Gianluca Gilardoni

Gianluca Gilardoni Riccagioia, 16 ottobre 2013

Bulbo olfattivo

Cavità nasale

Lingua

Aroma ortonasale

Gusto Aroma retronasale

Profumi, sapori e colori: aspetti chimici e sensoriali

SAPORE = GUSTO + AROMA RETRONASLE

Possiamo dividere le tecniche applicabili allo studio del gusto e degli aromi in:   CHIMICHE

cromatografia, spettrometria di massa

  SENSORIALI panel di rinoanalisti/assaggiatori

  COMBINATE GC-O, naso elettronico, bocca artificiale





Similar behavior is seen, except that the minimum apparent

size is slightly smaller and the EGCG/casein ratio at which

the minimum occurs is much higher, at approximately 10.

This matches the DLS results but not the SAXS and

viscometry results. It is striking that the two experiments in

which the solution was prefiltered give minima at a ratio of

10, while the other experiments give minima at a ratio of

approximately 0.25.

Discussion

This study is aimed at deriving a molecular description

of the interactions between proteins (particularly salivary

basic PRPs) and dietary polyphenols, which are thought to

be responsible for the astringent sensation of polyphenol-

rich foods and beverages such as red wine and tea. The

purification of single PRPs from saliva is laborious, so as a

substitute for PRPs, bovine !-casein was used. This is cheapand easily available and has a number of similarities with

PRPs: it shares a similar extended conformation, with

exposed prolines,25,26 it competes with gelatin to bind to

polyphenols,7 and it binds to wine polyphenols in a similar

way to the established model polyvinyl-polypyrrolidone,although there is a lower density of polyphenol binding

sites.45 Dephosphorylation removes the hydrophilic phos-

phates and, therefore, reduces the tendency of casein to bind

calcium and form micelles.46

Polyphenols are multidentate ligands able to bind simul-

taneously, via different phenolic groups, at more than one

point on the protein strand.1,47 It has previously been

suggested that polyphenol-protein precipitation occurs inthree stages.15 Here, we have confirmed and expanded the

three-stage model (Figure 8):

(1) The free proteins exist in a loose, randomly coiled

conformation. Simultaneous binding of the multidentate

polyphenols to several sites on the protein leads to coiling

of the protein around the polyphenols. This causes the

physical size to decrease and the structure of the protein to

become more compact and spherical. Chelated binding at

several sites increases the overall binding affinity: in support

of this statement, we note that the affinity of a full-length

PRP (70 residues) for polyphenols is much greater than that

of a 19-residue single PRP sequence.20

(2) As the polyphenol concentration rises, polyphenols

complexed onto the protein surface cross-link different

protein molecules and dimerization ensues, causing insolubil-

ity.15 This phenomenon is similar to the precipitation of

Figure 7. Hydrodynamic radius of EGCG/casein mixtures, deter-mined by NMR PGSE experiments. (A) Unfiltered solutions. The graphin the inset shows the integrated intensity of the casein NMR signalsrelative to the same concentration of free casein, which should beconstant as long as casein remains soluble. The dashed line indicatesthe ratio at which visible precipitation was first observed. (B) Filteredsolutions.

Figure 8. Proposed binding model: The original random coiled PRP binds to multidentate polyphenols on more than one site because eachproline and each aromatic ring represents a possible binding site. At a low polyphenol concentration, the protein binds in several places to thepolyphenol molecules leading to a contraction of the loose random coil and decrease in the molecular size of the protein. Upon addition of morepolyphenols, intermolecular cross-linking takes place and aggregates are formed that finally precipitate.

948 Biomacromolecules, Vol. 5, No. 3, 2004 Jobstl et al.

Molecular Model for Astringency Produced by Polyphenol/Protein Interactions

Elisabeth Jobstl,†,‡ John O’Connell,§ J. Patrick A. Fairclough,† and Mike P. Williamson*,‡

Department of Molecular Biology and Biotechnology and Department of Chemistry, University of Sheffield,Sheffield S10 2UH, United Kingdom, and Unilever Research, Colworth House, Sharnbrook,

Bedford MK44 1LQ, United Kingdom

Received December 8, 2003; Revised Manuscript Received February 6, 2004

Polyphenols are responsible for the astringency of many beverages and foods. This is thought to be caused

by the interaction of polyphenols with basic salivary proline-rich proteins (PRPs). It is widely assumed that

the molecular origin of astringency is the precipitation of PRPs following polyphenol binding and the

consequent change to the mucous layer in the mouth. Here, we use a variety of biophysical techniques on

a simple model system, the binding of !-casein to epigallocatechin gallate (EGCG). We show that at lowEGCG ratios, small soluble polydisperse particles are formed, which aggregate to form larger particles as

EGCG is added. There is an initial compaction of the protein as it binds to the polyphenol, but the particle

subsequently increases in size as EGCG is added because of the incorporation of EGCG and then to

aggregation and precipitation. These results are shown to be compatible with what is known of astringency

in foodstuffs.

Introduction

Polyphenols are widely distributed in the plant kingdom

and, therefore, commonly found in plant-based foods and

beverages.1 They are characterized by containing several

phenolic groups (often in the form of galloyl [3,4,5-

trihydroxybenzoyl] groups) and have been found to have a

variety of effects on animals including humans.2,3 Polyphe-

nols of intermediate size have the ability to bind to proteins

and precipitate them and, hence, are also known as tannins.1,2

They have been suggested to reduce the nutritional value of

some foodstuffs,4-7 but they are also important constituents

of many foods and beverages, such as red wine and tea,

because it is the astringency of the tannins in these beverages

that gives them many of their desirable qualities. It is widely

believed that salivary proteins may act as a primary defense

against harmful (mainly higher molecular weight) tannins

by forming insoluble complexes with them and preventing

their absorption from the intestinal canal and interaction with

other biological compounds.2,3,8 The interaction of polyphe-

nols with salivary proteins has long been thought to lead to

the sensation of astringency, which is generally recognized

as a feeling of puckeriness and dryness in the palate.2,5,6,9 It

is not confined to a particular region of the mouth but is a

diffuse surface phenomenon, characterized by a loss of

lubrication,10 which takes a time of the order of 15-20 s todevelop fully.11,12 It is, therefore, quite different from the

more well-known taste sensations.

A mucous layer composed of salivary proteins and

glycoproteins covers the exposed surface of the mouth to

maintain lubrication. The primary reaction leading to the

sensation of astringency is the precipitation of proteins and

mucins by polyphenolic compounds. The essential feature

is the cross-linking of polypeptides by surface-exposed

phenolic groups on the polyphenols, leading to aggregation

and precipitation and, therefore, the occurrence of the

astringent response.13-15 Saliva is produced by salivary glands

and contains a variety of proteins. The major protein

constituent of saliva is a group of proteins consisting of

multiple repeats of an unusual amino acid sequence contain-

ing a large amount of proline, commonly referred to as

proline-rich proteins (PRPs).16,17 Of the three groups of PRPs

(acidic, basic, and glycosylated), the main function of the

basic PRPs seems to be the complexation of polyphe-

nols.2,14,18

The molecular interaction of polyphenols with PRPs has

been studied using a peptide containing a typical repeat

sequence of a mouse PRP and the human basic PRP IB-

5.15,19,20 It was shown that the major requirement is for the

peptide to have an extended conformation and that the

principal binding sites on these peptides are prolines and the

preceding amide bonds together with the preceding amino

acid (see also ref 18). The pyrrolidine rings of the prolyl

groups act as potential binding sites and form “hydrophobic

sticky patches” that stack face to face with the galloyl rings

of the phenolic substrate. Other interactions including

hydrogen bonding interactions can further stabilize the

complex.21

There is, thus, a good deal known about the molecular

basis of polyphenol/protein interactions. However, the events

after binding are not as well understood, not least because

* To whom correspondence should be addressed. Fax+44 114 272 8697.E-mail [email protected].

† Department of Chemistry, University of Sheffield.‡ Department of Molecular Biology and Biotechnology, University of

Sheffield.§ Unilever Research.

942 Biomacromolecules 2004, 5, 942-949

10.1021/bm0345110 CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 03/13/2004

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)"#!6-,#!$53/,7!!!"#!/('"/!/1-&#,!%$!1-)!6-&&-1!%1!1/)5,#7!

O

OH

OHOH

O

O

OH

OH

OH

O

O

OH

OH

OH

OO

OH

OH

OH

O

O

OH

OH

OH

O

O

OH

OH

OH

OH

O

!-1,2,3,4,6-pentagalloyl-O-D-glucose

gallic acid

E%C#!/((!-4!)"#!3/((-)/11%1$2!>@@!"/$!&/1*!%$-&#,$7!!!"#!&-(#65(/,!F#%3")$!-4!/((!)"#!%$-&#,$!-4!>@@!

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6",-&/)-3,/'"%6!0#"/B%-,J!/1+!0%-6"#&%6/(!',-'#,)%#$!$56"!/$!/0%(%)*!)-!',#6%'%)/)#!',-)#%1J!/,#!

$),56)5,#"+#'#1+#1)7!!!

