what do metals tell us about wine?

Trends in Analytical Chemistry, Vol. 26, No. 9, 2007 Trends

What do metals tell us about wine?Pawel Pohl

This article focuses on analysis of metals in wine in the literature devoted to

analytical atomic spectrometry in the past decade. It emphasizes the role of

metals in wine and processes involved in winemaking. It covers endogenous

and exogenous sources of metals in wine, changes in metal composition

during vinification, methods of wine analysis, techniques of wine classifica-

tion corresponding to distinctive metal patterns, and, finally, approaches to

operationally-defined fractionation analysis of metals carried out to under-

stand their nutritional value and bioavailability in wine.

ª 2007 Elsevier Ltd. All rights reserved.

Keywords: Atomic spectrometry; Determination; Fractionation; Metal; Wine; Wine-

making

Pawel Pohl*

Wroclaw University of

Technology,

Faculty of Chemistry,

Wybrzeze Stanislawa

Wyspianskiego 27,

50-370 Wroclaw,

Poland

*Tel.: +48 71 320 3445;

Fax: +48 71 328 4330;

E-mail:

[email protected]

0165-9936/$ - see front matter ª 20070165-9936/$ - see front matter ª 2007

1. Sources and content of metals inwine

Wine is an alcoholic beverage, a productof yeast fermentation of natural sugarspresent in grape juice. Grape species usedfor winemaking should be chemicallybalanced to ferment without addition ofextra sugar, acid, enzymes or nutrients[1,2]. According to this definition, it canbe expected that the origin of metals inwine is two-fold (Fig. 1).

Metals of primary, natural origin comefrom soil on which vines are grown andreach wine through grapes. The concen-tration of primary metals is characteristicand comprises the largest part of the totalmetal content in wine [3,4]. It is con-nected with the maturity of the grapes,their variety, the type of soil in the vine-yard, and the climatic conditions duringtheir growth [5].

The contribution of metals of a second-ary origin is associated with externalimpurities that reach wine during growthof grapes or at different stages in wine-making (from harvesting to bottling andcellaring). During the growth of grapes ina vineyard, contaminations can be classi-fied as geogenic (originating in the soil),from protection and growing practices,or from environmental pollution [4].Accordingly, wines from vineyards in thevicinity of sea or ocean result in a higherNa content compared to wines from other

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2007.07.005Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2007.07.005

regions [5–8]. Wind from sea or oceanaffects the coastal region and vineyardslocated therein by a marine spray [5,9].Differences in K, Ca and Cu content can bedue to fertilizers used for cultivation [9].Application of pesticides, fungicides andfertilizers containing Cd, Cu, Mn, Pb andZn compounds during the growing seasonof vines leads to increases in the amountsof these metals in wine [3,10–15]. Winesfrom vineyards located close to road trafficor situated in industrial areas containhigher levels of Cd and Pb because ofvehicle-exhaust fumes or other emissionsto air, water and soil [10,12–14].

Finally, there is an enological (wine-making) source of metals, as contamina-tion may occur at different steps of wineproduction (Fig. 2). The reason for this isthe long contact of wine with materials(aluminum, brass, glass, stainless steel,wood) from which wine-making machin-ery and pipes, and casks and barrels usedfor handling and storing wine are made.This is the usual source of Al, Cd, Cr, Cu,Fe and Zn [3,12]. Contamination with Na,Ca or Al can be associated with fining andclarifying substances (flocculants, such asbentonites) added to wine to remove sus-pended solids after fermentation and toreduce turbidity [7,9,12,16]. Ca concen-tration can also be affected by addingCaCO3 or CaSO4 for de-acidification ofmust and wine or enhancement of acidityof grape juices, respectively, [12,15].

When considering winemaking andquality assurance (QA) of branded wines,knowledge of metal in wine is of specialeconomic importance. Major metals areCa, K, Na, and Mg [4], which are presentat levels in the range of 10–103lg/ml(Table 1). The concentration of K is thehighest [6,17]. Concentrations of Ca andMg are comparable, but lower than that ofK by an order of magnitude. Na concen-tration is correspondent to those of Ca andMg or lower [6]. Al, Cu, Fe, Mn, Rb, Sr and

941941

mailto:�[email protected]

Grapevariety

Soil ClimateCulti

vatio

n (soil f

ertil

izatio

n, foli

arsp

rays

)

Vinification (winemaking processes)

Pollution (air, water and soil)

Metals in wine

Figure 1. Endogenous (solid line) and exogenous (dotted line)sources of metals in wine.

