyrah Merlot and Cabernet Franc (B MP S M and CF)provided significant composition differences whereas thenegative ionization mode revealed differences in thecomposition of extracts from Montepila Merlot SyrahBobal and Pedro-Ximenez (MP M S BO and PX) vine

shoots The BPCs obtained from these extracts (and theirreplicates) do not reveal anomalous behaviors that could justifytheir labeling as laboratory outliers and for this reason thediscrimination should be linked to their compositionAfter this preliminary statistical analysis the next objective

was to apply an ANOVA test to identify MFs with the highestcontribution to explain the variability observed in PCA graphsThe MannminusWhitney test against zero was employed forestimation the p value computation was asymptotic and nomultiple testing correction was performed A key parameter tobe defined was the confidence level to set the number offeatures Thus the higher the confidence level the lowernumber of MFs A 95 confidence level involved a reductionfrom 68 and 190 to 47 and 27 MFs for the positive andnegative ionization modes respectively A 985 confidencelevel filtered MFs to 10 and 11 in the negative and positiveionization modes (Figure 3A) while a 99 confidence levelreduced them to 8 in global terms (Figure 3B)This test allowed labeling of the most significant features

contributing to explain the variability visualized for compositionof extracts from vine-shoot varieties Figure 3 shows the PCAscores plots resulting from the data set obtained after ANOVAwith a 985 confidence level which limited the number ofMFs to 10 Attending to this panel of compounds a maincluster was observed with other two additional groupings one

Figure 4 PCA scores plot for positive ionization mode using the original data set after filtration of those MFs not present in all samples belonging toeach class vine shoots or oak wood As can be seen there is a clear discrimination between extracts from oak wood (diamonds) and vine shoots(circles)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173414

formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173415

Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

frequency filter which involved the elimination of those MFsabsent in at least 20 of the samples In this way comparabilityof vine-shoot extracts is based on two fractions one of themcommon to all extracts and another one that could becharacteristic of each variety This data mining involved thereduction of the MFs to 68 in the negative mode and 190 in thepositive mode as the pretreated data set After this pretreat-ment the data matrix was mean-centered prior to statisticalanalysis which was initiated by nonsupervised analysis usingPCA both to evaluate the distribution of the samples and todetect clusters in the new space defined by principalcomponents Figure 2 shows the scores plots obtained fordata matrices acquired in the positive and negative ionizationmodes The variability explained by PC1 PC2 and PC3 was8968 and 8750 in positive and negative ionization modesrespectively As can be seen there is a random distribution ofthe vine-shoot varieties but some trends can be observedThere is a main cluster grouping most of the varieties but alsosome extracts present differences in their composition Thus inthe positive ionization mode extracts from Bobal MontepilaSyrah Merlot and Cabernet Franc (B MP S M and CF)provided significant composition differences whereas thenegative ionization mode revealed differences in thecomposition of extracts from Montepila Merlot SyrahBobal and Pedro-Ximenez (MP M S BO and PX) vine

shoots The BPCs obtained from these extracts (and theirreplicates) do not reveal anomalous behaviors that could justifytheir labeling as laboratory outliers and for this reason thediscrimination should be linked to their compositionAfter this preliminary statistical analysis the next objective

was to apply an ANOVA test to identify MFs with the highestcontribution to explain the variability observed in PCA graphsThe MannminusWhitney test against zero was employed forestimation the p value computation was asymptotic and nomultiple testing correction was performed A key parameter tobe defined was the confidence level to set the number offeatures Thus the higher the confidence level the lowernumber of MFs A 95 confidence level involved a reductionfrom 68 and 190 to 47 and 27 MFs for the positive andnegative ionization modes respectively A 985 confidencelevel filtered MFs to 10 and 11 in the negative and positiveionization modes (Figure 3A) while a 99 confidence levelreduced them to 8 in global terms (Figure 3B)This test allowed labeling of the most significant features

contributing to explain the variability visualized for compositionof extracts from vine-shoot varieties Figure 3 shows the PCAscores plots resulting from the data set obtained after ANOVAwith a 985 confidence level which limited the number ofMFs to 10 Attending to this panel of compounds a maincluster was observed with other two additional groupings one

Figure 4 PCA scores plot for positive ionization mode using the original data set after filtration of those MFs not present in all samples belonging toeach class vine shoots or oak wood As can be seen there is a clear discrimination between extracts from oak wood (diamonds) and vine shoots(circles)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173414

formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173415

Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

formed by Pedro-Ximenez Montepila Merlot GarnachaTintorera and Bobal (PX MP M G and B) and a secondone formed by Moscatel Chardonnay and Syrah (MG CHand S)This study can be completed with the PCA corresponding to