Copyright © 2002, 2010 by Ann E. Hagerman. All rights reserved. 2

Il colore del vino è dovuto alla presenza di composti non volatili, appartenenti alla classe delle antocianine.



O

OH

OH

OHOH

OH+

Cianidina: ROSSO pH < 3, VIOLA pH 7-8, BLU pH > 11

La tecnica analitica di elezione è la cromatografia liquida.

HPLC GC

composti volatili

composti volatili pesanti o polari derivatizzabili

composti non volatili


In generale:




METODI CHIMICI - LA CROMATOGRAFIA

La cromatografia è il metodo attualmente più diffuso

ed efficace per operare la separazione delle sostanze

in miscela a scopo analitico e preparativo.

Particolarmente utile per la separazione dei composti

organici, la cromatografia fu introdotta nel 1901 dal

botanico italo-russo Mikhail Semyonovich Tsvet

(1872-1919), che la applicò alla separazione di

pigmenti vegetali quali le clorofille ed i carotenoidi.



METODI CHIMICI - LA CROMATOGRAFIA

H. Wagner, S. Bladt, E. M. Zgainsky PLANT DRUG ANALYSIS – A Thin Layer Chromatography Atlas Springer-Verlag 1984



METODI CHIMICI - LA CROMATOGRAFIA Classificazione per stato fisico delle fasi e conformazione del supporto:   cromatografia liquida (LC): la fase mobile è liquida, la fase stazionaria può

essere solida, liquida o liquida supportata su solido;   cromatografia in fase supercritica (SFC): la fase mobile è un fluido

supercritico, la fase stazionaria è una specie organica supportata;   gascromatografia (GC): la fase mobile è gassosa, la fase stazionaria è un solido o

un liquido supportato (GLC);   cromatografia su colonna: la fase stazionaria è confinata all’interno di una

struttura tubolare;   cromatografia planare: la fase stazionaria è distesa su di una superficie piana.



METODI CHIMICI - LA CROMATOGRAFIA Classificazione per meccanismo di interazione:   cromatografia di ripartizione: si instaura tra le fasi un equilibrio analogo alla

solubilità   cromatografia di adsorbimento: si instaura tra le fasi un equilibrio basato su

interazioni aspecifiche tra analiti e struttura molecolare della fase stazionaria (solida)

  cromatografia ionica: si instaura tra le fasi un equilibrio basato su interazioni elettrostatiche tra analiti ionici e gruppi funzionali carichi elettricamente sulla fase stazionaria

  cromatografia di esclusione dimensionale: si instaura tra le fasi un equilibrio basato sull’analogia dimensionale tra le molecole degli analiti e le cavità della fase stazionaria

es. fase inversa (RP)

es. gel di silice

es. resine a scambio ionico

es. destrani (LH-20, G-20, ecc.)



METODI CHIMICI - LA CROMATOGRAFIA La cromatografia strumentale, sia preparativa che analitica, prevede che gli analiti in uscita dalla colonna cromatografica siano rivelati da opportuni dispositivi, detti rivelatori, e rappresentati graficamente su di un tracciato detto cromatogramma.

16.50 17.00 17.50 18.00 18.50 19.00 19.50

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Time-->

Abundance

TIC: 90316_01.DSull’asse delle ascisse è rappresentato il tempo trascorso dall ’ inizio dell’eluizione, sull’asse delle ordinate un valore proporzionale all’intensità del segnale elettrico del rivelatore. All’uscita di un analita dalla colonna, il rivelatore produce un picco di segnale che varia nel tempo con andamento gaussiano; il tempo a cui corrisponde il vertice della gaussiana è detto tempo di ritenzione.



LA GASCROMATOGRAFIA

Skoog D. A., Leary J. J. – CHIMICA ANALITICA STRUMENTALE – EdiSES 1995



LA GASCROMATOGRAFIA

Si O Si O Si

R

R

R

R

R

R

R

R

n

  lunghezza: 10 – 100 metri   diametro: 0.25 – 0.75 mm   spessore della fase stazionaria: 5 mµ≤



LA GASCROMATOGRAFIA

http://www.gerstel.com/pdf/TDS_eng.pdf

TDS: Thermal Desorption System CIS: Cooled Injection System




RIVELATORE VANTAGGI SVANTAGGI

FID

(Flame Ionization Detector) universale, economico

distruttivo, aspecifico,

richiede due gas di alimentazione

ECD

(Electron Capture Detector)

specifico per alogenati, non distruttivo, non

richiede gas

radioattivo, specifico

PID

(PhotoIonization Detector) non richiede gas,

parzialmente specifico

distruttivo, parzialmente

specifico

TCD

(Thermal Conductivity Detector) non richiede gas, non

distruttivo poco sensibile

MSD

(Mass Spectrometric Detector) permette

l’identificazione degli analiti senza standard

molto costoso, distruttivo




LA GASCROMATOGRAFIA – Rivelatore spettrometro di massa (MSD)


Diverse tipologie di spettrometri di massa possono essere accoppiati alla gascromatografia come rivelatori. Il più diffuso è lo spettrometro di massa a quadrupolo, grazie alle dimensioni ed ai costi relativamente contenuti, ma rivestono grande importanza anche gli spettrometri a settore magnetico ed a tempo di volo.

TABLE 4.1. Common GC / MS Conditions Used for Analysis of Grape Aroma Compounds

GC column Poly(ethylene)glycol (PEG) bound - phase fused - silica capillary (30 m ! 0.25 mm i.d.; 0.25 - µ m fi lm thickness)

Carrier gas He Column head pressure 12 psi Injector Temperature 200 ° C, sample volume injected 0.5 µ L, splitless

injection Oven program 60 ° C Isotherm for 3 min, 2 ° C/min to 160 ° C, 3 ° C/min to 230 ° C,

230 ° C Isotherm for 5 min MS conditions Ionization energy 70 eV, transfer line temperature 280 ° C, SCAN

mode

OH

OH

(E,E)-2,6-Dimethylocta-2,6-dien-2,8-diol

OH

OH

8-Hydroxy-dihydrolinalool

OH

OH

Hydroxy-geraniol

OH

OH

Hydroxy-nerol

030 40 50 60 70 80 90 100 110 120 130

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

30 40 50 60 70 80 90 100 110 120 130 1400

m/z-->

m/z-->m/z-->

m/z-->

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000Abundance

AbundanceAbundance

Abundance

43

5357

6477

93

107

97

121

137

31

44 5979

81

9695 109 111

121

55

43

71

84

68

40

31 53

93

83

43

59

69

121136

153111109

154 1751770

100002000030000400005000060000700008000090000

100000110000120000130000140000150000160000170000180000190000200000210000220000

60 80 100 120 140 160 18030

31

53

79

98

107 136

121

93

8169

59

43

40 50 60 70 80 90 100 110 120 130 1400

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

Figure 4.9. The GC/MS – EI (70 eV) mass spectra of principal terpenol and norisopren-oid compounds identifi ed in grape and not reported in the main libraries commercially available.

TABLE 4.1. Common GC / MS Conditions Used for Analysis of Grape Aroma Compounds

GC column Poly(ethylene)glycol (PEG) bound - phase fused - silica capillary (30 m ! 0.25 mm i.d.; 0.25 - µ m fi lm thickness)

Carrier gas He Column head pressure 12 psi Injector Temperature 200 ° C, sample volume injected 0.5 µ L, splitless

injection Oven program 60 ° C Isotherm for 3 min, 2 ° C/min to 160 ° C, 3 ° C/min to 230 ° C,

230 ° C Isotherm for 5 min MS conditions Ionization energy 70 eV, transfer line temperature 280 ° C, SCAN

mode

OH

OH

(E,E)-2,6-Dimethylocta-2,6-dien-2,8-diol

OH

OH

8-Hydroxy-dihydrolinalool

OH

OH

Hydroxy-geraniol

OH

OH

Hydroxy-nerol

030 40 50 60 70 80 90 100 110 120 130

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

30 40 50 60 70 80 90 100 110 120 130 1400

m/z-->

m/z-->m/z-->

m/z-->

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000Abundance

AbundanceAbundance

Abundance

43

5357

6477

93

107

97

121

137

31

44 5979

81

9695 109 111

121

55

43

71

84

68

40

31 53

93

83

43

59

69

121136

153111109

154 1751770

100002000030000400005000060000700008000090000

100000110000120000130000140000150000160000170000180000190000200000210000220000

60 80 100 120 140 160 18030

31

53

79

98

107 136

121

93

8169

59

43

40 50 60 70 80 90 100 110 120 130 1400

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

Figure 4.9. The GC/MS – EI (70 eV) mass spectra of principal terpenol and norisopren-oid compounds identifi ed in grape and not reported in the main libraries commercially available.

104 GRAPE AROMA COMPOUNDS

The dichloromethane solution is concentrated to 2 – 3 mL by distil-lation using a 40 - cm length Vigreux column, and fi nally to 200 µ L under a nitrogen fl ow prior to GC/MS analysis . The GC/MS profi le of free aroma compounds of a Muscat grape skin extract is shown in Fig. 4.6 .