Trends Trends in Analytical Chemistry, Vol. 26, No. 9, 2007

Zn are minor metals present at concentrations in therange 0.1–10 lg/ml (Tables 2 and 3). Trace metals,including Ba, Cd, Co, Cr, Li, Ni, Pb, V and others are in

Harvesting and transportation of grapes

Stemming and crushing

Pressing

Bottling

Storage

Filtration

Aging

Stabilization

Racking (separation of lees by decantation)

Fining

SulfitingBentonite

Gelatine, silica sol-gel

Malo-lactic fermentation

Alcoholic fermentation

Must

Pre-clarification

(settling, centrifugation, defecation)

Yeasts

Seeds

VIN

IFIC

AT

ION

Stems

Figure 2. Flow scheme of white-wine vinification (adapted from[1]). For red and rose wines, pressing and separation are performedafter fermentation.

942 http://www.elsevier.com/locate/trac

the range 0.1–100 ng/ml or lower (Tables 4 and 5). Inall specified metal groups, there are concentration dis-persions, but, as a rule, major metals are the less dis-persed while trace metals are the most dispersed,especially Ni and Pb [18].

2. Role of metals in winemaking and wine

Metals affect the organoleptic characteristics of wine,including flavor, freshness, aroma, color and taste,mainly due to precipitates being formed (yeast, finingand filtration sediments) or clouding during wine fer-mentation, maturation and storage [5,6,10,17].

Most metals are important for efficient alcoholic fer-mentation. Ca, K, Mg and Na take part in regulating thecellular metabolism of yeasts by maintaining adequatepH and ionic balance [19]. Minor metals (Cu, Fe, Mn,Zn) and some trace metals are also favorable for yeasts asthey are required for prosthetic metallo-enzyme activa-tion [19]. Precipitation of K and Ca tartrates changes pH,which enhances Cu and Fe oxidation in addition toforming Al, Cu and Fe clouding. Both oxidation andclouding affect wine conservation [16,19].

Cu, Fe and Mn are responsible for changes in stabilityof old wine and modification of the sensory quality ofwine after bottling [20]. This phenomenon is called‘‘browning’’, which involves a cascade of oxidationreactions of organic components of wine, leading to lossof freshness and aroma and appearance of condensedprecipitates of tannins [20–22]. The mechanism iscomplex; apparently, Cu2+, Fe3+ and Mn2+ activatemolecular oxygen and further oxidation reactionsof organic compounds to aldehydes and ketones byforming reactive oxygen species (hydroxyl radicals)[12,21,23,24]. In addition, Fe catalyses oxidation ofpolyphenolic substances while Mn encourages formationof acetaldehyde, which reacts with polyphenolics in Fe-mediated reactions to produce precipitate [21]. Resultantchanges in the composition of wine lower quality andstability.

Cu, Fe and Mn form stable complexes with aminoacids, polyphenols and melanoids. These occur duringwine maturation and storage and determine ageingcharacteristics, final aroma, taste and even the color ofthe wine. In addition, association of Cu, Fe and Mn cat-ions with organic chelating ligands is an important nat-ural anti-oxidative mechanism that decreases the rate offormation of reactive oxygen species responsible forreactions causing staleness and spoilage of wine [25,26].

The content of Cu(II) and Fe(III) ions (above 1 lg/mland 7 lg/ml, respectively) can give unpleasant, astrin-gent cupric and ferric tastes and be responsible for pro-ducing cupric and ferric cloudiness, especially when thecontent of tannic substances or pH is high [2,3].However, this effect does not occur when wine contains

Table 1. Concentration of major metals in wine

Wine Concentration, lg/ml Ref.