99 of the confidence level By analogy to the positiveionization modes differences in composition were found forextracts from Montepila and Moscatel (MP and MG) varietiesand to a lesser extent Petit Verdot Pedro-Ximenez GarnachaTintorera and Merlot (PV PX G and M) varieties as can beseen in Figure 3 The identification of these MFs withsignificant contribution explains the observed variabilityreported interesting compounds such as 5-hydroxymethylfur-fural pyrogallol acetovanillone coniferyl alcohol acetovera-trone homovanillic acid syringaldehyde desapidinol A or ethylprotocatechuateComparison of SHL Extracts from Vine Shoots and

Oak Wood Attending to Their LC-TOFMS Profiles Aftercomparison of global composition profiles of extracts fromdifferent varieties of vine shoots the following step was tocompare the similarity of these extracts with those obtainedfrom oak wood chips This study could provide informationabout those vine-shoot varieties with a more similarcomposition to that of oak wood as a preliminary test to

evaluate the applicability of vine shoots or their extracts withoenological purposes Thus the first statistical analysis with thisaim was to filter by frequency the original data set composed byMFs obtained from TOF analysis from both types of extractsThe restriction for this type of filter is crucial to ensurecomparability Thus if the filter limits MFs to those existing inall extracts of each type of sample vine shoots or oak woodchips the representativeness of each type of sample is ensuredThis filter reduced the data set to 69 and 37 MFs in positiveand negative ionization modes respectively and providedPCAs such as that illustrated in Figure 4 for the positiveionization mode As can be seen there is a clear discriminationbetween extracts from oak wood and extracts from vine shootsTaking into account that extracts from two different types of

samples are being compared a less restrictive filter should beemployed to estimate the similarity level between both rawmaterials Thus an additional analysis was carried out byfiltering the original data set to eliminate those MFs not presentin at least 40 of the extracts from vine shoots or from oakwood chips In this case the original data set was reduced to647 MFs in positive and negative ionization modes Table 2 inthe Supporting Information lists the compounds identified inthe extracts from American Fresh American Blend and French

Figure 5 PCA scores plot for negative ionization mode using the original data set after filtration of MFs not present in 40 of samples belonging toeach class vine shoots or oak wood Garnacha Tinta Malbec Sauvignon Blanc Petit Verdot Cabernet Sauvignon Pedro-Ximenez ChardonnayGarnacha Tintorera Merlot Montepila American Fresh oak American Blend oak French Spice oak French Intense oak and French Sweet oak(GT MA SB PV CS PX CH G M MP AF AB SP FI and SW)

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173415

Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

Intense oak varieties thus demonstrating the varied composi-tionThe scores graph obtained by principal component analysis

of this new data set shown in Figure 5 illustrates a clusterformed by extracts from different vine-shoot varieties with ahigh similarity to those from oak wood that remain groupedThe varieties were Garnacha Tinta Malbec CabernetSauvignon Sauvignon Blanc Petit Verdot Pedro-XimenezChardonnay Garnacha Tintorera Merlot and Montepila (GTMA CS SB PV PX CH G M and MP) According to sampledistribution two red grape varieties such as Garnacha Tintoreraand Merlot and a white grape variety such as Montepilareported extracts with the most similar composition to thatfrom oak chips Therefore the vine-shoot type in terms ofgrape color seems not to play a relevant role to yield extractswith similar composition to those provided by oak chips As canbe seen PC1 PC2 and PC3 explained more than 90 of thevariability observed in the scores graphThis study is the basis to support the valorization of this

agricultural residue produced in vineyards The extracts fromthis residue have been characterized in this research resulting insome vine cultivars in a composition similar to that of extractsfrom oak wood chips which are used with oenologicalapplications

ASSOCIATED CONTENTS Supporting InformationTables containing a list of compounds identified in the extractsfrom Garnacha Tintorera Merlot and Syrah vine-shootvarieties as well as in the extracts from American BlendAmerican Fresh and French Intense oak varieties This materialis available free of charge via the Internet at httppubsacsorg

AUTHOR INFORMATIONCorresponding AuthorTelFax +34957218615 E-mail qa1lucamucoesFundingThe Spanish Ministerio de Ciencia e Innovacion (MICINN)and FEDER program are acknowledged for financial supportthrough project CTQ2009-07430 FP-C is grateful toMICINN for a Ramon y Cajal contract (RYC-2009-03921)NotesThe authors declare no competing financial interest

REFERENCES(1) Jimenez L Rodriacuteguez A Perez A Moral A Serrano LAlternative raw materials and pulping process using clean technologiesInd Crop Prod 2008 28 11minus16(2) Moldes A B Torrado A M Barral M T Domiacutenguez J MEvaluation of biosurfactant production from various agriculturalresidues by lactobacillus pentosus J Agric Food Chem 2007 554481minus4486(3) Moldes A B Torrado A Converti A Domiacutenguez J MComplete bioconversion of hemicellulosic sugars from agriculturalresidues into lactic acid by lactobacillus pentosus Appl BiochemBiotechnol 2006 135 219minus227(4) Valente Nabais J M Laginhas C Carrott P J M RibeiroCarrott M M L Thermal conversion of a novel biomass agriculturalresidue (vine shoots) into activated carbon using activation with CO2J Anal Appl Pyrolysis 2010 87 8minus13(5) Luque-Rodriacuteguez J M Perez-Juan P Luque de Castro M DExtraction of polyphenols from vine shoots of Vitis vinifera bysuperheated ethanolminuswater mixtures J Agric Food Chem 2006 548775minus8781