4.2.3 Analysis of Glycoside Compounds

The methanolic solution is evaporated to dryness under vacuum at 40 ° C, the residue is dissolved in 5 mL of a citrate – phosphate buffer (pH 5), then it is added to 200 mg of a glycosidic enzyme with strong glycosidase activity (e.g., AR 2000, Gist Brocades) and kept at 40 ° C overnight (15 h). The next day the solution is centrifuged, added to 200 µ L of a 1 - octanol 180 - mg/L solution as an internal standard, and the resulting solution is passed through a 1 - g C 18 cartridge previously activated by passage of 6 - mL dichloromethane, 6 - mL methanol, and 6 - mL water. After cartridge washing with 5 - mL water, the fraction containing the aglycones is eluted with 6 mL of dichloromethane, dehydrated with sodium sulfate, and concentrated to 200 µ L with a nitrogen fl ow before analysis. A last fraction, containing the poten-tially aromatic precursor compounds, is recovered from the cartridge by elution with 5 - mL methanol. The GC/MS profi le of aglycones from hydrolysis of glycoside compounds of a Prosecco grape must is shown in Fig. 4.7 .

Figure 4.6. The GC/MS – EI (70 eV) chromatogram recorded in SCAN mode of free aroma compounds of a Muscat grape skins extract. I.S., internal standard (1 - heptanol); peak 1. linalool; peak 2. trans - pyranlinalool oxide; peak 3. cis - pyranlinalool oxide; peak 4. nerol; peak 5. geraniol; peak 6. Ho - diendiol I; peak 7. Ho - diendiol II; peak 8. hydroxycitronellol; peak 9. 7 - hydroxygeraniol; peak 10. ( E ) - geranic acid.

Abundance

900,000

800,000

700,000

600,000

500,000

400,000

300,000

200,000

100,000

0Time--> 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

I.S.1

2

3

4

5

6

7

8

9

10



LA GASCROMATOGRAFIA

MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY

RICCARDO FLAMINICRA, Centro di Ricerca per la Viticoltura, Conegliano (TV), Italy

PIETRO TRALDICNR, Istituto di Scienze e Tecnologie Molecolari, Padova, Italy

A JOHN WILEY & SONS, INC., PUBLICATION







LO SPAZIO DI TESTA STATICO

www.microglass.it/3_HAMILTON/3_HAMILTON_SCHEDE/SERIE_1000.htm

CH3SSCH3

CH3SSCH3

bagno termico

spazio di testa

matrice CH3SSCH3

CH3SSCH3



LO SPAZIO DI TESTA STATICO – SPME (Solid Phase MicroExtraction)

ulceet.com/site30.php

 polidimetilsilossano (PDMS) – apolare  poliacrilato (PA) – polare  carbopack (carbone grafitato) – polarità intermedia  carboxen/polidimetilsilossano (CAR/PDMS) – polarità

media e bassa  polidimetilsilossano/divinilbenzene (PDMS/DVB) –

apolare e aromatica  divinilbenzene/carboxen/polidimetilsilossano (DVB/

CAR/PDMS) – polivalente  carbowax/glicole polietilenico – polare  carbowax/resina templata (CW/TPR) - polare



SPME in matrici acquose La già trattata tecnica di microestrazione in fase solida, molto valida per l’analisi dello spazio di testa statico, è in teoria applicabile anche agli analiti organici in soluzione acquosa.

www2.mst.dk/common/Udgivramme/Frame.asp?http://www2.mst.dk/Udgiv/publikationer/2000/87-7944-147-5/html/bil02.htm

Si tratta di immergere direttamente la fibra nel campione liquido. VANTAGGI: •  permette di evidenziare le sostanze meno volatili, •  aumenta la sensibilità perché concentra gli analiti. SVANTAGGI: •  può adsorbire sostanze fisse che non si desorbono e si decompongono sulla fibra (accorciamento della vita).



SBSE (Stir Bar Sorptive Extraction) E’ una tecnica appositamente sviluppata per il campionamento delle sostanze organiche in campioni liquidi; si applica quindi alle stesse problematiche della SPME in matrici acquose, rispetto alla quale tuttavia è più efficiente.

Si tratta di un agitatore magnetico esternamente rivestito di una guaina in materiale adsorbente, normalmente PDMS. L’agitatote, messo in rotazione all’interno di un campione liquido sigillato, adsorbe gli analiti organici fino alla concentrazione di equilibrio. Al termine del campionamento, le sostanze adsorbite possono essere analizzate in GC per desorbimento termico.

www.gerstel.com/en/twister-stir-bar-sorptive-extraction.htm



LO SPAZIO DI TESTA DINAMICO

Nello spazio di testa dinamico il campione è arricchito dallo spostamento dell’equilibrio verso la fase aeriforme, come conseguenza del principio di Le Châtelier. Gli analiti sono asportati dallo spazio di testa da un flusso di azoto ed intrappolati nelle consuete fasi solide adsorbenti. LA SENSIBILITA’ RISPETTO A L L O S P A Z I O D I T E S T A S T A T I C O E ’ NOTEVOLMENTE INCREMENTATA: POSSONO ESSERE CAMPIONATI SVARIATI LITRI DI SPAZIO DI TESTA ANCHE PER CAMPIONI DI PICCOLO VOLUME. Se il campione è attraversato dal flusso di campionamento, il contributo all’arricchimento potrebbe derivare non solo dal principio di Le Châtelier ma anche dall’innalzamento della tensione di vapore degli analiti (vedi distillazione in corrente di vapore). In campioni liquidi il metodo è noto come “purge and trap”.



METODI SENSORIALI AROMA ORTONASALE – La misura sensoriale dell’odore si attua tramite la valutazione di un “panel” di analisti selezionati. Qualora lo scopo dell’analisi fosse la misura della soglia di percezione olfattiva di un campione aeriforme, il panel si dovrebbe avvalere di un olfattometro a diluizione dinamica.

Il campione, all’interno di sacchetti in Nalophan, viene proposto al panel a concentrazione crescente; i volontari segnalano con un pulsante la percezione dell’odore, evitando di segnalare i “bianchi” casuali introdotti dal programma. Il risultato è una c o n c e n t r a z i o n e d ’ o d o r e , o t t e n u t a statisticamente.

http://www.ecoma.de/en/index_frameset.php?id=198



METODI SENSORIALI AROMA ORTONASALE E RETRONASALE – Qualora lo scopo dell’analisi fosse una valutazione qualitativa dell’odore, il panel potrebbe procedere anche senza olfattometro, scomponendo mentalmente la percezione sensoriale in componenti descrivibili con altrettanti aggettivi detti descrittori. I descrittori e le relative intensità sono normalmente rappresentate in un grafico radiale, a dare un poligono la cui forma identifica sensorialmente il campione.

LO STESSO PROCEDIMENTO SI APPLICA A QUALUNQUE ANALISI SENSORIALE, IN PARTICOLARE AL SAPORE.

www.beverfood.com/v2/modules/smartsection/item.php?itemid=127



MEODI COMBINATI – IL NASO ELETTRONICO

IL NASO ELETTRONICO PERMETTE DI ASSOCIARE UN’EMISSIONE ODOROSA A D U N A S O R G E N T E , A L L ’ I N T E R N O D I U N G RU P P O D I C A M P I O N I DIFFERENZIATI.

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Resina di pino

basilico A

0

200

400

600

800

1000

1200

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cromatogramma aromagramma

solfuro Erba tagliata

muffa, erbaceo

Terra bagnata

Agrumato, menta

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METODI COMBINATI La gascromatografia-olfattometria è una tecnica che combina la separazione gascromatografica delle sostanze volatili alla possibilità di percepirne l’odore in tempo reale. L’analista segnala la percezione con un pulsante, generando un grafico detto aromagramma che può essere sovrapposto al cromatogramma.



LA CROMATOGRAFIA LIQUIDA AD ALTE PRESTAZIONI (HPLC)





RIVELATORE UV-VIS

DAD (Diode Array Detector)

MS RID (Refractive Index Detector)




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OSi CH3CH3

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SPE (Solid Phase Extraction)

www.biotage.com/DynPage.aspx?id=35833



Chemical components contributing to flavor

Grape and wine flavor is complex and many di!erent sensorymodalities and chemical compounds influence flavor percep-tion (Table 1).5 However, aroma (smell) is the major contri-butor to overall flavor perception and this review will focuslargely on the volatile aroma compounds that contribute togrape and wine flavor.The basic processes for producing red and white wines are

shown in Fig. 1, with the main distinction being that red winesare fermented with the skins present so that more chemicalcomponents from the skins (e.g., anthocyanins, polyphenols,flavor compounds) are extracted into the juice/wine during thefermentation. The complex aromas of the final wine aretherefore derived from the grape, the yeast fermentation(typically Saccharomyces cerevisiae), any secondary microbialfermentations that occur, and the aging/storage conditions.There are clear sensory di!erences in the aromas of most

grape varieties, however the overall volatile composition ofmost varieties is similar, with the varietal aroma derivinglargely from di!erences in relative ratios of many volatilecompounds, as further discussed below. In only a few caseshave individual character impact compounds (see Fig. 2) beenidentified and associated with specific varietal aroma attributes(Table 2) (an impact compound is a single compound thatconveys the named flavor6). Most of the impact compounds

that have been identified are present at low concentrations ingrapes and wines, however because of their very low (ng L!1)sensory thresholds they can have a large impact on the overallgrape/wine aroma.In general, the fermentation-derived volatiles make up the

largest percentage of the total aroma composition of wine.Fermentation by Saccharomyces cerevisiae leads to formationof many alcohols (predominantly ethanol and the C3–C5

straight chain and branched n-alcohols, and 2-phenylethanol)

Fig. 1 White and red wine production. 1Indicates steps that are optional and/or not done on every variety or wine style. 2If skins are removed

from red grape must, a blush or rose juice is obtained; color is dependant on grape varietal and contact time with skins.