K Na Ca Mg

Australian 383–1482 ND–276 7–139 [16]Austrian 118–140 68 [45]Argentinian 10–15 [12]Czech 553–3056 2.0–110 40–210 7.8–138 [3,34,45]French 265–426 7.7–14.6 65–161 55–96 [17,45]German 480–1860 6–25 58–200 56–105 [36,45]Hungarian 51–153 72–100 [45,47]Italian 88–151 53–60 [45]Macedonian 265–1100 5–310 30–120 64–718 [7]Portuguese 108–114 58–59 [45]Slovakian 62–236 57–145 [45]Spanish 338–2032 3.5–300 12–241 50–236 [5,6,8,9,14,15,17,18,30–32,45]

ND, Not detected.

Table 2. Al, Cu, Fe and Mn in wine


Al Cu Fe Mn

Argentinian 0.017–0.018 0.023–0.028 0.48–0.79 [12]Australian 0.20–6.37 0.10–0.42 0.06–11.49 [16]Czech 0.9–5.2 0.28–3.26 [3]French 0.56–1.27 ND–0.48 0.81–2.51 0.63–0.96 [17]German 0.02–0.71 0.4–4.2 0.5–1.3 [36]Greek 0.14–0.74 0.2–0.9 1.1–5.6 ND–2.3 [10]Hungarian 0.96–3.56 0.15–0.49 7.3–23.7 0.8–2.9 [47]Jordanian 0.03–2.60 [28]Macedonian ND–1.8 0.1–4.0 [7]Serbian 0.10–0.63 2.7–12.2 0.62–4.08 [14,27]Spanish 0.57–14.3 ND–3.1 0.4–17.4 0.1–5.5 [5,6,8,9,14,15,17,18,30–32]

ND, Not detected.

Table 3. Rb, Sr and Zn in wine


Rb Sr Zn

Argentinian 0.02–0.13 [12]Australian 0.39–0.43 [45]Austrian 0.40–0.46 [45]Czech 0.56–1.20 0.34–0.53 [45]French 0.64–0.72 0.22–0.47 0.44–0.74 [17,45]German 0.2–2.9 0.12–1.28 0.3–1.5 [36,45]Greek 0.3–8.9 [10]Hungarian 0.33–0.69 0.32–1.19 0.6–1.9 [45,47]Italian 0.50–9.90 0.40–1.16 [45]Jordanian 0.11–3.03 [28]Portuguese 1.23–1.53 0.57–0.85 [45]Slovakian 0.03–1.57 0.13–3.22 [45]Spanish 0.1–5.3 0.28–1.50 ND–4.63 [5,8,9,14,15,17,18,30–32,45]


a majority of Fe(II) ions and when the level of Cu, whichcatalyses Fe(II) oxidation, is low [5]. Al clouding isconsidered to form at Al concentrations above 10 lg/ml[10].

Metals also have a certain nutritional and/or toxiceffect on health [17]. Moderate wine consumption con-tributes daily nutritional requirements of many essentialmetals, including Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Ni

http://www.elsevier.com/locate/trac 943


and Zn [10,12]. However, it should be noted thatnutritional effect of wine and metal bioavailability arerelated to the physicochemical forms in which they arepresent [16]. To prevent excessive intake of metalsresulting in toxicosis (e.g., Cd and Pb [12]), metalcontent in wine is regulated. Accepted levels should berespected and controlled during vinification and in finalproducts, in line with recommendations given bynational laws or trade organizations (e.g., InternationalOrganization of Vine and Wine (OIV)) [27].

2.1. Changes in metal content during wine productionThe composition of metals in wine during fermentation,maturation and storage is not stable. In fermentedwines, metal concentrations are lower than those inrespective grape juices and musts, due to precipitation ofK and Ca tartrates [5,7,19] or other insoluble precipi-tates of Al, Cr, Cu, Fe, Mn, Ni, Pb and Zn [10,19]. Thisdeterioration is accelerated by increases in pH and theamount of alcohol in the fermenting media [19]. Infermented musts and wines, yeasts consume Ca, Cu, Fe,K, Mg and Zn, so their concentrations decrease [19].After fermentation, racking of yeast sediments and otherprecipitates is used to remove these metals. Only Nacontent is constant since it has less influence on yeastgrowth and performance [19].