(6) Max B Salgado J M Corte S Domiacutenguez J M Extraction ofphenolic acids by alkaline hydrolysis from the solid residue obtainedafter prehydrolysis of trimming vine shoots J Agric Food Chem 201058 1909minus1917(7) Pan G X Bolton J L Leary G J Determination of ferulic andp-coumaric acids in wheat straw and the amounts released by mild acidand alkaline peroxide treatment J Agric Food Chem 1998 46 5283minus5288(8) Luque de Castro M D Luque-Rodriacuteguez J M Japon-Lujan RExploitation of residues from vineyards olive groves and wine and oilproduction to obtain phenolic compound of high-added value InMethods of Analysis for Functional Foods and Nutraceuticals Hurst JEd Hershey Foods Techhnical Center Hershey PA 2007 pp 205minus244(9) Morales M L Beniacutetez B Troncoso A M Accelerated aging ofwine vinegars with oak chips evaluation of wood flavour compoundsFood Chem 2004 88 305minus315(10) Tesfaye W Morales M L Garciacutea-Parrilla M C Troncoso AM Evolution of phenolic compounds during an experimental aging inwood of Sherry vinegar J Agric Food Chem 2002 50 7053minus7061(11) Bautista-Ortiacuten A B Lencina A G Cano-Lopez M Pardo-Minguez F Lopez-Roca J M Gomez-Plaza E The use of oak chipsduring the aging of a red wine in stainless steel tanks or used barrelsEffect of the contact time and size of the oak chips on aromacompounds Aust J Grape Wine Res 2008 14 63minus70(12) Alamo del M Nevares I Gallego L Martiacuten C Merino SAging markers from bottled red wine aged with chips staves andbarrels Anal Chim Acta 2008 621 86minus99(13) Garde Cerdan T Rodriacuteguez Mozaz S Anciacuten Azpilicueta CVolatile composition of aged wine in used barrels of French oak andAmerican oak Food Res Int 2002 35 603minus610(14) Arapitsas P Antonopoulos A Stefanou E Dourtoglou V GArtificial aging of wines using oak chips Food Chem 2004 86 563minus570(15) Fernandez de Simon B Cadahiacutea E Alamo del M Nevares IEffect of size seasoning and toasting in the volatile compounds intoasted oak wood and in a red wine treated with them Anal ChimActa 2010 660 211minus220(16) Tesfaye W Morales M L Beniacutetez B Garciacutea-Parrilla M CTroncoso A M Evolution of wine vinegar composition duringaccelerated aging with oak chips Anal Chim Acta 2004 513 239minus245(17) Karvela E Makris D P Kelafas P Moutounet M Extractionof phenolics in liquid model matrices containing oak chips Kineticsliquid chromatography-mass spectroscopy characterization and asso-ciation with in vitro antiradical activity Food Chem 2008 110 263minus272(18) Ortega-Heras M Perez-Magarino S Cano-Mozo EGonzalez-San Jose M L Differences in the phenolic compositionand sensory profile between red wines aged in oak barrels and winesaged with oak chips LWT Food Sci Technol 2010 43 1533minus1541(19) Luque-Rodriacuteguez J M Luque de Castro M D Perez-Juan PDynamic superheated liquid extraction of anthocyanins and otherphenolics from red grape skins of winemaking residues BioresourTechnol 2007 98 2705minus2713(20) Eskin N A M Biochemistry of food processing Browningreactions in foods In Biochemistry of Foods 2nd ed Eskin N A MEd Academic Press London 1990 pp 239minus296(21) Pereira V Albuquerque F M Ferreira A C Cacho JMarques J C Evolution of 5-hydroxymethylfurfural (HMF) andfurfural (F) in fortified wines submitted to overheating conditionsFood Res Int 2011 44 71minus76(22) Report of the scientific papel on contaminants in the food chainon provisional findings of furan in food EFSA J 2004 137 1minus20Available at http httpwwwefsaeuropaeuenscdocsdoc137pdf(23) Jun H J Lee K G Lee Y K Woo G J Park Y S Lee S JCorrelation of urinary furan with plasma gamma-glutamyltranspepti-

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173416

dase levels in healthy men and women Food Chem Toxicol 2008 461753minus1759(24) Falcone P M Tagliazucchi D Verzelloni E Giudici P Sugarconversion induced by the application of heat to grape must J AgricFood Chem 2010 58 8680minus8691

Journal of Agricultural and Food Chemistry Article

dxdoiorg101021jf205337a | J Agric Food Chem 2012 60 3409minus34173417