Table 1 Sensory modalities and selected chemical components con-tributing to grape and wine flavor

Sensory modality AttributeExample chemicalcompounds in wine

Taste Sweet Glucose, fructose,glycerol, ethanol

Sour Tartaric acidSalty Sodium chloride,

potassium chlorideBitter Catechin

Smell/aroma Floral, lily-of-the valleyaroma

Linalool

Banana-like aroma Isoamyl acetateChemesthesis Mouth-warming/heat EthanolTactile Viscosity Glycerol,

polysaccharidesAstringency Tannins

Vision Red Malvidin-3-glucoside

This journal is "c The Royal Society of Chemistry 2008 Chem. Soc. Rev., 2008, 37, 2478–2489 | 2479

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Wine flavor: chemistry in a glass

Pavla Polaskova, Julian Herszage and Susan E. Ebeler*

Received 13th May 2008

First published as an Advance Article on the web 12th August 2008

DOI: 10.1039/b714455p

Although hundreds of chemical compounds have been identified in grapes and wines, only a few

compounds actually contribute to sensory perception of wine flavor. This critical review focuses

on volatile compounds that contribute to wine aroma and provides an overview of recent

developments in analytical techniques for volatiles analysis, including methods used to identify the

compounds that make the greatest contributions to the overall aroma. Knowledge of volatile

composition alone is not enough to completely understand the overall wine aroma, however, due

to complex interactions of odorants with each other and with other nonvolatile matrix

components. These interactions and their impact on aroma volatility are the focus of much

current research and are also reviewed here. Finally, the sequencing of the grapevine and yeast

genomes in the past B10 years provides the opportunity for exciting multidisciplinary studies

aimed at understanding the influences of multiple genetic and environmental factors on grape and

wine flavor biochemistry and metabolism (147 references).

Introduction

From Pasteur’s discoveries of the role of microorganisms infermentation and his studies on the analytical separations ofchiral organic acids in grape juice1,2 to Kepler’s developmentof early calculus theories to measure wine barrel volumes,3

grapes and wines have provided a rich basis for many dis-coveries that have had fundamental impacts on mathematics,microbiology, and chemistry over the past several centuries.The chemistry of grape and wine flavor, in particular, has beenthe focus of much research due to the complexity of thevolatile aromas that contribute to flavor and the nuancedvariations that arise from di!erent grape varieties, growingregions, and vintage years. In the 19th and early part of the20th centuries, much of the focus of wine flavor chemistryresearch was on measuring the major components that

contribute to taste and aroma (ethanol, organic acids, sugars),the compounds associated with protecting wine quality,4 andon those compounds associated with ‘‘defects’’ or undesirablearomas such as acetic acid (which results in a vinegar aroma).As fermentation technology improved, the incidence of defectsdecreased, and in the mid-1900s flavor chemists turned theirfocus toward understanding the chemical components thatcontribute to specific sensory attributes associated withdi!erent grapes and wines and di!erent wine styles (e.g., tablewines, port, Sauternes-style wines, etc.). These studies wereenabled by important advances in the development of gaschromatography (GC) in the 1950s and the introduction ofcommercial capillary GC columns in the 1980s. In this reviewwe will first summarize the components that contribute to wineflavor, focusing on aroma components, then we present anoverview of more recent developments in analytical techniquesfor the analysis of wine volatiles, methods for relatingchemical composition to sensory perception of aroma, andthe emerging role of genomics and proteomics for under-standing aroma development in grapes.

Department of Viticulture and Enology, University of California,Davis, One Shields Avenue, Davis, CA 95616, USA.E-mail: [email protected]; Fax: +1 530-752-0382;Tel: +1 530-752-0696

Pavla Polaskova earned her PhD degreein Analytical Chemistry in 2003 from theMasaryk University Brno in the CzechRepublik. Later she worked for two yearsas a postdoctoral scholar in the ChemistryDepartment at the University ofCalifornia Davis. Three years ago shejoined the Viticulture and EnologyDepartment in UC Davis as a post-doctoral scholar, where she is focusingon aroma compounds and flavorinteractions in wine.

In 2001 Julian Herszage earned his PhDdegree in Chemistry from the Universidadde Buenos Aires in Argentina. Followinghis degree he worked for two years as apostdoctoral scholar at the Scripps Insti-tution of Oceanography in San Diego,California. He joined the Viticulture andEnology Department in UC Davis as apostdoctoral scholar in 2006. His researchfocuses on detection and quantification ofvolatile sulfur compounds in wine andunderstanding the factors that influenceformation of these important wine aromacompounds.

Susan Ebeler is a professor in the depart-ment of Viticulture and Enology at UCDavis. Her research is focused on thedevelopment and application of analyticalchemistry techniques to study grape andwine flavor chemistry, understanding thephysico-chemical interactions of flavorswith nonvolatile food and beverage com-ponents, and elucidating the chemicalmechanisms for observed health e!ectsof grape and wine components. She tea-ches undergraduate and graduate classeson Grape and Wine Analysis and FlavorChemistry of Foods and Beverages.

2478 | Chem. Soc. Rev., 2008, 37, 2478–2489 This journal is !c The Royal Society of Chemistry 2008

CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews

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Linalool





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Introduction










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Profumi, sapori e colori: aspetti chimici e sensoriali MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY








HIGHER ALCOHOLS AND ESTERS FORMED FROM YEASTS 121

TABLE 5.2. Wine Volatiles Detectable by HS – SPME Using a CAR – PDMS – DVB Fiber and Their Principal m / z Signals a

Compound m/z Compound m/z

( E ) - 2 - Nonenal 70;41;83 Ethyl octanoate 88;101;127 ( E,E ) - 2,4 - Decadienal 81;41;39 Ethyl propanoate 29;57;27 1,1,6 - Trimethyl - 1,2 -

dihydronaphthalene 157;142;172 Ethyl lactate 45;29;75

1 - Hexanol 56;43;69 Geranyl ethylether 69;93;121 1 - Octen - 3 - ol 72;57;85 Hexanoic acid 60;73,87 2 - Methyl - 1 - butanol 57;41;70 Hexyl acetate 43;56;61 2 - Octanone 43;58;71 Isoamyl acetate 43;70;55 2 - Phenylethanol 91;92;122 Isoamyl alcohol 55;42;70 2 - Phenylethyl acetate 104;43;91 Isoamyl octanoate 70;127;43 3 - Methyl - 1 - butanol 55;42;70 Isobutyl alcohol 43;41;42 Acetic acid 43;45;60 Linalool 71;93;55 cis - 3 - Hexenol 67;41;55 Linalyl ethylether b 71;43;99 cis - Furanlinalool oxide 59;94;111 Methyl decanoate 74;87;155 Decanoic acid 60;73;129 Methyl heptanoate

(I.S.) 74;43;87

Diethylsuccinate 101;129;55 Methyl hexanoate 74;87;99 Ethyl 2 - methylbutanoate 102;85;74 Methyl octanoate 74;87;127 Ethyl 3 - hexenoate 69;41;68 Octanoic acid 60;73;101 Ethyl 9 - decenoate 41;55;88 Propanol 31;29;42 Ethyl acetate 61;70;73 trans - Furanlinalool

oxide 59;43;68

Ethyl butanoate 71;43;88 Vitispiranes 192;177;121 Ethyl decanoate 88;61;155 ! - Ionone 121;93;192 Ethyl dodecanoate 88;101;183 ! - Terpineol 59;93;136 Ethyl hexanoate 88;99;60 " - Damascenone 69;121;190 Ethyl isobutanoate 43;29;71 " - Ionone 177;178;135 Ethyl isovalerate 29;57;88 Furfural 96;95;39

a EI 70 eV. Versini et al., 2008 ; Ferreira and de Pinho, 2003 ; Bosch - Fust é , 2007. b Data kindly provided by Prof. R. Di Stefano.