During clarification and storage to stabilize and con-serve wine, metal content also decreases, the magnitudeof the decrease depending on harvest, grape quality andalcohol content, which normally increases withdecreasing solubility of salts [5]. At clarification, metalsare eliminated with sediments of proteins and sulphatedamino acids. During storage, they are precipitated assulfides (e.g., Cu) in reaction with H2S originating fromfree SO2 [5,19,28]. Further decrease of Ca and K contentis associated with lower solubility of their tartrates at thelow temperature used to stabilize wine [19]. A significantpart of Cu, Fe, Mn and Zn is removed by addingK4Fe(CN)6 and precipitating the respective ferrocyanidesto prevent wine from being susceptible to browning andformation of metal cloudiness [21,22]. Cation-exchang-ers, chelating resins and adsorbents are suitable for thistreatment, but the organoleptic quality of wine is alsochanged since polyphenolic species are also retained[20,22].

Increases in Cr, Cu, Fe and Zn contents have exoge-nous and/or endogenous origins. For red wines, it is aresult of long maceration [5]. Technological treatmentand long contact of wine with vineyard equipment alsoincrease metal concentrations, especially during matu-ration and ageing [5,7,10,28]. CuSO4 or FeSO4 added towine after fermentation to remove H2S, which reactswith organic components to form mercaptans and then,after their oxidation, respective disulfides (both produc-ing disagreeable odors), contribute to an increase of Cuand Fe content [10].


3. Total metal-content analysis

Information on total metal content in finished wines isessential for not only winemakers and wine merchantsbut also customers [8]. Primarily, metal concentrationshave to be controlled in line with health-protectionregulations [18,29]. Accepted tolerable limits for metalsin winemaking are critical for flavor, taste, color andlong-term stability of final products, so they have acommercial impact [7]. In this way, total metal contentsare crucial parameters contributing to wine quality,while metal analysis is a part of QA and quality control(QC). Finally, knowledge about metals is useful in testingwine authenticity and in investigating frauds or adul-terations by blending wines and admixing hazardoussubstances [29].

3.1. Sample preparationTypically, wine samples are analyzed after sampling.Otherwise, they are stored at a temperature of 3–4�C[7,16,30,31] or lower [32]. To ensure sample stabilityduring handling, toluene can be added to wine, pre-venting contact with air [5]. Wine can also be filtered toremove sediments and precipitates [10,33]. Sparklingwines are degassed [17,18].

As a sample, wine is a complex matrix comprisingwater, alcohol, sugars and other inorganic and organiccompounds [34,35], so, for metal determinations with-out any initial pre-treatment, usage of ethanol-contain-ing standards or the standard-addition method isrecommended to minimize physical and chemical inter-ferences [21,36–38].

Ca, K, Mg and Na are usually measured only afterdilution of original samples with water [6–9,19,22,30–32]. Al, Cd, Cu, Fe, Ni, Pb and Zn can be determinedwithout dilution [7,13,21,22,39], after dilution withHNO3 and HCl solutions and/or water [16,22,40–42], orfollowing acidification with concentrated HNO3 [3,28].Addition of HNO3 (to pH 1.5) is especially encouraged indirect analysis, since it prevents bacterial proliferationand sample spoilage [43]. Metals bound to organicligands are also readily released under these conditions.

Normally, it is necessary to decompose the wine be-cause of possible matrix interferences [8–10,13]. Thiscan be done by wet digestion on a hot plate or in amicrowave oven using concentrated HNO3 [6,8–10,40,44–46], a mixture of concentrated HNO3 and30% H2O2 [5,11,13,42], 30% H2O2 [30–32] or 30%H2O2 followed by adding concentrated H2SO4 [17,18] orHNO3 [15]. To catalyze oxidation, V2O5 can be added toreaction mixtures [40].

To remove alcohol, wine samples are evaporated todryness. The resultant residues are ashed in a mufflefurnace, and these ashes are dissolved in HNO3 solution[14] or digested with concentrated HNO3 and 30% H2O2

Table 4. Ba, Cd, Co and Cr in wines


Ba Cd Co Cr

Argentinian 0.0010–0.0047 ND–0.007 [12]Austrian 0.07–0.13 0.008–0.045 0.038–0.043 [45]Brazilian ND–0.0002 [40]Czech 0.09–0.12 ND–0.018 0.032–0.037 [45]French 0.025–0.24 ND–0.0002 0.004–0.011 0.030–0.057 [17,45]German 0.04–0.26 0.004–0.005 0.022–0.078 [36,45]Greek ND–0.006 ND–0.2 [10]Hungarian 0.11–0.33 0.035–0.054 0.003–0.009 0.032–0.062 [45,47]Italian 0.07–0.14 0.003–0.006 0.023–0.034 [45]Macedonian 0.0001–0.0009 [13]Portuguese 0.14–0.24 0.003–0.006 0.027–0.041 [45]Slovakian 0.10–0.48 ND–0.017 0.006–0.055 [45]Spanish 0.01–0.35 ND–0.019 ND–0.040 0.025–0.029 [14,15,17,18,30,32,45]

ND, Not detected.

Table 5. Li, Ni, Pb and V in wine


Li Ni Pb V

Argentinian ND 0.050–0.090 [12]Australian 0.009–1.1 [16]Austrian 0.014–0.016 0.07–0.17 [45]Brazilian 0.009–0.050 [11,40]Czech 0.015–0.052 0.020–0.054 [45]French 0.008–0.036 ND–0.052 0.006–0.023 0.06–0.23 [17,45]German 0.005–0.043 0.01–0.14 [36,45]Greek ND–0.5 ND–0.62 [10]Hungarian 0.013–0.028 0.01–0.40 [45]Jordanian 0.01–0.20 ND–0.11 [28]Portuguese 0.017–0.018 0.11–0.16 [45]Slovakian 0.005–0.098 ND–0.40 [45]Spanish 0.002–0.13 0.005–0.079 0.001–0.096 0.026–0.043 [5,8,9,11,14,17,18,30–32,45]

ND, Not detected.


[29]. Dry residues after evaporation can also be treatedwith concentrated HNO3 and H2O2, evaporated to dry-ness again and then dissolved in HNO3 solution [33,47].After evaporation to near dryness, the remaining liquidsare diluted with water [18] or HNO3 solutions [34]. Theycan be also heated with an HNO3–H2O2 mixture [19].

For oxidation of matrix during evaporation, H2O2 withconcentrated H2SO4 [36,38] or HClO4 [12] can beadded. In that case, the resultant residues are dissolvedin water [36,38] or in concentrated HNO3, then water[12].

3.2. Analysis and quality assuranceFor metal determinations in wine, atomic absorption andemission spectrometry (AAS and AES) are common[35,43]. Flame AAS (FAAS) [5–9,11,12,16,19–22,30–32,42,44,45] is applied for measurements of alkalis andalkaline earth metals and Cu, Fe, Mn and Zn. With the

same instrumentation as for FAAS but measuringemitted radiation or applying flame photometers, con-centrations of easily ionized alkali metals (K, Li, Na andRb) are determined [5,8,9,16,19,30–32]. Graphitefurnace AAS (GF-AAS) has found application todetermination of trace and sub-trace metals [10,12–14,16–18,22,28,40,45]. Inductively coupled plasmaAES (ICP-AES) facilitates multi-elemental analysis[12,14,17,18,22,29,33,34,36,38,44,46,47].

For testing method accuracy, results obtained arecompared with those achieved using reference-measurement techniques or different sample-preparationprocedures [13,29,36,37,40]. In addition, standard ref-erence materials of wine from European CommissionCommunity Bureau of Reference, including dry white(BCR C), sweet (BCR D) and red (BCR E), are analyzed[8,9,41] or recovery tests are performed [10,12,13,40]for QC of the whole analytical process.



4. Classifying wine according to metal content

When considering possible fraud involving brandedproducts and economic injury to producers, total metalcontents are useful for characterizing wine and classi-fying it according to geographical origin and assessmentof authenticity [30]. Differentiation of wines by theirgenuineness is rather difficult by simple comparison ofthe results of analyzing chemical composition. Basically,different statistical methods are applied to visualize datatrends, to establish criteria for quality and to retrieveinformation on variables that influence similarities anddifferences with respect to the wines being tested[15,17,32,48].

Analysis of variance (ANOVA) is the simplest ap-proach. Applied to all variables, it makes it easier toindicate the strength of correlation between two vari-ables of different categories. Often metal concentration isanalyzed versus place of geographical origin or ageingrepresented by year of harvest [6,8,9,34].

...

CM, NCM, 3CM, 2CM, 1M

...