TABLE 5.3. Wine Volatiles Detectable by HS – SPME Using a 100 - µ m PDMS Fiber and Their Principal m / z Signals a

Compound m/z Compound m/z

( E ) - 2 - Nonenal 70;41;83 Ethyl hexanoate 88;99;60 ( E ) - Cinnamaldehyde 131;103;51 Ethyl

isobutanoate 43;29;71

( E ) - " - Ocimene 93;91;73 Ethyl isovalerate 29;57;88 ( E,E ) - 2,4 - Decadienal 81;41;39 Ethyl lactate 45;29;75 ( Z ) - 3 - Hexen - 1 - ol 67;41;55 Ethyl nonanoate 88;101;73 ( Z ) - " - Ocimene 93;91;79 Ethyl octanoate 88;101;127 1,2 - Dihydro - 1,1,6 -

trimethylnaphthalene 157;142;172 Ethyl propanoate 29;57;27

1,3 - Butanediol 43;45;57 Ethyl sorbate 67;95;41

HIGHER ALCOHOLS AND ESTERS FORMED FROM YEASTS 119

example, by synthesis of ethyl esters by reaction of the corresponding organic acid with d 5 - ethanol (Siebert et al., 2005 ). Polydimethylsiloxane (PDMS) fi bers have high affi nity for high molecular weight nonpolar compounds, such as ethyl esters and higher alcohols (Bonino et al., 2003 , Vianna and Ebeler, 2001 ; Mart í et al., 2003 ), a mixed - fi ber carbowax – divinylbenzene (CW/DVB) for C 6 and higher aliphatic alcohols and monoterpenols (Bonino et al., 2003 ). The triphase CAR/PDMS/DVB fi ber is suitable for sampling of both lower molecular weight and more polar compounds (Howard et al., 2005 ), overcoming the lack of selectiv-ity toward some compounds of the one - or two - phase fi bers (Ferreira and de Pinho, 2003 ). The HS – SPME – GC/MS chromatogram relative to analysis of wine volatiles by triphase fi ber is shown in Fig. 5.1 , the experi-mental conditions are reported in Table 5.1 . A list of wine volatiles detectable by this method is reported in Table 5.2 .

Figure 5.1. HS (headspace) – SPME – GC/MS chromatogram recorded in the analysis of a Gew ü rztraminer wine volatiles performed using a CAR – PDMS – DVB fi ber and the experimental conditions reported in Table 5.1 . ( 1 ) ethyl hexanoate; ( 2 ) 2 - and 3 - methyl - 1 - butanol (isoamyl alcohols); ( 3 ) ethyl lactate; ( 4 ) 1 - hexanol; ( 5 ) ethyl octanoate; ( 6 ) 1 - heptanol (internal standard); ( 7 ) benzaldehyde; ( 8 ) linalool; ( 9 ) ethyl decanoate; ( 10 ) diethyl succinate; ( 11 ) ! - terpineol; ( 12 ) 2 - phenylethyl acetate; ( 13 ) 2 - phenylethanol; ( 14 ) octanoic acid.

1

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8,500,000

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Profumi, sapori e colori: aspetti chimici e sensoriali TABLE 5.8. Mean Contents of Carbonyl Compounds Identifi ed in Chardonnay and Cabernet Sauvignon Wines a and Grappa Samples b

Compound ( µ g/L) c

Wine Grappa

Cabernet Sauvignon Chardonnay Cabernet Sauvignon Chardonnay Before MLF After MLF Before MLF After MLF ( µ g/100 mL a.e.) ( µ g/100 mL a.e.)

( E ) - 2 - Hexenal 3.43 0.58 0.82 1.09 71.2 73.3 ( E ) - 2 - Nonenal trace 0.18 0.18 0.29 8.7 12.8 ( E ) - 2 - Octenal n.f. 4.4 ( E ) - 2 - Pentenal 15.7 28.5 ( E,E ) - 2,6 - Nonadienal 13.78 0.27 n.f. n.f. 2,3 - Butanedione 250 2820 50 940 179.2 189.4 3 - Hydroxy - 2 - butanone 1620 4560 5030 10910 892.1 384.9 Acetaldehyde (mg/L) 52.01 5.02 46.54 67.04 33.2 20.8 Benzaldehyde 1117.2 699.5 Butyraldehyde 12.36 19.99 8.48 5.08 Decanal 0.34 1.56 1.84 4.13 Glycolaldehyde d 48.43 391.6 38.51 110.87 6.1 6.0 Glyoxal 41.33 106.01 49.11 95.92 0.2 0.1 Heptanal 3.36 0.56 0.35 1.99 52.3 116.2 Hexanal 94.63 91.67 10.79 89.94 1093.5 1563.2 Isovaleraldehyde+ 50.6 148.6 2 - Methylbutyraldehyde 25.69 45.51 45.85 24.46 11.5 18.2 Methylglyoxal d 10.96 14.37 34.73 71.55 0.3 0.2 Nonanal 1.30 1.85 1.05 2.92 11.2 32.5 Octanal 0.82 0.77 0.39 0.80 4.7 13.6 Propanal 53.8 90.4 Vanillin 1.30 1.8

a Before and after Malolactic fermentation. b Not found, n.f., a.e., anhydrous ethanol. c Amounts expressed as internal standard o - chlorobenzaldehyde (I.S.). d Quantifi ed on basis of one of the two PFBOA syn – anti oxime peaks.

132










Profumi, sapori e colori: aspetti chimici e sensoriali 98 GRAPE AROMA COMPOUNDS

Figure 4.1. Principal monotepenes in grape and wine. ( 1 ) The cis - and trans - linalool oxide (5 - ethenyltetrahydro - ! , ! ,5 - trimethyl - 2 - furanmethanol) (furanic form); ( 2 ) lin-alool (3,7 - dimethyl - 1,6 - octadien - 3 - ol); ( 3 ) ! - terpineol ( ! , ! ,4 - trimethyl - 3 - cycloexene - 1 - methanol); ( 4 ) cis - and trans - ocimenol [( E - and Z - )2,6 - dimethyl - 5,7 - octen - 2 - ol]; ( 5 ) cis - and trans - linalool oxide (6 - ethenyltetrahydro - 2,2,6 - trimethyl - 2H - pyran - 3 - ol) (pyranic form); ( 6 ) hydroxycitronellol (3,7 - dimethyloctane - 1,7 - diol); ( 7 ) 8 - hydro-xydihydrolinalool (2,6 - dimethyl - 7 - octene - 1,6 - diol); ( 8 ) 7 - hydroxygeraniol [( E ) - 3,6 - dimethyl - 2 - octene - 1,7 - diol]; ( 9 ) 7 - hydroxynerol [( Z ) - 3,6 - dimethyl - 2 - octene - 1,7 - diol]; ( 10 ) cis - and trans - 8 - hydroxylinalool [( E - and Z - ) 2,6 - dimethyl - 2,7 - octadiene - 1,6 - diol];

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Figure 4.2. Principal norisoprenoid compounds in grape and wine. ( 29 ) TDN (1,1,6 - trimethyl - 1,2 - dihydronaphthalene); ( 30 ) ! - damascone; ( 31 ) ! - damascenone; ( 32 ) vom-ifoliol; ( 33 ) dihydrovomifoliol; ( 34 ) 3 - hydroxy - ! - damascone; ( 35 ) 3 - oxo - " - ionol; ( 36 ) 3 - hydroxy - 7,8 - dihydro - ! - ionol; ( 37 ) " - ionol; ( 38 ) ! - ionol; ( 39 ) " - ionone; ( 40 ) ! - ionone; ( 41 ) actinidols; ( 42 ) vitispiranes (spiro [4.5] - 2,10,10 - trimethyl - 6 - methylene - 1 - oxa - 7 - decene); ( 43 ) Riesling acetal (2,2,6 - tetramethyl - 7,11 - dioxatricyclo[6.2.1.0 1,6 ]undec - 4 - ene).

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Figure 4.3. Principal fl avoring benzenoid compounds in grape. ( 44 ) zingerone; ( 45 ) zingerol; ( 46 ) vanillin; ( 47 ) ethyl vanillate; and ( 48 ) methyl salicylate.

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Figure 4.2. Principal norisoprenoid compounds in grape and wine. ( 29 ) TDN (1,1,6 - trimethyl - 1,2 - dihydronaphthalene); ( 30 ) ! - damascone; ( 31 ) ! - damascenone; ( 32 ) vom-ifoliol; ( 33 ) dihydrovomifoliol; ( 34 ) 3 - hydroxy - ! - damascone; ( 35 ) 3 - oxo - " - ionol; ( 36 ) 3 - hydroxy - 7,8 - dihydro - ! - ionol; ( 37 ) " - ionol; ( 38 ) ! - ionol; ( 39 ) " - ionone; ( 40 ) ! - ionone; ( 41 ) actinidols; ( 42 ) vitispiranes (spiro [4.5] - 2,10,10 - trimethyl - 6 - methylene - 1 - oxa - 7 - decene); ( 43 ) Riesling acetal (2,2,6 - tetramethyl - 7,11 - dioxatricyclo[6.2.1.0 1,6 ]undec - 4 - ene).