CCa, NCCa, 3CCa, 2CCa, 1Ca

CBa, NCBa, 3CBa, 2CBa, 1Ba

CAl, NCAl, 3CAl, 2CAl, 1Al

N321Objects (wines)

Variables (metal concentrations) Data vector XN

Orthogonal linear transformation TX(M x N) T = Y(N x P)

Data matrix X(M x N)

PC3

PC2

PC1

Graphical representation of PCA (P=3, N=25)

N – Number of analyzed wines and data vectorsM – Metal, number of variablesCM, N – Concentration of metal M in wine NXN – Data vector for wine N comprising concentrations of determined metalsX(M x N) – Matrix comprising data vectors for analyzed winesY(N x P) – Matrix comprising principal components for analyzed wines P – Number of principal components PC

Principal components matrix Y(N x P)

...

CM, NCM, 3CM, 2CM, 1M

...




N321

Variables (metal concentrations) Data vector XN

Data matrix X(M x N)

PC3

PC2

PC1

PC3

PC2

PC1

Graphical representation of PCA (P=3, N=25)

N – Number of analyzed wines and data vectorsM – Metal, number of variablesCM, N – Concentration of metal M in wine NXN – Data vector for wine N comprising concentrations of determined metalsX(M x N) – Matrix comprising data vectors for analyzed winesY(N x P) – Matrix comprising principal components for analyzed wines P – Number of principal components PC

Principal components matrix Y(N x P)

Figure 3. Principal component analysis – reduction of a number ofvariables (M x N) by their transformation to a set of new coordi-nates (N x P) carrying on the greatest variance of the original data(adapted from [48]).

N – Number of analyzed winesM – Number of variablesCM, N – Concentration of metal M in wine Nd – DistanceS – Similarity

–

...

–dN,3dN,2dN,1N

...

d3,N–d3,2d3,23

d2,Nd2,3–d2,12

d1,Nd1,3d1,2–1

N321

Graphical representation of CA – dendogram with recognized 3 classes for 16 objects

Dis

tanc

e

Class 2 Class 3

...

CM, NCM, 3CM, 2CM, 1M...




N321

)max/dji,(d1ji,S

2)jM,CiM,C(...

2)jBa,CiBa,(C

2)j Al,Ci Al,(Cji,d

Class 1

N – Number of analyzed winesM – Number of variablesCM, N – Concentration of metal M in wine Nd – DistanceS – Similarity

Data matrix X(M x N) - variables (metal concentrations) in objects (wines)

–

...

–dN,3dN,2dN,1N

...

d3,N–d3,2d3,23

d2,Nd2,3–d2,12

d1,Nd1,3d1,2–1

N321

Proximities matrix - distances d or similarities S between objects

Dis

tanc

e

Class 2 Class 3

...

CM, NCM, 3CM, 2CM, 1M...




N321

)max/dji,(d1ji,S –=

2)jM,CM,C(...

2)jBa,CBa,(C

2)j Al,Ci Al,(Cji,d –++–+–=

Class 1

Figure 4. Cluster analysis classification – classification of objectsinto clusters according to their inter-object distance or similarity(adapted from [48]).


For multivariate data analysis, each wine sample isconsidered as an object of variables represented by totalmetal concentrations. These variables form a data vectorand all data vectors belonging to the same category areanalyzed using pattern-recognition techniques (e.g.,principal component analysis (PCA), linear discriminantanalysis (LDA), artificial neural networks (ANN), clusteranalysis (CA) or others (K-nearest neighbors (KNN), softindependent modeling of class analogy (SIMCA))[3,6,8,9,14,17,29–32,34,38,45]).

The most used, unsupervised PCA, is an orthogonallinear transformation (Fig. 3), which aims to find apattern in data of a high dimension expressed so as tohighlight similarities and/or differences. This can bedone by reducing a number of variables by transformingthem to a set of new coordinates for which the greatestvariance of the original data is carried on with a minimalloss of information [6,8,9,29,30,32–34,38,45]. PCA isvery practical for identification of clusters, outliers orother structures in data [34].


LDA is a supervised technique providing a number oforthogonal linear discriminant functions equal to thenumber of categories reduced by 1. It aims to maximizevariance between categories and minimize variancewithin categories, so it is valuable in exploring datastructure [6,8,9,30,32,34,36,38].

CA is an unsupervised classification method used forpartitioning the objects into subsets (clusters) by theirinter-object distance [3,8,29,30,38]. Such a classifica-tion is represented by a dendogram, useful for indicatinggroups with similar individuals (Fig. 4).

For wine, pattern recognition performed by selectingfeatures and using chemometric methods indicates thatmetal contents are satisfactory discriminatory variablesfor classifying wine according to its geographical origin[17]. Information on metals in finished wines is usefulfor their ‘‘fingerprinting’’, mostly because metals arestable and represent factors affecting wine composition[30]. In particular, Ba, Ca, Cr, Li, Mg, Mn, Na, Rb and Srconcentrations are the most discriminatory reflections ofthe composition of the solid characteristic of a distinctgeographical region [3,6,8,9,30,31,36,45]. Na contentreflects naturally occurring marine sprays [6,8,9]. Feamount is affected by both soil composition and tech-nological processes involved in winemaking [9,30,36].In addition, K and Mg contents are important variablesrelated to grape maturity, so they are useful in differ-entiating sweet and dry wines [8]. Cu concentration isan efficient variable for differentiating wines by enolog-ical treatment [36].

5. Metal species in wine

The extent of the effects of metals in wine is not welldetermined by their total metal content [41]. Other wineconstituents bind metals, limiting their availability, socalculation of dietary metal intake based on total contentis insufficient to estimate health or toxic effects of wineconsumption [49,50]. Accordingly, knowledge of thephysicochemical forms of metals in wine is moreadvantageous to understanding their nutritional and/ortoxic effects and the functions of processes involved inmaturation and ageing (e.g., cloud formation or in-creased oxidation rate) [16,41].

Because of the reductive conditions in wine, metals aremainly present in their lower oxidation states. They mayexist as free ions and complexes with organic acids, aminoacids and large molecular-mass species, namely polysac-charides, peptides, proteins and polyphenols [35]. Con-densed tannins and anthocyanins especially are the mostimportant metal ligands, since these species havenumerous coordination sites capable of binding metalcations [41] (e.g., cyanidin-3-glucoside anthocyanin withtwo -OH groups in the ortho position can complex Cu andZn and probably Fe and Mn at the ratio 2:1 [44]). Amino

acids and organic acids are weaker metal ligands and donot contribute significantly to their complexation [41].

Wine has high capacity for forming complexes withminor and trace-metal cations, but stability of thesecomplexes depends on pH and alcohol content[11,41,44,47]. Experiments with untreated wine passedthrough a minicolumn packed with polyurethane foammodified by 2-(2-benzothiazo-lylazo)-p-cresol [11] indi-cated that Pb(II) is strongly associated to other constit-uents, possibly bound to pectic polysaccharides and/orother related high-molecular-weight natural organicspecies [35], and cannot be retained by the sorbent.Other studies [41,42] conveyed that 40–50% of Cu inwine comprises labile metal species, while Cu bound totannins, anthocyanins and proteins accounts for 30–>40%. For Fe and Zn, labile metal species also predom-inate for Fe (>20–�60%) and Zn (50–>80%). Fractionsof Fe and Zn complexed by polyphenols and proteinswere in the range 10–30% [42]. Cu and Fe are alsopresent as negatively-charged species, while, for Zn, suchfraction has not been found [42].

For fermented wines, Cu and Fe are correlated withtannins and anthocyanins, so that, as concentrations ofpolyphenols increase, total concentrations of metals de-crease during vinification [44]. It is probable that Cu andFe participate in condensing polyphenols and/or arecomplexed by anthocyanins containing two o-hydroxylgroups.

5.1. Metal-fractionation analysis in wineWine complexity explains why metal-speciation analysisis a real challenge while there are few studies of it [42].Usually, it is easier and more practical to evaluate metaldistribution between distinct groups or classes of similarspecies, considering selected physical or chemicalfeatures.

A convenient approach to metal fractionation in wine isfiltration, enabling discrimination of metals in real solu-tion, colloid solution and suspension [47]. For separationof dissolved and suspended Al, Ca, Cd, Cu, Fe, Mn, Pb andZn, a paper filter can be used, while, for distinguishing thecolloidal metal fraction a 0.2 lm-pore size filter isrequired. When filtering wine, metal concentrationsdepending on enological treatment and wine storage [47].