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and esters (predominantly ethyl acetate and isoamyl acetate).The ester 3-methylbutyl acetate appears to be an importantaroma component of many varieties18,27 however, in general,most of the fermentation-derived compounds have relativelyhigh sensory thresholds and therefore do not individuallycontribute significantly to the aroma of wines. Combined,however, their impact may be important as shown in modelsystems.28

In addition to the primary yeast fermentation, some wines(e.g., Chardonnay in the US) undergo a secondary microbialfermentation with Oenococcus oeni (also called malolacticfermentation) and as a result may contain high concentrations

of diacetyl (2,3-butanedione), which contributes a butteryaroma to these wines. The e!ects of fermentation conditionsand reviews of the biochemical processes involved information of the fermentation-derived aromas have beenreviewed by others.29–31

Finally, changes in concentrations of many aromacompounds occur during storage and wine aging. Many winesare stored or fermented in oak barrels and one of the mostimportant volatiles extracted from the wood is b-methyl-g-octalactone (known as oak- or whiskey-lactone) which con-tributes a woody, oaky, coconut-like aroma to the wine. Thiscompound occurs as two isomers, cis- and trans-, and likemany isomeric compounds, the sensory properties are depen-dent on the isomeric structure. As reviewed by Waterhouseand Towey,32 the cis-oak lactone isomer has an aromathreshold reported as 92 mg L!1, compared to 460 mg L!1

for the trans-isomer and the ratio of the two isomers varieswith oak species and origin. Interestingly, several studies haveshown that the wood can also adsorb some aroma compounds(2-phenylethanol, ethyl decanoate)33–35 changing their concen-tration in solution. These adsorption reactions appear to be afunction of the ratio of wood surface area/solution volumeand are driven by acid–base and polar characteristics of thewood rather than solubility and hydrophobicity of the studiedaroma compounds.33 Wines can also be fermented and aged instainless steel tanks leading to wines that have simpler sensoryproperties mostly due to the lack of the compounds found inwine aged in oak barrels such as lactones and some phenoliccompounds.36,37

In addition to extraction of flavor compounds from oak,chemical and microbial (e.g., Acetobacter) oxidative reactionscan make significant contributions to the flavor of aged winesas a result of formation of compounds such as acetaldehyde(nutty, sherry-like aroma) and acetic acid (vinegar aroma).While acetaldehyde can contribute desirable characteristicaromas to aged wines and Sherries, if oxidative reactions areuncontrolled they can lead to very high concentrations ofacetaldehyde and acetic acid and the overall sensory impactis undesirable.

Fig. 2 Structures of compounds from Table 2: (a) linalool, (b) geraniol,

(c) nerol, (d) IBMP, (e) cis-Rose oxide, (f) Wine lactone, (g) oaminoaceto-

phenone, (h) 4-methyl-4-mercaptopentan-2-one, (i) 4-methyl-4-

mercaptopentan-2-one, (j) 3-mercapto-1-hexanol, (k) rotundone.

Table 2 Impact odorants contributing to varietal aromas of selected wines

Varietya Characteristic odorants Odor quality Sensory threshold Ref.

Muscat Linalool, Floral 170 ng L!1 (in water) 7,8Terpenols, e.g. geraniol, nerol Citrus, floral

Riesling TDN (1,1,6-trimethyl-1,2-dihydronaphthalene) Kerosene, bottle age 20 mg L!1 9,10Cabernet Sauvignon, Sauvignon blanc,Cabernet franc, Merlot, Carmenere

3-Isobutyl-2-methoxypyrazines (IBMP) Bell pepper 2 ng L!1 (in water) 11–14

Gewurztraminer cis-Rose oxide Geranium oil, carrotleaves

200 ng L!1 15–20

Wine lactone Coconut, woody, sweet 0.02 pg L!1 (in air)Vitis labrusca, Vitis rotundifolia o-Aminoacetophenone Foxy, sweet 400 ng L!1 21,22Sauvignon blanc, Scheurebe 4-Methyl-4-mercaptopentan-2-one Blackcurrant 0.6 ng L!1 in

water–ethanol(90 : 10, w/w)

16,18

Grenache rose, Sauvignon blanc,Semillon

3-Mercapto-1-hexanol Grapefruit/citrus peel(R isomer)

50 ng L!1 23

Passion fruit (S isomer) 60 ng L!1 24Shiraz Rotundone Black pepper 16 ng L!1 (in wine) 25,26a All varieties are Vitis vinifera except where indicated.

2480 | Chem. Soc. Rev., 2008, 37, 2478–2489 This journal is "c The Royal Society of Chemistry 2008

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Linalool





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200 ng L!1 15–20



16,18



50 ng L!1 23



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200 ng L!1 15–20



16,18



50 ng L!1 23



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200 ng L!1 15–20



16,18



50 ng L!1 23



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on a point interval scale as it elutes from the GC column. Anadvantage of the TI methods relative to other GC-O methodsis that more sni!ers/assessors can evaluate the same sample ina given time span since intensity is measured during a singleGC run while the dilution methods require multiple analysesof the same sample over several dilutions to calculate thearoma dilution or Charms values. In addition, the intensityratings from multiples assessors can be statistically evaluatedusing TI methods.Another final variation, the Detection Frequency Method,

is based on the frequency of odorant detection by a panel of8–12 persons who separately sni! the GC eluent of thenondiluted extract. The individual aromagrams are recordedand the odor’s intensity is estimated based on the number ofpanelists who detect the odor (detection frequency).87–89

Limitations to the GC-O methods have been reviewed.90

Most importantly, the GC-O techniques are based onseparation of mixtures into individual components, whilehuman sensory perception of the overall aroma of a wine orother food sample is integrative and takes into account thecombined sensations of all components, including any additiveor masking e!ects that may occur when the aroma of complexmixtures is smelled. In addition, odor quality of some

compounds can change with changing concentration so thatperceptual di!erences may occur as peaks elute from the GCcolumn and as odorant concentrations in solution change. Ifpeaks are poorly resolved, odor perception may be dependenton relative concentrations of the unresolved odorants as theyelute. Finally significant di!erences in individual sensitivitiesto odorants occur, requiring careful training and standardiza-tion of GC-O protocols.91–93 However, GC-O remains apowerful tool for identifying important odorants that contri-bute to grape and wine aroma and for relating the contribu-tions of individual odorants to the di!erences among di!erentwines samples (Table 3).

Reconstitution and omission tests

As discussed previously, one of the principal drawbacks ofGC-O approaches for identifying important odorants, is thatthey consider only the impact of isolated aroma compoundsand they overlook the additive (or masking) e!ects of aromacompounds in a mixture. Therefore, once a set of potentiallyimportant odorants are identified by GC-O, additional recon-stitution tests are often performed by mixing together theseodorants at the concentrations at which they are present in the

Table 3 Important odorants in several varietal wines identified using GC-O techniques as reported in selected literature sources

Variety Most important odorants identified by Various GC-O methods Ref.

Scheurebe 4-Mercapto-4-methylpentan-2-one, ethyl 2-methylbutyrate, 3-methylbutanol,2-phenylethanol, 3-ethylphenol, 3-hydroxy-4,5-dimethyl-2(5H)-furanoneand wine lactone

16

Gewurztraminer cis-Rose oxide, ethyl 2-methylbutyrate, 3-methylbutanol, 2-phenylethanol,3-ethylphenol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone and wine lactone

16

Grenache rose 3-Mercapto-1-hexanol, furaneol, homofuraneol 23Chardonnay Ethyl butanoate, octanoic acid, 2-phenylacetaldehyde, 4-vinyphenol,

d-decalactone, 2-methyltetrahydrothiophen-3-one, 3-methylbutyl acetate,decanoic acid, 4-vinyl-2-methoxyphenol and linalool

94,95

Spanish Rioja (blend of Tempranillo,Grenache and Graciano grape varieties)

4-Ethylguaiacol, (E)-whiskey lactone, 4-ethylphenol, b-damascenone,fusel alcohols, isovaleric and hexanoic acids, eugenol, fatty acid ethyl esters,ethyl esters of isoacids, furaneol, 2-phenylacetic acid and (E)-2-hexenal

96

Zalema Mainly fatty acids and their ethyl esters, b-damascenone andb-ionone, isoamyl alcohol and 2-phenylethanol, 4-mercapto-4-methyl-2-pentanone,3-mercaptohexyl acetate, 3-mercapto-1-hexanol, acetaldehyde and 2-phenylacetaldehyde

97

Castanal b-Ionone, 3-methyl-1-butanol, benzyl alcohol, 2-phenylethanol,ethyl acetate, isoamyl acetate, ethyl lactate, ethyl butyrate, ethyl hexanoateand ethyl octanoate

98

Pinot Noir 2-Phenylethanol, 3-methyl-1-butanol, 2-methylpropanoate,ethyl butanoate, 3-methylbutyl acetate, ethyl hexanoate, benzaldehyde,

70

Cabernet Sauvignon and Merlotfrom Bordeaux

Methylbutanols, 2-phenylethanol, 2-methyl-3-sulfanylfuran,acetic acid, 3-(methylsulfanyl)propanal, methylbutanoic acids,b-damascenone, 3-sulfanylhexan-1-ol, furaneol, homofuraneol

99

Cabernet Sauvignon and Merlotfrom USA and Australia

3-Methyl-1-butanol, 3-hydroxy-2-butanone, octanal, ethyl hexanoate,ethyl 2-methylbutanoate, b-damascenone, 2-methoxyphenol,4-ethenyl-2-methoxyphenol, ethyl 3-methylbutanoate, acetic acid and 2-phenylethanol

100

Madeira (Malvazia, Boal, Verdelhoand Sercial varieties)