Dissolved metal fraction (<0.45 lm) can be furtherfractionated using membrane ultrafilters with mass cut-offs of 1–100 kDa [16]. Such analysis indicates that Kand Na do not exhibit size partitioning and that they arepredominantly present as simple cations. Absence ofnoticeable Ca and Al size fractions shows that thesemetals are complexed by small molecular-mass com-pounds or that interactions of these metals with ligands(tartaric, malic, gallic and polyuronic acids, or poly-phenols) are weak.

An interesting protocol for Cu, Fe and Zn partition-ing according to chemical properties of metal species



has been proposed [42]. Wines were filtered at 0.2 lmto determine the colloidal metal fraction. Filtrates(dissolved metal fraction) were driven through mini-columns packed with Amberlite XAD8 adsorbent toretain polysaccharide-phenolic Cu, Fe and Zn com-plexes, subsequently eluted with 2 mol/l HNO3. Efflu-ents collected were then separately passed throughDowex 50W · 8 cation-exchanger, Dowex 1 · 8 anion-exchanger and Amberlite XAD2 adsorbent minicol-umns (in the last case after adding 1,10-phenantrolineto effluents), respectively, to sorb labile Cu, Fe(II, III)and Zn species, negatively charged metal species andlabile Fe(II) metal species. Respective fractions wererecovered using 2 mol/l HCl (for ion-exchangers) andmethanol. In addition, proteins and polysaccharideswere precipitated from effluent using acetone andmethanol, correspondingly. Precipitates separated weredissolved and analyzed to determine the contributionsof Cu, Fe and Zn bound to proteins and polysaccha-rides, respectively.

The examples given above demonstrate that simplefractionation analysis of metals in wine can indeed be ofgreat importance to enologists and winemakers.Assessment of colloidal and particle-metal fractionsaffecting wine quality can be practical for detecting andavoiding possible mistakes in vinification. However,identifying different metal groups can be useful forevaluation of bioavailability of metals in wine, so it iscrucial for consumers who are interested in the safetyand wholesomeness of wine.

6. Conclusions and future outlook

Considering vinification processes, metals determinewine quality and have a profound effect on humanhealth and well-being. Hence, knowledge of the metalcontent of wine is crucial for not only winemakers butalso consumers, while metal determination at variousstages of winemaking can be an important aspect of QAand QC. Certainly, because of the nutritional value ofmetals in wine, analysis of the total contents of major,minor and trace metals is of a particular interest withrespect to the wholesomeness and the safety of wine.

Identifying and quantifying the physicochemical formsof metals in wine enables their bioavailability to beestimated, and that is why studies focusing on this issuewill continue. Operationally-defined fractionation pro-cedures using sorbent columns for separation anddetection of distinct metal groupings would be advan-tageous in this respect because of the ease of operationand the low costs of materials required. Moreover,chemical classification based on different types of resins,including ion-exchangers, adsorbents and molecularsieves, can be easily integrated with physical partitioningby filtration and ultra-filtration to increase the number


of differentiated classes of metal species [51]. Also, mi-celle-mediated separation by sequential cloud-pointextraction enables selective isolation of different metalspecies in wine (e.g., soluble Fe(II,III) ions and tannin-bound Fe [52]), and may become very important formetal fractionation.

Indisputably, to provide a more adequate account ofmetal intake and bioavailability through wine con-sumption, research activity would be expanded to in-volve assessment of soluble and assimilable fractions ofmetals, by using in vitro and/or in vivo methods thatsimulate digestion conditions, including stomachal andintestinal phases [49,50].

Metals are very good indicators of wine origin and canbe used as criteria for guaranteeing authenticity, sincethey are not metabolized or modified during winemakingand reflect the average composition of vineyard soils.However, agricultural practices, climatic changes, envi-ronmental pollutions, and vinification processes, couldchange a primary pattern of metals in wine andendanger the relationship between wine and soil com-position. In this case, fingerprinting of wine provenancedue to certain geographic region would be difficult butchemometric pattern-recognition techniques offeradvantages in characterizing, classifying and authenti-cating wines.

Acknowledgement

Pawel Pohl acknowledges Alexander von HumboldtFoundation for the grant of a research fellowship.

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