Sotolon, 2-phenylacetaldehyde, (Z)-whiskey lactone 101

Riesling (from Croatia) 2-Phenylethanol, 3-methyl-1-butanol, 3-(methylthio)-1-propanol,ethyl propanoate, ethyl butanoate, ethyl 3-methylbutanoate, 3-methyl-1-butanol acetate,ethyl hexanoate, ethyl octanoate, ethyl 3-hydroxybutanoate, 2-phenylethyl acetate,hexanoic acid, 3-methylbutanoic acid, butanoic acid, b-damascenone,g-undecalactone and 4-vinylguaiacol

102

Riesling (from US) b-Damascenone, 2-phenylethanol, linalool, fatty acids,ethyl 2-methyl butyrate, trans-2-hexenol, cis-3-hexenol, geraniol, ethyl butyrate, carvone,ethyl hexanoate, isoamyl acetate

103

Seyval blanc o-Aminoacetophenone, b-damascenone, C4 fatty acids,linalool, 1-octen-3-ol, vanillin

103

Vidal blanc b-Damascenone, 2-phenylethanol, methyl anthranilate, vanillin 103Cayuga White b-Damascenone, vanillin, 2-phenylethanol, geraniol, hexanal 103


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INTRODUCTION 227

TABLE 7.1. The GC / MS Qualitative and Semiquantitative Data of Volatile Compounds Identifi ed in 50% Hydroalcohol Extract of Different Types of Wood Used in Making Barrels for Wine and Spirits Aging a

Compound Acacia Chestnut Cherry Mulberry Oak

Aldehydes and Ketones

Furfural * * * Benzaldehyde * * Methylbenzaldehyde * Hydroxybenzaldehyde * * Anisaldehyde * Cinnamaldehyde * Vanillin * * * * * * * * * * Syringaldehyde * * * * * * * * * * * Coniferaldehyde * * * * * * * Acetophenone * * * * * * * Benzophenone * * Acetovanillone * * * * 3 - Methoxyacetovanillone * * 2 - Butanone - 4 - guaiacol * * 2,4 - Dihydroxybenzaldeide * * * * * 3 - Buten - 2 - one - 4 - phenyl * *

Alcohols and Phenols

! - Terpineol * * * * * * 3 - Oxo - ! - ionol * * * * * * " - Phenylethanol * * * * * Benzenepropanol * ! - Methylbenzenepropanol * * Coniferyl alcohol * Benzotriazole * * * * * * * 4 - Methylphenol * 4 - Ethylphenol * 4 - Methylguaiacol * Ethylguaiacol * Vinylguaiacol * tr * Eugenol * * * tr tr * * * Methoxyeugenol * * * * * * * 3 - Methoxyphenol * Dimethoxyphenol * * Trimethoxyphenol * * * * * * * 1,2,3 - Trimethoxybenzene * *

and furan compounds (Puech et al., 1999 ; P é rez - Coello et al., 1999 ; Guichard et al., 1995 ; Feuillat et al., 1997 ; Masson et al., 1996 ; 2000 ; Matricardi and Waterhouse, 1999 ; Hale et al., 1999 ; Sauvageot and Feuillat, 1999 ; Ibern - G ó mez et al., 2001 ; Chatonnet et al., 1992 ). Main compounds characterized by sensorial proprieties are vanillin (45)

228 COMPOUNDS RELEASED IN WINE FROM WOOD

Compound Acacia Chestnut Cherry Mulberry Oak

Acids and Esters

Ethyl benzoate * 2,5 - Dihydroxy ethyl benzoate * * Methyl salicylate * trans - ! - Methyl - " - octalactone * * * cis - ! - Methyl - " - octalactone * * * Homovanillic acid * * * * * Capronic acid * * * * * * * * Caprylic acid * * * * * * Lauric acid * * * * * * * Myristic acid * * * * * * * * * Pentadecanoic acid * * * * * * * * Palmitic acid * * * * * * * * * * * * * * Margaric acid * * * * * * * Stearic acid * * * * * * * * * * Oleic acid * * * * * * * * Linoleic acid * * * * * * * * * * Linolenic acid * * *

a (Not subjected to any toasting treatment). Data expressed as µ g/g of 1 - heptanol (internal standard). * 0.1 – 0.9 µ g/g wood; * * 1 – 10 µ g/g wood; * * * > 10 µ g/g wood; tr, trace (De Rosso et al., 2008 ).

TABLE 7.1. (Continued)

(vanilla note; sensory threshold 0.3 ppm) and eugenol (35) (clove, spicy; sensory threshold 0.5 ppm; Boidron et al., 1988 ). Toasting of wood made for making barrels induces formation of a great number of vola-tile and odoriferous compounds. In general, furan and pyran deriva-tives formed with heating wood are characterized from a toasty caramel aroma (Cutzach et al., 1997 ; Chatonnet, 1999 ). Among the compounds formed with toasting were ( 1 ) 3,5 - dihydroxy - 2 - methyl - 4 H - pyran - 4 - one, ( 2 ) 3 - hydroxy - 2 - methyl - 4 H - pyran - 4 - one or maltol, ( 3 ) 2,3 - dihydro - 3,5 - dihydroxy - 6 - methyl - 4 H - pyran - 4 - one (DDMP), ( 4 ) 4 - hydroxy - 2,5 - dimethylfuran - 3(2 H ) - one (furaneol), ( 5 ) 2,3 - dihydro - 5 - hydroxy - 6 - methyl - 4 H - pyran - 4 - one (dihydromaltol), ( 6 ) 2 - hydroxy - 3 - methyl - 2 - cyclopenten - 1 - one (or cyclotene) and 5 - (acetoxymethyl)furfural. The structures of com-pounds 1 – 6 are shown in Fig. 7.1 . Formation of these molecules in the presence of proline infers that Maillard reactions occur. The GC/MS – EI (70 eV) mass spectra of some of them are reported in the Table 7.2 . A complete list of compounds identifi ed in toasted oak wood extracts is reported in Table 7.3 .









INTRODUCTION 229

Figure 7.1. Structures of volatile compounds characterized from “ toasty caramel ” aroma released in wine from toasted woods during aging. ( 1 ) 3,5 - dihydroxy - 2 - methyl - 4 H - pyran - 4 - one; ( 2 ) 3 - hydroxy - 2 - methyl - 4 H - pyran - 4 - one; ( 3 ) 2,3 - dihydro - 3,5 - dihydroxy - 6 - methyl - 4 H - pyran - 4 - one (DDMP); ( 4 ) 4 - hydroxy - 2,5 - dimethylfuran - 3(2 H ) - one (furaneol); ( 5 ) 2,3 - dihydro - 5 - hydroxy - 6 - methyl - 4 H - pyran - 4 - one (dihydro-maltol); ( 6 ) 2 - hydroxy - 3 - methyl - 2 - cyclopenten - 1 - one (or cycloteme) (Cutzach et al., 1997 ).

CH3

CH3CH3

O

O

1 2 3

4 5 6

O

O

OHHO

HO

CH3O

O

OHHO

CH3 CH3

O

O O

OHOH

CH3O

O

OH

TABLE 7.2. Principal Fragments Observed in the GC / MS – EI (70 eV) Mass Spectra of Compounds in Fig. 7.1 With Their Relative Abundance a

Compound ( m/z )

3,5 - Dihydroxy - 2 - methyl - 4 H - pyran - 4 - one ( 1 ) 142(100), 55(37), 68(30), 43(29), 85(18), 96(11)

2,3 - Dihydro - 3,5 - dihydroxy - 6 - methyl - 4 H - pyran - 4 - one ( 3 ) 43(100), 144(36), 101(32), 73(21), 55(18)

4 - Hydroxy - 2,5 - dimethylfuran - 3(2 H ) - one ( 4 ) 43(100), 128(71), 57(64), 85(21), 55(21)

2,3 - Dihydro - 5 - hydroxy - 6 - methyl - 4 H - pyran - 4 - one ( 5 ) 43(100), 128(76), 72(26), 57(24), 85(8)

a Cutzach et al., 1997 .

Some compounds from wood may induce defects to the wine. Carbonyl compounds, such as ( E ) - 2 - nonenal, ( E ) - 2 - octenal, 3 - octen - 1 - one, and 1 - decanal are responsible for the sawdust smell sometimes found in the wine after ageing in new 225 - L oak wood barrels (bar-riques); ( E ) - 2 - nonenal is reputed as being mainly responsible for the sawdust smell of wine (Chatonnet and Dubourdieu, 1998 ).


Profumi, sapori e colori: aspetti chimici e sensoriali 124 VOLATILE AND AROMA COMPOUNDS IN WINES

Figure 5.2. Volatile sulfur compounds of wines: ( 15 ) dimethyl sulfi de, ( 16 ) ethylmer-captan, ( 17 ) diethyl sulfi de, ( 18 ) methyl thioacetate, ( 19 ) dimethyl disulfi de, ( 20 ) ethyl thioacetate, ( 21 ) diethyl disulfi de, ( 22 ) 2 - mercaptoethanol, ( 23 ) 2 - (methylthio) - 1 - ethanol, ( 24 ) 3 - (methylthio) - 1 - propanol, ( 25 ) 4 - (methylthio) - 1 - butanol, ( 26 ) 3 - mercap-tohexan - 1 - ol, ( 27 ) 4 - methyl - 4 - mercaptopentan - 2 - one, ( 28 ) 3 - mercaptohexanol acetate, ( 29 ) benzothiazole, ( 30 ) 5 - (2 - hydroxyethyl) - 4 - methylthiazole, ( 31 ) trans - 2 - methylthio-phan - 3 - ol, ( 32 ) 2 - methyltetrahydrothiophen - 3 - one.

S HS S S

O

SS

S

O

SS

OHHS S

OH

SOH

S OH

OHS

O

O SH

HO

SH

N

S

N

SOH

S

OH

CH3

SO

15 16 17 18

20 21 22 23

19

262524

292827

323130

addition of 5 - g Na 2 SO 4 to 50 mL of the sample, extraction was per-formed with 2 ! 5 mL of ethyl acetate. The GC/MS – EI (70 eV) mass spectrum of bis(2 - hydroxyethyl)disulfi de is reported in Fig. 5.3 .

5.2.2 HS – SPME – GC / MS Analysis of Volatile Sulfur Compounds

The carboxen – polydimethylsiloxane – divinylbenzene (CAR/PDMS/DVB) 50 : 30 µ m and 2 - cm length resulted in the more effi cient fi ber for the extraction of sulfur compounds with the simple sampling condi-tions (e.g., ionic strength, sample temperature, and adsorption time) (Fedrizzi et al., 2007a ). A suitable HS – solution volumes ratio is 1 : 2 (Mestres et al., 2000 ). Figure 5.4 reports the HS – SPME – GC/MS chro-matograms relative to analyses of compounds used as internal stan-dards (a) and of analytes (b) using the SPME conditions reported









THE SPME–GC/MS/MS ANALYSIS OF TCA AND TBA IN WINE 253

8.2.2 The GC / MS Analysis

Chloroanisoles (2,4 - dichloroanisole, TCA, 2,3,4,6 - tetrachloroanisole, pentachloroanisole, TCP, 2,3,4,6 - tetrachlorophenol, pentachlorophe-nol) in wines or cork stopper extracts are usually analyzed using a 5% diphenyl – 95% dimethyl polysiloxane GC column (e.g., 30 m ! 0.25 mm i.d., 0.25 - µ m fi lm thickness). By using a singular quadrupole mass spec-trometer recording signals in SIM mode, TCA is quantifi ed on the sum of signals m/z 195+197+199+210+212+214, with the last two coming from molecules containing one or two 37 Cl atoms, respectively.

The signals recorded for analysis of TCP are at m/z 196, 198 and 200, for tetrachlorophenol at m/z 229, 231, 244 and 246. By performing SPME - GC/MS - SIM single quadrupole analysis the LOD and LOQ achieved for TCA are 0.2 and 0.4 ng/L, respectively (Lizarraga et al., 2004 ). The GC/MS – electron impact (EI 70 eV) fragmentation spectra of TCA is reported in Fig. 8.7 .

A GC/MS – EI chromatogram recorded in the analysis of TCA and TCP in wine is shown in Fig. 8.8 ; below the chromatographic conditions used are reported.

The TCA can be determined using an ion trap system performing collision - induced dissociation (CID). Quantifi cation is based on the daughter ion signals of the M + • species at m/z 210 and 212 used as precur-sor ions. Depending on the system used, CID can be performed in either resonant or non - resonant mode. In the former condition, the most intense daughter ions are at m/z 195 and 197, in non - resonant mode the principal signals are at m/z 167 and 169. The CID of a wine spiked with

Figure 8.7. The GC/MS – EI fragmentation spectrum (70 eV) of 2,4,6 - trichloroanisole.

230m/z

Rel

ativ

e ab

unda

nce

2202102001901801701601501401301201101009080706050403020

OCl Cl

Cl

H3C100

50

2936

49

62 74

83

97

109

132 145160

167

181

195

210

0

258 COMPOUNDS RESPONSIBLE FOR WINE DEFECTS

Figure 8.11. The GC/MS – EI (70 eV) mass spectrum of geosmin [octahydro - 4,8 a - dimethyl - 4 a (2 H ) - naphthalenol, C 12 H 22 O, MW 82.30248]. (Reproduced from Journal of Agricultural and Food Chemistry , 2000, 48 p. 4837, Darriet et al., with permission of American Chemical Society.)

50 75 100 125 150 175 m/z

Relativeintensity

100%

4155

67 8397

125

135 149 167 182

112

HO

at m/z 58 (Darriet et al., 2000 ). Performing MS/MS analysis of the signal at m/z 112 quantifi cation of geosmin is done on the daughter ion at m/z 97. The SPME – GC/MS analysis using geosmine - d 5 as internal standard gave a LOD of 5 ng/L (Dumoulin et al., 2004 ).

8.4 ANALYSIS OF 1 - OCTEN - 3 - ONE

1 - Octen - 3 - one is a compound that can be present in relevant levels in the must obtained from mildew - infected grapes. This compound is characterized by very low sensory thresholds that can be responsible for the fungus odor in the must (Darriet et al., 2002 ). Normally, during fermentation the molecule is completely reduced to the lesser powerful 3 - octanone by the yeasts, but it was found to be present in dry wines (Culle r e et al., 2006 ).

A method proposed for determination of 1 - octen - 3 - one in wines is by performing an on cartridge derivatization of the sample followed by GC/MS/MS analysis (Culler é et al., 2006 ). A 90 - mL volume of wine is passed through a 90 - mg ethylvinylbenzene – divinylbenzene copolymer SPE cartridge and, after removing the major volatiles by washing with 9 mL of a 40% methanol/water solution containing 1% NaHCO 3 , the analyte adsorbed on the stationary phase is derivatized by passing through the cartridge 2 mL of an O - (2,3,4,5,6 - pentafl uorobenzyl) hydroxylamine (PFBOA) 5 mg/mL aqueous solution. To allow the reaction to occur, the cartridge is kept imbibed with the reagent for 15 min at room

150 VOLATILE AND AROMA COMPOUNDS IN WINES

Figure 5.11. Mechanism of AAP formation proposed by Hoenicke ( 2002b ) . (Reprinted from Journal of Chromatography A 1150, Schmarr ( 2007 ) Analysis of 2 - aminoaceto-phenone in wine using a stable isotope dilution assay and multidimensional gas chromatography – mass spectrometry, p. 79, Copyright © 2006, with permission from Elsevier.)

Figure 5.12. The MS – EI (70 eV) mass spectrum of 2 ! - aminoacetophenone (AAP).

n - heptane as a cosolvent, then dichloromethane is evaporated at room temperature in order to have a residual extract in n - heptane (Schmarr et al., 2007 ).

Alternatively, a direct - immersion SPME method with DVB/CAR/PDMS fi ber (50/30 µ m, 2 cm length) has been proposed. An aliquot of 15 mL of wine is transferred into a 20 - mL vial and equilibrated at 30 ° C for 5 min, then the fi ber is immerged into the solution for 30 min under stirring. The fi ber is then desorbed into the GC injection port at 250 ° C (Fan et al., 2007 ).

ETHYL AND VINYL PHENOLS IN WINES 145

Couto et al., 2006 ). Figure 5.10 shows a scheme for formation of ethylphenols from hydroxycinnamic acids.

Volatile phenols greatly infl uence the aroma of wine, the most important are 4 - vinylphenol (4 - VP), 4 - vinylguaiacol (4 - VG), 4 - ethylphenol (4 - EP), and 4 - ethylguaiacol (4 - EG) (Chatonnet et al., 1992 ). The 4 - EP compound was reported in wine for the fi rst time in 1967 by Webb and co - workers and its presence, together with the other phenols cited, was confi rmed in 1970 by Dubois and Brul é (Webb, 1967 ; Dubois and Brul è , 1970 ).

Oligomer proanthocyanidins inhibit Saccharomyces cerevisiae cin-namate decarboxylase (Chatonnet et al., 1990 ), justifying the very low amounts of vinylphenols in red wines; on the contrary, Brettanomyces decarboxylase is not inhibited by proanthocyanidines (Chatonnet et al., 1993 ). Formation of stable vinylphenol – anthocyanin adducts can arrest the successive formation of ethylphenols in red wines (Fulcrand et al., 1996 ). Also, derivates of vinylcatechol (Schwarz et al., 2003 ; Hayasaka and Asenstorfer, 2002 ), were found in wine.

White wines can contain vinylphenols in concentrations up to several hundreds of a microgram per liter, but they usually lack ethylphenols. On the contrary, in red wine ethylphenols can reach some milligrams per liter (Chatonnet et al., 1992; 1993 ; Chatonnet, 1993 ).

In red wines, high levels of 4 - EP are associated with disagreeable odors described as “ phenolic ” , “ leather ” , “ horse sweat ” , “ stable ” , or

Figure 5.10. Formation of ethylphenols from hydroxycinnamic acids.

OHO

OH OH

CH3OCH3OCH3O

OH

OHO

OH OH OH

Ferulic acid 4-Vinylguaiacol 4-Ethylguaiacol

p-Coumaric acid 4-Vinylphenol 4-Ethylphenol

Hydroxycinnamatedecarboxilase

Vinylphenolreductase

Hydroxycinnamatedecarboxilase

Vinylphenolreductase

Grazie per l’attenzione

Gianluca Gilardoni

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